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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
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Remarks:
The study was conducted according to the guideline in effect at the time of study conduct.
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
Remarks:
The study was conducted according to the guideline in effect at the time of study conduct.
Qualifier:
according to guideline
Guideline:
other: EC Commission Directive 2000/32/EC Annex 4A-B10 No. L 136 (2000)
Deviations:
no
Remarks:
The study was conducted according to the guideline in effect at the time of study conduct
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl methacrylate
EC Number:
218-407-9
EC Name:
3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl methacrylate
Cas Number:
2144-53-8
Molecular formula:
C12H9F13O2
IUPAC Name:
3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl 2-methylprop-2-enoate
Details on test material:
- Purity: 97.59%

Test animals

Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 6 to 8 weeks
- Weight at study initiation: Pilot toxicity study: 28.8 – 32.6 g (males) and 25.1 – 26.9 g (females); Toxicity study: 28.3 – 33.2 g (males) and 21.1 – 30.0 (females); Definitive study: 29.2 – 36.0 g (males) and 23.0 – 27.4 g (females)
- Fasting period before study: No
- Housing: Mice of the same sex were housed up to five per rodent Micro-Barrier cage. Cages were placed on racks equipped with Micro-VENT full ventilation, HEPA filtered system.
- Diet (e.g. ad libitum): ad libitum (Harlan 2018C Certified Global Rodent Diet)
- Water (e.g. ad libitum): ad libitum
- Acclimation period: no less than 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72 ± 3°F (22.2 ± 3.6°C)
- Humidity (%): 50 ± 20%
- Air changes (per hr): at least 10 changes of fresh HEPA-filtered air every hour
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: Based on the workability of the test substance in the vehicle at a concentration of 200 mg/mL.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance dose formulations were prepared fresh for each phase of the study just prior to dose administration. The test substance formulations at 200 mg/mL (pilot toxicity study), 100 and 50 mg/mL (toxicity study) and 35, 17.5 and 8.75 mg/mL (definitive study) were prepared as follows: 1. Appropriate amount of the test substance was weighed into a container of appropriate size. 2. The vehicle (corn oil) was added to the test substance until the final volume was achieved, and the formulation was vortexed and stirred for several minutes. Formulations at 50 mg/mL and above appeared as yellow suspensions and formulations of 35 mg/mL and below appeared as yellow solutions. The dose volume was for all formulations 10 mL/kg.
Duration of treatment / exposure:
Single treatment
Frequency of treatment:
Once
Post exposure period:
48 hours
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
2000 mg/kg
Basis:
actual ingested
Pilot toxicity study
Remarks:
Doses / Concentrations:
500, 1000 mg/kg
Basis:
actual ingested
Toxicity study
Remarks:
Doses / Concentrations:
87.5, 175, 350 mg/kg
Basis:
actual ingested
Micronucleus assay
No. of animals per sex per dose:
Pilot toxicity study: 3 males and 3 females (2000 mg/kg)
Toxicity study: 3 males and 3 females (500 and 1000 mg/kg)
Micronucleus assay: 10 males and 10 females (vehicle control); 10 males and 10 females (87.5, and 175 mg/kg); 15 males and 15 females (350 mg/kg, includes 5 replacement mice/sex); 5 males and 5 females (positive control)
Control animals:
yes, concurrent vehicle
Positive control(s):
- Positive Control: Cyclophosphamide monohydrate (CP)
- Justification for choice of positive control(s): Not reported
- Route of administration: Oral gavage
- Doses / concentrations: One / 50 mg/kg

Examinations

Tissues and cell types examined:
Polychromatic erythrocytes (PCEs) and Normochromatic erythrocytes (NCEs) from bone marrow taken from one femur were used for the micronucleus assay. Bone marrow from the other femur was taken for the chromosome aberration assay.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Dose selection was based on the results of the pilot toxicity study and toxicity study.

TREATMENT AND SAMPLING TIMES: All dose formulations were administered by a single oral gavage at a dose volume of 10 mL/kg. Five mice per sex from the vehicle control, low dose, mid dose, high dose, and positive control groups were sacrificed 24 hours post-dosing. Five mice per sex from the vehicle control and high dose group were sacrificed 48 hours post-dosing.

DETAILS OF SLIDE PREPARATION: Immediately following sacrifice, marrow from one femur of each animal was aspirated and flushed into fetal bovine serum. The bone marrow cells were collected by centrifugation at approximately 100 x g for 5 minutes and the supernatant removed. The cells were resuspended and a small drop of bone marrow suspension spread onto a glass slide. Two slides were prepared from each mouse. The slides were fixed in methanol, stained with May-Gruenwald-Giemsa and permanently mounted.

METHOD OF ANALYSIS: Representative slides from each animal were examined in a blind manner using a light microscope and a medium magnification (400X). Only cells showing good morphology and staining were selected for scoring. Polychromatic erythrocytes (PCEs) were identified by their characteristic bluish staining; Normochromatic erythrocytes (NCEs) appeared pink (reddish). Two thousand PCEs per animal were scored for the presence of micronuclei resulting in evaluation of a total of 10000 PCEs per each treatment group. The number of NCEs and micronucleated NCEs (MNCEs) in the field of 1000 total erythrocytes (ECs) was determined for each animal in order to determine the proportion of polychromatic erythrocytes to total erythrocytes (PCEs/ECs). Micronuclei are round, darkly-staining nuclear (chromosome) fragments with a sharp contour and diameters usually from 1/20 to 1/5 of an erythrocyte. Micronuclei may occur in PCEs (MPCEs) or NCEs (MNCEs). The proportion of PCEs among one thousand erythrocytes was determined for each animal.
Evaluation criteria:
The test substance was considered to induce a positive response in the micronucleus assay if a dose-responsive increase in the incidence of micronucleated polychromatic erythrocytes was observed and one or more doses were statistically elevated relative to the vehicle control (p ≤ 0.05) at any sampling time.

Values that were statistically significant but did not exceed the range of historical vehicle controls were judged as not biologically significant/relevant.

The test substance was judged negative if no statistically significant increase in the incidence of micronucleated polychromatic erythrocytes above the concurrent vehicle control values and no evidence of dose response were observed at any sampling time.
Statistics:
The incidence of micronucleated polychromatic erythrocytes per 2000 PCEs for each mouse and per 10000 PCEs for each treatment group was determined. Statistical significance was determined using the Kastenbaum-Bowman tables which are based on the binomial distribution. All analyses were performed separately for each sex and sampling time. In order to quantify the proliferation state of the bone marrow as an indicator of bone marrow toxicity, the proportion of polychromatic erythrocytes to total erythrocytes was determined for each mouse and treatment group (PCEs/ECs ratio). The proportion of polychromatic erythrocytes to total erythrocytes in test substance-treated animals should not be less than 20% of the control value.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
clinical signs: piloerection and decreases of up to 24% in ratio of polychromatic erythrocytes to erythrocytes in test substance-treated groups relative to controls
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF PILOT TOXICITY STUDY
One of the male mice and all three female mice were found dead by Study Day 3 post-dose. The surviving male mice had piloerection (ruffled hair coat) and appeared lethargic.

RESULTS OF TOXICITY STUDY
All mice at 1000 mg/kg were lethargic with piloerection (ruffled hair coat). Diarrhea was observed in 2/3 male mice and convulsions were seen in one female mouse. All male mice and female mice were found dead on Study Day 2 and Study Day 3, respectively. At a dose of 500 mg/kg, piloerection (ruffled hair coat) was seen in 2/3 males and 2/3 females. One male mouse was convulsing on Study Day 2 and one female mouse was convulsing on Study Days 2 and 3. During the course of the study, 2/3 males and 2/3 females at 500 mg/kg were found dead. Loss in body weights was observed in all surviving animals on Study Days 1 and 3. Based on these results, the high dose for the micronucleus study was set at 350 mg/kg, which was estimated to be the maximum tolerated dose (MTD).

RESULTS OF MICRONUCLEUS STUDY
Reductions in the PCEs/ECs ratio of up to 24% were seen in the test substance treated groups relative to the respective vehicle control groups suggesting bioavailability and cytotoxicity. No statistically significant or biologically relevant increase in the incidence of MPCEs in the test substance treated groups relative to the respective vehicle control groups in male or female mice at 24 or 48 hours after dose administration (p > 0.05, Kastenbaum-Bowman Tables).
CP, the positive control, induced a statistically significant increase in the incidence of MPCEs (p ≤ 0.05, Kastenbaum-Bowman Tables) in both male and female mice. The number of MPCEs in the vehicle control groups did not exceed the historical vehicle control range.

No mortality was observed in test substance treated or control treated groups. All animals dosed with the controls, the test substance at 87.5 and 175 mg/kg, and 13/15 female mice at 350 mg/kg appeared normal. All male mice and 2/15 female mice at 350 mg/kg had piloerection (ruffled hair coat) after dose administration and during the course of the study.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the conditions of this assay, the test substance did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow of male and female mice.
This study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
Executive summary:

An oral micronucleus study was conducted in mice to determine whether the test substance induces an increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow. In this study, groups of male and female ICR mice were administered a single oral administration of the test substance at doses up to and including 350 mg/kg (the maximum tolerated dose). Bone marrow smears were prepared approximately 24 and 48 hours after treatment and 2000 polychromatic erythrocytes per animal were evaluated for the presence of micronuclei. The test substance did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes. Under the conditions of this study, the test substance is negative.