Reaction mass of (3E)-1,1,1,2,2,3,4,5,6,6,7,7,7-tridecafluoro-5-methoxyhept-3-ene and (2E)-1,1,1,2,3,4,5,5,6,6,7,7,7-tridecafluoro-4-methoxyhept-2-ene and (3E)-1,1,1,2,2,4,5,5,6,6,7,7,7-tridecafluoro-3-methoxyhept-3-ene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In Vitro (Mutagenic effects - bacterial): OECD 471; Bacterial reverse mutation assay. Negative. Reliability = 1

In Vitro (Clastogenic effects - mammalian): OECD 473; Chromosome aberrations in Chinese hamster ovary (CHO) cells. Negative. Reliability = 1

In vitro ( gene mutation study in mammalian cells): OECD 476;Mammalian Cell Forward Gene Mutation (CHO/HRPT) assay with dupplicate cultures. Negative. Reliability =1.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In vivo (Chromosome aberration – mammalian): OECD 474; Rat Micronucleus assay. Negative. Reliability = 1

Link to relevant study records

Reference

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: see 'Remark'

Remarks:

This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labeling and/or risk assessment. Although data provided have a report year after 2008, the study was performed to fulfill needs required by government regulators and/or for product stewardship purposes. DuPont’s stewardship principle states that “We will adhere to the highest standards for the safe operation of facilities and the protection of our environment, our employees, our customers and the people of the communities in which we do business”. The study was carried out in accordance with our internal Product Stewardship standard which is part of the American Chemical Council’s “Responsible Care Program”. This study was not performed to fulfill an information requirement under REACH, but since the test data were already available they were provided as part of the REACH submission.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Remarks:

The study was conducted according to guideline in effect at time of study conduct.

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Approximately 8 weeks
- Weight at study initiation: males 237.7-276.5 g; females 172.0-211.8 g
- Assigned to test groups randomly: Yes
- Fasting period before study: No
- Housing: Solid bottom cages with enrichment-containing bedding
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26°C
- Humidity (%):30-70%
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark

Route of administration:

oral: gavage

Vehicle:

None

Details on exposure:

Due to insolubility of the test substance in a solvent suitable for oral gavage, the test substance was dosed neat.

Duration of treatment / exposure:

Single dose

Frequency of treatment:

Single dose

Post exposure period:

2 days

Remarks:

Doses / Concentrations:
0, 500, 1000, 2000 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

5/sex/dose at 0, 500, and 1000 mg/kg
7/sex/dose at 2000 mg/kg

Control animals:

yes

Positive control(s):

Cyclophosphamide (CAS 6055-19-2)
- Justification for choice of positive control(s): not reported
- Route of administration: gavage
- Doses / concentrations: 10 mg/kg

Tissues and cell types examined:

Micronucleated reticulocytes in peripheral blood

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Dosing concentrations used were determined by the rangefinding results from an experiment using a group of 3 male animals. The highest dose used for the rangefinder for the in vivo micronucleus study was the limit dose of 2000 mg/kg bod weight administered by oral gavage. The animals were observed for clinical sign of toxicity and mortality immediately after dosing and daily thereafter for 2 days (until approximately 48 hours post-dosing). Additional rangefinding groups of 3 male animal were added if clinical signs of toxicity or mortality were noted at 2000 mg/kg. There were 3 test substance concentrations for the main study: a high dose, ½, and ¼ of the high-dose concentration.

TREATMENT AND SAMPLING TIMES: Based on the results from the rangefinder, each group of male and female animals were administered a single dose of the test substance at 500, 1000, or 2000 mg/kg body weight, or the negative or positive control substances for the main study. Due to insolubility of the test substance in a solvent suitable for oral gavage, the test substance-treated animals were dosed with the neat test substance. The volume of test substance administered was adjusted for each animal according to their most recent body weight and density of the compound. CP, the positive control, was administered at a concentration of 10 mg/kg body weight. The test substance, negative, and positive controls were administered by single oral gavage. The negative and positive controls were dosed at a volume of 10 mL/kg body weight. The negative and positive control groups were administered a single dose by oral gavage the same day as groups receiving test substance treatment. Additional male and female animals were treated with the highest concentration to allow for unexpected mortality. Approximately 24 and 48 hours after dose administration, peripheral blood samples were collected for micronucleus evaluation. All samples were disposed of after study completion.

DETAILS OF SLIDE PREPARATION: Peripheral blood samples were collected from all animals on study. The micronucleus evaluation was conducted by flow cytometry using the In Vivo MIcroFlow® Plus Rat Micronucleus assay kit. Approximately 3 drops (i.e., 60-120 μL) of blood were collected from each animal directly into a labeled microcentrifuge tube containing 350 μL anticoagulant/diluent (anticoagulant) found in the MicroFlow Plus kit. The tubes were capped and inverted several times to mix the blood with the anticoagulant. The blood/anticoagulant mixture may be stored at room temperature for up to 6 hours or refrigerated for up to 24 hours before fixing. The blood samples were fixed in duplicate (approximately 180 μL of blood/anticoagulant mixture each) in 2 mL ultra-cold reagent-grade methanol and stored below -75ºC until processed.

METHOD OF ANALYSIS: Evaluations were conducted on 5 animals/sex/group. At the highest concentration, 5 males and 5 females were selected for evaluation based on survival until the scheduled sacrifice and then cage order. Whenever feasible, at least 20000 RETs were analysed per blood sample for induction of micronuclei, and toxicity as indicated by the frequency of immature erythrocytes (%RETs) among the total (RETs plus NCEs). The samples were analysed and evaluated using the In Vivo MicroFlow Plus® Rat Micronucleus assay kit. The frequency of micronucleated reticulocytes (%MN-RETs) was used as a measure of induction of aneugenic or clastogenic alterations by the test substance. All groups from the 24-hour time point were analysed. For the 48-hour time point, groups 1, 4, and 5 were analysed. Groups 2 and 3 were not analysed since no positive response was observed for group 4.

Evaluation criteria:

The individual animal was considered the experimental unit. Data were evaluated using scientific judgment taking into account both statistical and biological significance. Results not meeting the indicated criteria for positive or negative findings were evaluated on a case-by-case basis. Further investigation of an equivocal result may be required to obtain a conclusive finding.

The test substance was judged negative if the following conditions were met: (1) No statistically significant dose-related increases in the group mean %MN-RETs above the concurrent negative control value occurred at any concentration of the test substance. (2) The %MN-RET values of the test substance-treated animals were within reasonable limits of the recent (past 2 years) laboratory historical control range.

The test substance was judged positive if the following conditions were met: (1) The group mean %MN-RETs showed a statistically significantly increase at one or more concentrations of the test substance compared to the concurrent negative control values. (2) An accompanying statistically significant dose-response increase in %MN-RETs was observed.

Statistics:

Data for the proportion of micronucleated RETs among 20000 immature erythrocytes and the proportion of RETs among total erythrocytes (MN-RET and RET frequency, respectively) were transformed prior to analysis using an arcsine square root or Freeman-Tukey function. This transformation was appropriate for proportions since the distribution of the transformed data more closely approximates a normal distribution than does the nontransformed proportion. Transformed data for RET and MN-RET frequencies were analysed separately for normality of distribution and equal variance using the Shapiro-Wilk and Levene’s tests, respectively. Data from the positive control group was not included in evaluating normality or variance homogeneity of distribution.

For those data that were normally distributed and had equal variance, parametric statistics (e.g., analysis of variance (ANOVA) and Dunnett’s test) were performed using the transformed data. For those data that were normally distributed but had unequal variance, a robust ANOVA and unequal-variance Dunnett test was done. For those data that were not normally distributed, nonparametric statistics (e.g., Kruskal-Wallis and Dunn’s tests) utilizing non-transformed data were performed. The individual animal was considered the experimental unit. All data analyses were one-tailed and conducted at a significance level of 5%.

No statistical analysis was conducted on body weights or clinical signs.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No statistically significant increase in the frequency of MN-RETs compared to the negative control group were observed in any test substance group at either time point. There were no statistically significant test substance-related decreases in %RET. The negative and positive control groups exhibited a response consistent wit the %MN-RETs historical control data.

As expected, statistically significant increases in %MN-RETs were found in the CP-treated animals of both sexes at both time points. A statistically significant depression in %RETs was observed in both male and female animals administered CP at the 48 hour time point.

Conclusions:

Interpretation of results (migrated information): negative
The test substance was negative in the in vivo rat micronucleus assay.

This study and the conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability).

Executive summary:

The test substance was evaluated for its ability to induce micronucleated reticulocytes (MN-RETs) in peripheral blood cells in male and female Crl:CD(SD) rats following oral intubation (gavage). Based on rangefinding results, doses of 0, 500, 1000, or 2000 mg/kg of the test substance were selected for the main study. Due to insolubility of the test substance in solvent suitable for oral gavage, the test substance-treated animals were dosed with the neat test substance. The volume of test substance to be administered was adjusted for each animal according to their most recent body weight and density of the compound. Concurrent control groups were administered deionized water (negative control), or 10 mg/kg of cyclophosphamide (positive control), at a volume of 10 mL/kg. All animals were given a single dose by oral gavage. The positive and negative control groups, as well as the low- and intermediate-dose groups contained 5 animals/sex. The high-dose group contained 7 animals/sex to allow for unexpected mortality. A total of 20000 reticulocytes (RETs) were analysed per animal for induction of micronuclei and toxicity as indicated by the frequency of immature erythrocytes (% reticulocytes, RETs) among the total (RETs plus normochromatic erythrocytes, NCEs). The frequency of micronucleated reticulocytes (%MN-RETs) was used as a measure of induction of aneugenic or clastogenic alterations by the test substance.

No clinical signs of toxicity or mortality were observed in the rangefinding experiment at 2000 mg/kg body weight. In the main study, no clinical signs of toxicity or mortality were observed at any dose level tested in male and female rats exposed to the test substance. No abnormalities were detected in the negative or positive control groups. There were no significant changes in body weight or body weight gain in either male or female animals administered the test substance. No statistically significant increases in the frequency of MN-RETs compared to the negative control group were observed in any test substance group at either time point. There were no statistically significant test substance-related decreases in %RET. The negative and positive control groups exhibited a response consistent with the %MN-RETs historical control data. All criteria for a valid study were met. Under the conditions of this study, this test substance did not induce biologically relevant increases in %MN-RETs in male or female rat peripheral blood cells. The test substance was concluded to be negative in this in vivo study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity:

Under in vitro conditions, the substance was not found to be mutagenic in bacterial cells, not clastogenic in a Chinese Hamster Ovary cell lineand was negative for the induction of forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system. The substance was negative in an in vivo rat micronucleus assay.

Based on the results of this battery of in vitro and in vivo studies, it is concluded that the substance is not genotoxic in cell culture or laboratory animals.

Justification for selection of genetic toxicity endpoint
Multiple OECD guideline, GLP studies have been identified as key for this endpoint. In addition the study selected above, an Ames assay and in vitro chromosome aberration assay are pertinent to the hazard conclusion for this endpoint.

Justification for classification or non-classification

The test substance did not produce mutagenicity or clastogenicity when evaluated in bacterial or mammalian cell cultures in vitro or in animals in vivo. The substance does not need to be classified for mutagenicity according the EU Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.