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Diss Factsheets

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13th April 2018 to 4th July 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Constituent 1
Reference substance name:
Diamino Trimethylphenylindane
IUPAC Name:
Diamino Trimethylphenylindane
Test material form:
solid: particulate/powder
Details on test material:
Purity:
97.6% (49.9% 6-4’ isomer, 47.7 5-4’ isomer; per Protocol)
Specific details on test material used for the study:
Identification:
Diamino Trimethylphenylindane
Synonym:
DAPI
Batch No.:
AEF0030300
Purity:
97.6% (49.9% 6-4’ isomer, 47.7 5-4’ isomer; per Protocol)
Molecular Weight:
266.38 g/mol
Description:
Off-white powder
Storage Conditions:
Room temperature, protected from light
Receipt Dates:
07 June 2017
09 February 2018

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 May 2018 (initial DRF), 01 June 2018 (repeat DRF) and 15 June 2018 (definitive assay).
Sex:
male
Details on test animals or test system and environmental conditions:
Since no mortality or significant differences in clinical observations were seen between the sexes in the DRF assay, only male rats were used in the definitive assay.

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 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. Animals were provided with environmental enrichment such as Nylabones®, as needed.
Bedding, Food and Water
Heat treated hardwood chips 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:
Corn Oil
8001-30-7
Supplier- Sigma
Lot MKCC0462
Expiry/Retest- 30th November 2020
Details on exposure:
Dose formulations were prepared prior to dose administration in each assay as follows:
A suitably sized amber glass vial with a PTFE stir bar, containing an appropriate amount of test substance 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 to achieve the final target volume, and the formulation was stirred magnetically. In addition, all formulations in the repeat DRF and definitive assays were sonicated, homogenized with a polytron and stirred until uniform. Per Protocol Amendment 4, the low and mid dose formulations in the definitive assay were prepared a second time on the same day because, initially, the low dose was cloudy and the mid dose was clear. The second preparations looked the same as the initial preparations and were, therefore, not sampled or used for dosing. Ultimately, after sonication, homogenization with a polytron and stirring, as indicated above, the initial low and mid dose formulations became clear solutions. Formulations were maintained at room temperature, protected from light until use.
Duration of treatment / exposure:
All dose formulations were administered at a volume of 10 mL/kg by single oral gavage using appropriately sized disposable polypropylene syringes with gavage needles.
Frequency of treatment:
24 and 48 hours. See additional table for further information.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
75 mg/kg bw/day
Dose / conc.:
150 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
No. of animals per sex per dose:
6
Control animals:
yes, concurrent vehicle

Examinations

Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
Femoral bone marrow was collected at approximately 24 hours and 48 hours after dose administration, as indicated above. 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. 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. Four slides were prepared from each animal, air dried and fixed by dipping in methanol. One set of two slides (including at least five positive control slides) was stained with acridine orange for microscopic evaluation. The other set of slides was kept as backup and were 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:
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.
BioReliance Study No. AE95CF.125M012.BTL 14
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.
Stained slides were discarded prior to report finalization.

A test substance was considered to have induced a positive response if:
a) at least one of the test substance doses exhibited a statistically significant increase when compared with the concurrent negative 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 substance 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 substance (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 were presented for each treatment group.
The use of parametric or non-parametric statistical methods in the evaluation of data was based on the variation between groups. The group variances for micronucleus frequency for the vehicle and test substance groups at the respective sampling time were compared using Levene’s test (significance 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.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Additional information on results:
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 groups, indicating the test substance did not induce cytotoxicity.
• Group variances for the mean 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.
BioReliance Study No. AE95CF.125M012.BTL 19
• A statistically significant increase in the incidence of MnPCEs was observed in the 300 mg/kg dosed animals at 48 hours relative to the vehicle control group (ANOVA followed by Dunnett’s post-hoc analysis, p < 0.05). However, the value was within the 95% historical vehicle control limit, and was therefore considered to not be biologically significant. No statistically significant increases in the incidence of MnPCEs were observed in the remaining treatment 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 (Appendix I).

Any other information on results incl. tables

The following clinical signs were observed:

 Dose Level (mg/kg)  Males
 150  Piloerection and hunched position
 300  Piloerection, hunched position, ireegular breathing, lethargy, ear discoloration (red on outer edge) and crusty nose

Applicant's summary and conclusion

Conclusions:
Under the conditions of the assay described in this report, Diamino Trimethylphenylindane was concluded to be negative for the induction of micronucleated polychromatic erythrocytes.
Executive summary:

The test substance, Diamino Trimethylphenylindane, was evaluated for its clastogenic activity and/or disruption of the mitotic apparatus by detecting micronuclei in polychromatic erythrocytes (PCEs) in rat bone marrow. Corn oil was used as the vehicle. Test and/or control substance formulations were administered at a dose volume of 10 mL/kg by single oral gavage.

In the dose range-finding assays (DRF), the maximum dose tested was 2000 mg/kg. The dose levels tested in the initial DRF were 500, 1000, and 2000 mg/kg and 150 and 300 mg/kg in the second DRF, and both assays consisted of 3 animals/sex/dose level. Based upon the results, the high dose for the definitive assay was 300 mg/kg, which was estimated to be the maximum tolerated dose (MTD).

No statistically significant increase in the incidence of MnPCEs was observed in the test substance treated groups at 24 hours relative to the vehicle control groups. There was a statistically significant increase in the test substance dosed animals at 48 hours; however, the value was within the 95% historical vehicle control limit, and was therefore considered to not be biologically significant. The positive control induced a statistically significant increase in the incidence of MnPCEs. The number of MnPCEs in the vehicle control groups did not exceed the historical control range.

Under the conditions of this study, the administration of Diamino Trimethylphenylindane at doses up to and including a dose of 300 mg/kg was concluded to be negative in the Micronucleus assay.