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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian germ cell study: gene mutation
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study follows GLP and OECD TG 488

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015

Materials and methods

Test guideline
Guideline:
other: OECD Guidelines for the Testing of Chemicals 488 (26 July 2013: Transgenic Rodent Somatic and Germ Cell Gene Mutation Assays
GLP compliance:
yes (incl. certificate)
Type of assay:
transgenic rodent mutagenicity assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- Name of test material (as cited in study report): Ethyl acrylate
- Physical state: liquid
- Analytical purity: 99.87wt% (GC)
- Lot/batch No.: 14352-14-273
- Stability under test conditions: The typical sheff-life is 12 months.
- Storage condition of test material: Room temperature (30"C or less)
- Other: Actual storage temperature and storage period: 9.2°C to 24.0"C January 13, 2015 to April 21, 2015 (from receipt to the final day of use)

Test animals

Species:
mouse
Strain:
other: C57BL/6JJmsSlc-Tg (gpt delta) [SPF]
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Japan SLC, Inc.
- Age at study initiation:
At the time of purchase: 8 weeks of age
At the time of assignment to groups: 9 weeks of age
- Weight at study initiation: 24.2 to 26.8 g
- Assigned to test groups: Animals were assigned to groups based on their body weights on Day 1 using LATOX-F/V5 (FFC) computer system package. The weight range of the animals used was within ±20% of the overall mean weight. Unselected animals were excluded from the study on Day 1.
- Housing: Three animals were housed in a polycarbonate cage (W 18.2 × D 26.0 × H 12.8 cm) with bedding (ALPHA-driTM; lot No. 04114, Shepherd Specialty Papers). However, animals were housed individually from Day -6 because aggressive behavior was observed.
- Diet (e.g. ad libitum): Animals were allowed access to pellet diet CRF-1 sterilized by radiation (lot No. 140703, Oriental Yeast) ad libitum. BSRC obtained the certificate of analysis (report No. AR-14-JP-002550-01: July 22, 2014) on the contaminant levels for this lot from the manufacture and confirmed that such levels were within the acceptable limits proposed by the Japan Experimental Animal Feed Association. The food was also provided to animals at the time of exchanging the feeders.
- Water (e.g. ad libitum): Animals were provided access to tap water from water bottles ad libitum. The drinking water was examined for the quality at another inspection agency in April 2015 according to the specifications of the Water Works Law. In March and May 2015, BSRC examined the water for bacteria (common bacteria and Escherichia coli). The analytical results (report No. K15-0033, in-house data Nos. GT15-03 and GT15-05) of these analyses indicated that the levels of contaminants in the water were within the acceptable limits of the Tap Water Quality Standard and that no bacteria were detected in the water
- Acclimation period: The quarantine and acclimation period was from Day -7 to 1

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 26°C (actual values: 22.8 to 23.1°C)
- Humidity (%): 35 to 70%RH (actual values: 42.2 to 58.3%RH)
- Air changes (per hr): 12 times or more/hour
- Photoperiod (hrs dark / hrs light): 12 hours (lights on: 7:00, lights off: 19:00)

IN-LIFE DATES: From: March 24,2015 To: June 5, 2015

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Corn oil (lot No. WEF2972), the vehicle to prepare the test substance formulations, was used
- Storage conditions: Room temperature
- Manufacturer: Wako Pure Chemical Industries, Ltd
- Lot/batch no. (if required): (lot No. WEF2972)
- Grade: For biochemistry
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The vehicle and the test substance were administered to mice orally once daily for 28 consecutive days at about 24-hour intervals using a disposable syringe with a Teflon sonde. The dosage volume (mL) was set at 0.1 mL per 10 g of body weight and was calculated on the basis of the most recent individual body weight measured in section 15.12.6.
The positive control substance was administered to mice orally once daily for 5 consecutive days at about 24-hour intervals using a disposable syringe with a Teflon sonde. The dosing volume was set at 0.1 mL per 10 g of body weight and was calculated on the basis of the most recent individual body weight measured.
Duration of treatment / exposure:
28 consecutive days at about 24-hour intervals
Frequency of treatment:
Once daily
Post exposure period:
3 days of manifestation period
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 8, 20, and 50 mg/kg/day
Basis:
nominal conc.
No. of animals per sex per dose:
To ensure that the data would be available for 5 animals in each group, the number of the treated animals was 6 in each group.
Control animals:
yes
yes, historical
Positive control(s):
Benzo[a]pyrene (B[a]P)
- Justification for choice of positive control(s): Considering information in the academic documents, the following substance was selected as the positive control
- Route of administration: The positive control substance was administered to mice orally once daily for 5 consecutive days at about 24-hour intervals using a disposable syringe with a Teflon sonde.
- Doses / concentrations: The dose was set at 125 mg/kg in reference to a literature

Examinations

Tissues and cell types examined:
The animals were necropsied after euthanasia by exsanguination under isoflurane anesthesia. The liver, stomach and testes were removed from the animals. The liver, stomach and testes were observed macroscopically. The organ weight of the liver was measured. The organ weight to body weight ratio (relative organ weight) was calculated from the body weight weighed on the day of necropsy and organ weight (absolute organ weight / body weight on the harvest day × 100). The liver was measured in grams (to 2 decimal places).
Details of tissue and slide preparation:
METHOD OF ANALYSIS: Pathological examinations consisted of macroscopic examination and histopathological examination. Because histopathological findings were observed in the forestomach in a carcinogenesis study[3], the stomach and liver were selected for histopathological examination. Fixed stomach and liver were embedded in paraffin, sectioned and stained with hematoxylin and eosin (H.E.) routinely
Histopathological examination was conducted using all prepared specimens. All histopathological observations were graded according to severity and recorded.
Evaluation criteria:
VALIDITY OF STUDY
Since the following conditions were satisfied, the test will be considered successfully performed:
- The mutation frequency for the liver in the positive control group markedly increases with a statistically significant difference from the negative control group. - The mutant frequency in the negative control group should be within the acceptable range calculated from our historical data.
Statistics:
The data on the mutant frequency from the negative control group and each test substance treated group were tested by Bartlett’s test for homogeneity of variance first. If homogeneity was determined (not significant on Bartlett’s test), Dunnett’s multiple comparison test was performed to assess the statistical significance of differences between the negative control group and each test substance treated group. If there was no homogeneity (significant on Bartlett’s test), Steel’s test was performed to analyze the differences.
The data on the mutant frequency from the negative control group and the positive control group were tested by F test for homogeneity of variance first. If homogeneity of variance was determined (not significant on F test), Student’s t test was performed to assess the statistical significance of differences between the negative control group and the positive control group. If there was no homogeneity (significant on F test), Aspin-Welch’s t test was performed to analyze the differences.
The significance level of 5% (two-sided) was selected for each test.
The results were evaluated as positive when the mutant frequency in the test substance treated group was significantly different from that in the negative control group. Final judgment was made in consideration of biological relevance under the test conditions.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
inflammatory cell infiltration (slight) in the forestomach
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF DEFINITIVE STUDY
Liver
In the negative control group, the mean±SD of mutant frequency among the individuals was 3.14±1.35 (×10-6).
The means±SD of mutant frequencies in the ethyl acrylate treated groups, 8.00, 20.0 and 50.0 mg/kg/day, were 2.29±1.51 (×10-6), 1.67±0.98 (×10-6) and 1.68±1.16 (×10-6), respectively, indicating that ethyl acrylate was negative for liver genotoxicity in the Spi- assay . Furthermore, no statistically significant increases were observed as compared with the negative control group.
In the positive control group, the mean±SD of mutant frequency among the individuals was 12.17±7.03 (×10-6) and a statistically significant increase was observed compared with the negative control group.
Stomach
The means±SD of mutant frequencies in the ethyl acrylate treated groups, 8.00, 20.0 and 50.0 mg/kg/day, were 4.02±3.32 (×10-6), 3.36±3.38 (×10-6) and 2.00±0.31 (×10-6), respectively, indicating that ethyl acrylate was negative for stomach genotoxicity in the Spi-assay. Furthermore, no statistically significant increases were observed as compared with the negative control group.
In the positive control group, the mean±SD of mutant frequency among the individuals was 21.86±8.81 (×10-6) and a statistically significant increase was observed compared with the negative control group.

Any other information on results incl. tables

Body weight and general conditions: Body weights in all the test substance treated groups were similar to those in the control group throughout the treatment periods. There was no change in the general condition in any of the test substance treated groups.

Liver weight and relative liver weight:There was no difference in the liver weight or relative liver weights of the test substance treated groups compared with those of the negative control group.

Macroscopic findings:There were no macroscopic findings related to test substance treatment in the liver, stomach or testis of the other animals.

Histopathological findings:Inflammatory cell infiltration (slight) in the forestomach was observed in 2 animals in the 8.00 mg/kg/day group and 1 animal in the 50.0 mg/kg/day group. Furthermore, inflammatory cell infiltration (slight) in the forestomach was observed in 4 animals in the positive control.There were no findings related to test substance treatment in the liver

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Ethyl acrylate did not induce gene mutation in transgenic mice under the conditions in this study.
Executive summary:

In 2014 IARC added Ethyl Acrylate as an agent for high priority reevaluation in 2015-2019.  Conduct of this study was required in preparation for IARC’s cancer classification reevaluation of Ethyl Acrylate. A gene mutation assay with transgenic mice (gpt delta mouse) was conducted to assess the potential of ethyl acrylate to induce gene point mutations and deletion mutations using the gpt gene and the red/gam genes (Spi⁻ selection), respectively, as mutation reporter genes in the liver and stomach.

In dose range-finding study in C57BL/6JJmsSlc mice treated for 28 days with 0, 25.0, 50.0, 100, or 200 mg/kg/day of ethyl acrylate in corn oil, gross observation revealed white nodules in the forestomach in all mice receiving 200 mg/kg/day and in one of the 3 mice receiving 100 mg/kg/day. Histopathological examination of animals in the 100 and 200 mg/kg/day groups revealed that there was an inflammatory response in the forestomach (consisting of squamous cell hyperplasia, fibrosis of mucosa, and infiltration of inflammatory cells). In addition, erosion was observed in 1 animal in the 200 mg/kg/day group. A local inflammatory response may interfere with the outcome of the mutagenicity data and should thus be avoided. Metabolism of ethyl acrylate occurs through carboxylesterases and through conjugation with glutathione. The latter metabolic reaction becomes saturated between 20 and 100 mg/kg, while dosages ≥ 100 mg/kg exhibit complete saturation. Therefore, a dosage of 50.0 mg/kg/day was selected as the high dose as this dose was without a potentially confounding inflammatory response and is anticipated to be at the threshold for saturation of GSH metabolism, based on previous rodent studies. Two additional dosage levels including 20.0 and 8.00 mg/kg/day were selected as lower doses in the present study.

The test substance was administered to male transgenic mice orally for 28 consecutive days by gavage and after 3 days of the manifestation period, the liver, stomach and testis were removed and mutant frequencies in the liver and stomach were determined.

The results showed that the mutant frequencies (6-thioguanine and Spi-selection) in the liver and stomach of all groups treated with ethyl acrylate were negative for genotoxicity under the conditions of this study . Furthermore, the mutant frequencies in the liver and stomach of all groups treated with ethyl acrylate did not show any statistically significant increases as compared with the negative control group.

The mutant frequencies in the liver and stomach of the positive control group, which was treated with benzo[a]pyrene (B[a]P, dosage level of 125 mg/kg/day), increased in both thegptand Spi-assays and these increases were statistically significant compared with that of the negative control group.

 

It is, therefore, concluded that ethyl acrylate did not induce gene mutation in transgenic mice (negative) under the conditions in this study.