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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian erythrocyte micronucleus test
Specific details on test material used for the study:
The test material in the Comet assay is presented with the CAS no. 1866-31-5, the generic, non-isomer specific CAS no for Allyl cinnamate. This CAS no was also used by the registrant in the pre-registration phase of this material. Recent analytical data with more specific detection of trans and cis isomerization has shown that Allyl cinnamate is purely a trans-isomer. In view of the substance being produced the same over all the years (esterification of trans-cinnamic acid with allyl alcohol), this generic, non-isomer specific CAS no is considered to be representative of the trans-isomer.
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS, Inc., Frederick, MD
- Age at study initiation: 6 weeks
- Weight at study initiation: DRF Males: 171.5 to 183.3g; DRF Females: 141.6 to 155.2g; Main study: 170.3 to 190.6g
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: Animals of the same sex were housed up to three per Micro-Barrier cage. Cages were placed on racks equipped with an automatic watering system and Micro-VENT full ventilation, HEPA filtered system. Heat treated hardwood chips (P.J. Murphy Forest Products) were used for bedding to absorb liquids.
- Diet: Certified laboratory rodent chow (Envigo 2018C Teklad Global 18% Protein Rodent Diet, ad libitum
- Water: Tap water, which met U.S. EPA drinking water standards [Washington Suburban Sanitary Commission (WSSC) Potomac Plant], ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.6 to 23.9
- Humidity (%): 50 ± 20%
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: olive oil
- Amount of vehicle: 10 mL/kg bw/dose
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Dosing formulations were adjusted to compensate for the purity (98.8%) of the test article using a correction factor of 1.012. Dose formulations were prepared prior to each dose administration as follows: A suitably sized amber glass vial with a PTFE stir bar, containing an appropriate amount of test article was calibrated to the target batch size. Approximately 70% of the total volume of vehicle was added to the vial and stirring was initiated. Additional vehicle was added until the final target volume was achieved, and the formulation was stirred magnetically until uniform. Formulations were maintained at room temperature until use.
Frequency of treatment:
Three consecutive days. Second dose approximately 24 hours after the first dose. Third dose approximately 20 hours after the second.
Post exposure period:
3-4 hours
Dose / conc.:
62.5 mg/kg bw/day (actual dose received)
Dose / conc.:
125 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
6
Control animals:
yes, concurrent vehicle
Positive control(s):
The scoring positive control slides (fixed and unstained), generated from a BioReliance Study No. AE93CB.125M012.BTL, were included to verify scoring in the Micronucleus assay. These slides were generated from male rats treated once with cyclophosphamide monohydrate (CP) at 40 mg/kg, and the bone marrow harvested 24 hours after treatment.
Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
DETAILS OF SLIDE PREPARATION:
The bone marrow was transferred to a centrifuge tube containing 1-3 mL fetal bovine serum, the cells were pelleted by centrifugation, and the supernatant was drawn off leaving a small amount of fetal bovine serum with the pellet. Cells were re-suspended and a small drop of the bone marrow suspension was spread onto a clean glass slide. At least four slides were prepared from each animal, air dried and fixed by dipping in methanol. One set of two slides (including at least 5 positive control slides) was stained with acridine orange for microscopic evaluation. The other set of slides was kept as backup and will be archived at report finalization. Each slide was identified by the harvest date, study number, and animal number. Slides were coded using a random number table by an individual not involved with the scoring process.

METHOD OF ANALYSIS:
Bone marrow was evaluated by fluorescent microscopy. The staining procedure permitted the differentiation by color of polychromatic and normochromatic erythrocytes (bright orange PCEs and ghost-like, dark green NCEs, respectively). The criteria for the identification of micronuclei are those of Schmid (1975). Micronuclei are brightly stained bodies that generally are round and that generally are between 1/20 and 1/5 the size of the PCE. Scoring was based upon the micronucleated cell, not the micronucleus; thus, occasional cells with more than one micronucleus were counted as one micronucleated PCE (MnPCE), not two (or more) micronuclei. At least 4000 PCEs/animal were scored for the presence of micronuclei (MnPCEs). In addition, at least 500 total erythrocytes (ECs; PCEs + NCEs) were scored per animal to determine the proportion of PCEs as an index of bone marrow cytotoxicity. PCE/EC proportions < 20% of the vehicle control value were considered excessively cytotoxic, and the animal data was excluded from evaluation. Stained bone marrow slides were discarded prior to report finalization.
Evaluation criteria:
Validity criteria
- The group mean frequency of MnPCEs for the vehicle control groups should ideally be within the 95% control limits of the distribution of the historical negative control database. If the concurrent negative control data fall outside the 95% control limits, they may be deemed acceptable as long as these data are not extreme outliers (indicative of experimental or human error).
- The frequency of MnPCEs for the positive controls must be significantly greater than the concurrent vehicle control (p ≤ 0.05) and should be compatible with those observed in the historical positive control database.
- At least three doses were tested. At least five animals/group were available for micronucleus analysis.
- At least 4000 PCEs/animal were scored for the presence of micronuclei (MnPCEs). In addition, at least 500 total erythrocytes (PCEs + NCEs) were scored per animal to determine the proportion of PCEs as an index of bone marrow cytotoxicity. A reduction in the PCE proportions to less
than 20% of the vehicle control was considered excessively cytotoxic, and the animal data were
excluded from evaluation.
- The maximum dose evaluated for micronucleus induction was the MTD.

Evaluation criteria
The test article was considered to have induced a positive response if:
a) at least one of the test article doses exhibited a statistically significant increase when compared with the concurrent vehicle control (p ≤ 0.05), and
b) when multiple doses were examined at a particular sampling time, the increase was dose-related (p ≤ 0.01 and R2≥70%), and
c) results of the group mean or of the individual animals in at least one group were outside the 95% control limit of the historical negative control data.
A test article was considered to have induced a clear negative response if none of the criteria for a positive response were met and there was evidence that the bone marrow was exposed to the test article (unless intravenous administration was used).
Statistics:
Statistical analysis was performed on the micronucleus frequency (MnPCE%) and PCE% using the animal as the unit. The mean and standard deviation of MnPCE% and PCE% was presented for each treatment group. The use of parametric or non-parametric statistical methods in evaluation of data was based on the variation between groups. The group variances for micronucleus frequency for the vehicle and test article groups at the respective sampling time were compared using Levene’s test (significant level of p ≤ 0.05). Since the variation between groups was found not to be significant, a parametric one-way ANOVA was performed followed by a Dunnett’s post-hoc analysis to compare each dose group to the concurrent vehicle control. A linear regression analysis was conducted to assess dose responsiveness in the test article treated groups (p ≤ 0.01 and R2≥70%). A pair-wise comparison (Student’s T-test, p ≤ 0.05) was used to compare the positive control group to the concurrent vehicle control group.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Dose Range Finder:
Mortality was observed in all animals at 500 and 1000 mg/kg bw/day. Animals in these two groups were not dosed on day 3 due to mortality. No clinical signs were observed in males of the 125 mg/kg bw/day dose group. Two females of this group showed piloerection on day 3. Piloerection, hunched position, and diarrhea were observed in the animals of the 250 mg/kg bw/day dose group on day 2 and 3. One female on day 3 also showed lethargy. In the 250 mg/kg bw/day dose group, piloerection, hunched position, diarrhea, and crusty eyes were observed in all animals. Piloerection, hunched position, diarrhea, crusty eyes, prostration, and irregular breathing were observed in the males of the high dose group. In the females of this group, piloerection, prostration, and crusty eyes were observed.

Main test:
No mortality occurred at any dose level during the course of the definitive assay. No appreciable reductions in mean group body weights were seen in the test article treated groups during the course of the study. All vehicle control animals appeared normal throughout the observation period. Piloerection was observed at all dose levels. In addition, lethargy, hunched position, crusty nose, and irregular breathing were observed in the 250 mg/kg bw/day animals.
The scoring results and a statistical analysis of data indicated the following:
- A statistically significant reduction in the PCEs/EC ratio was observed in the test article group at 250 mg/kg bw/day compared to the vehicle control group (ANOVA followed by Dunnett’s post-hoc analysis, p < 0.05), indicating the test article did induce cytotoxicity.
- Group variances for mean of means of the micronucleus frequency in the vehicle and test article groups were compared using Levene’s test. The test indicated that there was no significant difference in the group variance (p > 0.05); therefore, the parametric approach, ANOVA followed by Dunnett’s post-hoc analysis, was used in the statistical analysis of data.
- No statistically significant increase in the incidence of MnPCEs was observed in the test article treated groups relative to the vehicle control group (ANOVA followed by Dunnett’s post-hoc analysis, p > 0.05).
- The positive control, CP, induced a statistically significant increase in the incidence of MnPCEs (20,000 PCEs scored, 4000 PCEs/animal; Student’s t-test, p ≤ 0.05).
- The number of MnPCEs in the vehicle control groups did not exceed the historical control range
Based upon this, all criteria for a valid test were met as specified in the protocol.
Conclusions:
Under the conditions of this Micronucleus test (OECD 474 and GLP), the test substance was concluded to be negative for the induction of micronucleated polychromatic erythrocytes in male rat bone marrow.
Executive summary:

Method: The test article was evaluated for its genotoxic potential in bone marrow according to OECD TG 474 and in compliance with GLP. This test was combined with a Comet assay performed according to OECD 489. Sprague-Dawley rats were exposed to the test substance by oral gavage on three consecutive days. Three to four hours after the last treatment, the animals were euthanized. Olive oil was selected as the vehicle. Test and/or control article formulations were administered at a dose volume of 10 mL/kg bw/dose. In the dose range finding assay, the dose levels tested were 125, 250, 500 and 1000 mg/kg bw/day in 3 animals/sex. However, animals in the two high dose groups of 500 and 1000 mg/kg bw/day were not dosed on day 3 due to 100% mortality in these two dose groups. Based upon the results, the high dose for the definitive assay was 250 mg/kg bw/day, which was estimated to be the maximum tolerated dose because at this dose piloerection, lethargy, hunched posture, crusty nose and irregular breathing was observed. As no differences in clinical observations were seen between the sexes, only males were used in the definitive assay. The definitive assay dose levels tested were 0 (olive oil), 62.5, 125 and 250 mg/kg bw/day in six male rats per dose. The scoring positive control slides (fixed and unstained), generated from another study by the test lab, were included to verify scoring in the micronucleus assay. These slides were generated from male rats treated once with cyclophosphamide monohydrate (CP) at 40 mg/kg, and the bone marrow harvested 24 hours after treatment.

Results: Slight but significant decrease (-5%) in PCEs/EC ration was seen at the high dose of 250 mg/kg bw indicating that the substance has reached the bone marrow. No statistically significant increases in the incidence of MnPCEs were observed in the test article treated groups relative to the vehicle control group. Thus, the test article was negative (non-clastogenic). The positive control induced a statistically significant increase in the incidence of MnPCEs. The number of MnPCEs in the vehicle control group did not exceed the historical control range.

Conclusion: Under the conditions of this study, the administration of the test substance, at doses up to and including a dose of 250 mg/kg bw/day, did not induce a significant increase in the incidence of MnPCEs relative to the concurrent vehicle control. Therefore, the test substance was concluded to be negative in the in vivo micronucleus assay.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
GLP compliance:
yes
Type of assay:
mammalian comet assay

Test material

Reference
Name:
Unnamed
Test material form:
liquid
Specific details on test material used for the study:
The test material in the Comet assay is presented with the CAS no. 1866-31-5, the generic, non-isomer specific CAS no. for Allyl cinnamate. This CAS no. was also used by the registrant in the pre-registration phase of this material. Recent analytical data with more specific detection of trans and cis isomerization has shown that Allyl cinnamate is purely a trans-isomer. In view of the substance being produced the same over all the years (esterification of trans-cinnamic acid with allyl alcohol), this generic, non-isomer specific CAS no. is considered to be representative of the trans-isomer.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS, Inc., Frederick, MD
- Age at study initiation: 6 weeks
- Weight at study initiation: DRF Males: 171.5 to 183.3g; DRF Females: 141.6 to 155.2g; Main study: 170.3 to 190.6g; Repeat Comet: 165.8 to 184.7
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: Animals of the same sex were housed up to three per Micro-Barrier cage. Cages were placed on racks equipped with an automatic watering system and Micro-VENT full ventilation, HEPA filtered system. Heat treated hardwood chips (P.J. Murphy Forest Products) were used for bedding to absorb liquids.
- Diet: Certified laboratory rodent chow (Envigo 2018C Teklad Global 18% Protein Rodent Diet, ad libitum
- Water: Tap water, which met U.S. EPA drinking water standards [Washington Suburban Sanitary Commission (WSSC) Potomac Plant], ad libitum
- Acclimation period: 5 days (except animals of the repeat Comet. These were acclimatized for 6 days)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.6 to 23.9
- Humidity (%): 50 ± 20%
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: olive oil
- Amount of vehicle: 10 mL/kg bw/dose
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Dosing formulations were adjusted to compensate for the purity (98.8%) of the test article using a correction factor of 1.012. Dose formulations were prepared prior to each dose administration as follows: A suitably sized amber glass vial with a PTFE stir bar, containing an appropriate amount of test article was calibrated to the target batch size. Approximately 70% of the total volume of vehicle was added to the vial and stirring was initiated. Additional vehicle was added until the final target volume was achieved, and the formulation was stirred magnetically until uniform. Formulations were maintained at room temperature until use.
Frequency of treatment:
Three consecutive days. Second dose approximately 24 hours after the first dose. Third dose approximately 20 hours after the second.
Post exposure period:
3-4 hours
Doses / concentrationsopen allclose all
Dose / conc.:
62.5 mg/kg bw/day (actual dose received)
Dose / conc.:
125 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
6 (3 for positive control)
Control animals:
yes, concurrent vehicle
Positive control(s):
Ethylmethanesulphonate
- Route of administration: Via oral gavage, once on Study Day 3 approximately 3 to 4 hours prior to euthanasia.
- Doses / concentrations: The neat EMS was prepared in 0.9% saline. The dosing formulation was prepared at a concentration of 19.96 or 20 mg/mL, just prior to each use.

Examinations

Tissues and cell types examined:
liver and duodenum
Details of tissue and slide preparation:
DETAILS OF SLIDE PREPARATION:
- Comet Assay Tissue Collection and Processing: All animals were euthanized 3 to 4 hours after the last dose by CO2 asphyxiation, and then, the following was performed: Animals were dissected, and the liver and duodenum were extracted (removed) and collected. A section of the liver and duodenum was cut and placed in formalin for possible histopathology analysis. Another section of the liver and duodenum was placed in chilled mincing solution (Hanks’ balanced salt solution with EDTA and DMSO) and was used in preparation of cell suspensions and Comet slides.
- Preparation of Cell Suspensions and Comet Slides: A portion of each dissected liver was placed in cold mincing buffer, then the liver was finely cut (minced) with a pair of fine scissors to release the cells. The interior of the duodenum was scraped with a plastic spatula to remove the mucosa and rinsed with working mincing solution. The duodenum was placed in chilled mincing solution, and the surface of the duodenum was gently scraped to release the cells. Each cell suspension was strained through a Cell Strainer into a pre-labeled 50 mL polypropylene conical tube. An aliquot of the suspension was used to prepare the Comet slides. Preparation of Slides: From each liver suspension, an aliquot of 2.5 μL was mixed with 75 μL of low melting agarose (0.5%). From each duodenum suspension, an aliquot of 7.5 μL was mixed with 75 μL of low melting agarose (0.5%). The cell/agarose suspension was applied to commercially available pretreated multi-well microscope slides. These slides have 3 individual circular areas, referred to as wells in the text below. The slides were kept at 2 - 8°C for at least 15 minutes to allow the gel to solidify. Slides were identified with a random code that reflects the study number, group, animal number, and organ/tissue. At least two Trevigen, Inc. 3-well slides were prepared per animal per tissue. Three wells were used in scoring. The other wells were designated as a backup and scored as needed. Following solidification of the agarose, the slides were placed in jars containing lysis solution. After slides were prepared for all animals, ~1 mL of the remaining cell suspension was transferred to a cryovial labeled (at minimum) with Study Number, Animal Number, Organ, and Storage Conditions. The cell suspension samples were frozen at -60ºC or below until acceptance of scoring data by the Study Director. Unused cell suspensions were disposed of prior to report finalization. Lysis: Following solidification of agarose, the slides were submerged in a commercially available lysis solution supplemented with 10% DMSO on the day of use. The slides were kept in this solution at least overnight at 2-8°C. Unwinding: After cell lysis, slides/wells were washed with neutralization buffer (0.4 M tris hydroxymethyl aminomethane in purified water, pH ~7.5) and placed in the electrophoresis chamber. The chamber reservoirs were slowly filled with alkaline buffer composed of 300 mM sodium hydroxide and 1 mM EDTA (disodium) in purified water. The pH was > 13. All slides remained in the buffer for 20 minutes at 2-10°C and protected from light, allowing DNA to unwind. Electrophoresis: Using the same buffer, electrophoresis was conducted for 30 minutes at 0.7 V/cm, at 2-10°C and protected from light. The electrophoresis time was constant for all slides. Neutralization: After completion of electrophoresis, the slides were removed from the electrophoresis chamber and washed with neutralization buffer for at least 10 minutes. The slides (gels) were then dehydrated with 200-proof ethanol for at least 5 minutes, then air dried for at least 4 hours and stored at room temperature with desiccant. Staining: Slides were stained with a DNA stain (i.e., Sybr-gold™) prior to scoring. The stain solution was prepared by diluting 1 µL of Sybr-gold™ stain in 15 mL of 1xTBE (tris-boric acid EDTA buffer solution).

METHOD OF ANALYSIS:
Three slides/wells per organ/animal were used. Fifty randomly selected cells per slide were scored resulting in a total of 150 cells evaluated per animal. The following endpoints of DNA damage were assessed and measured: a) Comet Tail Migration; defined as the distance from the perimeter of the Comet head to the last visible point in the tail. b) % Tail DNA; (also known as % tail intensity or % DNA in tail); defined as the percentage of DNA fragments present in the tail. c) Tail Moment (also known as Olive Tail moment); defined as the product of the amount of DNA in the tail and the tail length [(% Tail DNA x Tail Length)/ 100; Olive et al. 1990)]. Each slide/well was also examined for indications of cytotoxicity. The rough estimate of the percentage of “clouds” was determined by scanning 150 cells per animal (percentage of “clouds” was calculated by adding the total number of clouds for all slides scored, dividing by the total number of cells scored and multiplying by 100). The “clouds,” also known as “hedgehogs,” are a morphological indication of highly damaged cells often associated with severe genotoxicity, necrosis or apoptosis. A “cloud” is produced when almost the entire cell DNA is in the tail of the Comet and the head is reduced in size, almost nonexistent (Collins et. al., 2004). “Clouds” with visible gaps between the nuclei and the Comet tail were excluded from Comet image analysis. In order to confirm the increase in % Tail DNA observed in liver cells in the repeat Comet assay, the high dose and vehicle control slides were rescored. These data were combined with the initial data from the repeat Comet assay and reported. The Comet slides, which are not permanent (the slides can be affected/damaged by environmental storage conditions), were discarded prior to report finalization.
Evaluation criteria:
The test article was considered to have induced a positive response if:
a) at least one of the test article doses exhibited a statistically significant increase when compared with the concurrent vehicle control (p ≤ 0.05), and
b) when multiple doses were examined at a particular sampling time, the increase was dose-related (p ≤ 0.01) and
c) results of the group mean or of the individual animals of at least one group were outside the distribution of the historical negative control database for that tissue.
The test article was considered to have induced a clear negative response if none of the criteria for a positive response were met and direct or indirect evidence supportive of exposure of, or toxicity to, the target tissue was demonstrated.
If the response was neither clearly positive nor clearly negative, or in order to assist in establishing the biological relevance of a result, the data were evaluated by expert judgment and/or further investigations. Any additional work was only carried out following consultation with, and at the request of, the Sponsor.
Biological significance of a positive, negative and equivocal result in the Comet assay portion was based on the information on cytotoxicity at the target tissue. Where positive or equivocal findings were observed solely in the presence of clear evidence of cytotoxicity (e.g. histopathology evaluation, changes in clinical chemistry measures), the study was concluded as equivocal for genotoxicity unless there was enough information that was supportive of a definitive conclusion. In case of a negative study outcome where there were signs of toxicity at all doses tested, further study at non-toxic doses may be advisable.
Statistics:
The median value of 150 counts of % Tail DNA, Tail moment and Tail migration were determined and presented for each animal in each treatment group for each organ. The mean and standard deviation of the median values only for % Tail DNA were presented for each treatment group. Statistical analysis was performed only for % Tail DNA. The use of parametric or non-parametric statistical methods in evaluation of data was based on the variation between groups. The group variances for % tail DNA generated for the vehicle and test article groups were compared using Levene’s test (significant level of p ≤ 0.05). The differences and variations between groups in the repeat Comet assay (initial slide scoring) were found to be significant for liver; therefore, the suitability of a transformation of the original data was evaluated (e.g. using logarithm, or BoxCox transformed values of the original data) in an attempt to meet the normality criteria. Afterwards, a parametric one-way ANOVA followed by a Dunnett’s post-hoc test was performed (significant level of p < 0.05). The differences and variations between groups in the initial Comet assay for liver and duodenum and the repeat Comet assay for liver (rescoring) and duodenum were found not to be significant; therefore, a parametric one-way ANOVA followed by a Dunnett’s post-hoc test was performed (significant level of p < 0.05). A linear regression analysis was conducted to assess dose responsiveness in the test article treated groups (p ≤ 0.01). A pair-wise comparison (Student’s T-test, p ≤ 0.05) was used to compare the positive control group to the concurrent vehicle control group.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Dose Range Finder:
Mortality was observed in all animals at 500 and 1000 mg/kg bw/day. Animals in these two groups were not dosed on day 3 due to mortality. No clinical signs were observed in males of the 125 mg/kg bw/day dose group. Two females of this group showed piloerection on day 3. Piloerection, hunched position, and diarrhea were observed in the animals of the 250 mg/kg bw/day dose group on day 2 and 3. One female on day 3 also showed lethargy. In the 250 mg/kg bw/day dose group, piloerection, hunched position, diarrhea, and crusty eyes were observed in all animals. Piloerection, hunched position, diarrhea, crusty eyes, prostration, and irregular breathing were observed in the males of the high dose group. In the females of this group, piloerection, prostration, and crusty eyes were observed.

Initial Comet Assay
No mortality occurred at any dose level during the course of the definitive assay. No appreciable reductions in mean group body weights were seen in the test article treated groups during the course of the study. All vehicle control animals appeared normal throughout the observation period. Piloerection was observed at all dose levels. In addition, lethargy, hunched position, crusty nose, and irregular breathing were observed in the 250 mg/kg bw/day animals.
- Liver: The scoring results and a statistical analysis of data indicated the following: The presence of ‘clouds’ in the test article groups was 20.8 to 28.3%, which was higher than the % of clouds in the vehicle control group (18.5%). Group variances for mean of medians of the % Tail DNA in the vehicle and test article groups were compared using Levene’s test. The test indicated that there was no significant difference in the group variance (p > 0.05); therefore, the parametric approach, ANOVA followed by Dunnett’s post-hoc analysis, was used in the statistical analysis of data. A statistically significant response in the % Tail DNA (DNA damage) was observed in the test article group at 62.5 mg/kg/day relative to the concurrent vehicle control group (ANOVA followed by Dunnett’s post-hoc analysis, p < 0.05). No dose-dependent increase in the % Tail DNA was observed across three test article doses (regression analysis, p > 0.01). The positive control, EMS, induced a statistically significant increase in the % Tail DNA in liver cells as compared to the vehicle control group (Student’s t-test, p ≤ 0.05). In the vehicle control group, % Tail DNA was outside of the historical vehicle control range for the liver.
- Duodenum: The scoring results and a statistical analysis of data indicated the following: The presence of ‘clouds’ in the test article groups was 86.6 to 100.0%, which was comparable to the % of clouds in the vehicle control group (100.0%). Group variances for mean of medians of the % Tail DNA in the vehicle and test article groups were compared using Levene’s test. The test indicated that there was no significant difference in the group variance (p > 0.05); therefore, the parametric approach, ANOVA followed by Dunnett’s post-hoc analysis, was used in the statistical analysis of data. No statistically significant response in the % Tail DNA (DNA damage) was observed in the test article groups relative to the concurrent vehicle control group (ANOVA followed by Dunnett’s post-hoc analysis, p > 0.05). No dose-dependent increase in the % Tail DNA was observed across three test article doses (regression analysis, p > 0.01). The positive control, EMS, did not induce a statistically significant increase in the % Tail DNA in liver cells as compared to the vehicle control group (Student’s t-test, p ≤ 0.05). In the vehicle control group, % Tail DNA was outside of the historical vehicle control range for the duodenum. Due to the vehicle control values in the initial Comet assay for both liver and duodenum, the Comet assay was repeated using the same dose levels, dosing regimen and assay procedures.

Repeat Comet Assay
No mortality occurred at any dose level during the course of the repeat Comet assay. No appreciable reductions in mean group body weights were seen in the test article treated groups during the course of the study. All vehicle control animals appeared normal throughout the observation period. Piloerection was observed at all dose levels. In addition, lethargy and hunched position were observed in the 250 mg/kg bw/day animals.
- Liver: The scoring results and a statistical analysis of data indicated the following: The presence of ‘clouds’ in the test article groups was 0.0 to 2.3%, which was comparable to the % of clouds in the vehicle control group (0.5%). Group variances for mean of medians of the % Tail DNA in the vehicle and test article groups were compared using Levene’s test. In the initial slide scoring, the test indicated that there was a significant difference in the group variance (p < 0.05); therefore, the data were log transformed and the parametric approach, ANOVA followed by Dunnett’s post-hoc analysis, was used in the statistical analysis of data. When the slides were rescored as indicated below, Levene’s test indicated that there was not a significant difference in the group variance (p < 0.05); therefore, ANOVA followed by Dunnett’s post-hoc analysis was also used in the statistical analysis of the rescored data. A statistically significant response in the % Tail DNA (DNA damage) was observed in the test article group at 250 mg/kg bw/day relative to the concurrent vehicle control group (ANOVA followed by Dunnett’s post-hoc analysis, p < 0.05). However, the value was within the Testing Facility’s historical range, and thus, was not considered biologically relevant. No dose-dependent increase in the % Tail DNA was observed across three test article doses (regression analysis, p > 0.01). The positive control, EMS, induced a statistically significant increase in the % Tail DNA in liver cells as compared to the vehicle control group (Student’s t-test, p ≤ 0.05). In the vehicle control group, % Tail DNA was within of the historical vehicle control range for the liver. In order to confirm the increase in % Tail DNA observed in liver cells, the high dose and vehicle control slides were rescored. No statistically significant or dose-dependent increase in % Tail DNA was observed at 250 mg/kg bw/day in liver cells compared to the vehicle control. The vehicle control % Tail DNA in liver cells was within the Testing Facility’s historical range.
- Duodenum: The scoring results and a statistical analysis of data indicated the following: The presence of ‘clouds’ in the test article groups was 4.3 to 8.2%, which was comparable to the % of clouds in the vehicle control group (3.5%). Group variances for mean of medians of the % Tail DNA in the vehicle and test article groups were compared using Levene’s test. The test indicated that there was no significant difference in the group variance (p > 0.05); therefore, the parametric approach, ANOVA followed by Dunnett’s post-hoc analysis, was used in the statistical analysis of data. No statistically significant response in the % Tail DNA (DNA damage) was observed in the test article groups relative to the concurrent vehicle control group (ANOVA followed by Dunnett’s post-hoc analysis, p > 0.05). No dose-dependent increase in the % Tail DNA was observed across three test article doses (regression analysis, p > 0.01). The positive control, EMS, induced a statistically significant increase in the % Tail DNA in liver cells as compared to the vehicle control group (Student’s t-test, p ≤ 0.05). In the vehicle control group, % Tail DNA was within of the historical vehicle control range for the duodenum.

Applicant's summary and conclusion

Conclusions:
Under the conditions of this Comet assay (OECD 489 and GLP), the test substance was concluded to be negative for DNA damage in liver and duodenum.
Executive summary:

Method: The test article was evaluated for its genotoxic potential in liver and duodenum according to OECD TG 489 and in compliance with GLP. This test was combined with a Micronucleus assay performed according to OECD 474. Sprague-Dawley rats were exposed to the test substance by oral gavage on three consecutive days. Three to four hours after the last treatment, the animals were euthanized. Olive oil was selected as the vehicle. Test and/or control article formulations were administered at a dose volume of 10 mL/kg bw/dose. In the dose range finding assay, the dose levels tested were 125, 250, 500 and 1000 mg/kg bw/day in 3 animals/sex. However, animals in the two high dose groups of 500 and 1000 mg/kg bw/day were not dosed on day 3 due to mortality in these two dose groups. Based upon the results, the high dose for the definitive assay was 250 mg/kg bw/day, which was estimated to be the maximum tolerated dose, because this dose piloerection, lethargy, hunched posture, crusty nose and irregular breathing was seen. At this dose there was also decrease in body weight (7.7%). As no differences in clinical observations were seen between the sexes, only males were used in the definitive assay. The definitive assay dose levels tested were 0 (olive oil), 62.5, 125 and 250 mg/kg bw/day in six male rats per dose. Ethyl methanesulfonate (EMS) was used as positive control. Two tests were performed because in the first test after the in vivo part, in the preparation of the slides, the voltage was possibly applied during the DNA denaturion instead of after denaturation. This may have caused the vehicle control the % tail DNA being outside the historical control for both liver and duodenum cells. And also the positive control did not result in positive result in the duodenum. Therefore the test was repeated, the results of this second valid test is presented in the result section.

Results in the liver: A statistically significant increase in % Tail DNA was observed at 250 mg/kg bw/day in liver cells compared to the vehicle control. However, the value was within the Testing Facility’s historical range, and thus, this increase was not considered biologically relevant. In order to confirm the absence of increase in % Tail DNA, the high dose and vehicle control slides were rescored with an additional 150 cells/animal. This additional scoring showed that there was no statistically significant or dose-dependent increase in % Tail DNA at 250 mg/kg bw/day in liver cells.

Results in duodenum: No statistically significant or dose-dependent increases in % Tail DNA were observed at any dose level in duodenum cells compared to the vehicle control.

Results controls: The positive controls in both liver and duodenum cells had a statistically significant increase in % Tail DNA compared to the vehicle control. The vehicle control % Tail DNA in both liver and duodenum cells was within the Testing Facility’s historical range. In order to confirm the increase in % Tail DNA observed in liver cells, the high dose and vehicle control slides were rescored with an additional 150 cells/animal. No statistically significant or dose-dependent increase in % Tail DNA was observed at 250 mg/kg bw/day in liver cells compared to the vehicle control. The vehicle control % Tail DNA in liver cells was within the Testing Facility’s historical range.

Conclusion: Under the conditions of this study, the administration of the test substance, at doses up to and including a dose of 250 mg/kg bw/day, did not induce a significant increase in DNA damage in liver or duodenum cells. Therefore, the test substance was concluded to be negative in the in vivo Comet assay.