Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vivo

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian bone marrow chromosome aberration test
Specific details on test material used for the study:
ID: 2,2'-[(1-Methylethylidene)bis(cyclohexane-4,1-diyloxymethylene)]-bisoxirane (EponexTM Resin 1510)
Batch No.: BS6E1006
Purity: 100% (per protocol)
Description: Clear colorless highly viscous liquid
Storage Conditions: Room temperature, protected from light upon receipt
Receipt Date: 17 February 2017
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
Test System
Sprague-Dawley (Hsd:SD) rats were received from Envigo RMS, Inc., Frederick, MD on 02 November 2018 (DRF assay; males and females) and 26 December 2018 (repeat definitive assay; males only).

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:

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

DRF Male 164.6 to 172.9 6 5
Female 136.0 to 142.0
Definitive Male 165.6 to 188.5 6 7

Justification for the Test System
This species has been routinely used as an animal model of choice for the mammalian bone marrow chromosomal aberration assay. This strain was an outbred strain that maximizes genetic heterogeneity and therefore tends to eliminate strain-specific response to the test substance.

Animal Welfare Provisions
This study is not duplicative or unnecessary. The number of animals, procedures, and design used for this study has been reviewed and were approved by the BioReliance Institutional Animal Care and Use Committee. Procedures involving animals performed at BioReliance follow the specifications recommended in the most current version of The Guide for the Care and Use of Laboratory Animals adopted by BioReliance (National Academy Press, Washington, D.C., 2011).

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 interrupted for other 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 three 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 were used for bedding to absorb liquids. A certified laboratory rodent chow (Teklad 2018C 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.
Randomization and Identification
Animals were assigned to groups using a randomization procedure within Microsoft Excel. At the time of randomization, the weight variation of animals did not exceed ±20% of the mean weight. Following randomization, animals were identified by sequentially numbered ear tags. The cage card contained, at least, the animal number(s), sex, study number, treatment group number, dose level, test substance ID and route of administration. Cage cards were color coded by treatment group. Raw data records and specimens were also identified by the unique animal number.
Route of administration:
oral: gavage
Vehicle:
Arachis Oil BP
Details on exposure:
Dose Administration
Test and/or control article formulations were administered at a dose volume of 20 mL/kg/dose (DRF and initial Chromosome Aberration assay), 13.33 mL/kg/dose (repeat Chromosome Aberration) and 10 mL/kg/dose (positive controls. All animals were administered by oral gavage for one day, using appropriately sized disposable polypropylene syringes with gavage needles. These routes have been routinely used and are widely-accepted for use in the In Vivo Bone Marrow Chromosome Aberration assay.

Dose Range-Finding Assay (DRF)
The assay design was as follows:
Group Treatment Dose LevelA (mg/kg/dose) Dose VolumeB (mL/kg/dose) Animals/Sex
1 Eponex 1510 500 20 3
2 Eponex 1510 1000 20 3
3 Eponex 1510 2000 20 3
A A maximum dose of 2000 mg/kg/day was used.
B Based upon individual body weight

Following the observation period (3 days post final dosing), all surviving animals were euthanized by carbon dioxide inhalation, and discarded without further examination.

Since no noticeable gender differences were observed during the dose range finding assay, only male rats were used in the Chromosomal Aberration Assay.

Chromosomal Aberration Assay
Dosing formulation stability was not confirmed and there was inconsistent metaphase observed on the slides in the initial assay; therefore, the dosing volume of test substance and vehicle were changed to 13.33 mL/kg/dose in the repeat assay.

The initial/repeat assays design was as follows:
Group Treatment Dose Level Test Substance Concentration Assay Dose VolumeA Euthanasia Time
(mg/kg/dose) (mg/mL) (mL/kg/dose) (hrs after treatment)
Initial (Repeat) Initial (Repeat) 18 42
Number of Male Rats
1 Vehicle 0 0 20 (13.33) 6 6
2 Eponex 1510 500 25 (37.5) 20 (13.33) 6 0
3 Eponex 1510 1000 50 (75) 20 (13.33) 6 0
4 Eponex 1510 2000 100 (150) 20 (13.33) 6 6
5 B CP 40 4 (4) 10 (10) 6 0
6B CP 50 5 (5) 10 (10) 6 0
A Based upon individual body weight
B All animals were dosed, harvested, and evaluated for mitotic index. Only 5 animals per group were evaluated for chromosomal aberrations. The 5 animals selected were with similar mitotic index. Only Group 5 positive control was evaluated for chromosomal aberration.
Duration of treatment / exposure:
1 day
Frequency of treatment:
Once
Post exposure period:
3 days
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
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide monohydrate (CP)
Tissues and cell types examined:
Blood Collection and Sample Handling
Group Treatment Dose Level Animals/ Timepoint Post-final Dose Collection Total Number of Males
(mg/kg/dose) Timepoint (hrs)*
7 Eponex 1510 500 3 Pre-dose, 18 6
3 2, 42
8 Eponex 1510 1000 3 Pre-dose, 18 6
3 2, 42
9 Eponex 1510 2000 3 Pre-dose, 18 6
3 2, 42

Frequency First day of dosing
Collection Site Retro-orbital Sinus
Target Volume 0.5 mL of whole blood
Anesthesia Animals were anesthetized prior to collection by 70% CO2/30% O2.
Anticoagulant K2EDTA
Sample Handling Blood samples were maintained on wet ice until centrifugation.
Centrifugation Blood samples were centrifuged for 5 minutes, 2-8°C, at 2000 g within 1 hour of collection, and plasma was harvested into two sets of approximately equal aliquots.
Sample Storage Plasma samples were stored at < -60°C until required for analysis.
Animal Disposition Animals were sacrificed by CO2 overdose after their last collection timepoint.

Bioanalysis (BioA)/Toxicokinetic Analysis (TK)
Plasma samples were taken as follows for potential TK analysis:
Sample 1 - before dosing (internal control value for each group)
Sample 2 - two hours after the initial dosing
Sample 3 - at the 18 hour sac (terminal sampling)
Sample 4 - at the 42 hour sac (terminal sampling)

Samples were stored at -60°C or below until required for analysis. If not required for analysis, samples will be disposed prior to report finalization.
Details of tissue and slide preparation:
Femoral bone marrow was collected at approximately 18 or 42 hours after the last dose administration, as indicated previously. To arrest the cells in metaphase, 3 hours ± 30 minutes prior to the scheduled bone marrow collection time, all animals received a single intraperitoneal injection of colchicine (2 mg/kg; prepared at 0.5 mg/mL in Hanks Balanced Salt Solution and administered at 4 mL/kg). 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.
Bone marrow suspensions were transferred into tubes containing 3 mL HBSS and the cells were collected by centrifugation (100 x g for 10 minutes at 2 to 8C). Cells were re suspended in 5 mL warm (371C) 0.075M KCl, and incubated in a water bath for 10 ± 1 minutes at 37±1°C to swell the cells. Several drops of fixative (methanol: acetic acid, 3:1 v/v) were added to each tube, and the contents were gently mixed by inverting the containers. The cells were then collected by centrifugation (100 x g for 5-10 minutes at 2 to 8C), re suspended in two consecutive changes of fixative (methanol: acetic acid, 3:1 v/v), capped, and stored at least overnight at 2 to 8°C.
To prepare the slides, the cells were collected by centrifugation (100 x g for 5-10 minutes at 2 to 8ºC) and re-suspended in fresh fixative. Two to four drops of fixed cells were dropped onto a wet slide and air-dried. At least two slides were prepared from each animal, air dried, stained with Giemsa and permanently mounted. Each slide was identified by the experiment and animal number. Slides were coded using a random number table by an individual not involved with the scoring process.
Evaluation criteria:
The mitotic index was recorded as the percentage of cells in mitosis. The mean mitotic index was calculated for each treatment group (including positive and negative control groups) and served as a parameter for inhibition of cell division and cytotoxicity. Five animals with closer mitotic index from each group were selected for analysis of chromosomal aberration. Metaphase spreads (cells) with 42 ± 2 centromeres were examined using a light microscope and under oil immersion (1000X) without prior knowledge of treatment groups. Where possible, a minimum of 200 metaphase spreads (100 per slide) containing 42 chromosomes, was examined from each animal and scored for chromatid-type and chromosome-type aberrations (Scott et al., 1990). Fewer cells (metaphase spreads) may be scored per animal/dose/treatment when a high percentage of aberrations, i.e. 10%, were observed during the scoring.
Chromatid-type aberrations include chromatid and isochromatid breaks and exchange figures such as quadriradials (symmetrical and asymmetrical interchanges), triradials, and complex rearrangements. Chromosome-type aberrations include chromosome breaks and exchange figures such as dicentrics and rings. Fragments (chromatid or acentric) observed in the absence of any exchange figure were scored as a break (chromatid or chromosome). Fragments observed with an exchange figure were not scored as an aberration but were considered part of the incomplete exchange. Pulverized cells and severely damaged cells (counted as 10 aberrations) were also recorded. Chromatid and isochromatid gaps were recorded but not included in the analysis. The XY vernier for each cell with a structural aberration was recorded. The mitotic index was recorded as the percentage of cells in mitosis per 1000 cells counted. The percent polyploid and endoreduplicated cells was evaluated per 200 cells for each animal.
Statistics:
Statistical analysis was performed on the % frequency of chromosome aberrations using the animal as the unit. The mean and standard deviation of chromosome aberrations were 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 chromosome aberration frequency for the vehicle and test substance 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 substance 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:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Reductions of 63% and 72% in the percentage of cells in mitosis per 1000 cells scored (mitotic index) were observed in the test article-treated groups at 2000 mg/kg/day at 18 and 42 hours post-final dose, respectively, relative to the vehicle control. The reduction in mitotic index in the bone marrow demonstrates that there was bioavailability of the test substance to the bone marrow.
No statistically significant increase in structural or numerical (polyploid or endoreduplicated cells) aberrations was observed in the test substance treated groups relative to the vehicle control group (ANOVA, Dunnett’s post-hoc analysis, p > 0.05). The positive control induced a statistically significant increase in the frequency of cells with structural chromosomal aberrations (excluding gaps; Student’s t test, p ≤ 0.05). The frequency of cells with structural chromosomal aberrations (excluding gaps) in the vehicle control group was within the 95% control limits of the distribution of the historical negative control database.
Based upon this, all criteria for a valid test were met as specified in the protocol.
Conclusions:
Under the conditions of the assay described in this report, EponexTM Resin 1510 was concluded to be negative for the induction of structural and numerical chromosomal aberrations in the bone marrow of male rats following oral gavage for one day up to and including a dose of 2000 mg/kg/day.
Executive summary:

The test substance, 2,2'-[(1-Methylethylidene)bis(cyclohexane-4,1-diyloxymethylene)]-bisoxirane (EponexTM Resin 1510), was evaluated for its clastogenic activity by detecting structural and/or numerical chromosome aberrations in the bone marrow cells of rats.  Arachis oil BP was selected as the vehicle.  Test and/or control substance formulations were administered at a dose volume of 20 mL/kg/dose (DRF and initial Chromosome Aberration assay), 13.33 mL/kg/dose (repeat Chromosome Aberration assay) and 10 mL/kg/dose (positive controls), were administered by oral gavage once.

In the dose range-finding assay, the dose levels tested were 500, 1000 and 2000 mg/kg/day in 3 animals/sex.  Based on the results, the maximum dose in the definitive assay was also 2000 mg/kg/day, which was the guideline recommended limit dose.

The dose levels tested in the chromosomal aberration assays were 500, 1000 and 2000 mg/kg/day in males only.  In the initial assay, dosing formulation stability was not confirmed and there was inconsistent metaphase observed on the slides.  Based on revised stability information of the test article in vehicle and stock dosing formulation, the study was repeated with a dose volume of 13.33 mL/kg/dose at concentration of 37.5, 75 and 150 mg/mL (500, 1000 and 2000 mg/kg/day, respectively). In this report, only the repeat chromosomal aberration assay is presented in the data tables.

At 18 and 42 hours post-final dose, cytotoxicity was observed at 2000 mg/kg/day (-63% and -72%, respectively).  No statistically significant increase in structural or numerical (polyploid or endoreduplicated cells) aberrations was observed in the test substance treated groups relative to the vehicle control group.  The positive control induced a statistically significant increase in the frequency of cells with structural chromosomal aberrations (excluding gaps).  The frequency of cells with structural chromosomal aberrations (excluding gaps) in the vehicle control group was within the 95% control limits of the distribution of the historical negative control database.

Under the conditions of the assay described in this report, EponexTM Resin 1510 was concluded to be negative for the induction of structural and numerical chromosomal aberrations in the bone marrow of male rats following oral gavage for one day up to and including a dose of 2000 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Under the conditions of the assay described in this report, EponexTM Resin 1510 was concluded to be negative for the induction of structural and numerical chromosomal aberrations in the bone marrow of male rats following oral gavage administration for one day up to and including a dose of 2000 mg/kg/day.

Endpoint Conclusion: No Adverse effect observed (negative)

Justification for classification or non-classification

Under the conditions of the assay described in this report, EponexTM Resin 1510 was concluded to be negative for the induction of structural and numerical chromosomal aberrations in the bone marrow of male rats following oral gavage administration for one day up to and including a dose of 2000 mg/kg/day.