Registration Dossier

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:
2019
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)
Version / remarks:
2016
Deviations:
no
Remarks:
No deviations ocurred that negatively impacted the integrity of the study.
GLP compliance:
yes
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Constituent 1
Reference substance name:
Novec 7300
IUPAC Name:
Novec 7300
Details on test material:
- Name of test material (as cited in study report): Novec 7300
- Physical state: Colorless, clear liquid
- Analytical purity: 99.85%
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
- Expiration date of the lot/batch:
- Purity test date:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity:
- Specific activity:
- Locations of the label:
- Expiration date of radiochemical substance:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
- Stability under test conditions:
- Solubility and stability of the test substance in the solvent/vehicle:
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material)

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)

OTHER SPECIFICS:

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Per OECD 474
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS, Inc.
- Age at study initiation: 6
- Weight at study initiation: Male: 173.5-187.8, Female: 136.5-144.2
- Assigned to test groups randomly: Yes
- Fasting period before study: None
- Housing: Animals of the same sex were housed three per Micro-Barrier cage.
- Diet (e.g. ad libitum): Certified laboratory rodent chow (Envigo 2018C Teklad Global 18% Protein Rodent Diet) was provided ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.6-23.9
- Humidity (%): 30-70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 24 January, 2019 To: 30 April, 2019

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: None
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Dosed neat
Duration of treatment / exposure:
Test substance formulations were administered once per day for two consecutive days at a dose volume based on body weights via oral gavage
Frequency of treatment:
Daily
Post exposure period:
24 hours
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
Control
Dose / conc.:
500 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
Dose / conc.:
2 000 mg/kg bw/day
No. of animals per sex per dose:
3/sex in the range-finding study. As no differences in toxicity were observed between sexes in the range-finder, males were used in the definitive assay (6 per dose)
Control animals:
yes, concurrent no treatment
Positive control(s):
Cyclophosphamide
- Justification for choice of positive control(s): Per OECD 474

Examinations

Tissues and cell types examined:
Bone marrow - polychromatic erythrocytes (PCEs)
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Based on a range-finding assay

TREATMENT AND SAMPLING TIMES: Animals were dosed daily for two days and euthanized (and bone marrow removed) 24 hours following the final dose.

DETAILS OF SLIDE PREPARATION: Femoral bone marrow was collected at approximately 24 hours after dose administration. 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 5 positive control slides) were stained with acridine orange for microscopic evaluation. The other set of slides was kept as backup and were archived. Each slide was identified by the harvest date, study number, and animal number (or slide number for positive control slides). Slides were coded using a random number table by an individual not involved with the scoring process.

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).
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.
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.

Evaluation criteria:
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 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
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 untreated 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 untreated group.
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 untreated control group.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
No toxicity observed but tested up to the limit dose of 2,000 mg/kg/day
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 1500 and 2000 mg/kg/day were tested. No toxicity observed at the limit dose.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): A statistically significant increase in the incidence of MnPCEs was observed in the test substance treated group at 1000 and 2000 mg/kg/day relative to the untreated group (ANOVA followed by Dunnett’s post-hoc analysis, p ≤0.05). However, as these values are within the 95% control limit of the historical control data, they are considered to not be of biological significance.
- Ratio of PCE/NCE (for Micronucleus assay): No appreciable reductions in the PCE/EC ratio were observed in the test substance groups compared to the untreated group, indicating the test substance did not induce cytotoxicity.

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the administration of MTDID 665 at dose levels up to and including a dose level of 2000 mg/kg was concluded to be negative in the micronucleus assay.
Executive summary:

The test substance MTDID 665 was evaluated for its clastogenic potential in the micronucleus assay in male Sprague Dawley rats. The study was conducted according to OECD 474 in compliance with OECD GLP. Test substance formulations were administered once per day for two consecutive days at a dose volume based on body weights via oral gavage. No vehicle control was used in the study; animals in Group 1 in the definitive assay were untreated. In the dose range-finding assay (DRF), the dose levels tested were 1500 and 2000 mg/kg (dose volume 0.91 and 1.21 mL/kg, respectively) in 3 animals/sex. Based upon the results, the high dose for the definitive assay was 2000 mg/kg, which is the guideline recommended limit dose. The definitive dose levels were 500, 1000, and 2000 mg/kg/ (dose volume 0.30, 0.60 and 1.21 mg/kg respectively). There were significant increases in the incidence of micronuclei in the test substance dosed animals as compared to the untreated control in the mid and high dose levels (1000 and 2000 mg/kg/day), however, as these values are within the 95% control limit of the historical control data, they are considered to not be of biological significance. Positive and untreated control values were within the expected range and there was a statistically significant increase in micronuclei in the positive control group as compared to the vehicle control. All criteria for a valid assay were met. Under the conditions of this study, the administration of MTDID 665 at dose levels up to and including a dose level of 2000 mg/kg was concluded to be negative in the micronucleus assay.