Safety evaluation of ice-structuring protein (ISP) type III HPLC 12 preparation. Lack of genotoxicity and subchronic toxicity

Food and Chemical Toxicology 42 (2004) 321–333 www.elsevier.com/locate/foodchemtox

Safety evaluation of ice-structuring protein (ISP) type III HPLC 12 preparation. Lack of genotoxicity and subchronic toxicity T. Hall-Manninga,*, M. Spurgeona, A.M. Wolfreysa, A.P. Baldrickb a

SEAC—Safety & Environmental Assurance Centre, Unilever Colworth, Colworth House, Sharnbrook, Bedfordshire MK44 1LQ, UK b Scientific & Regulatory Consultancy, Covance Laboratories Ltd, Otley Road, Harrogate, North Yorkshire HG3 1PY, UK Received 11 June 2003; accepted 9 September 2003

Abstract Ice-structuring proteins (ISPs) naturally occur in a range of species (including edible plants and fish) that need to protect themselves against freeze damage. ISPs have potential applications in a number of areas including cryopreservation and frozen foods manufacture. However, these materials are not currently generally available for commercial use. ISP type III HPLC 12 is of particular interest and although it is likely to be consumed naturally, its toxicological safety has not previously been assessed. This paper presents data from a set of in vitro and in vivo genotoxicity assays (bacterial mutation, chromosome aberration, mammalian cell gene mutation and rat bone marrow micronucleus) and a 3-month repeat-dose gavage study in the rat using high levels of ISP type III HPLC 12 preparation produced by recombinant baker’s yeast. No evidence was seen of a genotoxic potential (using levels accepted as limit concentrations for the assays used) or notable subchronic toxicity following oral administration for 3 months in the rat at up to 580 mg ISP type III HPLC 12/kg/day, the highest dose tested (which was considered to be a NOAEL). # 2004 Elsevier Ltd. All rights reserved. Keywords: Ice-structuring protein; Genotoxicity; Toxicity; Rat; Food material

1. Introduction Ice structuring proteins (ISPs), originally termed antifreeze or thermal hysteresis proteins, are naturally occurring proteins which have evolved to protect organisms from freeze damage. They function by binding directly to ice thereby influencing the growth and shape of ice crystals (Barrett, 2001; Clarke et al., 2002). They were first identified over 30 years ago in the blood of fish living in areas where the sea freezes, such as cod and herring (DeVries and Wohlschlag, 1969; Fletcher et al., 1999). Since then, ISPs have been found in a wide variety of organisms that need to protect themselves against freeze damage, including many plants (such as oats, rye, barley, wheat carrot and potato), insects, fungi and bacteria (Crevel et al., 2002; Duman and Olsen, 1993; Griffith and Ewart, 1995) Only limited data are available on the ISP content of different organisms. However, it is known that relatively * Corresponding author. E-mail address: [email protected] (T. Hall-Manning). 0278-6915/$ - see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2003.09.007

high concentrations occur naturally. Levels of up to 0.307 mg/g fresh weight have been reported in the leaves of winter rye (Griffith, 1999). More data are available on fish, with blood levels of about 30 mg/ml reported in ocean pout for ISP type III and 7–14 mg/ml in Atlantic cod for the related material, ice-structuring glycoprotein (Kao and Fletcher, 1988). Using a blood volume of 30– 70 ml/kg for a teleost (Olson, 1992), the ISP content of an ocean pout and cod is estimated at approximately 900–1200 and 210–980 mg/kg, respectively. ISP type III was originally isolated in the mid-1980s from the blood of the ocean pout (Macrozoarces americanus), a cold water fish found off the northeast coast of North America (Hew et al., 1984; Li et al., 1985). High performance liquid chromatography (HPLC)-extraction has revealed 12 isoforms for this protein type of which ISP type III HPLC 12 has been shown to be the most functionally active in in vitro ice-structuring studies. Sequencing work by Hew et al. (1988) has shown that ISP type III HPLC 12 is composed of 66 amino acids, all of which are commonly found in dietary proteins (see Fig. 1).The ISP type III HPLC 12 (hereafter referred to as ISP type III) preparation used in this study was pro-

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Fig. 1. Amino acid structure of ISP Type III HPLC 12 (Based on Hew et al., 1998).

duced by fermentation using recombinant baker’s yeast (Saccharomyces cerevisiae). The amino acid sequence of the expressed ISP type III is identical to the sequence found in the native ocean pout protein. Although present in biological systems, no safety data appear to exist for ISPs and specifically for ISP type III although recent studies have indicated that they are unlikely to present a potential sensitisation/allergenicity hazard (Baderschneider et al., 2002; Bindslev-Jensen et al., 2002; WHO/FAO, 2001). Therefore, the mutagenicity and subchronic toxicity of this preparation was assessed in a series of genotoxicity assays and a 3-month oral gavage study in rats.

2. Material and methods 2.1. Materials The preparation produced by yeast fermentation is a cell-free brown liquid consisting of functionally active ISP type III, inactive mannose-conjugated (glyco-) ISP type III, proteins and peptides from the yeast fermentation and, sugars, acids and salts, commonly found in food. The preparation is stabilised at pH3.0  0.5 using citrate buffer (10 mM). Two ISP preparations were used in this study. The first was a liquid preparation containing 29 g/l ISP type III to be used for the animal feeding study and the second was a freeze-dried preparation used for the genotoxicity studies (Table 1). Both materials were maintained frozen. Upon thawing, stability data (not shown) confirmed that both the concentrated and freeze-dried materials were stable over 6 months under frozen conditions; formulations of the concentrate were also shown to be homogeneous. Total protein was determined by Kjeldahl nitrogen methodology while protein characterisation was carried

out using HPLC; specific separation and measurement of glyco-ISP type III and yeast protein/peptide involved the use of gel filtration chromatography (GFC). 2.1.1. Genotoxicity studies Freeze-dried (FD) test material was dissolved in sterile purified water in all four studies. For the bacterial mutation assay, the highest concentration used was 5000 mg total solids/plate in the plate incorporation and pre-incubation experiments (although 8000 mg total solids/plate was investigated in an additional experiment); dilutions were made with water. For the chromosome aberration and mammalian cell gene mutation assays, the highest concentration used were limited by toxicity but did not exceed 5000 mg total solids/ml; dilutions were made with water. For the rat bone marrow micronucleus study, dose levels of 500, 1000 and 2000 mg total solids/kg were administered by oral gavage at 20 ml/kg using direct dilution with water. The studies were validated using the positive controls 2nitrofluorene (2-NF), 9-aminoacridine (AAC), glutaraldehyde (GLU) and benzo[a]pyrene (B[a]P) (Aldrich Chemical Co., Gillingham, UK) and sodium azide (NaN3) and 2-aminoanthracene (AAN) (Sigma Chemical Co., Poole, UK) for the bacterial mutation assay. As necessary, formulations were prepared in water or dimethyl sulphoxide (DMSO) (Sigma Chemical Co.). 4Nitroquinoline 1-oxide (NQO) (Aldrich Chemical Co.) and cyclophosphamide (CPA) (Sigma Chemical Co.) were used in the chromosome aberration assay, NQO and B[a]P were used in the mammalian cell gene mutation assay and CPA was used in the rat micronucleus study. 2.1.2. 3-Month oral rat study Concentrated test material was administered by oral gavage at 20 ml/kg to give ISP type III levels of 58, 290 and 580 mg/kg/day. The highest level also represented a dose of 4000 mg total solids/kg/day of which the total

Table 1 Protein profiles of the liquid and freeze dried test materials Component

Liquid material

Freeze dried material

Total Kjeldahl protein (% w/w) ISP type III HPLC 12 content (% w/w) Glyco-ISP III HPLC 12 content (% w/w)a Yeast proteins and peptides (% w/w)a

7.71 2.73 1.85 3.1

41.1 13.7 9.9 17.7

a

Estimated by GFC.

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ISP level was 960 mg/kg/day. The lower dose levels were prepared by dilution of the high dose level. Two further groups were included, one receiving ultra-purified water and another receiving a 0.12% citric acid solution. The latter group was included to allow comparison with a solution of equivalent pH (and citric acid level) to that of the ISP type III preparation (pH 2.5–3.5). Concentration analysis for ISP type III levels on four occasions from samples used during weeks 10–12 of the study showed values of 90–96, 84–96 and 83–92% of expected concentration (3.05, 14.7 and 29 mg/ml, respectively) for the three treatment groups (data not shown). 2.2. Genotoxicity assays Genotoxicity investigations were performed in 2001– 2002 at Covance Laboratories Ltd (Harrogate, UK), with some supporting investigations conducted at Safety and Environmental Assurance Centre, Unilever Colworth. All investigations followed UK and OECD Good Laboratory Practice (GLP) requirements. All four assays were in compliance with OECD Test Guidelines (OECD, 1997a–d). 2.2.1. Bacterial mutation assay The mutagenic activity of ISP Type III preparation was assessed in 5 histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium both using the freeze-dried preparation in the absence and in the presence of metabolic activation (10%). The metabolic activation was derived from an Aroclor 1254-induced rat liver post-mitochondrial fraction. Experiment 1 (plate incorporation method) used concentrations of 1.6–5000 mg total solids/plate while a narrowed dose range of 156.25–5000 mg total solids/ plate and a repeat of the plate incorporation method for TA1535 only was used for Experiment 2 (pre-incubation). Due to evidence of a small increase in revertant colonies and some concern over potential microbial contamination, a third experiment, involving testing of TA1535 (both plate incorporation and pre-incubation), at concentrations of 1250–8000 mg total solids/plate was conducted. The identity of the colonies on the plates at 2500 and 5000 mg/plate in Experiment 3 were investigated using a Salmonella Latex Kit (Oxoid, UK) and data were re-analysed for biological and statistical significance once any non-Salmonella colonies had been removed from the total colony counts. Evidence of mutagenic activity was defined as a two-fold, dose-related increase in the number of revertant colonies per plate above the negative control values with supporting statistical significance. Data were evaluated using standard statistical tests (m-statistic, Dunnett’s test and linear regression analysis) with statistical significance expressed at P < 0.01.

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2.2.2. In vitro chromosome aberration assay The ability of ISP type III preparation to induce chromosome aberrations was assessed in human peripheral blood lymphocytes using the freeze dried material. Duplicate whole blood cultures (pooled from three donors) were used both in the absence and in the presence of 2% S-9 in two separate experiments. Chromosome aberrations were analysed at three dose levels (2048, 3200 and 5000 mg total solids/ml in the absence of S-9 and 1311, 2048 and 2560 mg total solids/ml in the presence of S-9) with treatment for 3 h followed by a 17h recovery period in Experiment 1. In Experiment 2, chromosome aberrations were analysed at 371.3, 513.8 and 836.7 mg total solids/ml in the absence of S-9 with continuous treatment for 20 h or at 1603, 2219, 2610 and 4250 mg total solids/ml in the presence of S-9 with treatment for 3 h followed by a 17-h recovery period. Toxicity was determined by calculating the mitotic index (number of dividing cells per 1000 cells) and the maximum concentration tested resulted in approximately 50% reduction in the mitotic index. A total of 200 cells (100 per replicate dose) were scored for chromosome aberrations. Evidence of mutagenic potential was defined as a statistically and biologically significant increase in the percentage of cells with aberrations. Data (excluding aberrant cells with gaps) were analysed using Fisher’s exact test with significance expressed at P < 0.05. In addition, the number of polyploid and endoreduplicated cells per 1000 mitotic cells (2000 per dose) was assessed, to assess the ability of the ISP Type III preparation to cause changes in the chromosome number. 2.2.3. In vitro mammalian cell gene mutation assay The ability of ISP type III preparation to induce mutations at the tk+/ (thymidine kinase) locus was assessed in mouse lymphoma L5178Y cells using a microtitre fluctuation method. Two separate experiments, using duplicate cultures with freeze dried preparation both in the absence and in the presence of 1% S-9 were performed. Cells were exposed to 250–5000 mg total solids/ml in the absence and presence of S-9 (with treatment for 3 h) in Experiment 1. In Experiment 2, exposure occurred at 250–2500 mg total solids/ml in the absence of S-9 (with treatment for 20 h) or at 1000–5000 mg total solids/ml in the presence of S-9 (with treatment for 3 h). Toxicity was determined by measuring the percentage relative survival of cells following treatment. The expression period was 2 days, following which the cells were plated in the presence of the toxic base analogue 5-trifluorothymidine (5-TFT) at 3 mg/ml. Mutant frequency (tk / cells) was determined by counting the number of mutant colonies after 10–14 days. Evidence of mutagenic potential was defined as a statistically and biologically significant, dose-related increase in the mutant frequency (the number of 5-TFT resistant cells/106 viable

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cells). Data were analysed using Dunnett’s test for multiple comparisons and a test for linear trend with significance expressed at P < 0.05. 2.2.4. In vivo bone marrow micronucleus assay The potential mutagenic activity of ISP type III preparation was investigated in vivo by determining the incidence of micronuclei in bone marrow polychromatic erythrocytes (PCE) in the rat, an established species for this test using the freeze dried material. Rats of the Crl:HanWist (Glx:BRL) BR strain and approximately 7 weeks old were obtained from Charles River UK Ltd (Margate, UK). In a preliminary range-finder investigation, a dose level of 2000 mg total solids/kg/day administered by oral gavage for two consecutive days to three males and three females was well-tolerated with mild clinical signs of hyperactivity, piloerection and protruding eyes. Owing to the lack of toxicity observed and the similarity in response between the sexes, a dose level of 2000 mg total solids/kg (given at 20 ml/kg) was selected as the high dose for the main study using males only . Lower dose levels were 1000 and 500 mg total solids/kg/day; the vehicle control was purified water and the positive control, CPA was administered orally at 20 mg/kg (and a dose volume of 10 ml/kg). Groups of six males were treated for two consecutive days (other than CPA which was given once) and 24 h after the last dose, animals were killed and bone marrow slides were prepared from a single femur. Slides were stained in 12.5 mg/ml acridine orange. Toxicity was assessed from the relative proportion of PCEs and normochromatic erythrocytes (NCEs) per 1000 cells. The incidence of micronuclei was measured in 2000 PCEs per animal and the group means were determined. Evidence of mutagenic activity was defined by a statistically and biologically significant increase in micronuclei. Data were analysed by chi-squared analysis with significance expressed at P < 0.05. Dose confirmation analysis was performed. 2.3. Ninety day repeat dose rat gavage study 2.3.1. Study design Groups of 20 male and 20 female Wistar rats were administered test material equivalent to 58, 290 and 580 mg ISP type III/kg/day (Groups 3, 4 and 5) at 20 ml/kg each day for 3 months. Two other identically-sized groups received water (negative control Group 1) or 0.12% citric acid solution (vehicle control Group 2). Dose levels were based on a 2-week preliminary range-finder investigation which showed no adverse toxicity. Measured parameters comprised clinical observations, food consumption, neurobehavioural testing, ophthalmoscopic examination, clinical pathology, gross necropsy, selected organ weights and histopathology of specified organs/tissues. The study was

conducted at Covance Laboratories Ltd (Harrogate, UK) in 2001–2002, followed UK and OECD Good Laboratory Practice (GLP) requirements and was in accordance with OECD Test Guidelines (OECD, 1998). 2.3.2. Test animals and husbandry Wistar rats of the Crl:HanWist (Glx:BRL) BR strain were obtained from Charles River UK Ltd (Margate, UK). The strain is routinely used in the testing facility for this type of study. Following an acclimation period of approximately 2 weeks, rats commenced treatment at approximately 6 weeks of age. Animals were allocated (males and females separately) to the various study groups by a randomisation procedure based on stratified bodyweight and individually identified by electronic implant. Treatment group positions in the cage battery were assigned using a set of random letter permutations. Animals were maintained under conditions of 22  3  C, 40–70% relative humidity and a 12-h light/ dark cycle. Diet (Special Diets Services Ltd, Witham, UK) and water (mains source, supplied in bottles) were available ad libitum (other than during certain laboratory investigations) throughout the study. Rats were housed one to a cage. 2.3.3. Clinical observations and ophthalmoscopy All animals were observed daily for signs of ill health or overt toxicity. In addition, each animal was given a detailed physical examination at weekly intervals. An ophthalmoscopic examination of all animals occurred prior to the start of the study. The eyes of rats from the control and high dose groups were also examined during week 12. Examination was carried after administration of a mydriatic agent into the eyes. 2.4. Bodyweights and food consumption Bodyweights were recorded before treatment, on the first day of dosing and weekly thereafter. Food consumption was measured on a weekly basis and was expressed as g/rat/week. 2.5. Neurobehavioural testing Testing comprised measurements from a functional observational battery (FOB) and motor activity assessment on 10 animals/sex/group (lowest identity numbers). Detailed clinical (neurobehavioural) observations were performed prior to study start and on a weekly basis thereafter. During week 13, sensory reactivity to stimuli testing, grip-strength measurement and motor activity assessment were performed in addition to the detailed clinical observations. Details of the tests performed are available elsewhere (e.g., Moser and MacPhail, 1992; Moser, 2000).

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2.6. Clinical pathology Blood samples for haematology and clinical chemistry assessment were collected during weeks 4 and 8 (10 animals/sex/group with the highest identity numbers) and prior to necropsy at the end of the study (all surviving animals). Samples were collected in EDTA and lithium heparin tubes, respectively. Collection was from the lateral caudal vein. Food was withdrawn towards the end of the working day prior to blood sampling on the following morning. The list of parameters examined is given in Tables 6 and 7. During week 12, 10 rats/sex/group (highest identity numbers) were moved to metabolism cages towards the end of the working day and urine was collected overnight. Animals were deprived of food and water during this period. From the urine samples, the following parameters were measured: volume, specific gravity, pH, protein, glucose, reducing substances, ketones, urobilinogen, bilirubin, blood and microscopy of the sediments.

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of variances. A Dunnett’s pairwise comparison test was then performed if there was no evidence heterogeneity. Parameters with significantly different variances (P < 0.01) were re-evaluated with Kruskal–Wallis nonparametric anova, the Terpstra–Jonckheere test for trend and pairwise comparison by the Wilcoxon rank sum test. Organ weights were analysed using analysis of covariance (ANCOVA) and Dunnett’s test, for each sex separately, with the necropsy bodyweight as covariate. Levene’s test was used to confirm heterogeneity; if this was not seen a one-way ANOVA was performed. The negative control group (Group 1) was also compared with the vehicle control using pairwise comparisons but was excluded from tests of dose-related trends as the two groups were not shown to be different. Probability values of P < 0.05 and P < 0.01 were considered statistically significant.

3. Results 3.1. Genotoxicity assays

2.7. Pathology examinations Rats were sacrificed after 3 months of treatment by exsanguination following an overdose of intraperitoneal sodium pentobarbitone then examined grossly for pathological changes. Selected organs were weighed (paired organs together) and are listed in Table 8. Following processing to 5 mm sections and staining with haematoxylin and eosin for light microscopy, the following organs/tissues were examined histopathologically for all animals of the negative control, vehicle control and high dose groups as well as gross lesions for all rats: adrenals, aorta, brain, caecum, colon, eyes, femur (with bone marrow) and articular surface, heart, kidneys, liver, lungs with mainstem bronchi, lymph nodes (mandibular and mesenteric), mammary gland (female), muscle (quadriceps), oesophagus, optic nerves, ovaries, pancreas, parathyroid, Peyer’s patches, pituitary, prostate, rectum, salivary glands, sciatic nerves, seminal vesicles, skin, small intestine (duodenum, ileum, jejunum), spinal cord (at three levels), spleen, sternum (with bone marrow), stomach, testes and epididymides, thymus, thyroids and parathyroids, trachea, urinary bladder, uterus and vagina. 2.8. Statistical procedures Statistical analysis involved comparison of ISP type III-treated groups (Groups 3–5) with those receiving vehicle control (Group 2). Bodyweight gain, necropsy bodyweights, food consumption, neurobehavioural variables and clinical pathology parameters were analysed using a one-way analysis of variance (ANOVA), separately by sex, followed by Levene’s test for equality

3.1.1. Bacterial mutation assay Maximum concentrations used were not limited by solubility or cytotoxicity and so were set by the maximum level used in this type of study (5000 mg total solids/plate). Results from the plate incorporation test (Experiment 1) are shown in Table 2; results from the pre-incubation test (Experiment 2) were similar and are therefore not shown. Small increases in revertant numbers were observed in strain TA1535 in both experiments although the increases were inconsistent, did not always show a dose response and showed variation in response in either the absence or presence of S-9. In a third experiment, concentrations up to 8000 mg total solids/plate were investigated with strain TA1535 only and excessive microbial contamination was observed at concentrations including and above 6000 mg/plate, which precluded scoring of the revertant colonies. The identity of the revertant colonies at 2500 and 5000 mg/ plate were investigated and a small number of revertant colonies were found not to be Salmonella species. Thus the revertant colony counts originally scored were not representative of the true colony counts for Salmonella. Data were re-analysed, removing the non-Salmonella colony counts from the counts for TA1535. On re-analysis, there was no evidence of a statistically or biologically significant increase in revertant colony counts for TA1535 indicating a lack of mutagenic activity. Other statistically significant findings included small increases in revertant numbers for TA102 (at an intermediate concentration in the presence of S-9) and for TA1537 (at the maximum concentration in the absence of S-9) in Experiment 2. However, given the lack of reproducibility, the lack of dose response and the low actual

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Table 2 Bacterial mutation assay (plate incorporation assay)a Dose level (mg total solids /plate)

Mean revertant colony counts per platestandard deviationb

Rat liver S9 (%)

TA98

TA100

TA1535

TA1537

TA102

Water (100 ml/plate) 1.6 8 40 200 1000 5000 DMSO 2NF(5) NaN3 (2) AAC (50) GLU (25)

0 0 0 0 0 0 0 0 0 0 0 0

308 435 3512 421 407 396 448 337 822103 – – –

117 19 103 17 109 19 115 14 98 28 100 10 110 11 – – 678 41 – –

214 185 224 153 223 276 419*** – – 61539 – –

11 3 14 5 10 1 7 3 15 2 16 3 15 3 – – – 222 38 –

364 46 329 36 339 28 360 49 305 42 305 24 272 68 – – – – 877 65

Water (100 ml/plate) 1.6 8 40 200 1000 5000 DMSO B[a]P (10) AAN (5) AAN (20)

10 10 10 10 10 10 10 10 10 10 10

406 377 284 392 417 368 536* 347 29933 – –

138 14 115 15 118 8 120 5 141 13 135 4 144 8 154 13 – 1730 61 –

224 271 212 174 236 278 343* 203 – 14311 –

16 7 11 1 12 1 10 1 12 1 25 4 21 8 19 4 – 218 14 –

293 41 288 26 277 50 304 29 299 9 301 36 223 12 290 37 – – 1682 143

a

Results for the preincubation test are not presented but gave similar results. Mean of five plates (water and DMSO) or three plates (all other treatments). * P <0.05. *** P <0.001. b

Table 3 In vitro chromosome aberration assay in human peripheral blood lymphocytes Concentration Without S9 (mg total solids /ml) No of aberrant cells/200 cells

Concentration With S9 (mg total solids /ml) MI Polyploidy+ (mean) endoreduplication/ 2000 cells (mean%)

No of aberrant cells 200 cells

Excluding Including gaps gaps

Excluding Including gaps gaps

Experiment 1 0 (water) 2048 3200 5000 NQO (5)

3-h treatment and 17-h recovery 4 3 10.6 0 0 10.2 7 5 6.7 2 1 4.5 20a – 22a

Experiment 2 0 (water) 371.3 513.8 836.7

20-h treatment 4 2 4 3 4 4 4 2

16.4 14.4 10.7 7.3

NQO (2.5)

35a

–

30a

MI (mean) Polyploidy+ endoreduplication/ 2000 cells (mean%)

4 426(0.2) 4 426(0.2) 10 (0.5) 4 (0.2) –

4 4 8 3 –

MI=mitotic index (frequency/1000 cells counted). a P40.001.

(0.2) (0.2) (0.4) (0.15)

0 (water) 1311 2048 2560 CPA (6.25)

3-h treatment and 17-h recovery 4 4 13.9 6 6 11.3 13 13 8.1 7 7 7.6 93a 90a –

4 5 8 10 –

(0.2) (0.25) (0.4) (0.5)

0 (water) 1603 2219 2610 4250 CPA (6.25)

3-h treatment and 17-h recovery 4 1 16.8 3 1 15.3 4 1 8.0 5 2 9.0 10 3 7.7 82a 68a –

9 13 14 8 8 –

(0.45) (0.65) (0.7) (0.4) (0.4)

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T. Hall-Manning et al. / Food and Chemical Toxicology 42 (2004) 321–333 Table 4 In vitro mammalian cell gene mutation asay in L5178Y tk+/ cells Concentration (mg total solids/ml)

Without S9

Concentration (mg total solids/ml)

% RS

RTG

MF

Experiment 1 0 (water) 250 500 1000 2000 3000 4000 5000 NQO (0.05) NQO (0.1)

3-h treatment 100 104 99 110 81 60 46 37 94 97

1.00 1.03 0.91 1.03 0.92 0.68a 0.48a 0.27 1.05 1.00

79.24 76.18 68.34 77.91 93.59 72.68a 64.97a 82.33 155.67b 215.92b

Experiment 2 0 (water) 250 500 1000 1250 1500 1750 2000 2250 2500 NQO (0.02) NQO (0.04)

24-h treatment 100 93 91 74 61 48 48 40 24 15 99 87

1.00 0.97 1.19 0.99 0.89 0.69 0.65 0.49 0.41 0.29 1.05 1.03

87.89 70.76 63.78 74.80 61.32 67.92 62.51 81.03 96.24 126.23* 164.8b 232.8b

With S9 % RS

RTG

MF

0 250 500 1000 2000 3000 4000 5000 B[a]P(2) B[a]P(3)

3-h treatment 100 90 81 82 86 77 60 42 61 39

1.00 0.74 0.76 0.76 0.74 0.57 0.33 0.24 0.42 0.25

67.68 63.28 85.38 66.27 61.32 75.75 74.59 74.03 601.53b 755.94b

0 1000 2000 3000 4000 5000 B[a]P(2) B[a]P(3)

3-h treatment 100 108 93 76 54 40 86 73

1.00 0.83 0.89 0.72 0.36 0.22 0.49 0.53

86.55 73.25 67.23 65.24 75.90 67.68 496.62b 517.71b

% RS=Percentage relative survival in each test culture. RTG=Relative total growth (calculated from suspension growthplating efficiency). MF=5-TFT resistant mutants/106 viable cells 2 days after treatment. a Based on one replicate only (other replicate discarded due to duplication error). b Statistical programme does not include analyses of positive control groups. * P <0.05 for linear trend.

Table 5 In vivo bone marrow micronucleus study in rats Group total solids (mg total solids/kg/day)

Mean ratio PCE/NCE

Group mean ratio of micronucleated PCE (per 1000 cells) S.D.

Vehicle control (water) 500 1000 2000 CPA (20)a

0.98 1.42 1.57 1.36 0.49

0.500.55 0.330.41 0.330.26 0.080.20 7.083.53b

S.D.=standard deviation. a Administered as a single dose. b P40.001.

increases in revertant colony counts, these results were not considered to be biologically significant. There were no other statistically significant increases in revertant colony numbers following treatment with ISP type III. 3.1.2. In vitro chromosome aberration assay Maximum concentrations used were set either by the maximum level used in this type of study (5000 mg total solids/ml) or by a mitotic inhibition of approximately

50%. Results are presented in Table 3 and indicate that there was no biological or statistically significant increase in the percentage of cells with chromosome aberrations (including and excluding gaps) in the treated cultures in comparison with the negative solvent control cultures. In addition, there was no evidence of increased frequencies of polyploid and endoreduplicated cells for any of the treatment regimens. 3.1.3. In vitro mammalian cell gene mutation assay Maximum concentrations used were set either by the maximum level used in this type of study (5000 mg total solids/ml) or a percentage relative survival of greater than 10%. Results are presented in Table 4. No evidence of an increase in mutant frequency was observed at any concentration either in the absence or presence of metabolic activation. A weak linear trend was seen in the absence of S-9 following 24 h of treatment but this increase in mutant frequency was not considered biologically significant as there was no evidence of a dose response, the increase occurred in one replicate only (at the highest test concentration) and it failed to meet the criteria for a mutagenic response.

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Table 6 Haematology Parameter

(a) Males Haemoglobin concentration (g/dl) Red blood cell count (mil/cmm) Packed cell volume (%) Reticulocytes (%) Mean cell volume (fl) Mean cell haemoglobin (pg) Mean cell haemoglobin concentration (g/dl) Platelets (1000/cmm) Prothrombin time (s) Activated partial thromboplastin time (s) White blood cell count (1000/cmm) Neutrophils (1000/cmm) Lymphocytes (1000/cmm) Monocytes (1000/cmm) Eosinophils (1000/cmm) Basophils (1000/cmm) (b) Females Haemoglobin concentration (g/dl) Red blood cell count (mil/cmm) Packed cell volume (%) Reticulocytes (%) Mean cell volume (fl) Mean cell haemoglobin (pg) Mean cell haemoglobin concentration (g/dl) Platelets (1000/cmm) Prothrombin time (s) Activated partial thromboplastin time (s) White blood cell count (1000/cmm) Neutrophils (1000/cmm) Lymphocytes (1000/cmm) Monocytes (1000/cmm) Eosinophils (1000/cmm) Basophils (1000/cmm)

Group (meanstandard error) Negative control

Vehicle control

58 mg ISP type III/kg/day

290 mg ISP type III/kg/day

580 mg ISP type III /kg/day

16.20.5 8.910.50 48.72.6 2.70.3 54.71.2 18.30.8 33.41.3

16.2 0.6 8.90 0.26 48.4 1.6 2.4 0.4 54.4 1.2 18.2 0.6 33.5 1.1

16.60.4 8.790.40 48.61.8 2.60.3 55.41.3 18.90.8* 34.11.1

16.6 0.5 9.16 0.46 49.9 2.9 2.60.4 54.5 1.6 18.1 0.6 33.2 1.0

16.5 0.5a* 9.09 0.49a* 49.9 2.9a* 2.5 0.3 54.9 1.5 18.2 0.8 33.2 1.1

931138 20.11.2 21.12.8

952 145 19.9 1.5 20.9 2.3

925131 20.10.9 21.42.9

917 94 20.2 1.6 21.9 2.0

952 94 19.8 1.4 21.4 2.7

6.71.6 1.50.5 4.71.3 0.20.1 0.20.1 0.10.1

6.2 1.7 1.7 0.7 4.0 1.4 0.2 0.1 0.2 0.1 0.0 0.0

7.22.0 1.80.5 5.01.5 0.20.1 0.20.1 0.10.1

7.01.5 1.70.6 4.91.0 0.20.1 0.20.1 0.00.1

6.4 1.1 1.4 0.4 4.6 0.9 0.2 0.1 0.2 0.1 0.0 0.1

15.90.6 8.370.50 47.12.2 2.90.7 56.21.5 19.00.9 33.80.9

16.1 0.7 8.35 0.40 47.5 2.9 2.7 0.6 56.9 1.8 19.3 0.7 34.0 1.2

16.30.5 8.460.28 48.11.6 2.70.4 56.80.9 19.30.6 34.01.1

16.0 0.5 8.46 0.45 48.1 2.6 2.80.5 56.9 1.8 18.9 0.7 33.3 0.9

16.1 0.6 8.47 0.51 47.7 2.7 2.6 0.5 56.3 1.6 19.0 0.8 33.8 1.1

930107 19.90.8 18.82.2

859 99 19.7 0.8 19.1 2.3

940146 20.20.9 19.21.6

935 135 20.2 1.0 19.4 1.6

856 82 20.2 0.8 19.4 1.7

3.60.8 0.80.3 2.60.7 0.10.0 0.10.0 0.00.0

3.5 0.9 0.7 0.3 2.6 0.8 0.1 0.1 0.1 0.0 0.0 0.0

3.30.6 0.60.2 2.50.6 0.10.0 0.10.0 0.00.0

4.00.7 0.80.3 3.00.6 0.10.1 0.10.0 0.00.0

4.1 1.2a** 0.9 0.6 3.0 0.7a* 0.1 0.0 0.1 0.0 0.0 0.0

a

Significant dose–response test. * P <0.05. ** P <0.01.

3.1.4. In vivo bone marrow micronucleus assay At up to 2000 mg total solids/kg/day, the group mean frequencies of micronucleated PCE were similar to the vehicle control with no statistically significant difference observed, indicating a lack of mutagenic activity. Results are presented in Table 5. Some increases in the PCE:NCE ratio were observed but these were not dose related and thus were not considered indicative of toxicity to the bone marrow. Doses administered were shown to be within 10% of the nominal dose.

3.2. Ninety day repeat dose rat gavage study 3.2.1. Clinical observations and ophthalmoscopy One male in the high dose group was killed during week 10 due to deterioration in condition, characterised by laboured and noisy respiration plus a bodyweight loss. It is possible that the findings were related to incubation damage although the cause of morbidity was not determined as no significant changes in the blood or during macroscopic and microscopic examination were

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Group (meanstandard error) Negative control

Vehicle control

58 mg ISP type III/kg/day

290 mg ISP type III/kg/day

580 mg ISP type III/kg/day

(a) Males Aspartate aminotransferase (IU/l) Alanine aminotransferase (IU/l) Gamma glutamyl transferase (IU/l) Alkaline phosphatase (IU/l) Sodium (mmol/l) Potassium (mmol/l) Chloride (mmol/l) Calcium (mmol/l) Inorganic phosphorus (mmol/l) Urea (mmol/l) Total bilirubin (mmol/l) Creatinine (mmol/l) Total protein (g/l) Albumin (g/l) Globulin (g/l) Albumin/globulin ratio Total cholesterol (mmol/l) Glucose (mmol/l)

6210 4010 10 18543 1422 4.40.6 1042 2.590.07 1.70.2 7.91.5 2.00.3 647 664 432 233 1.80.2 1.60.2 7.20.9

617 429 10 17833 1411 4.30.4 1042 2.590.06 1.60.3 7.91.4 1.90.4 607 662 432 232 1.90.2 1.60.3 7.01.1

646 4613 10 21062 1421 4.40.4 1042 2.620.08 1.70.2 8.31.5 2.20.5 626 673 432 242 1.80.2 1.60.3 6.70.8

68 7 44 9 1 0 225 49* 141 1 4.6 0.3 104 2 2.64 0.06 1.7 0.2 8.7 1.6 2.2 0.5 62 6 68 2* 43 2 25 3** 1.7 0.2* 1.6 0.3 6.9 0.8

67 10 36 8 1 0 213 57 142 2 4.6 0.4a* 104 2 2.64 0.1a* 1.8 0.3 8.5 1.3 2.4 0.6a* 60 7 66 3 43 2 23 3 1.9 0.3 1.4 0.3 7.0 0.7

(b) Females Aspartate aminotransferase (IU/l) Alanine aminotransferase (IU/l) Gamma glutamyl transferase (IU/l) Alkaline phosphatase (IU/l) Sodium (mmol/l) Potassium (mmol/l) Chloride (mmol/l) Calcium (mmol/l) Inorganic phosphorus (mmol/l) Urea (mmol/l) Total bilirubin (mmol/l) Creatinine (mmol/l) Total protein (g/l) Albumin (g/l) Globulin (g/l) Albumin/globulin ratio Total cholesterol (mmol/l) Glucose (mmol/l)

658 389 10 10824 1412 4.20.5 1043 2.640.07 1.30.3 9.01.1 2.10.4 708 673 452 212 2.20.3 1.40.3 5.20.7

6915 4415 10 10628 1422 4.10.4 1052 2.660.08 1.10.3 9.01.1 2.20.4 718 693 482 211 2.30.2 1.40.3 5.40.6

6512 419 10 13246 1421 4.20.5 1053 2.640.06 1.20.2 9.51.3 2.20.3 748 693 471 222 2.10.2 1.40.3 5.10.7

65 10 43 11 1 0 127 29 142 2 4.2 0.5 105 2 2.66 0.08 1.3 0.2 9.6 1.6 2.1 0.5 68 5 68 3 45 2*** 23 2 2.0 0.2** 1.4 0.3 5.2 0.9

69 12 40 8 1 0 119 51 141 2 4.1 0.3 104 2 2.62 0.07 1.2 0.2 9.7 1.7 2.1 0.4 66 6a** 67 3a* 45 2*** 22 3 2.1 0.3* 1.4 0.4 5.4 0.8

a

Significant dose–response test. * P <0.05. ** P <0.01. *** P <0.001.

noted for this animal. Increased salivation, observed from week 7 onwards, was seen among rats of both sexes at the high dose group immediately after dosing but was generally absent by 30 min post-dose. There were no other effects on survival and treatment-related clinical observations. No treatment-related ocular changes were seen at week 13. 3.2.2. Bodyweights and food consumption Bodyweight changes are shown in Fig. 2. Although values were generally similar for all groups throughout the study, calculation of group mean bodyweight gains from

the start to the end of treatment for the five groups showed a slight increase in gain for the two higher dose groups (200.9 19.7, 203.2 21.1, 205.5 17.1, 215.0 27.1 and 213.8 26.5 for males and 75.6 9.6, 82.5 11.5, 81.0 8.7, 87.6 12.9 and 89.8 11.5 for females, respectively). There was no effect on food consumption (see Fig. 3). 3.2.3. Neurobehavioural testing An extensive assessment of neurobehavioural observations and motor activity did not indicate any neurotoxic potential in any of the treated groups (results not shown).

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Table 8 Organ weights (g) (males)a Organ

Adrenals Kidneys Spleen Liver Heart Brain Thymus Testes/epididymides

Group mean Negative control

Vehicle control

58 mg ISP type III HPLC 12/kg/day

290 mg ISP type III HPLC 12/kg/day

580 mg ISP type III HPLC 12/kg/day

0.068 1.812 0.684 8.236 1.010 2.005 0.358 5.267

0.068 1.790 0.649 8.318 0.984 1.996 0.325 5.020

0.072 1.789 0.704 8.156 0.981 1.984 0.334 5.099

0.070 1.844 0.714 8.606 1.037 2.001 0.332 5.227

0.075 1.849 0.715 8.696 1.073** 1.976 0.329 5.078

a

Values are for actual organ weights; values adjusted for final bodyweight are not presented as they are almost identical. ** P <0.01.

Fig. 2. Bodyweights.

3.2.4. Clinical pathology The results of haematology and clinical chemistry parameter assessment for week 13 are shown in Table 6a and b and Table 7a and b, respectively. ISP type IIItreated groups did not show any findings that were considered real in comparison with the negative and vehicle control groups. Results from weeks 4 and 8 (data not shown) reveal the same picture. The minor statistical changes in examined clinical pathology parameters seen on isolated occasions showed no pattern across gender, nor sampling week and were considered

co-incidental. There was no treatment-related effect on examined urine parameters (data not shown). 3.2.5. Pathology examinations Organ weight results are shown in Table 8. The only notable changes were slightly increased mean heart weight for both sexes at the intermediate and high dose levels (which reached statistical significance only for females at the intermediate group and males at the high dose group) and a statistically increased mean spleen weight for females at the high dose level in comparison

T. Hall-Manning et al. / Food and Chemical Toxicology 42 (2004) 321–333

331

Fig. 3. Food consumption (mean).

with the vehicle control group. Analysis of individual data shows a general similarity of spleen weights between the two groups and, in light of no other associated findings, the latter finding was considered coincidental. At termination, there were no macroscopic findings at necropsy considered to be related to treatment with ISP type III. Histopathological examination did not reveal any treatment-related changes. The occurrence of adrenal agonal congestion/haemorrhage (6/39 animals) at the high dose level (and correlated with a macroscopic finding of dark adrenal) was considered a non-specific, random event.

4. Discussion The genotoxic potential and sub-acute toxicity of a preparation of ISP Type III made by a conventional fermentation process using bakers’ yeast, were assessed. Studies showed that a high level of ISP type III preparation was not genotoxic when assessed in three in vitro and one in vivo genotoxicity assays (bacterial mutation, chromosome aberration, mammalian cell gene mutation and rat bone marrow micronucleus). In

addition, the ingredient did not produce any notable signs of systemic toxicity following subchronic administration by oral gavage at levels up to 580 mg ISP type III/kg/day (or 960 mg/kg/day for total ISP type III and glyco-ISP type III) for 3 months in the rat. To our knowledge, this is the first time the toxicological properties of any ice-structuring protein have been investigated. In the bacterial mutation assay, small increases in revertant numbers were observed in one strain of Salmonella typhimurium (TA1535) although the increases were not consistent, did not always show a dose response and showed variation in response in either the absence or presence of S9. Further evaluation showed that a low level of contaminating non-Salmonella bacteria were present in the experimental plates and had been scored as revertant colonies. When these colonies were removed from the total counts and the results were re-calculated for true Salmonella organisms, no statistically or biologically significant increases in revertant colony counts were observed indicating that there was no mutagenic activity with TA1535. The findings were restricted to TA1535 which due to the low spontaneous reversion counts associated with this strain is particularly sensitive as small increases in the number of

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colonies rapidly gave rise to an apparently significant increase in revertant colonies. For the other strains, the spontaneous reversion rate is higher and thus a few additional colonies per plate did not move the total colony counts into numbers which would indicate concern. Oral administration of high levels of ISP type III preparation was well tolerated and did not produce any obvious toxicity over a period of 90 days. There were no effects on food consumption, ophthalmoscopy, neurobehavioural tests, clinical pathology parameters, gross necropsy and histopathology. One male rat in the high dose group was killed humanely due to poor condition but there was no evidence that the deterioration in its condition was related to treatment. Transient post-dose increased salivation was seen among animals of the high dose group and may have been related to the taste of the formulation. However, the finding did not appear until week 7 and so remains unexplained, but unlikely to be toxicologically significant due to a lack of other associated study findings. Over the course of the study, body weight gains were slightly higher (up to 9%) among rats receiving the two higher doses of ISP type III. This finding is almost certainly related to the higher level of proteineous material available at these doses. An apparent slight increase in mean heart weight (up to 9%) was seen for both sexes at the two higher dose levels. Analysis of individual data indicated a few animals with heart weight higher than the greatest value seen in the control group. However, the lack of a dose response in females and any other associated findings (especially no histopathological cardiac changes), makes this observation of doubtful biological significance and, again, probably related to the consumption of higher levels of proteineous material. It can therefore be concluded that the NOAEL is at least 580 mg/kg body weight per day, giving an Acceptable Daily Intake in man of 5.8 mg/kg body weight per day, using a conventional 100-fold safety factor. Our findings thus formally confirm the conclusions of the review by Crevel et al (2002). A particular concern with novel proteins is the possibility that they may provoke reactions in individuals allergic to proteins from the same source. Related studies have demonstrated that ISP type III does not provoke allergic reactions in individuals with diagnosed fish allergy (Bindslev-Jensen et al., 2002). Also, ISP type III preparation did not provoke the formation of IgE or IgG antibodies specific for ISP type III, when administered to volunteers daily for 8 weeks. Furthermore, ISP type III was shown to be structurally unrelated to any known allergen, and to degrade readily in simulated gastric fluid (Baderschneider et al., 2002). Taken together, these data indicate a very low potential to sensitise potentially susceptible individuals. Ice structuring proteins occur naturally in many foods consumed by man, although human exposure has not

been directly measured. Substantial amounts are likely to be consumed in most northerly and temperate regions. Much of this intake is likely to be from edible plants, given their importance in the diet, but in some regions, intake from fish will be significant (Crevel et al., 2002; DeVries and Wohlschlag, 1969; Duman and Olsen, 1993; Fletcher et al.,1999; Griffith and Ewart, 1995). Crevel et al. (2002) estimated that a portion of cod, for instance, may contain up to 196 mg of icestructuring glycoprotein, while up to 420 mg ISP type III could be present in the same weight of ocean pout. Based on ISP concentrations in the blood of cold-water fish and the landings of such fish, the same authors also estimated the average available fish ISP in the diet at approximately 1–10 mg/day in the US and 50–500 mg/ day in Iceland, although they acknowledged these figures to be subject to considerable uncertainty. These data provide circumstantial evidence that, at these levels of consumption, ice-structuring proteins produce no adverse effects on health. Our investigation thus confirms the conclusions reached from those published data and indeed indicates that considerably higher levels of ISPs could be consumed without adverse effects on health. In conclusion, this paper shows that ISP preparation did not possess genotoxic activity and showed no evidence of subchronic toxicity when administered orally to the rat for 90 days at up to 580 mg ISP type III/kg/day.

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