Registration Dossier

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
Includes OECD 474 Micronucleus Assay
Deviations:
yes
Remarks:
See Method
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian comet assay

Test material

Constituent 1
Chemical structure
Reference substance name:
4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with fatty acids, C18-unsatd., dimers
EC Number:
500-180-5
EC Name:
4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with fatty acids, C18-unsatd., dimers
Cas Number:
67989-52-0
Molecular formula:
(C15H16O2.C3H5ClO.Unspecified)x
IUPAC Name:
2-(chloromethyl)oxirane; 4-[2-(4-hydroxyphenyl)propan-2-yl]phenol
Test material form:
liquid: viscous
Details on test material:
As per IUCLID Sections 1.1. 1.2. and 4.1.
Specific details on test material used for the study:
The analytical method for the determination of 4,4’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with fatty acids, C18-unsatd., dimers (ERRCF #62) was not validated using mass spectrometry (MS). The study was terminated since the methodology did not work with the test substance. An ionizable compound was not detected and thus the study did not generate suitable method.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Sprague-Dawley (Hsd:SD) rats were received from Envigo RMS, Inc., Frederick, MD on 09 November 2016 (DRF) and 23 November 2016 (definitive assay).
The age at time of initiation, as well as the body weights and days of acclimation of the rats assigned to the study groups at randomization are indicated below:

Body Weight Range at
Randomization Age at Initiation Days of
Study Sex (grams) (weeks) Acclimation

DRF Male 162.4-172.6 6 5
Female 134.8-140.8

Definitive Male 188.7-219.2 6 5
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animal Receipt and Acclimation
Virus antibody-free (VAF) animals were acclimated as noted above and were judged to be healthy prior to utilization in the study.

Housing
Animals were housed in a controlled environment at 72 ± 3 F and 50 ± 20% relative humidity with a 12-hour light/dark cycle. The light cycle was not 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 five per Micro-Barrier cage. Cages were placed on racks equipped with an automatic watering system and Micro-VENT full ventilation, HEPA filtered system.

Bedding, Food and Water
Heat treated hardwood chips (P.J. Murphy Forest Products) were used for bedding to absorb liquids. A certified laboratory rodent chow (Envigo 2018C Teklad Global 18% Protein Rodent Diet) was provided ad libitum. The food was analyzed by the manufacturer for the concentrations of specified heavy metals, aflatoxin, chlorinated hydrocarbons, organophosphates and specified nutrients. Animals had free access to tap water, which met U.S. EPA drinking water standards [Washington Suburban Sanitary Commission (WSSC) Potomac Plant]. Drinking water was monitored at least annually for levels of specified microorganisms, pesticides, heavy metals, alkalinity and halogens. The results of bedding, food and water analyses are on file at BioReliance. There were no contaminants in the bedding, feed and water that were expected to interfere with the study.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
PEG400 (Polyethylene Glycol 400)
PEG 400 was the vehicle of choice based on the solubility of the Test Substance. The Test Substance was soluble in PEG 400 at a concentration of approximately 400 mg/mL, the maximum concentration tested.
Details on exposure:
Test substance and vehicle control substance dose formulations were administered at a volume of 5 mL/kg by oral gavage using appropriately sized disposable polypropylene syringes with gastric intubation tubes (needles). The positive control substance formulation was administered at a volume of 10 mL/kg by oral gavage. The route has been routinely used and is widely-accepted for use in the mammalian bone marrow erythrocyte Micronucleus assay.

In order to allow both endpoints to be examined in a combined multi-end point in vivo assay, marginal modifications to the recommended guidance for the Micronucleus and Comet assays were undertaken in respect of dosing. The combination protocol design used here involved animals dosed at 0, 24 and 48 hours and sampled 51 hours (i.e. 3 (or 4) hours after the final dose). Using this dosing regimen, only the first two doses (at 0 and 24 hours) could impact upon the bone marrow micronucleus endpoint, as the final dose is given too close to the sampling time to have any effect on micronuclei production. Bone marrow was sampled effectively 24 hours after the second dose, and therefore conforms to the OECD 474 recommendations. Comet tissue sampling was undertaken 3 or 4 hours after the final dose or ~24 hours after the second dose which also conforms to OECD 489 recommendations
Duration of treatment / exposure:
3 days
Frequency of treatment:
Once per day
Post exposure period:
NA
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
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:
6 male
Control animals:
yes, concurrent vehicle
Positive control(s):
Ethyl methanesulfonate

Examinations

Tissues and cell types examined:
All animals were euthanized 3 to 4 hours after the last dose (Study Day 3) by CO2 asphyxiation, and then, the following was performed:
• Animals were dissected and the liver and stomach was extracted (removed) and collected.
• A section of the organ was cut and placed in formalin for possible histopathology analysis.
• Another section of the organ 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.

Micronucleus Assay Bone Marrow Collection and Processing
Femoral bone marrow was collected, at approximately 24 hours after the final dose, as previously indicated. Animals were euthanized by carbon dioxide inhalation. Immediately following euthanasia, the femurs were exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing fetal bovine serum.
Details of tissue and slide preparation:
Comet Assay Tissue Collection and Processing
All animals were euthanized 3 to 4 hours after the last dose (Study Day 3) by CO2 asphyxiation (See Deviations), and then, the following was performed:
• Animals were dissected and the liver and stomach was extracted (removed) and collected.
• A section of the organ was cut and placed in formalin for possible histopathology analysis.
• Another section of the organ 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. A portion of each dissected stomach was placed in cold mincing buffer; then, the stomach was 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. An aliquot of the suspension was used to prepare the Comet slides.
Preparation of Slides
From each organ suspension, an aliquot of 2.5 µL (liver) or 7.5 µL (stomach) was mixed with 75 µL (0.5%) of low melting agarose (0.5%). The cell/agarose suspension was applied to microscope slides commercially available pre-treated. The slides were kept at 2 - 8°C for at least 15 minutes to allow the gel to solidify. At least two Trevigen, Inc 3-well slides were prepared per animal per tissue. Two wells were used in scoring and the other wells were designated as a backup.
Lysis
Following solidification of agarose, all slides (irrespective of the tissue examined) were submerged in a commercially available lysis solution supplemented with 10% DMSO on the day of use. The slides were kept in this solution for two days at 2-8oC.
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 (irrespective of the tissue examined) 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 (irrespective of the tissue examined).
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 overnight 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).

Preparation of Micronucleus Slides
The bone marrow was transferred to a centrifuge tube containing 2 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.
Evaluation criteria:
Scoring of Comet Slides
Three slides/wells per organ/animal were used. Fifty randomly selected cells per slide were scored blind resulting in a total of 150 cells evaluated per animal.
The following endpoints of DNA damage were assessed and measured:
• Comet Tail Migration; defined as the distance from the perimeter of the Comet head to the last visible point in the tail.
• % Tail DNA; (also known as % tail intensity or % DNA in tail); defined as the percentage of DNA fragments present in the tail.
• 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). 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.
The Comet slides, which are not permanent (the slides can be affected/damaged by environmental storage conditions), will be discarded prior to report finalization.

Histopathology Evaluation
A portion of each dissected tissue was placed in formalin (10 % neutral-buffered formalin) for possible histopathology analysis. Per the study protocol, histopathology evaluation was not performed since biologically significant increases in DNA damage were not observed.
All unused tissue samples saved for histopathology will be discarded prior to finalization of the report.
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 substance groups were compared using Levene’s test (significant level of p ≤ 0.05). For the liver, 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). For the stomach, Levene’s test indicated heterogeneous group variances (p ≤ 0.05), the suitability of a transformation of the original data was evaluated (e.g. using logarithm transformed values of the original data) in an attempt to meet the normality criteria. Afterwards, statistical analysis was performed using the parametric tests described above. If parametric tests were not acceptable, non-parametric statistical methods, (Kruskal Wallis and/or Mann Whitney test) may have been used in evaluation of data.
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 resultsopen allclose all
Key result
Sex:
male
Genotoxicity:
negative
Remarks:
Comet Assay
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Sex:
male
Genotoxicity:
negative
Remarks:
Micronucleus Assay
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Comet Assay
Liver
The scoring results and a statistical analysis of data indicated the following:
• The presence of ‘clouds’ in the test substance groups was ≤ 1.8%, which was higher than the % of clouds in the vehicle control group (0.2%).
• 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.

Stomach
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 higher than the % of clouds in the vehicle control group (7.0%).
• Group variances for mean of medians of the % Tail DNA in the vehicle and test substance groups were compared using Levene’s test. The indicated heterogeneous group variances (p ≤ 0.05), the suitability of a transformation of the original data was evaluated (e.g. using logarithm transformed values of the original data) in an attempt to meet the normality criteria. Since the transformed data were also heterogeneous, statistical analysis was performed using the non-parametric test (Kruskal Wallis).
• 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 stomach 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 stomach.

These results indicate that all criteria for a valid test, as specified in the protocol, were met, with the specificity and sensitivity of the endpoint demonstrated.

Micronucleus Assay
The scoring results and a statistical analysis of data indicated the following:
• No appreciable reductions in the PCEs/EC ratio were observed in the test substance groups compared to the vehicle control group, indicating the test substance did not induce cytotoxicity.
• Group variances for mean of means of the micronucleus frequency 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 increase in the incidence of MnPCEs was observed in the test substance 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, with the specificity and sensitivity of the endpoint demonstrated.

Any other information on results incl. tables

See attached data tables.

Applicant's summary and conclusion

Conclusions:
• In vitro testing (bacterial mutagenicity and chrom ab) were positive without metabolic activation thus prompting the in vivo Comet and micronucleus assay.

• Oral is the only route of administration practical with this material. Dermal absorption is unlikely because of the size of the molecule(s) and other phsy/chem properties. Because of the low vapor pressure (de minimis), administration by inhalation is not justifiable.

• The test substance was administered orally at the maximum level (2000 mg/kg) as defined by the ECHA guidance and the protocol.

• There was no evidence of genetoxicity in cells from the stomach, liver or bone marrow.

• The cells harvested from the stomach for the assay had direct contact with the test substance because the route of administration was oral. Bioanalytical evaluation of plasma did not produce clear evidence of systemic exposure of the parent compound at any of the dose levels administered in this study.

Therefore, within the limits of our ability to test this material, 4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with fatty acids, C18-unsatd., dimers was not genotoxic in vivo at the maximum allowable dose.
Executive summary:

The test substance, 4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with fatty acids, C18-unsatd., dimers (ERRCF #62), was evaluated for its genotoxic potential in bone marrow of male rats and in the Comet assay to induce DNA damage in liver and stomach cells. PEG400 (Polyethylene Glycol 400) was selected as the vehicle. Test and/or control substance formulations were administered at a dose volume of 5.0 mL/kg/day by oral gavage once per day for three consecutive days. 

 

In the dose range finding assay (DRF), the maximum dose tested was 2000 mg/kg/day. The dose levels tested were 500, 1000, or 2000 mg/kg bw/day in 6 animals. 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. 

 

The definitive assay dose levels tested were 500, 1000, and 2000 mg/kg/day in males only as no sex-related differences in toxicity were observed in the DRF.

 

In the Micronucleus assay, no statistically significant increase in the incidence of MnPCEs was observed in the test substance treated groups relative to the vehicle control groups. Thus, the test substance was negative (neither clastogenic nor aneugenic). The positive control induced a statistically significant increase in the incidence of MnPCEs, thus demonstrating the sensitivity and specificity of the assay endpoint. The number of MnPCEs in the vehicle control groups did not exceed the historical control range. 

 

In the Comet assay, the test substance gave a negative (non-DNA damaging) response in stomach and liver cells for male rats in % Tail DNA. None of the test substance treated animal slides had significant increases in the % Tail DNA compared to the respective vehicle controls. Although bioanalytical evaluation of plasma did not produce clear evidence of systemic exposure of the parent compound at any of the dose levels administered in this study, the cells harvested from the stomach for the assay did have direct contact with the test substance because the route of administration was oral. 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 demonstrating the sensitivity and specificity of the assay endpoint. Thus, all criteria for a valid assay were met for stomach and liver.

 

Under the conditions of this study, the administration of 4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with fatty acids, C18-unsatd., dimers (ERRCF #62), at doses up to and including a dose of 2000 mg/kg bw/day (the maximum recommended dose for short term in vivo testing) following oral gavage dosing at 0, 24 and 48 hours with harvesting of tissues 3 to 4 hours later, did not induce a significant increase in the incidence of MnPCEs relative to the concurrent vehicle control; nor did it induce a significant increase in DNA damage in liver or stomach cells of male rats. Overall, it is concluded that 4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with fatty acids, C18-unsatd., dimers (ERRCF #62) is negative in the combined in vivo Micronucleus-Comet assay.