Genotoxicity of styrene-7,8-oxide and styrene in Fisher 344 rats: A 4-week inhalation study

Toxicology Letters 211 (2012) 211–219

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Genotoxicity of styrene-7,8-oxide and styrene in Fisher 344 rats: A 4-week inhalation study Laurent Gaté ∗ , Jean-Claude Micillino, Sylvie Sébillaud, Cristina Langlais, Frédéric Cosnier, Hervé Nunge, Christian Darne, Yves Guichard, Stéphane Binet Institut National de Recherche et Sécurité, Rue du Morvan, CS 60027, 54519 Vandoeuvre les Nancy Cedex, France

a r t i c l e

i n f o

Article history: Received 3 October 2011 Received in revised form 20 March 2012 Accepted 21 March 2012 Available online 7 April 2012 Keywords: Genotoxicity Styrene Styrene-7,8-oxide Comet assay Micronucleus assay Inhalation

a b s t r a c t The cytogenetic alterations in leukocytes and the increased risk for leukemia, lymphoma, or all lymphohematopoietic cancer observed in workers occupationally exposed to styrene have been associated with its hepatic metabolisation into styrene-7,8-oxide, an epoxide which can induce DNA damages. However, it has been observed that styrene-7,8-oxide was also found in the atmosphere of reinforced plastic industries where large amounts of styrene are used. Since the main route of exposure to these compounds is inhalation, in order to gain new insights regarding their systemic genotoxicity, Fisher 344 male rats were exposed in full-body inhalation chambers, 6 h/day, 5 days/week for 4 weeks to styrene-7,8-oxide (25, 50, and 75 ppm) or styrene (75, 300, and 1000 ppm). Then, the induction of micronuclei in circulating reticulocytes and DNA strand breaks in leukocytes using the comet assay was studied at the end of the 3rd and 20th days of exposure. Our results showed that neither styrene nor styrene-7,8-oxide induced a significant increase of the micronucleus frequency in reticulocytes or DNA strand breaks in white blood cells. However, in the presence of the formamidopyridine DNA glycosylase, an enzyme able to recognize and excise DNA at the level of some oxidized DNA bases, a significant increase of DNA damages was observed at the end of the 3rd day of treatment in leukocytes from rats exposed to styrene but not to styrene-7,8-oxide. This experimental design helped to gather new information regarding the systemic genotoxicity of these two chemicals and may be valuable for the risk assessment associated with an occupational exposure to these molecules. © 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Styrene (Fig. 1) is one of the most widely used industrial chemicals. However, increased risk for leukemia, lymphoma, or all lymphohematopoietic cancer among styrene-exposed workers in the reinforced-plastics industries has been suggested in epidemiological studies (Kogevinas et al., 1994). For this reason, styrene has been recently listed in the Twelfth Edition of the National Toxicology Report on Carcinogens (NTP, 2011) as reasonably anticipated to be a human carcinogen and has been classified as possibly carcinogenic to humans (Group 2B) by the International Agency on Cancer Research (IARC, 2002). These classifications are also based on in vivo carcinogenesis studies in experimental animals, and supporting data on mechanisms of carcinogenesis.

Abbreviations: PBPK, physiologically-based pharmacokinetic; SO, styrene-7,8oxide; ENU, N-ethyl-N-nitrosourea; Fpg, formamidopyrimidine DNA glycosylase; Gr, granulocyte; ACGIH, American Conference of Governmental Industrial Hygienists. ∗ Corresponding author. Tel.: +33 0 3 83 50 85 04; fax: +33 0 3 83 50 20 96. E-mail address: [email protected] (L. Gaté). 0378-4274/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxlet.2012.03.796

Styrene which induces lung cancer in mouse but not in rat, is metabolized into styrene-7,8-oxide (SO) by various cytochrome P450 isoenzymes including CYP2E1 and CYP2F (Fig. 1) (Carlson, 2004). SO is an electrophilic compound which can react with nucleophilic groups of biological macromolecules including DNA and proteins. In vitro studies have suggested that these interactions can lead to deleterious cellular effects including cell death (Boccellino et al., 2003), DNA strand breaks (Bastlova et al., 1995; Laffon et al., 2001) and mutations (Shield and Sanderson, 2004). Following oral administration, SO also induces tumors in rat and mouse models (IARC, 1994). Because of its genotoxic and carcinogenic properties in these species, this epoxide has been classified as probably carcinogenic to human (Group 2A) by the International Agency on Cancer Research (IARC, 1994), as a suspected carcinogen (Category 1B) in the European Union (European Commission, 2008), and as reasonably anticipated to be a human carcinogen in the Twelfth Edition of the National Toxicology Report on Carcinogens (NTP, 2011). Although SO is used in various industrial processes, the few occupational exposure investigations done so far, have only been performed in workplaces where polyester-based reinforced plastics are made (Nylander-French et al., 1999; Rappaport et al., 1996). In such industries, where styrene is used as a cross-linking agent,

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L. Gaté et al. / Toxicology Letters 211 (2012) 211–219 compound within Phosphate-buffered saline pH 6.0 (PBS) (Invitrogen, Cergy Pontoise, France). 2.2. Animal husbandry and exposure

Fig. 1. Chemical structures of styrene and styrene-7,8-oxide.

relatively high atmospheric concentrations of this compound are found (40–400 mg/m3 ) along with much lower but not negligible levels of SO (<1 mg/m3 ) which may come from styrene oxidation (Miller et al., 1994; Pfaffli and Saamanen, 1993). Even though contradictory or equivoqual results are found in the literature, some epidemiological studies have shown that cytogenetic changes including chromosome aberration, micronuclei induction and sister chromatid exchange were detected in leukocytes from styrene-exposed workers from reinforced-plastic industries (Henderson and Speit, 2005). It has been suggested that these genotoxic effects were associated with the hepatic metabolisation of styrene into SO (Henderson and Speit, 2005). This, however, does not take into account the presence of SO as an airborne contaminant. Although the atmospheric SO levels are about 1/500 to 1/1500 times lower than those of styrene, a physiologically-based pharmacokinetic (PBPK) suggested that inhaled SO contributed 3640 times more to the SO blood content than an equivalent amount of inhaled styrene (Tornero-Velez and Rappaport, 2001). Despite the fact that the main route of exposure to styrene-7,8-oxide is inhalation, only few data regarding the in vivo genotoxicity of inhaled SO are available. Inhalation studies performed so far showed that inhalation exposure of mice to SO at a concentration of 72 ppm induced a slight increase in sister chromatid exchange (SCE) in hepatocytes and alveolar macrophages but not in bone marrow cells (IARC, 1994). In addition, no increase in the incidence of SCE was observed in bone marrow cells of Chinese hamster exposed to SO at concentrations of 25, 50, 75 or 100 ppm for 2, 4 and 21 (25 ppm only) days (Norppa et al., 1979). Most of the data available were obtained following oral administration or intraperitoneal injection and showed that this oxide can induce DNA strand breaks (assessed by the comet assay) in female C57BL/6 mice following intraperitoneal injection of 100–200 mg/kg (Vaghef and Hellman, 1998). Since styrene and styrene-7,8-oxide exposure may occur in the reinforced plastic industry and, from epidemiological studies, may lead to cytogenetic changes in leukocytes and be related to an increased risk of lymphohematopoietic cancer, it was of interest to investigate the systemic genotoxicity of inhaled SO and styrene in an experimental animal model widely used in regulatory genetic toxicology. Thus, we exposed Fisher 344 male rats 6 h/day, 5 days/week for 4 weeks to different concentrations of styrene and styrene-7,8-oxide and examined the genotoxic effects of these substances by analyzing the induction of micronuclei in circulating reticulocytes, and DNA strand breaks using the comet assay in circulating leukocytes. The study of these genetic toxicology parameters was completed by the analysis of styrene and styrene-7,8-oxide blood levels and hematological profiles. 2. Materials and methods 2.1. Chemicals Styrene (Reference S4972), Styrene-7,8-oxide (Reference S5006) and N-ethyl-Nnitrosourea (Reference N3385) were purchased from Sigma Aldrich (Saint Quentin Fallavier, France). ENU stock solution was made by dissolving this alkylating

All experiments involving animals were conducted in the INRS laboratory animal facility approved by the French Ministry of Agriculture according to French regulations regarding the protection of animals used for experimental and other scientific purposes. 6-week old F344 male rats (n = 6 per group) were purchased from Charles River Laboratories (Saint Germain sur l’Arbresle, France). The animals were housed into polycarbonate cages covered with spun-bonded polyester cage filter and fed with standard pellet food and water ad libitum. For the genotoxicity assays, the rats were exposed to styrene or styrene-7,8-oxide vapors using a whole-body exposure system. Animals were exposed to 75, 300 or 1000 ppm of styrene or 25, 50 or 75 ppm of styrene-7,8-oxide 6 h/day, 5 days/week, for 4 weeks, from 09:00 am to 03:00 pm in 200 L glass/stainless-steel inhalation chambers. The unexposed rats (controls) were maintained in similar chambers but exposed to clean air. During the 6-h exposure period, the rats did not have access neither to food nor water. In the inhalation chambers, the animals were separated from each other by small wire cloth enclosures. The chambers were designed to sustain a dynamic and adjustable airflow (5–6 m3 /h) and were maintained at a negative pressure of no more than 5 mm H2 O in order to prevent any leakage of the test atmospheres. The input air was filtered and conditioned at a temperature of 22 ± 1 ◦ C and a humidity of 55 ± 10%. Styrene and styrene-7,8-oxide were generated using a thermoregulated glass streamer. The chemicals, brought by a pump, were instantaneously vaporized by contact with the heated surface and carried out, with the help of an additional airflow, through the streamer, into the main air inlet pipe of the exposure chambers. Exposure levels were measured three times during the 6 h-exposure period by collecting atmosphere samples through glass tubes packed with activated charcoal 18–35 mesh (VWR International S.A.S, Fontenay Sous Bois, France) or with TENAX TA 60/80 (Sigma Aldrich, Saint Quentin Fallavier, France) for styrene and styrene7,8-oxide respectively. Styrene was then desorbed from activated charcoal with carbon disulfide containing toluene as the internal standard and analyzed on a Shimadzu GC8 gas chromatograph equipped with a flame ionization detector. Styrene samples were chromatographed on a 10 m × 0.53 mm (2 ␮m film thickness) CP-Sil 5 column, using nitrogen as the carrier gas at a constant flow of 15 mL/min. The column temperature was kept at 100 ◦ C while the injector and the detector were maintained at 220 ◦ C. Styrene-7,8-oxide was desorbed from TENAX with hexane containing o-xylene as the internal standard and analyzed on a Varian CP-3800 gas chromatograph equipped with a flame ionization detector. Styrene-7,8-oxide samples were chromatographed on a 30 m × 0.53 mm (1.5 ␮m film thickness) CP-Sil 5 column, using nitrogen as the carrier gas at a constant flow of 3 mL/min. The column temperature program was: 70 ◦ C for 1 min then increased to 110 ◦ C at a rate of 5 ◦ C/min. The injector (Flash 1061) and the detector were maintained at 240 ◦ C and 250 ◦ C respectively. These analyses allowed carrying out daily calibrations. A third GC was also used to perform online concentration measurements every minute in order to follow the stability of the vapor generation throughout the exposure. This GC was also equipped with a flame ionization detector and an automatic gas-sampling valve. 2.3. Blood levels of styrene-7,8-oxide and styrene 6-week old rats with a catheter located into their carotid artery were purchased from Charles River Laboratories (L’Arbresle, France). The animals (n = 6) were placed into plethysmographs and exposed nose-only to styrene-7,8-oxide (25, 50 or 75 ppm) or styrene (1000 ppm) for 6 h. Blood collections through the catheter were performed just before the end of exposure. Blood samples were collected into tubes containing K3 EDTA as an anticoagulant. For the measurement of blood styrene-7,8-oxide, after a brief homogenization, blood samples were transferred into tubes containing hexane and butylbenzene used as an internal standard, mixed for 5 min at room temperature and then centrifuged for 10 min at 2500 × g at −4 ◦ C. The subsequent organic phases were concentrated by evaporation. For the measurement of styrene, blood was mixed with carbon disulfide containing toluene as an internal standard. The tubes were then homogenized and centrifuged as described above. The concentrations of styrene-7,8-oxide or styrene in the final organic solution were measured by gas chromatography coupled with a flame ionization detector. 2.4. Hematology Blood samples (100 ␮L) were collected, at the end of the 3rd and 20th days of inhalation, at the tail vein using a Terumo 24G SurFlo I.V. catheter (Laboratoires TERUMO France S.A., Guyancourt France) and immediately transferred into tubes containing 10 ␮L of a Na2 EDTA solution (20 mg/mL) and gently homogenized by pipeting. Total numbers of erythrocytes, leukocytes and platelets were determined with a Scil Vet abc hematology analyzer (Scil animal care company, Altorf, France). Blood smears were made and stained using the May-Grunwald Giemsa staining technique. Leukocyte differential cell counts were then performed by counting 1000 white blood cells per animal.

L. Gaté et al. / Toxicology Letters 211 (2012) 211–219

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Fig. 2. Measurement of blood styrene-7,8-oxide concentrations in rats exposed to styrene-7,8-oxide or styrene for 6 h. Histograms represent the mean and standard deviation for styrene-7,8-oxide concentrations in each treatment group. Blood concentration of styrene in animals exposed to styrene 1000 ppm was 85.5 ± 11.3 ␮g/g of blood. In the control group, blood levels of styrene-7,8-oxide and styrene were below the quantification limits of the analytical methods (15 and 80 ng/g of blood respectively).

2.5. Micronucleus assay on circulating red blood cells The micronucleus assay was performed following the OECD guideline no. 474 (OECD, 1997) using the MicroFlowPLUS® kit from Litron Laboratories (distributed by BD Biosciences, Le Pont de Claix, France) according to the manufacturer’s instructions. Briefly, blood samples were collected at the end of the 3rd and 20th days of inhalation, at the tail vein using a Terumo 24G SurFlo I.V. catheter (Laboratoires TERUMO France S.A, Guyancourt France) and immediately transferred into tubes containing an anticoagulant solution provided with the kit. Samples were then fixed in methanol kept at −80 ◦ C and stored at this temperature until red blood cell labeling. The day of the micronucleus analysis, blood samples were removed from the −80 ◦ C freezer and washed once with the buffer solution provided with the kit. Samples were then labeled with a FITC-conjugated anti-rat CD71 antibody and a PE-conjugated anti-rat platelet antibody and the RNA degraded using an RNAse solution. Cell DNA was finally stained with propidium iodide before analysis by flow cytometry using a BD FACS Canto II flow cytometer coupled with the FACS Diva software (version: 6.1.2). Prior to each experiment, flow cytometry analysis was calibrated with blood samples obtained from rats infected by Trypanosoma brucei and provided with the kit. Proper cytometer settings were further validated with well-defined blood samples also provided with the kit. Results were expressed as the percentage of reticulocytes (CD71+ cells) within the whole red blood cell population and the percentage of micronucleated reticulocytes among the whole reticulocyte population. Groups of age-matched rats which received, 48 and 3 h prior to blood collection, intraperitoneal injections of N-ethyl-N-nitrosourea (40 mg/kg) or vehicle (PBS pH 6.0) were used as positive and negative controls respectively.

considered as a statistical unit and the median of the 100 analyzed comets was used to represent the extent of DNA damages in each sample. Groups of age-matched rats which received, 48 and 3 h prior to blood collection, intraperitoneal injections of N-ethyl-N-nitrosourea (40 mg/kg) or vehicle (PBS pH 6.0) were used as positive and negative controls respectively.

3. Results 3.1. Blood concentrations of styrene-7,8-oxide Fig. 2 shows that the average blood level in styrene-7,8-oxide increases with the inhalation chamber level of this chemical (0.24 ± 0.10 ␮g/g of blood at 25 ppm, 0.60 ± 0.20 ␮g/g of blood at 50 ppm and 0.86 ± 0.43 ␮g/g of blood at 75 ppm). The statistical analysis of these results shows a moderate but significant relationship between the blood concentration of styrene-7,8-oxide and its atmospheric level (simple linear regression followed by an ANOVA and Fisher test, p < 0.05; r2 = 0.46). In addition, the average blood concentration of styrene-7,8-oxide in animals exposed to styrene 1000 ppm (0.37 ± 0.08 ␮g/g of blood) was between those measured when animals were exposed to 25 and 50 ppm of atmospheric SO.

2.6. Single cell electrophoresis assay (comet assay)

3.2. Blood cell counts

Immediately after the end of the 3rd and 20th days of inhalation, blood samples (20 ␮L) were collected at the tail vein using a Terumo 24G SurFlo I.V. catheter (Laboratoires TERUMO France S.A., Guyancourt, France) and quickly transferred into tubes containing 2 ␮L of a Na2 EDTA solution (20 mg/mL) and gently homogenized by pipeting. 500 ␮L of ice-cold PBS was then added to the blood samples and centrifuged 5 min at 400 × g at 4 ◦ C. The pellets were resuspended into 50 ␮L of PBS before being mixed with 1% low-gelling agarose (Sigma Aldrich) and poured onto microscope glass slides pre-coated with 1% general routine agarose (Sigma Aldrich). Microscope slides were laid onto an ice bed to let the agarose solidified. The slides were then immersed into the ice-cold lysis buffer (2.5 mM NaCl; 100 mM Na2 EDTA; 10 mM Tris base; 10% DMSO and 1% Triton; pH 10) overnight at 4 ◦ C. For each animal 2 slides were made: one for the regular single gel electrophoresis assay and one for the formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay. For the Fpg-modified comet assay, following lysis, slides were washed three times 5 min with the fpg incubation buffer (Hepes 40 mM, KCl 0.1 M, EDTA 0.5 mM, bovine serum albumin 0.2 mg/mL; pH 8) at 4 ◦ C and then incubated for 30 min at 37 ◦ C with 5 U/mL of Fpg (Sigma Aldrich) in the Fpg incubation buffer. For the regular comet assay, slides were not treated with Fpg. The slides were then immersed into the ice-cold alkaline electrophoresis buffer (300 mM NaOH; 1 mM Na2 EDTA; pH > 13) for 20 min and then submitted to electrophoresis for 40 min at 0,9 V/cm. At the end of the electrophoresis, slides were immersed into the neutralization buffer (Tris base 0.4 M; pH 7.5) for 15 min at 4 ◦ C. Slides were then washed twice with ultrapure water for 5 min and dehydrated for 10 min into 75% ethanol before being dried at 45 ◦ C and stored in the dark at room temperature. For fluorescent microscopy analysis, slides were rehydrated 10 min with ultrapure water and then stained with propidium iodide 2.5 ␮g/mL in PBS. For each sample, 100 cells were analyzed using the Comet Assay IV software (Perceptive Instruments, Suffolk, UK) and the percentage of DNA in the tail of each comet was measured. For the statistical analysis, each blood sample was

The results presented in Table 1A show that after 3 days of treatment, the animal exposure to styrene-7,8-oxide leads to a significant decrease of the total leukocyte number. In animals exposed to 50 or 75 ppm of SO, this is also associated with a modification of the white blood cell differential count demonstrated by a drop in the percentage of neutrophil granulocytes and an elevation of that of lymphocytes. In the case of animals exposed to styrene, only the highest concentration (1000 ppm) induces a significant decrease of the white blood cell number without modification of the percentages of each sub-population. At the end of the 20th day of exposure, the number of circulating leukocytes is still lower in animals exposed to styrene-7,8-oxide and to 1000 ppm of styrene (Table 1B). However, while for the control and the groups exposed to the two lowest dose of styrene (75 and 300) the total leukocyte numbers are similar between days 3 and 20, they are statistically higher in SO-exposed animals at day 20 than at day 3 (Student T-test, p < 0.05). This is also true for animals exposed to 1000 ppm of styrene. In addition, at the end of the 20th day of exposure no difference is observed in the percentages of each leukocyte sub-population between untreated and exposed animals. Finally, a significant change in the white blood cell differential count is observed in the control group between days 3 and 20 (Tables 1A and 1B). The numbers of platelets and red blood cells

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Table 1A Blood cell count at the end of the 3rd day of exposure to styrene or styrene-7,8-oxide. The percentages of each leukocyte subpopulation are expressed between brackets. Leukocytes

Erythrocytes 3

Control SO 25 ppm SO 50 ppm SO 75 ppm Styrene 75 ppm Styrene 300 ppm Styrene 1000 ppm

3

3

3

3

3

Lymphocytes (×10 /␮L)

Neutrophil Gr. (×10 /␮L)

Eosinophil Gr. (×10 /␮L)

Basophil Gr. (×10 /␮L)

Monocytes (×10 /␮L)

Total (×10 /␮L)

7.88 ± 1.00 (71.1 ± 2.5) 5.36 ± 0.57* (73.6 ± 3.7) 4.39 ± 0.36* (80.8 ± 2.5)* 4.25 ± 0.66* (80.4 ± 4.8)* 7.84 ± 0.27 (74.0 ± 4.5) 7.80 ± 0.64 (71.8 ± 4.5) 4.51 ± 0.33* (72.2 ± 2.5)

2.84 ± 0.41 (25.6 ± 2.5) 1.72 ± 0.28* (23.6 ± 3.5) 0.89 ± 0.15* (16.4 ± 2.9)* 0.86 ± 0.20* (16.8 ± 5.3)* 2.38 ± 0.73 (22.0 ± 4.8) 2.72 ± 0.55 (24.8 ± 3.6) 1.57 ± 0.25* (25.0 ± 2.6)

0.08 ± 0.03 (0.7 ± 0.3) 0.04 ± 0.04 (0.6 ± 0.4) 0.02 ± 0.01 (0.3 ± 0.1) 0.01 ± 0.00 (0.3 ± 0.1) 0.11 ± 0.06 (1.0 ± 0.6) 0.07 ± 0.05 (0.7 ± 0.4) 0.03 ± 0.01 (0.4 ± 0.2)

0.01 ± 0.01 (0.1 ± 0.1) 0.00 ± 0.00 (0.1 ± 0.1) 0.00 ± 0.00 (0.0 ± 0.0) 0.00 ± 0.01 (2.5 ± 0.7) 0.01 ± 0.01 (0.1 ± 0.1) 0.00 ± 0.00 (0.0 ± 0.0) 0.01 ± 0.01 (0.1 ± 0.1)

0.28 ± 0.11 (2.6 ± 1.1) 0.16 ± 0.03 (2.2 ± 0.2) 0.14 ± 0.03 (2.5 ± 0.4) 0.14 ± 0.05 (2.5 ± 0.7) 0.30 ± 0.07 (2.9 ± 0.8) 0.30 ± 0.08 (2.8 ± 0.7) 0.15 ± 0.05 (2.3 ± 0.7)

11.08 7.29 5.43 5.27 10.64 10.89 6.26

± ± ± ± ± ± ±

1.27 0.72* 0.36* 0.66* 0.82 0.92 0.52*

6

(×10 /␮L) 7.5 8.0 8.7 8.9 7.7 7.8 7.8

± ± ± ± ± ± ±

0.5 0.6 0.4 0.5 0.4 0.5 0.3

Platelets (×103 /␮L) 757.7 733.0 733.0 773.7 696.3 730.5 732.0

± ± ± ± ± ± ±

46.7 55.4 44.1 82.2 77.2 51.8 15.0

L. Gaté et al. / Toxicology Letters 211 (2012) 211–219

For each group, results are expressed as the mean ± standard deviation. Gr: granulocytes. * Significantly different from the control group (ANOVA, Dunnett test, p < 0.05%).

Table 1B Blood cell count at the end of the 20th day of exposure to styrene or styrene-7,8-oxide. The percentages of each leukocyte subpopulation are expressed between brackets. Leukocytes

Control SO 25 ppm SO 50 ppm SO 75 ppm Styrene 75 ppm Styrene 300 ppm Styrene 1000 ppm

Lymphocytes(×103 /␮L)

Neutrophil Gr. (×103 /␮L)

Eosinophil Gr. (×103 /␮L)

Basophil Gr. (×103 /␮L)

Monocytes (×103 /␮L)

Total (×103 /␮L)

7.78 ± 0.73 (62.2 ± 3.5) 6.59 ± 0.97* (67.4 ± 3.4) 5.78 ± 0.80* (58.7 ± 3.3) 5.50 ± 0.82* (57.5 ± 6.0) 7.40 ± 0.49 (66.4 ± 5.0) 8.29 ± 0.50 (66.7 ± 2.6) 5.59 ± 1.20* (59.6 ± 5.4)

4.26 ± 0.61 (34.0 ± 3.5) 2.83 ± 0.48* (29.0 ± 3.4) 3.67 ± 0.41 (37.5 ± 3.9) 3.72 ± 0.67 (39.0 ± 6.6) 3.33 ± 0.55* (29.8 ± 4.2) 3.64 ± 0.44 (29.2 ± 2.9) 3.35 ± 0.42* (36.9 ± 6.2)

0.08 ± 0.03 (0.7 ± 0.3) 0.07 ± 0.02 (0.7 ± 0.2) 0.06 ± 0.03 (0.6 ± 0.3) 0.06 ± 0.02 (0.6 ± 0.2) 0.08 ± 0.04 (0.7 ± 0.3) 0.12 ± 0.04 (1.0 ± 0.4) 0.07 ± 0.04 (0.8 ± 0.3)

0.01 ± 0.01 (0.1 ± 0.1) 0.01 ± 0.01 (0.1 ± 0.1) 0.01 ± 0.01 (0.1 ± 0.1) 0.00 ± 0.00 (0.0 ± 0.0) 0.01 ± 0.01 (0.1 ± 0.1) 0.02 ± 0.01 (0.1 ± 0.1) 0.00 ± 0.01 0.1 ± 0.1

0.38 ± 0.08 (3.1 ± 0.6) 0.27 ± 0.09 (2.8 ± 0.9) 0.30 ± 0.06 (3.1 ± 0.5) 0.27 ± 0.06 (2.9 ± 0.7) 0.34 ± 0.11 (3.0 ± 1.0) 0.37 ± 0.06 (3.0 ± 0.3) 0.26 ± 0.10 (2.7 ± 0.8)

12.52 9.77 9.82 9.56 11.17 12.43 9.28

For each group, results are expressed as the mean ± standard deviation. Gr: granulocytes. * Significantly different from the control group (ANOVA, Dunnett test, p < 0.05%).

± ± ± ± ± ± ±

1.06 1.24* 1.00* 0.86* 0.45 0.72 1.41*

Erythrocytes

Platelets

(×106 /␮L)

(×103 /␮L)

8.3 8.8 9.3 9.5 8.7 8.6 8.8

± ± ± ± ± ± ±

0.5 0.3 0.4 0.3 0.4 0.7 0.4

664.7 653.2 661.7 670.7 700.3 697.5 707.0

± ± ± ± ± ± ±

34.0 50.8 44.2 43.7 90.0 73.9 43.2

L. Gaté et al. / Toxicology Letters 211 (2012) 211–219 Table 2A Frequency of micronuclei in circulating reticulocytes following exposure to styrene7,8-oxide or styrene. Blood collection was made at the end of the 3rd day of exposure. Reticulocytes %

Mean ± S.D.

3.00 4.31 4.28 3.82 4.16 3.96

3.92 ± 0.11

3.33 2.87 3.39 2.88 2.99 2.94

3.06 ± 0.07*

2.94 2.69 2.84 3.14 2.91 2.80

2.89 ± 0.07*

2.66 2.28 2.29 1.91 1.89 2.77

2.30 ± 0.07*

Styrene 300 ppm

2.44 2.46 3.65 2.82 2.93 2.95

2.88 ± 0.06*

Styrene 1000 ppm

2.42 2.27 2.71 2.59 3.17 2.74

2.65 ± 0.14*

Control ENU

2.95 2.91 3.55 3.47

3.22 ± 0.05

0.98 0.97 1.22 1.23

1.10 ± 0.11**

Control

SO 25 ppm

SO 50 ppm

SO 75 ppm

ENU 40 mg/kg

* **

MN-reticulocytes %

Table 2B Frequency of micronuclei in circulating reticulocytes following exposure to styrene7,8-oxide or styrene. Blood collection was made at the end of the 20th day of exposure. Reticulocytes

Mean ± S.D.

0.12 0.27 0.29 0.20 0.41 0.11

0.23 ± 0.11

0.04 0.25 0.13 0.10 0.14 0.14

0.13 ± 0.07

0.22 0.14 0.08 0.08 0.22 0.05

0.13 ± 0.07

0.37 0.17 0.27 0.21 0.23 0.25

0.25 ± 0.07

0.10 0.12 0.05 0.22 0.07 0.18

0.12 ± 0.06

0.12 0.06 0.10 0.16 0.43 0.09

0.16 ± 0.14

0.19 0.22 0.12 0.11

0.16 ± 0.05

0.77 0.65 0.52 0.55

0.62 ± 0.11**

3.3. Micronucleus assay Intraperitoneal injections of the positive control N-ethyl-Nnitrosourea induce a significant increase of the frequency of micronucleated reticulocytes as compared to the untreated group that received intraperitoneal injections of the vehicle (0.62 ± 0.11% for day 3 and 0.43 ± 0.14% for day 20 for ENU vs. 0.16 ± 0.05% for day 3 and 0.13 ± 0.05% for day 20 for the untreated group). Furthermore, this amount of ENU induces a 3-fold drop of the percentage of blood reticulocytes (Tables 2A and 2B). In blood samples collected at the end of the 3rd and 20th days of exposure to styrene-7,8-oxide or styrene, no significant change in the frequency of micronucleated reticulocytes is observed in exposed animals as compared to the untreated ones (0.23 ± 0.11%

MN-reticulocytes

%

Mean ± S.D.

%

Mean ± S.D.

Control

2.00 2.10 2.22 2.48 2.22 2.14

2.19 ± 0.05

0.22 0.14 0.14 0.17 0.15 0.06

0.15 ± 0.05

SO 25 ppm

1.71 2.09 2.13 2.16 2.15 1.88

2.02 ± 0.03

0.14 0.16 0.18 0.21 0.14 0.12

0.16 ± 0.03

SO 50 ppm

1.51 1.64 1.87 1.45 1.85 1.52

1.64 ± 0.10*

0.07 0.17 0.10 0.22 0.09 0.34

0.17 ± 0.10

SO 75 ppm

1.21 1.48 1.46 1.52 1.28 1.49

1.41 ± 0.05*

0.08 0.06 0.06 0.15 0.08 0.16

0.10 ± 0.05

Styrene 300 ppm

2.31 2.36 2.17 2.53 2.49 2.21

2.34 ± 0.10

0.30 0.11 0.26 0.10 0.06 0.13

0.16 ± 0.10

Styrene 1000 ppm

2.25 2.54 2.48 2.53 2.56 2.11

2.41 ± 0.11

0.09 0.15 0.12 0.15 0.41 0.15

0.18 ± 0.11

Control ENU

1.74 1.79 2.25 1.43 1.73 2.25

1.91 ± 0.05

0.14 0.16 0.07 0.11 0.21 0.11

0.13 ± 0.05

ENU 40 mg/kg

0.51 1.10 1.11 0.49 0.66 0.47

0.72 ± 0.14**

0.50 0.26 0.32 0.58 0.34 0.57

0.43 ± 0.14**

Statistically different from the control group (ANOVA, Dunnett test, p < 0.05). Statistically different from the control ENU group (Student T test, p < 0.05).

are however not affected by rat exposure to styrene-7,8-oxide or styrene at both time points (Tables 1A and 1B).

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* **

Statistically different from the control group (ANOVA, Dunnett test, p < 0.05). Statistically different from the control ENU group (Student T test, p < 0.05).

on day 3 and 0.15 ± 0.05% on day 20 for the untreated group vs. 0.25 ± 0.07% on day 3 and 0.10 ± 0.05% on day 20 for SO 75 ppm and 0.16 ± 0.14% on day 3 and 0.18 ± 0.11% on day 20 for styrene 1000 ppm) (Tables 2A and 2B). However in the same blood samples, the percentage of reticulocytes is significantly decreased on day 3 for animals exposed to styrene-7,8-oxide or styrene as compared to the untreated group (3.92 ± 0.11% for the untreated group vs. 2.30 ± 0.07% for SO 75 ppm and 2.65 ± 0.14% for styrene 1000 ppm) (Table 2A). At the end of the 20th day of exposure, only the two highest doses of styrene-7,8-oxide still lead to a significant fall of the percentage of reticulocytes (2.19 ± 0.05% for the untreated group vs. 1.64 ± 0.10% and 1.41 ± 0.05% for SO 50 and 75 ppm respectively) (Table 2B).

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significant increase of DNA strand breaks is observed in cells collected at the end of the 3rd day from the animals exposed to styrene whatever the dose tested (0.59 ± 0.37% for the untreated group vs. 5.20 ± 2.08%, 4.37 ± 2.25% and 4.21 ± 1.37%; for styrene 75, 300 and 1000 ppm respectively). No such a modification is found neither in the animals exposed to styrene-7,8-oxide for 3 or 20 days nor the animals exposed to styrene for 20 days (1.15 ± 0.81% for the untreated group vs. 0.39 ± 0.17% for SO 75 ppm and 0.26 ± 0.35% for styrene 1000 ppm on day 20).

4. Discussion

Fig. 3. Comet assay performed on circulating leukocytes. Animal blood was collected at the end of the 3rd (A) and 20th (B) days of exposure to styrene-7,8-oxide or styrene. DNA damage extent in each sample was represented by the median of tail DNA percentages from 100 analyzed comets. For each treatment group, histograms represent the mean and standard deviation of these medians. *Statistically different from the control group (ANOVA, Dunnett test; p < 0.05). **Statistically different from the control ENU group (Student T test, p < 0.05).

3.4. Single cell electrophoresis assay The comet assay which was carried out on circulating white blood cells was also verified using ENU as a positive control (Fig. 3A and B). The results show a significant increase of DNA strand breaks (expressed as the percentage of the tail DNA) in the animals exposed to this alkylating agent as compared to the control group in the presence or absence of Fpg (11.91 ± 2.47% vs. 0.18 ± 0.07% at the 3rd day and 8.39 ± 1.36% vs. 0.06 ± 0.10% at the 20th day respectively without Fpg and 20.44 ± 1.72% vs. 2.46 ± 1.24% on day 3 and 13.16 ± 3.68% vs. 0.10 ± 0.07% on day 20 respectively with Fpg). In the absence of Fpg, the assays performed on the animals exposed to styrene or styrene-7,8-oxide immediately at the end of the 3rd and the 20th days of exposure do not show any increase of the percentage of tail DNA in the circulating leukocytes coming from the animals exposed to either chemical as compared to the control group (1.65 ± 1.81% on day 3 and 0.04 ± 0.04% on day 20 for the untreated group vs. 0.51 ± 0.20% at day 3 and 0.09 ± 0.07% on day 20 for SO 75 ppm and 0.70 ± 0.49% on day 3 and 0.09 ± 0.09% at day 20 for styrene 1000 ppm). However, in slides treated with Fpg, a

Increased risk for lymphohematopoietic cancers (NTP, 2011) and cytogenetic alterations in leukocytes (Henderson and Speit, 2005) have been observed in workers exposed to styrene and styrene-7,8-oxide in reinforced plastic industries where the levels of exposure to styrene may sometime be higher than the threshold limit values (with a 8 h-Time Weighed Average of 50 ppm in France, 20 ppm in Germany and for the ACGIH in the United States of America). However, despite the occupational exposure to styrene-7,8-oxide, no threshold limit value has been defined for this chemical (Nylander-French et al., 1999; Rappaport et al., 1996). Since the main route of exposure to such volatile organic compounds is inhalation, we decided to investigate the systemic genotoxic properties of inhaled styrene and styrene-7,8-oxide in rats. This route of exposure has been rarely used to evaluate the genotoxicity properties of this latter chemical. Among the studies available, one showed that styrene-7,8-oxide at a concentration of 100 ppm 7 h/day, 5 days/week for 3 weeks induced 16% of mortality in female Wistar rats (Sikov et al., 1986). In order to limit the toxicity and the lethality induced by this compound, we exposed the animals to a maximum concentration of 75 ppm, 6 h/day, 5 days/week for 4 weeks. We observed that this concentration did not induce any animal death. Regarding styrene, we decided to use, as the highest concentration, the same as the one tested in rat chronic inhalation study in rat (i.e. 1000 ppm) (Cruzan et al., 1998). In addition, the study of hematological parameters showed that rat exposure to styrene-7,8-oxide or styrene (1000 ppm) led to a significant decrease in circulating leukocytes and for some concentrations, a modification of the white blood cell differential counts after 3 days of exposure. In these exposed animals, leukocyte total counts were higher at the end of the 20th day of exposure as compared to the 3rd one. These results suggest that continuous exposure to such high levels of these organic compounds may lead to a hematopoietic adaptation, a resistance to styrene or SO, or an increased detoxicification of reactive and toxic metabolites of these compounds. This hypothesis is strengthened by the data from Cruzan et al. (1998) who did not observe any hematological modifications after 13 weeks of exposure to styrene in a chronic inhalation study with CD rats. In addition, since many chemotherapeutic molecules and industrial chemicals share the same metabolic pathways as well as mechanisms of genotoxicity, one may assume that cellular adaptation to exogenous stress induced by anticancer drugs is not different from that presumably triggered by styrene and SO (Tiligada, 2006). Various compounds are able to alter hematological parameters (Fliedner et al., 1990), however numerous mechanisms may lead to such modifications including myelotoxicity. Differences in white blood cell counts in the control group between days 3 and 20 is also difficult to explain but might be associated with the fact that animals were restrained in inhalation chambers or that blood collections may have affected leukocytes production. Since styrene-7,8-oxide is a metabolite of styrene that has been hypothesized to be responsible for the cytogenetic changes observed in blood cells from workers and exposed animals, it was important to assess the blood levels of styrene-7,8-oxide in

L. Gaté et al. / Toxicology Letters 211 (2012) 211–219

Fig. 4. Schematic fate of styrene and styrene-7,8-oxide following inhalation. ST: styrene, SO: styrene-7,8-oxide, SG: styrene glycol, SOSG: glutathione conjugate, CYP: cytochromes P450, EH: epoxide hydrolase, GST: glutathione S-transferases.

rats. Since the blood half-life of SO is extremely short which is probably associated with its high reactivity with biological macromolecules (DNA, proteins) (Kessler et al., 1990), we decided to collect blood samples at the end of a 6-h exposure period while animals were still maintained inside contention tubes inserted into the inhalation chamber. For this, we used animals equipped with a catheter placed into the carotid artery. This experimental animal model, somewhat different from the one used for the genotoxicity assays, was expected to be more relevant to quantify the blood styrene-7,8-oxide. In addition, following SO inhalation, blood collection from the carotid gave us the opportunity to measure the SO directly coming from the lungs and avoids the hepatic first-pass metabolism (Fig. 4). The data showed that the styrene7,8-oxide blood concentration was correlated with the amount of this molecule found in the inhalation chamber. The blood concentration of styrene-7,8-oxide determined in animals exposed to 1000 ppm of styrene was between those obtained from animals exposed to 25 or 50 ppm of styrene-7,8-oxide. Following styrene inhalation, blood SO may originate from the liver and lung metabolism of the parent compound by cytochrome P450 isoenzymes (IARC, 2002; Sumner and Fennell, 1994). SO has probably exited the liver and lungs before being further metabolized by epoxide hydrolase or glutathione S-transferases in these organs (Fig. 4). These results also showed that, in rats, the dose of styrene present in the inhalation chamber required to obtain an equivalent concentration of blood styrene-7,8-oxide would be 20–40 times higher than that of styrene-7,8-oxide. These figures are very different from the ones obtained with from the PBPK model of Tornero-Velez and Rappaport (2001) showed that in humans, inhaled SO would contribute 3640 times more to the blood SO

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concentration than inhaled styrene. This is explained by the fact that the kinetics of styrene and styrene-7,8-oxide metabolism are different between rats and humans (Csanady et al., 2003); indeed the activity of enzymes involved in styrene and styrene-7,8-oxide metabolism is higher in rodents than in humans. In addition, the glutathione conjugation of metabolites from these chemicals is much higher in rats (Sumner and Fennell, 1994). Although we have exposed animals to 75 ppm of styrene, the method was not sensitive enough to quantify styrene-7,8-oxide in blood at such a low concentration of atmospheric styrene. Other methods including the quantification of serum protein adducts might be more appropriate for such assessments with low styrene concentrations (Teixeira et al., 2007). The systemic genotoxicity of styrene and styrene-7,8-oxide was evaluated by different methods: the micronucleus assay on reticulocytes from circulating blood following the OECD guideline no. 474 (OECD, 1997) and the comet assay on circulating leukocytes. The micronucleus assay, performed on circulating reticulocytes is a good alternative to the standard bone marrow version since it allows to perform the experiment on a larger number of events (20,000 instead of 2000 respectively), may limit errors due to the operator (De Boeck et al., 2005; Torous et al., 2005) and appears to be as reliable as the test performed on bone marrow (Fiedler et al., 2010). The results showed that while ENU induced a significant increase of the micronucleated reticulocytes as compared to the control group, exposure to styrene or styrene-7,8-oxide did not induce any significant increase of the micronucleated reticulocytes whatever the concentration or the exposure time considered (3 or 20 days). The test was performed at two different time points in order to limit false negatives due to variations in the frequency of micronucleated cells during animal exposure (Hamada et al., 2001). The concentrations of styrene-7,8-oxide and styrene have an effect on the erythropoiesis as demonstrated by the fall of the percentage of circulating reticulocytes. These data suggest that the two compounds, or their metabolites, reach the bone marrow where they can alter the hematopoiesis. This level of hematopoietic toxicity was however within the tolerated range of the OECD no. 474 guideline (Dertinger et al., 2006; OECD, 1997). To our knowledge, this work was the first to study the ability of styrene-7,8-oxide to induce micronucleus in vivo following inhalation according to the OECD guidelines. The few available data which were obtained by intraperitoneal injection were also negative (Fabry et al., 1978; Morita et al., 1997). The results of this study regarding styrene exposure are also in good agreement with those obtained in mice exposed to 750 and 1500 mg/m3 of styrene (about 115 and 350 ppm) up to 21 days (Engelhardt et al., 2003). The authors did not observe any significant increase of the micronuclei frequency. In a first attempt, we performed the in vivo erythrocyte peripheral blood micronucleus assay as a recommended assay for the evaluation of chemicals genotoxicity regarding the European regulation. However, this assay may not be the most appropriate to assess styrene-induced genotoxicity because it is limited to the erythropoietic lineage within the bone marrow and cannot assess effects in other target organs. Thus it requires to be completed by another in vivo assay. A recent review of the literature showed that among 218 rodent carcinogens, 120 gave negative or equivocal results with the bone marrow micronucleus assay (Kirkland and Speit, 2008). We have then decided to use the comet assay which is considered by some authors as a good alternative to the in vivo unscheduled DNA synthesis currently used in the genotoxicity testing scheme (Burlinson et al., 2007; Cimino, 2006; Kirkland and Speit, 2008). The reliability of the test, performed on circulating leukocytes, was confirmed with rats exposed to ENU by intraperitoneal injection but gave negative results following 3 and 20 days of exposure to styrene or styrene-7,8-oxide. In contrast, a study performed in mice exposed by inhalation 6 h/day to 350 ppm

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styrene showed a significant increase of DNA damages in circulating leukocytes after 7 days of exposure but surprisingly not after 1 or 21 days of treatment (Vodicka et al., 2001). In addition, intraperitoneal administration of styrene (500 mg/kg) or styrene7,8-oxide (200 mg/kg) to mice lead to a significant increase of DNA strand breaks 4 and 16 h after the injections; the extent of the DNA damages was lower after 16 h (Vaghef and Hellman, 1998). The differences between our results and those from the literature may be associated with the difference in sensitivity of the two species to the genotoxic effects of styrene and its epoxide but also the blood concentrations reached following intraperitoneal injections. Studies have shown that styrene and styrene-7,8-oxide were more pneumo- and hepatotoxic in mice than in rats, these observations were associated with a higher glutathione depletion in mouse lung and liver (Carlson, 2004). In addition, comparative toxicokinetic data suggest that styrene-7,8-oxide blood concentrations are higher in mice than in rats following inhalation of styrene, this may be probably due to a higher cytochrome P450 activity in mice (Csanady et al., 2003). In our study, the comet assay performed with Fpg (an enzyme able to recognize and excise some oxidized DNA bases) showed a significant increase of DNA strand breaks in animals exposed for 3 days to styrene but interestingly not to styrene-7,8-oxide. However, this increase was not observed after 20 days of exposure to either styrene or styrene-7,8-oxide suggesting a possible cellular adaptation to genotoxic insults. This hypothesis is also in good agreement with the work of Vodicka et al. (2004) suggesting that specific DNA repair mechanisms may be induced by styrene exposure. Indeed they observed in peripheral lymphocytes from styrene-exposed workers a significant increase of the repair rates of DNA single-strand breaks induced by ␥-rays in vitro with the increase of blood or atmospheric styrene concentrations. The data obtained with the Fpg-modified comet assay may also suggest that styrene inhalation may be responsible for an oxidative stress in white blood cells. Styrene has been shown to induce oxidative stress in human lung epithelial cells (Roder-Stolinski et al., 2008) and in rodent Clara cells (Harvilchuck et al., 2009) probably through the formation of metabolites. However, styrene-7,8-oxide which is one of them, is also an inducer of oxidative stress in the same Clara cells (Harvilchuck et al., 2009), while in our study, it does not induce any increase of DNA strand breaks in leucocytes in the presence of Fpg. These data suggest that another metabolite with more potent oxidative properties may be involved in the case of styrene inhalation. The main candidate is 4-vinylphenol, a minor metabolite of styrene (Sumner and Fennell, 1994), which has been identified as a highly reactive molecule. It is able to induce an important oxidative stress and is more hepatotoxic and pneumotoxic than styrene-7,8-oxide (Carlson, 2002; Carlson et al., 2002). Additional studies may however be required to prove this hypothesis. Furthermore, in order to better understand the genotoxicity of styrene and its epoxide it would be interesting to perform the comet assay on additional organs including the lung (the first main organ in contact with these chemicals) and the liver (the key organ for the metabolism of these compounds). Studies performed on isolated human peripheral blood lymphocytes have shown, using the comet assay, that styrene-7,8-oxide induced DNA damages at concentrations higher than 50 ␮M (Bastlova et al., 1995; Laffon et al., 2002). Then, in our model, one can assume that the amount of styrene-7,8-oxide reaching the leukocytes following inhalation of this chemical might not be high enough to induce any significant DNA damages and may explain the lack of positive response. In conclusion, in our experimental conditions, inhalation of styrene-7,8-oxide does not induce any significant systemic increase of DNA damages in rats as demonstrated with the peripheral red blood cell micronucleus assay and the comet assay performed on

circulating leukocytes. However, from these results, it is still difficult to conclude on the possible role of styrene-7,8-oxide on the cytogenetic changes observed in reinforced-plastic industry workers since the toxicokinetics of these compounds are different from one species to the other and genetic polymorphism may also play a role. Indeed studies have shown that polymorphism in enzymes involved in styrene metabolism (Teixeira et al., 2007) or DNA repair (Godderis et al., 2006) may modify the cell capabilities to counteract the deleterious effects of styrene and/or SO. Further studies including the genotoxic assessment of other styrene metabolites and/or with other target organs such as lung and liver may give additional information in order to better understand the toxicological properties of styrene and its metabolites. Conflict of interest None declared. Acknowledgments We would like to thank Aurélie Remy for the statistical analysis and Marie-Josèphe Décret, Lionel Dussoul and Sylvie Michaux from the Laboratory Animal Facility for their help in rat handling and husbandry. References Bastlova, T., Vodicka, P., Peterkova, K., Hemminki, K., Lambert, B., 1995. Styrene oxide-induced HPRT mutations, DNA adducts and DNA strand breaks in cultured human lymphocytes. Carcinogenesis 16, 2357–2362. Boccellino, M., Cuccovillo, F., Napolitano, M., Sannolo, N., Balestrieri, C., Acampora, A., Giovane, A., Quagliuolo, L., 2003. Styrene-7,8-oxide activates a complex apoptotic response in neuronal PC12 cell line. Carcinogenesis 24, 535–540. Burlinson, B., Tice, R.R., Speit, G., Agurell, E., Brendler-Schwaab, S.Y., Collins, A.R., Escobar, P., Honma, M., Kumaravel, T.S., Nakajima, M., Sasaki, Y.F., Thybaud, V., Uno, Y., Vasquez, M., Hartmann, A., 2007. Fourth International Workgroup on Genotoxicity testing: results of the in vivo comet assay workgroup. Mutation Research 627, 31–35. Carlson, G.P., 2002. Effect of the inhibition of the metabolism of 4-vinylphenol on its hepatotoxicity and pneumotoxicity in rats and mice. Toxicology 179, 129–136. Carlson, G.P., 2004. Comparison of the susceptibility of wild-type and CYP2E1 knockout mice to the hepatotoxic and pneumotoxic effects of styrene and styrene oxide. Toxicology Letters 150, 335–339. Carlson, G.P., Ullman, M., Mantick, N.A., Snyder, P.W., 2002. 4-Vinylphenol-induced pneumotoxicity and hepatotoxicity in mice. Toxicologic Pathology 30, 565–569. Cimino, M.C., 2006. Comparative overview of current international strategies and guidelines for genetic toxicology testing for regulatory purposes. Environmental and Molecular Mutagenesis 47, 362–390. European Commission, 2008. Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2006 (text with EEA relevance). Cruzan, G., Cushman, J.R., Andrews, L.S., Granville, G.C., Johnson, K.A., Hardy, C.J., Coombs, D.W., Mullins, P.A., Brown, W.R., 1998. Chronic toxicity/oncogenicity study of styrene in CD rats by inhalation exposure for 104 weeks. Toxicological Sciences 46, 266–281. Csanady, G.A., Kessler, W., Hoffmann, H.D., Filser, J.G., 2003. A toxicokinetic model for styrene and its metabolite styrene-7,8-oxide in mouse rat and human with special emphasis on the lung. Toxicology Letters 138, 75–102. De Boeck, M., van der Leede, B.J., Van Goethem, F., De Smedt, A., Steemans, M., Lampo, A., Vanparys, P., 2005. Flow cytometric analysis of micronucleated reticulocytes time- and dose-dependent response of known mutagens in mice, using multiple blood sampling. Environmental and Molecular Mutagenesis 46, 30–42. Dertinger, S.D., Bishop, M.E., McNamee, J.P., Hayashi, M., Suzuki, T., Asano, N., Nakajima, M., Saito, J., Moore, M., Torous, D.K., Macgregor, J.T., 2006. Flow cytometric analysis of micronuclei in peripheral blood reticulocytes. I. Intra- and interlaboratory comparison with microscopic scoring. Toxicological Sciences 94, 83–91. Engelhardt, G., Gamer, A., Vodicka, P., Barta, I., Hoffmann, H.D., Veenstra, G., 2003. A re-assessment of styrene-induced clastogenicity in mice in a subacute inhalation study. Archives of Toxicology 77, 56–61. Fabry, L., Leonard, A., Roberfroid, M., 1978. Mutagenicity tests with styrene oxide in mammals. Mutation Research 51, 377–381. Fiedler, R.D., Weiner, S.K., Schuler, M., 2010. Evaluation of a modified CD71 MicroFlow® method for the flow cytometric analysis of micronuclei in rat bone marrow erythrocytes. Mutation Research 703, 122–129.

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