Registration Dossier

Administrative data

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 June 2017 to 29 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian comet assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Esterification products of acrylic acid and 4,4'-isopropylidenediphenol ethoxylated
EC Number:
701-362-9
Molecular formula:
(C2 H4 O)x (C2 H4 O)y C21 H20 O4
IUPAC Name:
Esterification products of acrylic acid and 4,4'-isopropylidenediphenol ethoxylated
Details on test material:
SID Change
Previous CAS: 64401-02-1
Previous EC: 613-584-2

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
This species has been routinely used as an animal model of choice for the mammalian alkaline Comet assay. This strain is an outbred strain that maximizes genetic heterogeneity, and therefore, tends to eliminate strain-specific responses to the test article.
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS, Inc., Frederick, MD
- Age at study initiation: 6 - 7 weeks
- Weight at study initiation:
Dose Range Finder: Males - 186.2 to 210.4 grams; Females - 146.9 to 160.1 grams.
Initial Definitive: Males - 198.8 to 227.3 grams;
Repeat Definitive: Males - 154.3 to 169.7 grams
- Assigned to test groups randomly: Yes, animals were assigned to groups using a randomization procedure. At the time of randomization, the weight variation per sex of all animals assigned to the study did not exceed +/-20% of the mean weight. A randomization function in Excel was used to achieve random placement of animals throughout all groups.
- Fasting period before study: None - Housing: Animals were housed in a controlled environment at 72+/-3 degrees F and 50+/-20% relative humidity with a 12-hour light/dark cycle. The light cycle may have been interrupted for study-related activities. The animal rooms were supplied with at least 10 changes of fresh HEPA-filtered air per hour. Animals of the same sex were housed up to 5 per Micro-Barrier cage.
- Diet (e.g. ad libitum): A certified laboratory rodent chow (Envigo 2018C Teklad Global 18% Protein Rodent Diet) was provided ad libitum. - Water (e.g. ad libitum): Animals had free access to tap water, which met US EPA drinking water standards.
- Acclimation period: 5-6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72+/-3 degrees F
- Humidity (%): 50+/-20% relative humidity
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12-hour light/dark cycle

IN-LIFE DATES:
Dose Range Finder From: 05 July 2017 To: 12 July 2017
Repeat Definitive From: 25 Oct 2017 To: 31 Oct 2017

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil for test substance; 0.9% Saline for injection, USP for positive control
- Justification for choice of solvent/vehicle: The vehicle was chosen based on its solubility with the test substance and compatibility with the target cells.
- Amount of vehicle : 10 mL/kg/day - Lot/batch no. of test substance vehicle: MKCC0462
Details on exposure:
Preparation of Control Article
The neat EMS was prepared in 0.9% saline for injection. The dosing formulation was prepared at a concentration of 20 mg/mL, just prior to use. Preparation of Test Substance Dose FormulationsThe test substance dose formulations were prepared fresh on each day of use. Each concentration was prepared by calibrating a suitable size amber vial with a PTFE stir bar to target batch size, which holds the pre-weighed amount of the test substance. An appropriate amount of the test substance was combined with approximately 70% of the total volume of vehicle and stirred with the PTFE stir bar. The vehicle was added to the QS line, sealed and mixed magnetically until homogenous in appearance. Formulations were stirred continuously after their preparation and throughout the dosing procedure. The final dose formulation was stored at room temperature.

All dose formulations were administered at a volume of 10 mL/kg/day by oral gavage using appropriately sized disposable polypropylene syringes with gastric intubation tubes (needles). The route has been routinely used and is widely-accepted for use in the comet assay.
Duration of treatment / exposure:
All animals in Groups 1 through 4 were dosed on 2 consecutive days with the vehicle and test substance. The second dose occurred approximately 21 hours after the first dose and 3-4 hours before euthanasia. Two animals in group 2 (males) were euthanized 4-6 minutes earlier than the 3-hour euthanasia time due to a time scheduling error. This deviates from the protocol, which states the animals were to be euthanized 3-4 hours after the 21-hour dose on Day 3.Animals in Group 5 were dosed with the positive control once approximately 3-4 hours prior to euthanasia on Day 2.
Frequency of treatment:
see above
Post exposure period:
All animals were euthanized 3-4 hours after the last dose (Study Day 2) for tissue collection, except as indicated above.
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
DRF: 3 animals/sex
Definitive: 6 male rats per dose (Groups 1 to 4), two additional animals were dosed in Group 4 to cover for any possible mortality, but were euthanized and discarded without evaluation because no mortality was observed; 3 males (Group 5)
Control animals:
yes, concurrent vehicle
Positive control(s):
Ethyl methanesulfonate
- Route of administration: oral gavage
- Doses / concentrations: 200 mg/kg/day / 10 mL/kg/day

Examinations

Tissues and cell types examined:
Liver, stomach, and duodenum
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: In the DRF assay, 3 animals/sex were exposed to 2000, 1000, and 500 mg/kg/day of test substance. A vehicle control was also included. Animals were dosed on two consecutive days. The second dose occurred approximately 21 hours after the first dose. Clinical observations were seen in the high dose group in both males and females. Following the last observation, animals were euthanized by exposure to CO2 and discarded without further examination after tissues were collected for histopatholgy. No mortality or differences in clinical observations were seen between the sexes, therefore only males were used in the definitive assay. In the Definitive assay, dose levels were based on the results from the DRF assay and histopathology.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): Animals were dosed at a dose volume of 10 mL/kg/day by oral gavage for two consecutive days. All animals were euthanized 3-4 hours after the last dose for tissue collection, except as previously indicated.

DETAILS OF SLIDE PREPARATION: A portion of each dissected liver was placed in 3 mL of cold mincing buffer, then the liver was finely cut (minced) with a pair of fine scissors to release the cells. A portion of each dissected interior glandular stomach and duodenum were placed in 1 mL of cold mincing buffer then glandular stomach and duodenum were scraped using a plastic spatula to release the cells. Each cell suspension was strained through a Cell Strainer into a pre-labeled 50 mL polypropylene conical tube and the resulting liver, stomach and duodenum cell suspensions were placed on wet-ice. An aliquot of the suspensions were used to prepare the comet slides. From each liver, stomach and duodenum suspensions, an aliquot of 2.5 µL (liver) and 7.5 µL (stomach and duodenum) were mixed with 75 µL (0.5%) of low melting agarose. The cell/agarose suspension was applied to microscope slides commercially available pre-treated multi-well slides. Commercially purchased multi-well slides were used and 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 20-well slides were prepared per animal per tissue. Three wells were used in scoring and the other wells were designated as a backup. Following solidification of agarose, the slides were placed in jars containing lysis solution.Following solidification of agarose, the slides were submerged in commercially available lysis solution supplemented with 10% DMSO on the day of use. The slides were kept in this solution at least overnight at 2-8oC.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 2-10oC and protected from light, allowing DNA to unwind.Using the same buffer, electrophoresis was conducted for 30 minutes at 0.7 V/cm, at 2-10oC and protected from light. The electrophoresis time was constant for all slides.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. Slides were stained with a DNA stain (i.e., Sybr-gold) prior to scoring. The stain solution was prepared by diluting 1 uL of Sybr-gold stain in 15 mL of 1xTBE (tris-boric acid EDTA buffer solution).

METHOD OF ANALYSIS: Slides were evaluated for DNA damage using a fully validated automated scoring system (Comet Assay IV) from Perceptive Instruments Ltd. (UK).
Evaluation criteria:
A test article was considered to have induced a positive response if:a) at least one of the group means for the % Tail DNA of the test article doses exhibited a statistically significant increase when compared with the concurrent negative control (p
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. In order to quantify the test substance effects on DNA damage, the following statistical analysis was performed: •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 substance groups were compared using Levene’s test (significant level of p < 0.05). If the differences and variations between groups were found not to be significant, 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 substance 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
Remarks:
Clinical signs of piloerection were observed at 1000 and 2000 mg/kg/day in the Definitive Assay.
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: in liver, stomach and duodenum cells
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
No mortality occurred at any dose level during the course of the dose range finding assay. No considerable reductions in mean group body weights were seen in the test substance treated groups during the course of the study.The following clinical signs were observed at 2000 mg/kg : Lethargy, Crusty Nose in males, and Hunched Position in females.

RESULTS OF DEFINITIVE STUDY
In the Initial Comet Assay, the vehicle control values were not within the expected range, thus not all criteria for a valid test were met. Therefore, in consultation with the Sponsor, the assay was repeated. The results reported are for the Repeat 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 substance treated groups during the course of the study.
The following clinical signs were observed:Piloerection at 1000 and 2000 mg/kg

In liver cells :
The scoring results and a statistical analysis of data indicated the following:
• The presence of ‘clouds’ in the test substance groups was = 0.3%, which was comparable with the % of clouds in the vehicle control group (0.3%).
• Group variances for mean of medians of the % Tail DNA in the vehicle and test substance 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 substance 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 substance 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 groups (Student’s t test, p = 0.05).
• In the vehicle control group, % Tail DNA was within the historical vehicle control range for the liver (Appendix I).
These results indicate that all criteria for a valid test, as specified in the protocol, were met.

In stomach cells :
The scoring results and a statistical analysis of data indicated the following:
• The presence of ‘clouds’ in the test substance groups was = 49.5%, which was lower than the % of clouds in the vehicle control group (59.0%).
• Group variances for mean of medians of the % Tail DNA in the vehicle and test substance 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 substance 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 substance doses (regression analysis, p > 0.01).
• The positive control, EMS, induced a statistically significant increase in the % Tail DNA in glandular stomach cells as compared to the vehicle control groups (Student’s t test, p = 0.05).
• In the vehicle control group, % Tail DNA mean was within the historical vehicle control range for the stomach (Appendix I).
These results indicate that all criteria for a valid test, as specified in the protocol, were met.

In duodenum cells:
The scoring results and a statistical analysis of data indicated the following:
• The presence of ‘clouds’ in the test substance groups was = 10.8%, which was lower than the % of clouds in the vehicle control group (18%).
• Group variances for mean of medians of the % Tail DNA in the vehicle and test substance 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 substance 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 substance doses (regression analysis, p > 0.01).
• The positive control, EMS, induced a statistically significant increase in the % Tail DNA in duodenal cells as compared to the vehicle control groups (Student’s t test, p = 0.05).
• In the vehicle control group, % Tail DNA was within the historical vehicle control range for the duodenum (Appendix I).
These results indicate that all criteria for a valid test, as specified in the protocol, were met.

Applicant's summary and conclusion

Conclusions:
Under the conditions of the assay described in this report, Ethoxylated (3) Bisphenol A Diacrylate was concluded to be negative for the induction of DNA damage in liver, glandular stomach and duodenum.
Executive summary:

The test substance,Ethoxylated (3) Bisphenol A Diacrylate, was evaluated for its genotoxic potential in the Comet assay to induce DNA damage in liver, glandular stomach, and duodenum cells of male rats. Corn oil was selected as the vehicle. Test and/or control substance formulations were administered at a dose volume of 10 mL/kg/day by oral gavage.

In the dose range finding assay (DRF), the maximum dose tested was 2000 mg/kg/day. The additional dose levels tested were 1000 and 500 mg/kg/day in 3 animals/sex. Based upon the results, the high dose for the definitive assay was 2000 mg/kg/day, which is the highest guideline recommended dose for this assay, which was estimated to be the maximum tolerated dose (MTD). 

The definitive assay dose levels tested were 500, 1000, and 2000 mg/kg/day.

The definitive assay was repeated due to vehicle values in the initial assay not being within the expected range. Thus, the initial assay did not meet the criteria for a valid assay and was repeated. The results and discussion will only include data from the repeated definitive assay. The test substance gave a negative (non-DNA damaging) response in this assay in liver, glandular stomach, and duodenum cells for males in % Tail DNA. None of the test substance treated animal slides had significant increases in the % Tail DNA compared to the respective vehicle controls. The vehicle control % Tail DNA was within the Testing Facility’s historical range, and the positive control had a statistically significant increase in % Tail DNA compared to the vehicle control. Thus, all criteria for a valid assay were met for liver, glandular stomach, and duodenum cells.

All critiera for a valid study were met as described in the protocol.

Under the conditions of this study, the administration of Ethoxylated (3) Bisphenol A Diacrylate at doses up to and including a dose of 2000 mg/kg/day did not cause a significant increase in DNA damage in liver, glandular stomach, and duodenum cells relative to the concurrent vehicle control. Therefore, Ethoxylated (3) Bisphenol A Diacrylate was concluded to be negative in the in vivo Comet Assay.