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Toxicological information

Genetic toxicity: in vivo

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
September, 2014
GLP compliance:
yes
Type of assay:
other: mammalian erythrocyte micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
4,4'-isopropylidenedi-2,6-xylol
EC Number:
227-033-5
EC Name:
4,4'-isopropylidenedi-2,6-xylol
Cas Number:
5613-46-7
Molecular formula:
C19H24O2
IUPAC Name:
4,4'-isopropylidenedi-2,6-xylol
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): 4,4’ – (1-methylethylidene)-bis(2,6-dimethylphenol) 4,4’-Isopropylidenebis(2,6-dimethylphenol)
- Purity: 99.77%
- Storage conditions: room temperature and protected from light

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD (SD)
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (Stoneridge, New York)
- Age at study initiation: 7 weeks
- Weight at study initiation: 161.6 - 214.1 g
- Assigned to test groups randomly: yes
- Housing: Micro-Barrier cages, up to 5 animals per 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 (met U.S. EPA drinking wtaer standards); ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 72 ± 3
- 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 used: corn oil
- Amount of vehicle: 10 mL/kg/day
- Supplier: MP Biomedical, LCC
- Lot/Batch No.: M6581
- Expiration date: 31 January 2017
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Dose formulations were prepared daily. A suitable sized vial with a PTFE stir bar was calibrated to the final batch size and an appropriate amount of test article was transferred to the vial. An appropriate amount of vehicle was added until approximately 70% of the target volume was reached. The suspension was stirred magnetically for 12 to 57 minutes. Then, the appropriate amount of vehicle was added until the target volume was achieved.
Duration of treatment / exposure:
3 days
Frequency of treatment:
Daily
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:
5 (males)
Control animals:
yes, concurrent vehicle
Positive control(s):
- Positive control: Cyclophosphamide monohydrate for micronucleus assay; Ethyl methanesulfonate (EMS) and vinblastine sulfate (VB) for potential CREST analysis
- Doses / concentrations: EMS: 200 mg/kg/day; VB: 4 mg/kg/day
- Administration: EMS/VB: once daily on Study Day 2 and approximately 3 to 4 hours prior to euthanasia on Study Day 3.
- Other: Scoring positive control slides (fixed and unstained), generated from a different study, 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

Examinations

Tissues and cell types examined:
Polychromatic erythrocytes derived from femoral bone marrow were evaluated on micronuclei.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: the selected doses were based on a range-finding study. The dose levels tested in the dose range finding assay (DRF) were 500, 1000, and 2000 mg/kg/day in 3 animals/sex. Based upon the results, the high dose for the definitive assay was 2000 mg/kg/day in males only, which is the highest guideline recommended dose for this assay.

TREATMENT AND SAMPLING TIMES: Daily, euthanisia by carbon dioxide inhalation took place 3 - 4 hours after the last treatment. 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 SLIDE PREPARATION:
- 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 two slides were prepared from each animal, air dried and fixed by dipping in methanol. One set of two slides (including at least 6 positive control (CP) slides) was stained with acridine orange for microscopic evaluation. 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.
- After preparation of the two slides for micronucleus evaluation, the remaining bone marrow suspension, including that from the VB-treated animals, was processed through a cellulose column containing a 50:50 mixture of cellulose type 50:alpha-cellulose (one gram per column, one column per animal). The bone marrow suspension was eluted through the cellulose column using 4 mL of fetal bovine serum. The eluted cells were pelleted by centrifugation, and the supernatant 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 two slides for CREST were prepared from each animal, air dried and fixed by dipping in methanol. Each slide was identified by the harvest date, study number and animal number. Slides were placed in a slide box within a sealed plastic bag purged with nitrogen. The CREST slides were stored at -10 to -30ºC until disposal prior to finalization.

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). 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), whenever possible. 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. PCE/EC proportions <20% of vehicle control value were considered excessively cytotoxic and the animal data was excluded from evaluation.
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 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:
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). 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:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
- Ratio of PCE/NCE: A statistically significant reduction in the PCEs/EC ratio was observed in male rats in the test article group treated with 2000 mg/kg/day compared to the vehicle control group. However, this reduction was determined to be biologically insignificant, and all dose levels could be scored.
- 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 (24,000 PCEs scored, 4000 PCEs/animal; Student’s t-test, p ≤ 0.05).
- The number of MnPCEs in the vehicle control group did not exceed the historical control range.

Any other information on results incl. tables

CLINICAL SIGNS

No mortality occurred. The only clinical effect observed was piloerection in all animals from the highest dose group, 2 hours after the latest dose on day 3. Animals gained weight during the study.

VALIDITY

All criteria for a valid test were met as mentioned under 'Any other information on materials and methods incl tables'. There were some deviations from the protocol, but these had no adverse impact on the integrity of the data or the validity of the study conclusion as determined by the study director.

Applicant's summary and conclusion

Conclusions:
The test substance was not genotoxic in the in vivo Micronucleus assay.

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