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2,4-Decadienal does not induce genotoxic effects in in vivo micronucleus studies
Mutat Res Gen Tox En 846 (2019) 503082
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2,4-Decadienal does not induce genotoxic effects in in vivo micronucleus studies
T
Maria Bastakia, Vivian Lua, Michel Aubanelb, Thierry Cachetc, Jan Demyttenaered, Maodo Malick Diope, Sylvain Etterf, Xing Hang, Christie L. Harmanh, Shim-mo Hayashii, ⁎ Zena Keig-Shevlinj, Gerhard Krammerk, Kevin J. Renskersl, Jürgen Schnabelm, Sean V. Taylora, a
International Organization of the Flavor Industry, 1101 17th Street N.W., Suite 700, Washington, DC 20036, USA Kerry Flavours France, Zl du Plan BP 82067, 63 Avenue Jean Maubert, 06131 Grasse Cedex, France c International Organization of the Flavor Industry, Avenue des Arts 6, B-1210 Brussels, Belgium d European Flavour Association, Avenue des Arts 6, B-1210 Brussels, Belgium e V. Mane Fils, 620 Route de Grasse, 06620 Le Bar-sur Loup, France f Firmenich SA, Rue de la Bergère 7, P.O. Box 148, CH-1217 Meyrin 2, Switzerland g International Flavors & Fragrance Inc., 800 Rose Lane, Union Beach, NJ 07735, USA h Flavor and Extract Manufacturers Association, 1101 17th Street N.W., Suite 700, Washington, DC 20036, USA i Japan Flavor and Fragrance Materials Association, Sankeinihonbashi Bldg. 6F, 4-7-1 Nihonbashi-Honcho, Chuo-ku, Tokyo 103-0023, Japan j Covance Laboratories, Ltd., Otley Road, Harrogate, North Yorkshire, HG3 1PY, United Kingdom k Symrise AG, Muehlenfeldstrasse 1, 37603 Holzminden, Germany l Takasago International Corporation, 4 Volvo Drive, Rockleigh, NJ 07647, USA m Givaudan International SA, Winterthurerstrasse, 8310 Kemptthal, Switzerland b
ARTICLE INFO
ABSTRACT
Keywords: Flavoring ingredient FEMA GRAS Genotoxicity 2,4-decadienal Rat Micronucleus
2,4-Decadienal (E,E-) occurs naturally in foods and is also used as a flavoring ingredient. In vivo micronucleus studies were used to evaluate the potential for 2,4-decadienal to cause genotoxic effects. Male Han Wistar rats were dosed either by intraperitoneal injection or by gavage in two independent studies. The animals (12/group) received 25, 50, or 100 mg/kg bw of 2,4-decadienal via intraperitoneal injection, or 350, 700, or 1400 mg/kg bw via gavage. Dose-dependent decreases in the percentages of peripheral blood reticulocytes were observed in both studies, indicating that the target tissue was exposed to toxic levels of 2,4-decadienal. No induction of micronuclei in the bone marrow polychromatic erythrocytes or the peripheral blood reticulocytes was observed in either study. These results, coupled with previous mutagenicity studies, support the overall conclusion that 2,4decadienal does not present a concern for genotoxicity.
1. Introduction
Panel first evaluated 2,4-decadienal (FEMA No. 3135) under its conditions of intended use as a flavoring in 1970 [7], and re-evaluated the safety of 2,4-decadienal in 2008 as part of its cyclical reassessment of the safety of flavorings [8]. Similarly, the Joint FAO/WHO Expert Committee on Food Additives (JECFA) evaluated 2,4-decadienal (JECFA No. 1190) and concluded that it does not pose a safety concern at current estimated levels of intake [9]. Recently, the European Food Safety Authority (EFSA) evaluated 2,4decadienal [FL-no. 05.140] as a flavoring substance and concluded that
2,4-Decadienal (E,E-) (Fig. 1) is a flavoring substance with a sweet, citrus aroma. It occurs naturally in a variety of foods, including chamomile, citrus fruits, pecans, and vanilla up to concentrations of 3000 ppm [1–4]. 2,4-Decadienal is also formed endogenously in animals through lipid peroxidation of polyunsaturated fatty acids [5] and as lipid peroxidation product of vegetable, meat, and fish oils [6]. The Flavor and Extract Manufacturers Association (FEMA) Expert
Abbreviations: bw, body weight; EFSA, the European Food Safety Authority; FEMA, the Flavor and Extract Manufacturers Association; GRAS, generally recognized as safe; GSH, glutathione; ip, intraperitoneal; JECFA, the Joint FAO/WHO Expert Committee on Food Additives; MN, micronucleus (micronuclei) or micronucleated; MTD, maximum tolerated dose; NTP, National Toxicology Program; OECD, Organisation for Economic Co-Operation and Development; PCE, polychromatic erythrocytes; RET, reticulocytes ⁎ Corresponding author. E-mail address: [email protected] (S.V. Taylor). https://doi.org/10.1016/j.mrgentox.2019.503082 Received 13 May 2019; Received in revised form 8 August 2019; Accepted 8 August 2019 Available online 11 August 2019 1383-5718/ © 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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92.9% of trans,trans-isomer. The test substance complied with EFSA and JECFA specifications (purity > 89%). The test substance purity is representative of the material in commerce and consistent with the material tested by the NTP (> 93% purity of sum of isomers; no information was provided on the relative trans, trans- to trans, cisfractions) [11]. The test substance was stored at 2–8 °C under nitrogen and protected from light. The stability of the test substance formulations in the vehicle (corn oil) was not tested.
Fig. 1. Chemical structure of (2E,4E)-decadienal.
based on the available genotoxicity data at the time and published toxicity studies by the National Toxicology Program (NTP), concern for genotoxicity of 2,4-decadienal could not be eliminated [10]. The reason for concern was based on mixed and equivocal findings in the two rodent in vivo micronucleus assays reported by NTP [11]. Specifically, the frequency of micronucleated bone marrow polychromatic erythrocytes (PCE) was increased in male rats 24 h following single intraperitoneal injections of 100, 200, 400 mg/kg bw (but not 600 mg/kg bw), with no follow up confirmatory test. In male mice treated with single intraperitoneal injections of 400 or 600 mg/kg bw, increased micronuclei frequencies were reported in peripheral blood erythrocytes and bone marrow PCE at 600 mg/kg bw, 48 h later. A standard three-day injection protocol in a separate set of mice, at dose levels of 25 to 200 mg/kg bw/day, resulted in a slight and dose-related but not statistically significant increase in micronuclei in bone marrow PCE, 24 h after the last injection (peripheral blood erythrocytes were not evaluated). A micronucleus assay incorporated into the subchronic toxicity study in mice (dose range 50–800 mg/kg bw/day) produced clear negative findings in peripheral blood erythrocytes (bone marrow was not evaluated). Independent mutagenicity studies conducted by NTP in two separate laboratories showed no evidence of mutagenic activity for 2,4decadienal in four (TA100, TA1535, TA97, TA98) or six (additional strains TA102 and TA104) strains of Salmonella typhimurium with and without metabolic activation with 10% and 30% microsomal fraction from rat or hamster liver. Taking into consideration all genotoxicity data, NTP concluded that 2,4-decadienal was not mutagenic in vitro or in vivo [11]. In the NTP subchronic toxicity study in rats and mice gavaged with 2,4-decadienal up to 800 mg/kg bw/day, regenerative lesions reported in the forestomach and olfactory epithelium were associated with inflammatory activity and a NOAEL of 100 mg/kg bw/day was determined for both species [11]. The studies presented here were conducted in response to EFSA’s request for additional genotoxicity data to firmly demonstrate the absence of genotoxic potential for 2,4-decadienal. Upon review of the data in these studies in 2018, EFSA concluded that the concern for genotoxic potential could be ruled out for the flavoring substance. Additionally, EFSA noted that the dose-related decrease in the percentage of peripheral blood reticulocytes in treated rats in both studies was indicative of target tissue (bone marrow) exposure [12]. The results of these studies are discussed in the context of the overall weight of evidence for the genotoxic potential of 2,4-decadienal.
2.2.1. Dose preparations Appropriate concentrations of 2,4-decadienal were added to the corn oil vehicle and thoroughly mixed. Dose formulations were prepared fresh, stored at 15–25 °C, protected from light, and used within two hours of preparation. Dose formulations calculations were not adjusted to account for test substance purity but reflect the test material as it meets the specifications. Dose bottles were stirred continuously by magnetic stirrer before and throughout dosing to ensure homogeneity. Control animals received corn oil vehicle alone. 2.3. Animals Han Wistar rats for both administration studies were obtained from Charles River Ltd. (Margate, UK) at approximately 7–9 weeks of age. Following a five-day acclimation period, animals were randomized into two subgroups of six per dosing group (one subgroup for bone marrow analysis and one subgroup for peripheral blood analysis). Individual animals did not exceed ± 20% of the mean weight. The body weights ranged from 250.0 g to 264.8 g at study initiation for the intraperitoneal administration study, while the body weights ranged from 251.0 to 271.8 g at study initiation for the oral gavage study. The animals were housed in solid bottom cages with three animals per cage at a room temperature of 20–24 °C with a relative humidity of 45–65% and a 12-h light/12-h dark cycle. Animals were provided 5LF2 EU Rodent Diet and filtered tap water ad libitum. 2.4. Experimental design 2.4.1. Intraperitoneal administration study The dose levels tested in the range-finding study were based on the study that was previously conducted at NTP, in which statistically significant increases in micronuclei were observed in rats intraperitoneally injected once at the three lowest of the four test doses (up to 400 mg/kg bw) [11]. Therefore, in the present preliminary range-finding study, groups of Han Wistar rats (3/sex/group) were given two injections of 400 mg/kg bw at 0 and 24 h. Based on toxicity observed, subsequent lower doses of 100 and 200 mg/kg bw/day were tested (all doses were given in a volume of 5 mL/kg bw), and 100 mg/ kg was estimated to be the maximum tolerated dose (MTD) [13]. No gender differences were observed in the intraperitoneal administration dose range-finding study, and therefore only male rats were used in the main study. In the main micronucleus study, groups of male rats (12/ group) were administered doses of 0 (control), 25, 50, or 100 mg/kg bw of 2,4-decadienal in corn oil viaintraperitoneal injection at 0 and 24 h. Half of the animals in each dose group were euthanized and sampled 24 h after the final dose for bone marrow evaluation (6/group) and the other half of the animals were euthanized and sampled for peripheral blood evaluation (6/group) 48 h after the final dose. A group of 3 male rats was included in the highest dose group to be used as satellite animals for possible bioanalysis. Positive control animals (6/group) were injected with 10 mg/kg bw of cyclophosphamide at 0 and 24 h and were sampled at 24 h after the final dose for both bone marrow and peripheral blood [13].
2. Methods and materials 2.1. Study compliance The studies [13,14] were conducted at Covance Laboratories (Harrogate, United Kingdom) according to OECD Guideline 474 [15] and Standard Operating Procedure. They were also compliant with the United Kingdom Good Laboratory Practice (GLP) Monitoring Authority, Medicines and Healthcare products Regulatory Agency (MHRA) Statutory Instrument 1999/3106 as amended by Statutory Instrument 2004/ 994 (UK [16]) and OECD Principles of Good Laboratory Practice [17]. 2.2. Test substance 2,4-Decadienal [IUPAC name: (2E,4E)-deca-2,4-dienal; MW 152.23; CAS No. 25152-84-5] was supplied by the International Organization of the Flavor Industry (Geneva, Switzerland) with a purity of 97.8% by gas chromatography-mass spectrometry for the sum of two isomers, with
2.4.2. Oral gavage administration study The dose levels tested in the range-finding study were based on previously conducted NTP toxicity studies [11]. An initial dose of 2
Mutat Res Gen Tox En 846 (2019) 503082
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2000 mg/kg bw/day was administered in a volume of 10 mL/kg bw. Based on observed toxicity, one lower dose of 1400 mg/kg bw was tested and was determined to be the maximum tolerated dose (MTD). No gender differences were observed in the gavage administration dose range-finding study, and therefore only male rats were used in the main study. In the main study, groups of male rats (12/group) were administered doses of 0 (control), 350, 700, or 1400 mg/kg bw of 2,4decadienal viaoral gavage at 0 and 24 h. Bone marrow was sampled from half of the animals in each dose group (6/group), euthanized 24 h after the final dose. The other half of the animals were euthanized and sampled 48 h after the final dose for peripheral blood evaluation (6/ group). A group of 3 male rats was included in the highest dose group to be used as satellite animals for possible bioanalysis. Positive control animals (6/group) were gavaged with 10 mg/kg bw of cyclophosphamide at 0 and 24 h and were sampled at 24 h after the final dose for both bone marrow and peripheral blood [14].
2.7. Peripheral blood analysis Peripheral blood samples were collected (0.2 mL of whole blood collected in K2EDTA-containing tubes) viathe abdominal aorta. Blood samples were mixed with anticoagulant (sodium heparin in PBS) and fixed in ultra-cold methanol following the precautions and processing instructions as detailed in the protocol of the manufacturer of the MicroFlow®PLUS micronucleus analysis kit (Litron Laboratories, NY). Further post-fixation sample processing was also conducted according to manufacturer protocol, including fixative removal, transfer to longterm storage solution (LTSS), further storage in LTSS until analysis as needed, labeling, and analysis. Before analysis, LTSS samples were rewashed with HBSS containing 1% fetal bovine serum, centrifuged, and re-suspended in a small volume of remaining supernatant, and then MicroFlow®PLUS micronuclei labeling solution was added. The samples were incubated at 2–8 °C for 30 min, 37 °C for 15 min, and then at room temperature for 15 min before analysis. DNA was stained with propidium iodide solution. Blood samples were analyzed by high-speed flow cytometry using a Becton Dickinson FACSCanto II cytometer. Cells labeled with CD71-FITC antibody with green fluorescence were identified as reticulocytes (highly stained) or normochromatic erythrocytes (weakly stained); platelets were labeled with CD61-PE antibody with yellow fluorescence and were excluded from analysis. Debris was excluded from counting based on forward angle light and side scatter profiles. Where possible, at least 20,000 reticulocytes in the peripheral blood were analyzed for each sample; the number of mature erythrocytes (ME), micronucleated ME, reticulocytes (RET), micronucleated (MN) RET, and total red blood cells were counted per sample.
2.5. Observations In the main micronucleus studies of gavage and intraperitoneal administrations, the animals were examined twice daily for overall gross toxicity and general signs of good health. Clinical observations were made prior to dosing and at 0, 0.5, 1, 2, 4–6 and 8 h after dosing; clinical signs were also noted twice a day after. Body weights were recorded once during the acclimation period, once before dosing, and once before necropsy. 2.6. Bone marrow analysis The bone marrow erythrocytes of animals were collected and analyzed following the same experimental procedures for both the intraperitoneal and the oral gavage micronucleus studies. The bone marrow of both femurs was isolated with a flush solution of fetal bovine serum. The samples were filtered through cellulose columns, containing 50 mg/mL equal mix of type 50 and α-cellulose [18]. Once the majority of the 2 mL had passed through the column, a further 4 mL of serum was added to the sample tubes and loaded onto the columns. After collection through the column, the bone marrow samples were centrifuged to collect the pellets. The pellets were re-suspended in fetal bovine serum and centrifuged again. The resulting pellet was mixed with a small volume of serum to make a smear slide. A smear was made from the drop by drawing the end of a clean slide along the labeled slide. Slides were air dried, then fixed for 10 min in absolute methanol and rinsed several times in distilled water. One slide per animal was immediately stained for 5 min in 12.5 μg/mL acridine orange made up in 0.1 M phosphate buffer pH 7.4. Slides were rinsed in phosphate buffer, then dried and stored protected from light at room temperature before analysis. Unstained slides were air-dried and stored at < 10 °C with desiccant. Slides from the vehicle and positive control animals were processed in the same manner. The slides were assigned a random code and analysis was blinded with respect to treatment. The slides of at least five animals per dose group were analyzed using fluorescence microscopy; polychromatic erythrocytes (PCE) and normochromatic erythrocytes (NCE) were counted until a total of 500 PCE and NCE was reached. The following criteria were used for analysis of slides: (1) cells were of normal cell morphology; (2) areas where erythrocytes overlap were to be ignored; (3) a micronucleus (MN) was to be round or oval in shape; (4) a cell containing more than one MN was scored as a single micronucleated cell; and (5) MN which were refractive, improperly stained or not in the focal plane of the cell were judged to be artifacts and were not scored. Initially, the relative proportions of polychromatic erythrocytes (PCE) and normochromatic erythrocytes (NCE) were determined until a total of at least 500 cells (PCE plus NCE) had been analyzed. Afterward, at least 4000 PCE per animal were analyzed for an increase in micronucleus induction.
2.8. Necropsy Following both range-finding studies, rats were euthanized by intraperitoneal injection of sodium pentobarbitone followed by cervical dislocation. Any moribund animals were euthanized in the same manner and underwent macroscopic necropsy to determine the cause of morbidity/death. No further testing was conducted. At the termination of the main micronucleus studies, all animals were euthanized by exsanguination under isoflurane anesthesia and underwent necropsies in dose order. Any moribund animals were humanely sacrificed by intraperitoneal injection of sodium pentobarbitone followed by cervical dislocation. Moribund animals euthanized before study termination underwent macroscopic necropsy to determine the cause of morbidity/mortality. Aside from the bone marrow and peripheral blood samples, no other tissues were analyzed or retained for either study. The serum collected from the peripheral blood samples was frozen at −80 °C for potential future bioanalysis. 2.9. Statistical analysis For each group, inter-individual variation in the numbers of micronucleated polychromatic erythrocytes was estimated using a heterogeneity chi-square calculation [19]. The numbers of MN PCE or percentages of MN RET in each treated group were compared with the numbers in vehicle control groups by use of the Wilcoxon rank sum test [20]. The tests were interpreted with one-sided risk for increased frequency with increasing dose. Probability values of p ≤ 0.05 were accepted as significant. Also, the Jonckheere-Terpstra test for dose-response was performed [21,22]. 3. Results 3.1. Observations and body weights 3.1.1. Intraperitoneal administration study In the preliminary range-finding experiment, marked clinical signs 3
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including arched gait, ataxia, piloerection, and ptosis were observed following the first administration at the dose level of 400 mg/kg bw/ day. On Day 2, mortality was observed in all three females and two males, and the remaining male was humanely euthanized. Following the first administration of 200 mg/kg bw/day, piloerection was seen in all animals, but all rats returned to a normal state by 8 h post-dosing. Following the second administration, piloerection returned, and decreased activity was also noted. At 8 h after the second administration, the females were normal; however, piloerection was still observed in the males. On Day 3, mortality was observed in a single male at the 200 mg/kg bw/day dose level. In the remaining animals, clinical observations including piloerection, decreased activity, vocalization, ptosis, hunched posture, and lethargy, which persisted for the remaining observation period. Surviving animals showed minor decreases in body weight. At 100 mg/kg bw/day dose level, no clinical signs of toxicity were noted following the first administration. Following the second administration, clinical signs, including piloerection, decreased activity, and sunken flanks (females), were seen immediately after administration. All animals returned to a normal state by 8 h after administration. Decreases in body weight were observed in males and females at 10–14% and 4–7%, respectively. The statistical significance of the body weight decreases was not determined. From these range-finding results, 100 mg/kg bw/day was considered to be an appropriate estimate of the MTD and was therefore selected as the top dose for the main study. Two lower dose levels of 50 and 25 mg/kg bw/day were also selected. While signs of toxicity differed slightly no substantial toxicity differences1 were observed between male and female animals in the preliminary test, and only male animals were used in the main study. For all three dose groups of the main experiment, there were no signs of clinical toxicity after 2,4-decadienal administration. Mortality was observed for one vehicle control animal, although no cause of death was determined. Treatment-related changes in group mean body weights were noted for both subgroups at all dose levels compared to the vehicle controls, but statistical significance was not determined2 (Table 1a).
Table 1 Change in the subgroup mean ( ± SD) body weightsa of male Wistar rats (n = 6) administered 2,4-decadienal by a) intraperitoneal injection or b) oral gavage. Change in Body Weight (%) Dose Levels (mg/kg bw/day) a. Intraperitoneal Administration Vehicle (0) 25 50 100 b. Oral Gavage Administration Vehicle (0) 350 700 1400
Bone Marrow Subgroup
Peripheral Blood Subgroup
1.4 ± 1.35 1.2 ± 0.65 −2.7 ± 3.93 −11.1 ± 1.01
3.1 ± 2.12 2.1 ± 0.39 −0.8 ± 1.78 −8.5 ± 6.40
3.4 ± 0.76 2.4 ± 0.79 −2.1 ± 2.30 −9.3 ± 2.42
1.7 ± 0.94 3.2 ± 2.17 0.6 ± 1.91 −5.3 ± 1.35
SD, standard deviation. a Change in reported group mean body weight from Day 1 to Day 3 (bone marrow) or Day 4 (peripheral blood).
to be an appropriate estimate of the MTD for 2,4-decadienal and was therefore selected as the highest dose level for the main study. Two lower doses of 350 and 700 mg/kg bw/day were also selected. While signs of toxicity differed slightly between male and female animals in the preliminary experiment, no substantial difference in toxicity was observed, and only male animals were used in the main study2. In the main micronucleus experiment, there were no signs of clinical toxicity at any point after 2,4-decadienal administration for the 350 mg/kg bw/day dose group. Skin and fur discoloration and soft feces were observed for both subgroups dosed with 700 or 1400 mg/kg bw/ day after the second administration. No deaths were observed for any of the dose levels (including the control). Treatment-related changes in group mean body weights were noted for both subgroups compared to the vehicle controls, but statistical significance was not determined3 (Table 1b).
3.1.2. Oral gavage administration study In the preliminary range-finding experiment, no signs of toxicity were observed following the first administration at the dose level of 2000 mg/kg bw/day. Following the second administration of this dose level, prolonged clinical signs of toxicity were observed, which included anogenital soiling, piloerection, arched gait, diarrhea, and discolored feces. The male animals returned to a normal state on Day 3; however, the females still showed signs of anogenital soiling at the end of the observation period. A decrease in body weight (7–9%) was observed in all animals. At the lower dose level of 1400 mg/kg bw/day, no clinical signs of toxicity were noted following the first administration. Following the second administration, piloerection was noted in male and female animals shortly after dosing, and anogenital soiling immediately after dosing in males and 8 h later in females. While all animals returned to a normal state within 24 h (by Day 3 of dosing), they all showed a decrease in body weight between 2–10%. The statistical significance of the body weight decreases was not determined. From these results, including the severity of toxic effects seen at 2000 mg/kg bw/day, the dose of 1400 mg/kg bw/day was considered
3.2. Micronucleus analysis 3.2.1. Intraperitoneal administration study Rats administered 2,4-decadienal had group mean %PCE and %MN PCE values that were similar to the vehicle control group (Table 2a) and comparable with the laboratory’s historical vehicle control range. There was no evidence of any test substance-induced toxicity to the bone marrow from these data and no effect of 2,4-decadienal treatment on micronuclei induction in the bone marrow of treated rats. There were biologically relevant, dose-related decreases in %RET of treated rats when compared to the concurrent vehicle control group (Table 2b). These data were considered indicative of test substanceinduced toxicity to the bone marrow, where RET are produced, confirming exposure of the target cells. Group mean frequencies of %MN RET were similar to the vehicle control group for all dose levels and consistent with historical vehicle control distribution ranges. There were no statistically significant increases in micronucleus frequency for any of the 2,4-decadienal groups compared to those of the vehicle control, confirming that 2,4-decadienal did not induce MN in the reticulocytes of treated rats.
1 While differences in clinical signs were noted in the dose range-finding experiment, they were not considered significant enough to warrant the use of both sexes in the main definitive study. Significant sex differences in systemic toxicity or dose tolerability are characterized by a two-fold or greater difference in incidence. 2 Statistical significance of mean body weights is not required to be determined according to the OECD testing guideline 474 [15]. Instead, the percent change in the group mean body weights over the duration of the study are presented to show the treatment-related effects on body weight.
3.2.2. Oral gavage administration study Rats treated with 2,4-decadienal had group mean %PCE values and %MN PCE values that were similar to the vehicle control group (Table 3a) and comparable with the laboratory’s historical vehicle control range. There was no evidence of any test article-induced toxicity to the bone marrow from these data and no effect of 2,4-decadienal treatment on micronuclei induction in the bone marrow of treated rats. 4
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for potential genotoxicity when EFSA evaluated the safety of this substance. The studies presented here were conducted according to OECD Guidelines to probe the conflicting results of the NTP genotoxicity testing further and to address EFSA’s remaining concerns. In the NTP tests, 2,4-decadienal was not mutagenic in the Ames assay. In the in vivo micronucleus tests, increased frequency of MN PCE in the bone marrow was reported 24 h after single intraperitoneal injections of 2,4-decadienal in male rats and after 48 h in male mice, but not after three consecutive intraperitoneal injections in male mice. A dose-dependent but slight increase in the frequency of MN RET in the peripheral blood of mice 48 h after single injections was also reported. The increase in MN RET frequency in peripheral blood (although statistically significant) was too small to indicate a positive response. Since peripheral blood is a more appropriate tissue to assess MN at 48 h after dosing than bone marrow [23–26], and since there was no repeat testing for bone marrow assessment in either rats or mice under the same conditions to confirm the respective responses, the results of both tests were judged to be inconclusive [11]. Furthermore, no increase in the frequency of micronucleated normochromatic erythrocytes in peripheral blood was detected in mice after three months of gavage administration of 2,4-decadienal [11]. Based on these data, EFSA could not exclude potential genotoxicity and requested additional testing [10]. The studies presented here were designed to examine effects from both routes of administration (gavage and intraperitoneal), in both target tissues (bone marrow and peripheral blood), and at optimum sampling times (24 h for bone marrow and 48 h for peripheral blood). Under all test conditions, no evidence of micronuclei induction was detected up to the estimated MTD of 1400 mg/kg bw/day by gavage or up to the estimated MTD of 100 mg/kg bw/day by intraperitoneal injection. These results indicate that 2,4-decadienal does not cause chromosomal damage regardless of the route of administration in tests conducted according to OECD guidelines. A difference in MTD between studies using different routes of administration of the same test substance is not uncommon, but consistent with the more rapid and efficient systemic availability of the test substance when administered intraperitoneally compared to the oral route. The lack of genotoxic activity, clastogenic or aneugenic as indicated by the micronucleus assay, in tests using either route of administration is evidence that neither the parent compound nor any potential metabolites have genotoxic potential in vivo. Furthermore, the intraperitoneal administration also bypasses absorption barriers and reflects the activity of the substance that is ∼100% systemically available at the nominal dose levels, whereas the systemic availability of the substance administered orally is dependent on the gastrointestinal absorption efficiency. There are no available data for the gastrointestinal absorption rate of 2,4-decadienal. The manifestation of clinical signs of toxicity in the range-finding studies at a lower intraperitoneal dose level compared to the oral dose level is consistent with a considerably lower bioavailability of 2,4-decadienal from the oral route. Based on the ratio of the respective MTDs for oral and intraperitoneal administrations (1400 mg/kg bw and 100 mg/kg bw) determined in the studies presented here, the bioavailable dose is approximately 14-fold lower than the nominal dose from oral intake. While the contribution of absorption and metabolic rates cannot be parsed out without more specific data from a bioavailability study, it is possible that the difference in the MTD of 2,4-decadienal administered orally (1400 mg/kg bw) from the MTD when administered by intraperitoneal injection (100 mg/kg bw) reflects a combined impact of likely lower gastrointestinal absorption and higher hepatic sequestration with glutathione (GSH) following oral administration. Binding and sequestration of 2,4-decadienal and similar aldehydes by GSH has been previously reported [27]. Considering that the oral route is the relevant route of human exposure to 2,4-decadienal either as a natural constituent of foods or when used as a flavoring substance, the difference in MTD implies that the internal dose may be 14-fold lower than the dose estimated with the dietary methods of
Table 2 Mean ( ± SD) percentage of micronucleated a) bone marrow polychromatic erythrocytes (PCE) or b) peripheral blood reticulocytes (RET) of male Wistar rats (n = 6) after intraperitoneal administration of 2,4-decadienala. Dose Levels (mg/kg bw/day) a. Bone Marrow (PCE) Vehicle (0) 25 50 100 CPA (10) b. Peripheral Blood (RET) Vehicle (0) 25 50 100 CPA (10)
%PCE or RET (Mean ± SD)
%MN PCE or RET (Mean ± SD)
Statistical Significance
46.55 46.87 45.70 44.17 45.53
0.11 0.15 0.12 0.10 2.75
± ± ± ± ±
0.05 0.08 0.08 0.08 0.28
– NS NS NS p ≤0.01
0.12 0.10 0.11 0.09 1.05
± ± ± ± ±
0.04 0.02 0.04 0.02 0.30
– NS NS NS p ≤0.01
2.29 1.95 1.71 1.23 0.77
± ± ± ± ±
± ± ± ± ±
2.84 5.38 3.15 2.26 7.09
0.25 0.27 0.32 0.56 0.08
SD, standard deviation; NS, not significant; CPA, cyclophosphamide (positive control). a Laboratory historical control ranges (mean ± SD): % PCE = 46.55 ± 8.696; %MN PCE = 0.12 ± 0.103; %RET = 2.17 ± 0.904; % MN RET = 0.10 ± 0.041. Table 3 Mean ( ± SD) percentage of micronucleated a) bone marrow polychromatic erythrocytes (PCE) or b) peripheral blood reticulocytes (RET) of male Wistar rats (n = 6) after gavage administration of 2,4-decadienala. Dose Levels (mg/kg bw/day) a. Bone Marrow (PCE) Vehicle (0) 350 700 1400 CPA (10) b. Peripheral Blood (RET) Vehicle (0) 350 700 1400 CPA (10)
%PCE or RET (Mean ± SD)
%MN PCE or RET (Mean ± SD)
Statistical Significance
45.50 50.30 50.47 44.83 42.83
0.14 0.21 0.13 0.17 2.99
± ± ± ± ±
0.04 0.11 0.07 0.10 0.63
– NS NS NS p ≤0.01
0.14 0.15 0.15 0.06 1.22
± ± ± ± ±
0.02 0.04 0.05 0.04 0.44
– NS NS NS p ≤0.01
2.09 1.92 1.65 0.83 0.97
± ± ± ± ±
± ± ± ± ±
5.85 6.09 9.73 7.79 10.79
0.34 0.39 0.34 0.38 0.39
SD, standard deviation; NS, not significant; CPA, cyclophosphamide (positive control). a Laboratory historical control ranges (mean ± SD): % PCE = 46.55 ± 8.696; %MN PCE = 0.12 ± 0.103; %RET = 2.17 ± 0.904; % MN RET = 0.10 ± 0.041.
There were biologically relevant, dose-related decreases in %RET observed in animals gavaged with 2,4-decadienal when compared to the concurrent vehicle control group (Table 3b). As stated above, these data were considered indicative of test substance-induced toxicity to the bone marrow, confirming exposure of the target cells. Group mean frequencies of %MN RET were similar to the vehicle control group for all dose levels and were consistent with laboratory historical vehicle control distribution ranges. There were no statistically significant increases in micronuclei frequency for any of the 2,4-decadienal dose groups compared to those of the vehicle control. 4. Discussion and conclusion 2,4-Decadienal occurs naturally in a large variety of foods that are consumed widely. Genotoxicity tests conducted as part of the NTP safety testing reported conflicting results, and although the NTP concluded that 2,4-decadienal was not genotoxic, the data raised concern 5
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exposure assessment. Compared to the dose levels tested in the studies presented here, the intraperitoneal dose levels administered to animals in the NTP study were considerably higher (100, 200, 400, and 600 mg/kg bw vs. the MTD of 100 mg/kg bw in our study). In our study, toxicity at the 400 mg/kg bw was severe after a single administration and resulted in significant mortality. Toxicity was notable at 200 mg/kg bw after the first administration and, although it initially subsided slightly, it was prominent on the third day and one animal died. It is not clear whether any toxicity was observed at the dose levels tested in the NTP study. At 100 mg/kg bw/day, toxicity was manifested only after the second administration and persisted for about 8 h, but no deaths occurred. These signs of toxicity observed at 100 and 200 mg/kg bw in our rangefinding study would not have been observed after a single intraperitoneal administration in the NTP study. Indeed, the dose levels in the 3-injections protocol in the NTP study were substantially lower (25, 50, 100, and 200 mg/kg bw) and within the range of the tolerated dose levels determined in our study. No further comparisons of toxicity can be made as no detailed information about signs of toxicity was provided in the NTP studies either for rats or for mice, except that preliminary range-finding studies were conducted prior to the main micronucleus studies. In the NTP studies, neither the frequency of PCE in the bone marrow of animals treated intraperitoneally nor the frequency of RET in mice treated orally were reported to indicate the degree of target tissue toxicity. The only other obvious factor that may at least partially explain the different dose range tolerance between ours and the NTP studies is the different strains of rats used (Han Wistar in our study compared to F344/N in the NTP rat study), and it is possible that there are toxicokinetic and/or toxicodynamic differences in sensitivity to the toxicity of 2,4-decadienal between the two strains. Regardless of the species difference, it is important that genotoxicity was assessed up to the MTD in the present study. The micronucleus studies presented here included more extensive analysis compared to those reported by the NTP. First, micronucleus analysis was conducted in both bone marrow and peripheral blood cells at optimum sampling times, while the NTP studies examined only the bone marrow in rats with 24-h sampling, or both tissues in mice but both assessed at the 48 h time point (NTP, 2003). Second, larger numbers of bone marrow cells (4000 PCE/animal) were scored for micronuclei in our studies, compared to the analysis in the NTP studies (2000 PCE/animal). Third, the analysis in peripheral blood reticulocytes relies on capturing cells released from the bone marrow carrying chromosomal damage. Because micronucleated immature erythrocytes are filtered in the spleen and the fraction that escaped filtering is released into peripheral blood is lower than the total events that occurred in the bone marrow tissue, scoring in peripheral blood must compensate by scoring a larger cell population to capture lowfrequency events. However, the number of immature erythrocytes in the peripheral blood of mice that were scored in the NTP study was too low (only 1000 cells) to capture such rare events. In our studies, the flow cytometry method used for the detection of MN RET in peripheral blood allowed scoring of significantly larger numbers of cells (20,000 cells/animal). This offers increased power to detect low-frequency events compared to microscopy-based scoring. In addition, sensitivity to detect MN RET was further enhanced by scoring the younger fraction of immature erythrocytes identified by the transferrin receptor expression (CD71+ labeling) compared to the less sensitive scoring of a broader immature erythrocyte population identified by acridine orange stain, as indicated in the OECD guideline [15]. In conclusion, two robust, OECD guideline-compliant genotoxicity studies using the in vivo micronucleus assay in rats were performed to test the ability of 2,4-decadienal to cause chromosomal damage, following administration by the oral and intraperitoneal routes. No evidence of genotoxicity was found at doses up to the MTD for each route of administration and analysis of both bone marrow and peripheral blood of treated animals at optimum time points, using protocols with a
higher power to detect low incidence events of genotoxicity compared to previous reports. Under the conditions of these studies, 2,4-decadienal is not genotoxic. Additionally, based on these data, EFSA ruled out the genotoxicity concern from the intake of 2,4-decadienal when used as a flavoring substance [12]. Acknowledgments This work was supported by the International Organization of the Flavor Industry. The authors declare that there are no conflicts of interest. The authors thank Dr. Xiaodong Li for his assistance in the preparation of this manuscript. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.mrgentox.2019. 503082. References [1] C. Bruschwig, S. Rochard, A. Pierrat, A. Rouger, P. Senger-Emonnot, G. George, P. Raharivelomanana, Volatile composition and sensory properties of Vanilla x tahitensis bring new insights for vanilla quality control, J. Sci. Food Agric. 96 (2016) 848–858, https://doi.org/10.1002/jsfa.7157. [2] K.R. Cadwallader, H. Kim, S. Puangpraphant, Y. Lorjaroenphon, Changes in the aroma components of pecans during roasting, in: I. Blank, M. Wüst, C. Yeretzian (Eds.), Expression of Multidisciplinary Flavour Science, Interlaken, Switzerland, 2009, pp. 301–304, , https://doi.org/10.1021/jf902402s. [3] F. Darriet, M. Bendahou, J. Costa, A. Muselli, Chemical compositions of the essential oils of the aerial parts of Chamaemelum mixtum (L.) Alloni, J. Agric. Food Chem. 60 (2012) 1494–1502, https://doi.org/10.1021/jf203872z. [4] C.E. Quijano, J.A. Pino, Volatile compounds of round kumquat (Fortunella japonica Swingle) peel oil from Colombia, J. Essent. Oil Res. 21 (2009) 483–485, https://doi. org/10.1080/10412905.2009.9700224. [5] E.N. Frankel, W.E. Neff, D.D. Brooks, K. Fujimoto, Fluorescence formation from the interaction of DNA with lipid oxidation degradation products, Biochim. Biophys. Acta (BBA) - Lipids Lipid Metab. 919 (1987) 239–244, https://doi.org/10.1016/ 0005-2760(87)90263-3. [6] J.M. Snyder, E.N. Frankel, E. Selke, Capillary gas chromatographic analyses of headspace volatiles from vegetable oils, J. Am. Oil Chem. Soc. 62 (1985) 1675–1679, https://doi.org/10.1007/BF02541664. [7] R.L. Hall, B.L. Oser, Recent progress in the consideration of flavoring ingredients under the food additives amendment. 4. GRAS Substances, Food Technol. 24 (1970) 25–34. [8] T.B. Adams, C.L. Gavin, S.V. Taylor, W.J. Waddell, S.M. Cohen, V.J. Feron, J. Goodman, I.M. Rietjens, L.J. Marnett, P.S. Portoghese, R.L. Smith, The FEMA GRAS assessment of alpha, beta-unsaturated aldehydes and related substances used as flavor ingredients, Food Chem. Toxicol. 46 (2008) 2935–2967, https://doi.org/ 10.1016/j.fct.2008.06.082. [9] JECFA, Evaluation of Certain Food Additives and Contaminants (Sixty-first Report of the Joint FAO/WHO Expert Committee on Food Additives), (2004). [10] EFSA, Scientific Opinion on Flavouring Group Evaluation 203, Revision 1 (FGE.203Rev1): alpha,beta-Unsaturated aliphatic aldehydes and precursors from chemical subgroup 1.1.4 of FGE.19 with two or more conjugated double-bonds and with or without additional non-conjugated double bonds, EFSA J. 3626 (2014), https://doi.org/10.2903/j.efsa.2014.3626. [11] NTP, NTP Technical Report on the Toxicity Studies of 2,4-Decadienal (CAS No. 25152-84-5) Administered by Gavage to F344/N Rats and B6C3F1 Mice. National Toxicology Program Toxicity Report Series Number 76, National Institutes of Health, Public Service, US Department of Health and Human Services, 2011. [12] EFSA, Scientific Opinion on the Flavouring Group Evaluation 203, Revision 2 (FGE.203Rev2): alpha,beta-Unsaturated aliphatic aldehydes and precursors from chemical subgroup 1.1.4 of FGE.19 with two or more conjugated double-bonds and with or without additional non-conjugated double-bonds, EFSA J. 16 (2018), https://doi.org/10.2903/j.efsa.2018.5322. [13] Z. Keig-Shevlin, 2,4-Decadienal: Intraperitoneal in Vivo Micronucleus Study in the Bone Marrow and Peripheral Blood of Treated Rats, Unpublished Report, Study no. 8321404, Covance Laboratories, Harrogate, UK, 2016, pp. 1–51. [14] Z. Keig-Shevlin, 2,4-Decadienal: Oral Gavage in Vivo Micronucleus Study in the Bone Marrow and Peripheral Blood of Treated Rats, Unpublished Report, Study no. 8321403, Covance Laboratories, Harrogate, UK, 2016, pp. 1–73. [15] OECD, Test No. 474: Mammalian Erythrocyte Micronucleus Test, OECD Publishing, 2014. [16] U.K. MHRA, The Good Laboratory Practice (Codification Amendments etc.) Regulations 2004, Statutory Instrument No. 2004/994 (2004). [17] OECD, Principles of Good Laboratory Practice (GLP), OECD Publishing (revised in 1997), 1998. [18] J.T. Sun, M.J. Armstrong, S.M. Galloway, Rapid method for improving slide quality
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in the bone marrow micronucleus assay; an adapted cellulose column procedure, Mutat. Res. Toxicol. Environ. Mutagen. 439 (1999) 121–126, https://doi.org/10. 1016/S1383-5718(98)00175-2. D.P. Lovell, D. Anderson, R. Albanese, G.E. Amphlett, G. Clare, R. Ferguson, M. Richold, D.G. Papworth, J.R.K. Savage, Statistical analysis of in vivo cytogenetic assays, in: D.J. Kirkland (Ed.), Statistical Evaluation of Mutagenicity Test Data (UKEMS Sub-Committee on Guidelines for Mutagenicity Testing. Report. Part III), Cambridge University Press, 1989pp 184 232. E.L. Lehmann, Nonparametrics: Statistical Methods Based on Ranks, Ch. 1, McGraw-Hill, New York, 1975. A.R. Jonckheere, A distribution-free k-sample test against ordered alternatives, Biometrika 41 (1954) 133–145, https://doi.org/10.2307/2333011. T.J. Terpstra, The asymptotic normality and consistency of Kendall’s test against trend, when ties are present in one ranking, Indagations Math. 14 (1952) 327–333. Collaborative Study Group for the Micronucleus Test, Protocol recommended by the CSGMT/JEMS.MMS for the short-term mouse peripheral blood micronucleus test, Mutagenesis 10 (1995) 153–159, https://doi.org/10.1093/mutage/10.3.153.
[24] M. Hayashi, Micronucleus test with mouse peripheral blood erythrocytes by acridine orange supravital staining: the summary report of the 5th collaborative study of CSGMT/JEMS multiplied by MMS, Mutat. Res. Toxicol. 278 (1992) 83–98, https://doi.org/10.1016/0165-1218(92)90215-L. [25] M. Hayashi, T. Morita, Y. Kodama, T. Sofuni, M. Ishidate Jr, The micronucleus assay with mouse peripheral blood reticulocytes using acridine orange-coated slides, Mutat. Res. Lett. 245 (1990) 245–249, https://doi.org/10.1016/0165-7992(90) 90153-B. [26] K.H. Mavournin, D.H. Blakely, M.C. Cimino, M.F. Salamone, J.A. Heddle, The in vivo micronucleus assay in mammalian bone marrow and peripheral blood. A report of the US Environmental Protection Agency Gene-Tox Program, Mutat. Res. Genet. Toxicol. 239 (1990) 29–80, https://doi.org/10.1016/0165-1110(90) 90030-F. [27] C. Jankowski, V. Glaab, C. Mueller, U. Straesser, H.G. Kamp, G. Eisenbrand, Α,βUnsaturated carbonyl compounds: induction of oxidative DNA damage in mammalian cells, Mutagenesis 18 (2003) 465–470, https://doi.org/10.1093/mutage/ geg018.
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2,4-Decadienal does not induce genotoxic effects in in vivo micronucleus studies
T
Maria Bastakia, Vivian Lua, Michel Aubanelb, Thierry Cachetc, Jan Demyttenaered, Maodo Malick Diope, Sylvain Etterf, Xing Hang, Christie L. Harmanh, Shim-mo Hayashii, ⁎ Zena Keig-Shevlinj, Gerhard Krammerk, Kevin J. Renskersl, Jürgen Schnabelm, Sean V. Taylora, a
International Organization of the Flavor Industry, 1101 17th Street N.W., Suite 700, Washington, DC 20036, USA Kerry Flavours France, Zl du Plan BP 82067, 63 Avenue Jean Maubert, 06131 Grasse Cedex, France c International Organization of the Flavor Industry, Avenue des Arts 6, B-1210 Brussels, Belgium d European Flavour Association, Avenue des Arts 6, B-1210 Brussels, Belgium e V. Mane Fils, 620 Route de Grasse, 06620 Le Bar-sur Loup, France f Firmenich SA, Rue de la Bergère 7, P.O. Box 148, CH-1217 Meyrin 2, Switzerland g International Flavors & Fragrance Inc., 800 Rose Lane, Union Beach, NJ 07735, USA h Flavor and Extract Manufacturers Association, 1101 17th Street N.W., Suite 700, Washington, DC 20036, USA i Japan Flavor and Fragrance Materials Association, Sankeinihonbashi Bldg. 6F, 4-7-1 Nihonbashi-Honcho, Chuo-ku, Tokyo 103-0023, Japan j Covance Laboratories, Ltd., Otley Road, Harrogate, North Yorkshire, HG3 1PY, United Kingdom k Symrise AG, Muehlenfeldstrasse 1, 37603 Holzminden, Germany l Takasago International Corporation, 4 Volvo Drive, Rockleigh, NJ 07647, USA m Givaudan International SA, Winterthurerstrasse, 8310 Kemptthal, Switzerland b
ARTICLE INFO
ABSTRACT
Keywords: Flavoring ingredient FEMA GRAS Genotoxicity 2,4-decadienal Rat Micronucleus
2,4-Decadienal (E,E-) occurs naturally in foods and is also used as a flavoring ingredient. In vivo micronucleus studies were used to evaluate the potential for 2,4-decadienal to cause genotoxic effects. Male Han Wistar rats were dosed either by intraperitoneal injection or by gavage in two independent studies. The animals (12/group) received 25, 50, or 100 mg/kg bw of 2,4-decadienal via intraperitoneal injection, or 350, 700, or 1400 mg/kg bw via gavage. Dose-dependent decreases in the percentages of peripheral blood reticulocytes were observed in both studies, indicating that the target tissue was exposed to toxic levels of 2,4-decadienal. No induction of micronuclei in the bone marrow polychromatic erythrocytes or the peripheral blood reticulocytes was observed in either study. These results, coupled with previous mutagenicity studies, support the overall conclusion that 2,4decadienal does not present a concern for genotoxicity.
1. Introduction
Panel first evaluated 2,4-decadienal (FEMA No. 3135) under its conditions of intended use as a flavoring in 1970 [7], and re-evaluated the safety of 2,4-decadienal in 2008 as part of its cyclical reassessment of the safety of flavorings [8]. Similarly, the Joint FAO/WHO Expert Committee on Food Additives (JECFA) evaluated 2,4-decadienal (JECFA No. 1190) and concluded that it does not pose a safety concern at current estimated levels of intake [9]. Recently, the European Food Safety Authority (EFSA) evaluated 2,4decadienal [FL-no. 05.140] as a flavoring substance and concluded that
2,4-Decadienal (E,E-) (Fig. 1) is a flavoring substance with a sweet, citrus aroma. It occurs naturally in a variety of foods, including chamomile, citrus fruits, pecans, and vanilla up to concentrations of 3000 ppm [1–4]. 2,4-Decadienal is also formed endogenously in animals through lipid peroxidation of polyunsaturated fatty acids [5] and as lipid peroxidation product of vegetable, meat, and fish oils [6]. The Flavor and Extract Manufacturers Association (FEMA) Expert
Abbreviations: bw, body weight; EFSA, the European Food Safety Authority; FEMA, the Flavor and Extract Manufacturers Association; GRAS, generally recognized as safe; GSH, glutathione; ip, intraperitoneal; JECFA, the Joint FAO/WHO Expert Committee on Food Additives; MN, micronucleus (micronuclei) or micronucleated; MTD, maximum tolerated dose; NTP, National Toxicology Program; OECD, Organisation for Economic Co-Operation and Development; PCE, polychromatic erythrocytes; RET, reticulocytes ⁎ Corresponding author. E-mail address: [email protected] (S.V. Taylor). https://doi.org/10.1016/j.mrgentox.2019.503082 Received 13 May 2019; Received in revised form 8 August 2019; Accepted 8 August 2019 Available online 11 August 2019 1383-5718/ © 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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92.9% of trans,trans-isomer. The test substance complied with EFSA and JECFA specifications (purity > 89%). The test substance purity is representative of the material in commerce and consistent with the material tested by the NTP (> 93% purity of sum of isomers; no information was provided on the relative trans, trans- to trans, cisfractions) [11]. The test substance was stored at 2–8 °C under nitrogen and protected from light. The stability of the test substance formulations in the vehicle (corn oil) was not tested.
Fig. 1. Chemical structure of (2E,4E)-decadienal.
based on the available genotoxicity data at the time and published toxicity studies by the National Toxicology Program (NTP), concern for genotoxicity of 2,4-decadienal could not be eliminated [10]. The reason for concern was based on mixed and equivocal findings in the two rodent in vivo micronucleus assays reported by NTP [11]. Specifically, the frequency of micronucleated bone marrow polychromatic erythrocytes (PCE) was increased in male rats 24 h following single intraperitoneal injections of 100, 200, 400 mg/kg bw (but not 600 mg/kg bw), with no follow up confirmatory test. In male mice treated with single intraperitoneal injections of 400 or 600 mg/kg bw, increased micronuclei frequencies were reported in peripheral blood erythrocytes and bone marrow PCE at 600 mg/kg bw, 48 h later. A standard three-day injection protocol in a separate set of mice, at dose levels of 25 to 200 mg/kg bw/day, resulted in a slight and dose-related but not statistically significant increase in micronuclei in bone marrow PCE, 24 h after the last injection (peripheral blood erythrocytes were not evaluated). A micronucleus assay incorporated into the subchronic toxicity study in mice (dose range 50–800 mg/kg bw/day) produced clear negative findings in peripheral blood erythrocytes (bone marrow was not evaluated). Independent mutagenicity studies conducted by NTP in two separate laboratories showed no evidence of mutagenic activity for 2,4decadienal in four (TA100, TA1535, TA97, TA98) or six (additional strains TA102 and TA104) strains of Salmonella typhimurium with and without metabolic activation with 10% and 30% microsomal fraction from rat or hamster liver. Taking into consideration all genotoxicity data, NTP concluded that 2,4-decadienal was not mutagenic in vitro or in vivo [11]. In the NTP subchronic toxicity study in rats and mice gavaged with 2,4-decadienal up to 800 mg/kg bw/day, regenerative lesions reported in the forestomach and olfactory epithelium were associated with inflammatory activity and a NOAEL of 100 mg/kg bw/day was determined for both species [11]. The studies presented here were conducted in response to EFSA’s request for additional genotoxicity data to firmly demonstrate the absence of genotoxic potential for 2,4-decadienal. Upon review of the data in these studies in 2018, EFSA concluded that the concern for genotoxic potential could be ruled out for the flavoring substance. Additionally, EFSA noted that the dose-related decrease in the percentage of peripheral blood reticulocytes in treated rats in both studies was indicative of target tissue (bone marrow) exposure [12]. The results of these studies are discussed in the context of the overall weight of evidence for the genotoxic potential of 2,4-decadienal.
2.2.1. Dose preparations Appropriate concentrations of 2,4-decadienal were added to the corn oil vehicle and thoroughly mixed. Dose formulations were prepared fresh, stored at 15–25 °C, protected from light, and used within two hours of preparation. Dose formulations calculations were not adjusted to account for test substance purity but reflect the test material as it meets the specifications. Dose bottles were stirred continuously by magnetic stirrer before and throughout dosing to ensure homogeneity. Control animals received corn oil vehicle alone. 2.3. Animals Han Wistar rats for both administration studies were obtained from Charles River Ltd. (Margate, UK) at approximately 7–9 weeks of age. Following a five-day acclimation period, animals were randomized into two subgroups of six per dosing group (one subgroup for bone marrow analysis and one subgroup for peripheral blood analysis). Individual animals did not exceed ± 20% of the mean weight. The body weights ranged from 250.0 g to 264.8 g at study initiation for the intraperitoneal administration study, while the body weights ranged from 251.0 to 271.8 g at study initiation for the oral gavage study. The animals were housed in solid bottom cages with three animals per cage at a room temperature of 20–24 °C with a relative humidity of 45–65% and a 12-h light/12-h dark cycle. Animals were provided 5LF2 EU Rodent Diet and filtered tap water ad libitum. 2.4. Experimental design 2.4.1. Intraperitoneal administration study The dose levels tested in the range-finding study were based on the study that was previously conducted at NTP, in which statistically significant increases in micronuclei were observed in rats intraperitoneally injected once at the three lowest of the four test doses (up to 400 mg/kg bw) [11]. Therefore, in the present preliminary range-finding study, groups of Han Wistar rats (3/sex/group) were given two injections of 400 mg/kg bw at 0 and 24 h. Based on toxicity observed, subsequent lower doses of 100 and 200 mg/kg bw/day were tested (all doses were given in a volume of 5 mL/kg bw), and 100 mg/ kg was estimated to be the maximum tolerated dose (MTD) [13]. No gender differences were observed in the intraperitoneal administration dose range-finding study, and therefore only male rats were used in the main study. In the main micronucleus study, groups of male rats (12/ group) were administered doses of 0 (control), 25, 50, or 100 mg/kg bw of 2,4-decadienal in corn oil viaintraperitoneal injection at 0 and 24 h. Half of the animals in each dose group were euthanized and sampled 24 h after the final dose for bone marrow evaluation (6/group) and the other half of the animals were euthanized and sampled for peripheral blood evaluation (6/group) 48 h after the final dose. A group of 3 male rats was included in the highest dose group to be used as satellite animals for possible bioanalysis. Positive control animals (6/group) were injected with 10 mg/kg bw of cyclophosphamide at 0 and 24 h and were sampled at 24 h after the final dose for both bone marrow and peripheral blood [13].
2. Methods and materials 2.1. Study compliance The studies [13,14] were conducted at Covance Laboratories (Harrogate, United Kingdom) according to OECD Guideline 474 [15] and Standard Operating Procedure. They were also compliant with the United Kingdom Good Laboratory Practice (GLP) Monitoring Authority, Medicines and Healthcare products Regulatory Agency (MHRA) Statutory Instrument 1999/3106 as amended by Statutory Instrument 2004/ 994 (UK [16]) and OECD Principles of Good Laboratory Practice [17]. 2.2. Test substance 2,4-Decadienal [IUPAC name: (2E,4E)-deca-2,4-dienal; MW 152.23; CAS No. 25152-84-5] was supplied by the International Organization of the Flavor Industry (Geneva, Switzerland) with a purity of 97.8% by gas chromatography-mass spectrometry for the sum of two isomers, with
2.4.2. Oral gavage administration study The dose levels tested in the range-finding study were based on previously conducted NTP toxicity studies [11]. An initial dose of 2
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2000 mg/kg bw/day was administered in a volume of 10 mL/kg bw. Based on observed toxicity, one lower dose of 1400 mg/kg bw was tested and was determined to be the maximum tolerated dose (MTD). No gender differences were observed in the gavage administration dose range-finding study, and therefore only male rats were used in the main study. In the main study, groups of male rats (12/group) were administered doses of 0 (control), 350, 700, or 1400 mg/kg bw of 2,4decadienal viaoral gavage at 0 and 24 h. Bone marrow was sampled from half of the animals in each dose group (6/group), euthanized 24 h after the final dose. The other half of the animals were euthanized and sampled 48 h after the final dose for peripheral blood evaluation (6/ group). A group of 3 male rats was included in the highest dose group to be used as satellite animals for possible bioanalysis. Positive control animals (6/group) were gavaged with 10 mg/kg bw of cyclophosphamide at 0 and 24 h and were sampled at 24 h after the final dose for both bone marrow and peripheral blood [14].
2.7. Peripheral blood analysis Peripheral blood samples were collected (0.2 mL of whole blood collected in K2EDTA-containing tubes) viathe abdominal aorta. Blood samples were mixed with anticoagulant (sodium heparin in PBS) and fixed in ultra-cold methanol following the precautions and processing instructions as detailed in the protocol of the manufacturer of the MicroFlow®PLUS micronucleus analysis kit (Litron Laboratories, NY). Further post-fixation sample processing was also conducted according to manufacturer protocol, including fixative removal, transfer to longterm storage solution (LTSS), further storage in LTSS until analysis as needed, labeling, and analysis. Before analysis, LTSS samples were rewashed with HBSS containing 1% fetal bovine serum, centrifuged, and re-suspended in a small volume of remaining supernatant, and then MicroFlow®PLUS micronuclei labeling solution was added. The samples were incubated at 2–8 °C for 30 min, 37 °C for 15 min, and then at room temperature for 15 min before analysis. DNA was stained with propidium iodide solution. Blood samples were analyzed by high-speed flow cytometry using a Becton Dickinson FACSCanto II cytometer. Cells labeled with CD71-FITC antibody with green fluorescence were identified as reticulocytes (highly stained) or normochromatic erythrocytes (weakly stained); platelets were labeled with CD61-PE antibody with yellow fluorescence and were excluded from analysis. Debris was excluded from counting based on forward angle light and side scatter profiles. Where possible, at least 20,000 reticulocytes in the peripheral blood were analyzed for each sample; the number of mature erythrocytes (ME), micronucleated ME, reticulocytes (RET), micronucleated (MN) RET, and total red blood cells were counted per sample.
2.5. Observations In the main micronucleus studies of gavage and intraperitoneal administrations, the animals were examined twice daily for overall gross toxicity and general signs of good health. Clinical observations were made prior to dosing and at 0, 0.5, 1, 2, 4–6 and 8 h after dosing; clinical signs were also noted twice a day after. Body weights were recorded once during the acclimation period, once before dosing, and once before necropsy. 2.6. Bone marrow analysis The bone marrow erythrocytes of animals were collected and analyzed following the same experimental procedures for both the intraperitoneal and the oral gavage micronucleus studies. The bone marrow of both femurs was isolated with a flush solution of fetal bovine serum. The samples were filtered through cellulose columns, containing 50 mg/mL equal mix of type 50 and α-cellulose [18]. Once the majority of the 2 mL had passed through the column, a further 4 mL of serum was added to the sample tubes and loaded onto the columns. After collection through the column, the bone marrow samples were centrifuged to collect the pellets. The pellets were re-suspended in fetal bovine serum and centrifuged again. The resulting pellet was mixed with a small volume of serum to make a smear slide. A smear was made from the drop by drawing the end of a clean slide along the labeled slide. Slides were air dried, then fixed for 10 min in absolute methanol and rinsed several times in distilled water. One slide per animal was immediately stained for 5 min in 12.5 μg/mL acridine orange made up in 0.1 M phosphate buffer pH 7.4. Slides were rinsed in phosphate buffer, then dried and stored protected from light at room temperature before analysis. Unstained slides were air-dried and stored at < 10 °C with desiccant. Slides from the vehicle and positive control animals were processed in the same manner. The slides were assigned a random code and analysis was blinded with respect to treatment. The slides of at least five animals per dose group were analyzed using fluorescence microscopy; polychromatic erythrocytes (PCE) and normochromatic erythrocytes (NCE) were counted until a total of 500 PCE and NCE was reached. The following criteria were used for analysis of slides: (1) cells were of normal cell morphology; (2) areas where erythrocytes overlap were to be ignored; (3) a micronucleus (MN) was to be round or oval in shape; (4) a cell containing more than one MN was scored as a single micronucleated cell; and (5) MN which were refractive, improperly stained or not in the focal plane of the cell were judged to be artifacts and were not scored. Initially, the relative proportions of polychromatic erythrocytes (PCE) and normochromatic erythrocytes (NCE) were determined until a total of at least 500 cells (PCE plus NCE) had been analyzed. Afterward, at least 4000 PCE per animal were analyzed for an increase in micronucleus induction.
2.8. Necropsy Following both range-finding studies, rats were euthanized by intraperitoneal injection of sodium pentobarbitone followed by cervical dislocation. Any moribund animals were euthanized in the same manner and underwent macroscopic necropsy to determine the cause of morbidity/death. No further testing was conducted. At the termination of the main micronucleus studies, all animals were euthanized by exsanguination under isoflurane anesthesia and underwent necropsies in dose order. Any moribund animals were humanely sacrificed by intraperitoneal injection of sodium pentobarbitone followed by cervical dislocation. Moribund animals euthanized before study termination underwent macroscopic necropsy to determine the cause of morbidity/mortality. Aside from the bone marrow and peripheral blood samples, no other tissues were analyzed or retained for either study. The serum collected from the peripheral blood samples was frozen at −80 °C for potential future bioanalysis. 2.9. Statistical analysis For each group, inter-individual variation in the numbers of micronucleated polychromatic erythrocytes was estimated using a heterogeneity chi-square calculation [19]. The numbers of MN PCE or percentages of MN RET in each treated group were compared with the numbers in vehicle control groups by use of the Wilcoxon rank sum test [20]. The tests were interpreted with one-sided risk for increased frequency with increasing dose. Probability values of p ≤ 0.05 were accepted as significant. Also, the Jonckheere-Terpstra test for dose-response was performed [21,22]. 3. Results 3.1. Observations and body weights 3.1.1. Intraperitoneal administration study In the preliminary range-finding experiment, marked clinical signs 3
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including arched gait, ataxia, piloerection, and ptosis were observed following the first administration at the dose level of 400 mg/kg bw/ day. On Day 2, mortality was observed in all three females and two males, and the remaining male was humanely euthanized. Following the first administration of 200 mg/kg bw/day, piloerection was seen in all animals, but all rats returned to a normal state by 8 h post-dosing. Following the second administration, piloerection returned, and decreased activity was also noted. At 8 h after the second administration, the females were normal; however, piloerection was still observed in the males. On Day 3, mortality was observed in a single male at the 200 mg/kg bw/day dose level. In the remaining animals, clinical observations including piloerection, decreased activity, vocalization, ptosis, hunched posture, and lethargy, which persisted for the remaining observation period. Surviving animals showed minor decreases in body weight. At 100 mg/kg bw/day dose level, no clinical signs of toxicity were noted following the first administration. Following the second administration, clinical signs, including piloerection, decreased activity, and sunken flanks (females), were seen immediately after administration. All animals returned to a normal state by 8 h after administration. Decreases in body weight were observed in males and females at 10–14% and 4–7%, respectively. The statistical significance of the body weight decreases was not determined. From these range-finding results, 100 mg/kg bw/day was considered to be an appropriate estimate of the MTD and was therefore selected as the top dose for the main study. Two lower dose levels of 50 and 25 mg/kg bw/day were also selected. While signs of toxicity differed slightly no substantial toxicity differences1 were observed between male and female animals in the preliminary test, and only male animals were used in the main study. For all three dose groups of the main experiment, there were no signs of clinical toxicity after 2,4-decadienal administration. Mortality was observed for one vehicle control animal, although no cause of death was determined. Treatment-related changes in group mean body weights were noted for both subgroups at all dose levels compared to the vehicle controls, but statistical significance was not determined2 (Table 1a).
Table 1 Change in the subgroup mean ( ± SD) body weightsa of male Wistar rats (n = 6) administered 2,4-decadienal by a) intraperitoneal injection or b) oral gavage. Change in Body Weight (%) Dose Levels (mg/kg bw/day) a. Intraperitoneal Administration Vehicle (0) 25 50 100 b. Oral Gavage Administration Vehicle (0) 350 700 1400
Bone Marrow Subgroup
Peripheral Blood Subgroup
1.4 ± 1.35 1.2 ± 0.65 −2.7 ± 3.93 −11.1 ± 1.01
3.1 ± 2.12 2.1 ± 0.39 −0.8 ± 1.78 −8.5 ± 6.40
3.4 ± 0.76 2.4 ± 0.79 −2.1 ± 2.30 −9.3 ± 2.42
1.7 ± 0.94 3.2 ± 2.17 0.6 ± 1.91 −5.3 ± 1.35
SD, standard deviation. a Change in reported group mean body weight from Day 1 to Day 3 (bone marrow) or Day 4 (peripheral blood).
to be an appropriate estimate of the MTD for 2,4-decadienal and was therefore selected as the highest dose level for the main study. Two lower doses of 350 and 700 mg/kg bw/day were also selected. While signs of toxicity differed slightly between male and female animals in the preliminary experiment, no substantial difference in toxicity was observed, and only male animals were used in the main study2. In the main micronucleus experiment, there were no signs of clinical toxicity at any point after 2,4-decadienal administration for the 350 mg/kg bw/day dose group. Skin and fur discoloration and soft feces were observed for both subgroups dosed with 700 or 1400 mg/kg bw/ day after the second administration. No deaths were observed for any of the dose levels (including the control). Treatment-related changes in group mean body weights were noted for both subgroups compared to the vehicle controls, but statistical significance was not determined3 (Table 1b).
3.1.2. Oral gavage administration study In the preliminary range-finding experiment, no signs of toxicity were observed following the first administration at the dose level of 2000 mg/kg bw/day. Following the second administration of this dose level, prolonged clinical signs of toxicity were observed, which included anogenital soiling, piloerection, arched gait, diarrhea, and discolored feces. The male animals returned to a normal state on Day 3; however, the females still showed signs of anogenital soiling at the end of the observation period. A decrease in body weight (7–9%) was observed in all animals. At the lower dose level of 1400 mg/kg bw/day, no clinical signs of toxicity were noted following the first administration. Following the second administration, piloerection was noted in male and female animals shortly after dosing, and anogenital soiling immediately after dosing in males and 8 h later in females. While all animals returned to a normal state within 24 h (by Day 3 of dosing), they all showed a decrease in body weight between 2–10%. The statistical significance of the body weight decreases was not determined. From these results, including the severity of toxic effects seen at 2000 mg/kg bw/day, the dose of 1400 mg/kg bw/day was considered
3.2. Micronucleus analysis 3.2.1. Intraperitoneal administration study Rats administered 2,4-decadienal had group mean %PCE and %MN PCE values that were similar to the vehicle control group (Table 2a) and comparable with the laboratory’s historical vehicle control range. There was no evidence of any test substance-induced toxicity to the bone marrow from these data and no effect of 2,4-decadienal treatment on micronuclei induction in the bone marrow of treated rats. There were biologically relevant, dose-related decreases in %RET of treated rats when compared to the concurrent vehicle control group (Table 2b). These data were considered indicative of test substanceinduced toxicity to the bone marrow, where RET are produced, confirming exposure of the target cells. Group mean frequencies of %MN RET were similar to the vehicle control group for all dose levels and consistent with historical vehicle control distribution ranges. There were no statistically significant increases in micronucleus frequency for any of the 2,4-decadienal groups compared to those of the vehicle control, confirming that 2,4-decadienal did not induce MN in the reticulocytes of treated rats.
1 While differences in clinical signs were noted in the dose range-finding experiment, they were not considered significant enough to warrant the use of both sexes in the main definitive study. Significant sex differences in systemic toxicity or dose tolerability are characterized by a two-fold or greater difference in incidence. 2 Statistical significance of mean body weights is not required to be determined according to the OECD testing guideline 474 [15]. Instead, the percent change in the group mean body weights over the duration of the study are presented to show the treatment-related effects on body weight.
3.2.2. Oral gavage administration study Rats treated with 2,4-decadienal had group mean %PCE values and %MN PCE values that were similar to the vehicle control group (Table 3a) and comparable with the laboratory’s historical vehicle control range. There was no evidence of any test article-induced toxicity to the bone marrow from these data and no effect of 2,4-decadienal treatment on micronuclei induction in the bone marrow of treated rats. 4
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for potential genotoxicity when EFSA evaluated the safety of this substance. The studies presented here were conducted according to OECD Guidelines to probe the conflicting results of the NTP genotoxicity testing further and to address EFSA’s remaining concerns. In the NTP tests, 2,4-decadienal was not mutagenic in the Ames assay. In the in vivo micronucleus tests, increased frequency of MN PCE in the bone marrow was reported 24 h after single intraperitoneal injections of 2,4-decadienal in male rats and after 48 h in male mice, but not after three consecutive intraperitoneal injections in male mice. A dose-dependent but slight increase in the frequency of MN RET in the peripheral blood of mice 48 h after single injections was also reported. The increase in MN RET frequency in peripheral blood (although statistically significant) was too small to indicate a positive response. Since peripheral blood is a more appropriate tissue to assess MN at 48 h after dosing than bone marrow [23–26], and since there was no repeat testing for bone marrow assessment in either rats or mice under the same conditions to confirm the respective responses, the results of both tests were judged to be inconclusive [11]. Furthermore, no increase in the frequency of micronucleated normochromatic erythrocytes in peripheral blood was detected in mice after three months of gavage administration of 2,4-decadienal [11]. Based on these data, EFSA could not exclude potential genotoxicity and requested additional testing [10]. The studies presented here were designed to examine effects from both routes of administration (gavage and intraperitoneal), in both target tissues (bone marrow and peripheral blood), and at optimum sampling times (24 h for bone marrow and 48 h for peripheral blood). Under all test conditions, no evidence of micronuclei induction was detected up to the estimated MTD of 1400 mg/kg bw/day by gavage or up to the estimated MTD of 100 mg/kg bw/day by intraperitoneal injection. These results indicate that 2,4-decadienal does not cause chromosomal damage regardless of the route of administration in tests conducted according to OECD guidelines. A difference in MTD between studies using different routes of administration of the same test substance is not uncommon, but consistent with the more rapid and efficient systemic availability of the test substance when administered intraperitoneally compared to the oral route. The lack of genotoxic activity, clastogenic or aneugenic as indicated by the micronucleus assay, in tests using either route of administration is evidence that neither the parent compound nor any potential metabolites have genotoxic potential in vivo. Furthermore, the intraperitoneal administration also bypasses absorption barriers and reflects the activity of the substance that is ∼100% systemically available at the nominal dose levels, whereas the systemic availability of the substance administered orally is dependent on the gastrointestinal absorption efficiency. There are no available data for the gastrointestinal absorption rate of 2,4-decadienal. The manifestation of clinical signs of toxicity in the range-finding studies at a lower intraperitoneal dose level compared to the oral dose level is consistent with a considerably lower bioavailability of 2,4-decadienal from the oral route. Based on the ratio of the respective MTDs for oral and intraperitoneal administrations (1400 mg/kg bw and 100 mg/kg bw) determined in the studies presented here, the bioavailable dose is approximately 14-fold lower than the nominal dose from oral intake. While the contribution of absorption and metabolic rates cannot be parsed out without more specific data from a bioavailability study, it is possible that the difference in the MTD of 2,4-decadienal administered orally (1400 mg/kg bw) from the MTD when administered by intraperitoneal injection (100 mg/kg bw) reflects a combined impact of likely lower gastrointestinal absorption and higher hepatic sequestration with glutathione (GSH) following oral administration. Binding and sequestration of 2,4-decadienal and similar aldehydes by GSH has been previously reported [27]. Considering that the oral route is the relevant route of human exposure to 2,4-decadienal either as a natural constituent of foods or when used as a flavoring substance, the difference in MTD implies that the internal dose may be 14-fold lower than the dose estimated with the dietary methods of
Table 2 Mean ( ± SD) percentage of micronucleated a) bone marrow polychromatic erythrocytes (PCE) or b) peripheral blood reticulocytes (RET) of male Wistar rats (n = 6) after intraperitoneal administration of 2,4-decadienala. Dose Levels (mg/kg bw/day) a. Bone Marrow (PCE) Vehicle (0) 25 50 100 CPA (10) b. Peripheral Blood (RET) Vehicle (0) 25 50 100 CPA (10)
%PCE or RET (Mean ± SD)
%MN PCE or RET (Mean ± SD)
Statistical Significance
46.55 46.87 45.70 44.17 45.53
0.11 0.15 0.12 0.10 2.75
± ± ± ± ±
0.05 0.08 0.08 0.08 0.28
– NS NS NS p ≤0.01
0.12 0.10 0.11 0.09 1.05
± ± ± ± ±
0.04 0.02 0.04 0.02 0.30
– NS NS NS p ≤0.01
2.29 1.95 1.71 1.23 0.77
± ± ± ± ±
± ± ± ± ±
2.84 5.38 3.15 2.26 7.09
0.25 0.27 0.32 0.56 0.08
SD, standard deviation; NS, not significant; CPA, cyclophosphamide (positive control). a Laboratory historical control ranges (mean ± SD): % PCE = 46.55 ± 8.696; %MN PCE = 0.12 ± 0.103; %RET = 2.17 ± 0.904; % MN RET = 0.10 ± 0.041. Table 3 Mean ( ± SD) percentage of micronucleated a) bone marrow polychromatic erythrocytes (PCE) or b) peripheral blood reticulocytes (RET) of male Wistar rats (n = 6) after gavage administration of 2,4-decadienala. Dose Levels (mg/kg bw/day) a. Bone Marrow (PCE) Vehicle (0) 350 700 1400 CPA (10) b. Peripheral Blood (RET) Vehicle (0) 350 700 1400 CPA (10)
%PCE or RET (Mean ± SD)
%MN PCE or RET (Mean ± SD)
Statistical Significance
45.50 50.30 50.47 44.83 42.83
0.14 0.21 0.13 0.17 2.99
± ± ± ± ±
0.04 0.11 0.07 0.10 0.63
– NS NS NS p ≤0.01
0.14 0.15 0.15 0.06 1.22
± ± ± ± ±
0.02 0.04 0.05 0.04 0.44
– NS NS NS p ≤0.01
2.09 1.92 1.65 0.83 0.97
± ± ± ± ±
± ± ± ± ±
5.85 6.09 9.73 7.79 10.79
0.34 0.39 0.34 0.38 0.39
SD, standard deviation; NS, not significant; CPA, cyclophosphamide (positive control). a Laboratory historical control ranges (mean ± SD): % PCE = 46.55 ± 8.696; %MN PCE = 0.12 ± 0.103; %RET = 2.17 ± 0.904; % MN RET = 0.10 ± 0.041.
There were biologically relevant, dose-related decreases in %RET observed in animals gavaged with 2,4-decadienal when compared to the concurrent vehicle control group (Table 3b). As stated above, these data were considered indicative of test substance-induced toxicity to the bone marrow, confirming exposure of the target cells. Group mean frequencies of %MN RET were similar to the vehicle control group for all dose levels and were consistent with laboratory historical vehicle control distribution ranges. There were no statistically significant increases in micronuclei frequency for any of the 2,4-decadienal dose groups compared to those of the vehicle control. 4. Discussion and conclusion 2,4-Decadienal occurs naturally in a large variety of foods that are consumed widely. Genotoxicity tests conducted as part of the NTP safety testing reported conflicting results, and although the NTP concluded that 2,4-decadienal was not genotoxic, the data raised concern 5
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exposure assessment. Compared to the dose levels tested in the studies presented here, the intraperitoneal dose levels administered to animals in the NTP study were considerably higher (100, 200, 400, and 600 mg/kg bw vs. the MTD of 100 mg/kg bw in our study). In our study, toxicity at the 400 mg/kg bw was severe after a single administration and resulted in significant mortality. Toxicity was notable at 200 mg/kg bw after the first administration and, although it initially subsided slightly, it was prominent on the third day and one animal died. It is not clear whether any toxicity was observed at the dose levels tested in the NTP study. At 100 mg/kg bw/day, toxicity was manifested only after the second administration and persisted for about 8 h, but no deaths occurred. These signs of toxicity observed at 100 and 200 mg/kg bw in our rangefinding study would not have been observed after a single intraperitoneal administration in the NTP study. Indeed, the dose levels in the 3-injections protocol in the NTP study were substantially lower (25, 50, 100, and 200 mg/kg bw) and within the range of the tolerated dose levels determined in our study. No further comparisons of toxicity can be made as no detailed information about signs of toxicity was provided in the NTP studies either for rats or for mice, except that preliminary range-finding studies were conducted prior to the main micronucleus studies. In the NTP studies, neither the frequency of PCE in the bone marrow of animals treated intraperitoneally nor the frequency of RET in mice treated orally were reported to indicate the degree of target tissue toxicity. The only other obvious factor that may at least partially explain the different dose range tolerance between ours and the NTP studies is the different strains of rats used (Han Wistar in our study compared to F344/N in the NTP rat study), and it is possible that there are toxicokinetic and/or toxicodynamic differences in sensitivity to the toxicity of 2,4-decadienal between the two strains. Regardless of the species difference, it is important that genotoxicity was assessed up to the MTD in the present study. The micronucleus studies presented here included more extensive analysis compared to those reported by the NTP. First, micronucleus analysis was conducted in both bone marrow and peripheral blood cells at optimum sampling times, while the NTP studies examined only the bone marrow in rats with 24-h sampling, or both tissues in mice but both assessed at the 48 h time point (NTP, 2003). Second, larger numbers of bone marrow cells (4000 PCE/animal) were scored for micronuclei in our studies, compared to the analysis in the NTP studies (2000 PCE/animal). Third, the analysis in peripheral blood reticulocytes relies on capturing cells released from the bone marrow carrying chromosomal damage. Because micronucleated immature erythrocytes are filtered in the spleen and the fraction that escaped filtering is released into peripheral blood is lower than the total events that occurred in the bone marrow tissue, scoring in peripheral blood must compensate by scoring a larger cell population to capture lowfrequency events. However, the number of immature erythrocytes in the peripheral blood of mice that were scored in the NTP study was too low (only 1000 cells) to capture such rare events. In our studies, the flow cytometry method used for the detection of MN RET in peripheral blood allowed scoring of significantly larger numbers of cells (20,000 cells/animal). This offers increased power to detect low-frequency events compared to microscopy-based scoring. In addition, sensitivity to detect MN RET was further enhanced by scoring the younger fraction of immature erythrocytes identified by the transferrin receptor expression (CD71+ labeling) compared to the less sensitive scoring of a broader immature erythrocyte population identified by acridine orange stain, as indicated in the OECD guideline [15]. In conclusion, two robust, OECD guideline-compliant genotoxicity studies using the in vivo micronucleus assay in rats were performed to test the ability of 2,4-decadienal to cause chromosomal damage, following administration by the oral and intraperitoneal routes. No evidence of genotoxicity was found at doses up to the MTD for each route of administration and analysis of both bone marrow and peripheral blood of treated animals at optimum time points, using protocols with a
higher power to detect low incidence events of genotoxicity compared to previous reports. Under the conditions of these studies, 2,4-decadienal is not genotoxic. Additionally, based on these data, EFSA ruled out the genotoxicity concern from the intake of 2,4-decadienal when used as a flavoring substance [12]. Acknowledgments This work was supported by the International Organization of the Flavor Industry. The authors declare that there are no conflicts of interest. The authors thank Dr. Xiaodong Li for his assistance in the preparation of this manuscript. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.mrgentox.2019. 503082. References [1] C. Bruschwig, S. Rochard, A. Pierrat, A. Rouger, P. Senger-Emonnot, G. George, P. Raharivelomanana, Volatile composition and sensory properties of Vanilla x tahitensis bring new insights for vanilla quality control, J. Sci. Food Agric. 96 (2016) 848–858, https://doi.org/10.1002/jsfa.7157. [2] K.R. Cadwallader, H. Kim, S. Puangpraphant, Y. Lorjaroenphon, Changes in the aroma components of pecans during roasting, in: I. Blank, M. Wüst, C. Yeretzian (Eds.), Expression of Multidisciplinary Flavour Science, Interlaken, Switzerland, 2009, pp. 301–304, , https://doi.org/10.1021/jf902402s. [3] F. Darriet, M. Bendahou, J. Costa, A. Muselli, Chemical compositions of the essential oils of the aerial parts of Chamaemelum mixtum (L.) Alloni, J. Agric. Food Chem. 60 (2012) 1494–1502, https://doi.org/10.1021/jf203872z. [4] C.E. Quijano, J.A. Pino, Volatile compounds of round kumquat (Fortunella japonica Swingle) peel oil from Colombia, J. Essent. Oil Res. 21 (2009) 483–485, https://doi. org/10.1080/10412905.2009.9700224. [5] E.N. Frankel, W.E. Neff, D.D. Brooks, K. Fujimoto, Fluorescence formation from the interaction of DNA with lipid oxidation degradation products, Biochim. Biophys. Acta (BBA) - Lipids Lipid Metab. 919 (1987) 239–244, https://doi.org/10.1016/ 0005-2760(87)90263-3. [6] J.M. Snyder, E.N. Frankel, E. Selke, Capillary gas chromatographic analyses of headspace volatiles from vegetable oils, J. Am. Oil Chem. Soc. 62 (1985) 1675–1679, https://doi.org/10.1007/BF02541664. [7] R.L. Hall, B.L. Oser, Recent progress in the consideration of flavoring ingredients under the food additives amendment. 4. GRAS Substances, Food Technol. 24 (1970) 25–34. [8] T.B. Adams, C.L. Gavin, S.V. Taylor, W.J. Waddell, S.M. Cohen, V.J. Feron, J. Goodman, I.M. Rietjens, L.J. Marnett, P.S. Portoghese, R.L. Smith, The FEMA GRAS assessment of alpha, beta-unsaturated aldehydes and related substances used as flavor ingredients, Food Chem. Toxicol. 46 (2008) 2935–2967, https://doi.org/ 10.1016/j.fct.2008.06.082. [9] JECFA, Evaluation of Certain Food Additives and Contaminants (Sixty-first Report of the Joint FAO/WHO Expert Committee on Food Additives), (2004). [10] EFSA, Scientific Opinion on Flavouring Group Evaluation 203, Revision 1 (FGE.203Rev1): alpha,beta-Unsaturated aliphatic aldehydes and precursors from chemical subgroup 1.1.4 of FGE.19 with two or more conjugated double-bonds and with or without additional non-conjugated double bonds, EFSA J. 3626 (2014), https://doi.org/10.2903/j.efsa.2014.3626. [11] NTP, NTP Technical Report on the Toxicity Studies of 2,4-Decadienal (CAS No. 25152-84-5) Administered by Gavage to F344/N Rats and B6C3F1 Mice. National Toxicology Program Toxicity Report Series Number 76, National Institutes of Health, Public Service, US Department of Health and Human Services, 2011. [12] EFSA, Scientific Opinion on the Flavouring Group Evaluation 203, Revision 2 (FGE.203Rev2): alpha,beta-Unsaturated aliphatic aldehydes and precursors from chemical subgroup 1.1.4 of FGE.19 with two or more conjugated double-bonds and with or without additional non-conjugated double-bonds, EFSA J. 16 (2018), https://doi.org/10.2903/j.efsa.2018.5322. [13] Z. Keig-Shevlin, 2,4-Decadienal: Intraperitoneal in Vivo Micronucleus Study in the Bone Marrow and Peripheral Blood of Treated Rats, Unpublished Report, Study no. 8321404, Covance Laboratories, Harrogate, UK, 2016, pp. 1–51. [14] Z. Keig-Shevlin, 2,4-Decadienal: Oral Gavage in Vivo Micronucleus Study in the Bone Marrow and Peripheral Blood of Treated Rats, Unpublished Report, Study no. 8321403, Covance Laboratories, Harrogate, UK, 2016, pp. 1–73. [15] OECD, Test No. 474: Mammalian Erythrocyte Micronucleus Test, OECD Publishing, 2014. [16] U.K. MHRA, The Good Laboratory Practice (Codification Amendments etc.) Regulations 2004, Statutory Instrument No. 2004/994 (2004). [17] OECD, Principles of Good Laboratory Practice (GLP), OECD Publishing (revised in 1997), 1998. [18] J.T. Sun, M.J. Armstrong, S.M. Galloway, Rapid method for improving slide quality
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