Registration Dossier

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other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
TNO Quality of Life, Zeist, The Netherlands
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): EEA-NH4
- Chemical name: acetic acid, 2,2-difluoro-2-[1,1,2,2-tetrafluoro-2-(1,1,2,2,2-pentafluoroethoxy)ethoxy]-,ammonium salt
- Other name: perflouro-3,6-dioxaoctanoic acid ammonium salt
- Physical state: white powder
- Analytical purity: 99.56%
- Lot/batch No.: 070921
- Storage condition of test material: ambient temperature

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 7 weeks (dose range finding study), 8 weeks (main study)
- Weight at study initiation: 201.5 - 264.9 g (males), 140.4 - 171.0 g (females)
- Assigned to test groups randomly: yes
- Fasting period before study: yes
- Housing: groups of five, separated by sex, in macrolon cages (type IV and III for the dose range finding and main study, respectively) with wood shavings (Lignocel, 3/4) as bedding material and strips of paper as environmental enrichment (Enviro-dri)
- Diet (e.g. ad libitum): cereal-based (closed formula) rodent diet (Rat & Mouse No. 3 Breeding Diet, RM3; pelleted) from a commercial supplier (SDS Special Diets Services, Witham, England)
- Water (e.g. ad libitum): domestic mains tap-water
- Acclimation period: 1 week (dose range finding study), 2 weeks (main study)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 40 - 70 %
- Air changes (per hr): about 10

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
sterile water for injection
Details on exposure:
Prior to each dosing, the test substance was freshly dissolved in sterile water for injection.
Duration of treatment / exposure:
2 consecutive days
Frequency of treatment:
once daily
Post exposure period:
24 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 125, 250 and 500 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Mitomycin C

Examinations

Tissues and cell types examined:
bone marrow cells of one of the femurs
Details of tissue and slide preparation:
At 24 hours following the final administration, all animals were weighed and killed. Immedialtely following sacrifice, the bone marrow cells of one of the femurs were collected into foetal calf serum and processed into glassdrawn smears according to the method described by Schmid (1976). Two bone marrow smears per animal were prepared, air-dried and fixed in methanol. One smear per animal was stained with a May-Grünwald Giemsa solution. The other unstained fixed smear was kept in reserve.
Evaluation criteria:
The study was considered valid if the positive controls showed a statistically significant increase in the mean number of MPE/2000 PE and if the negative controls were within the historical range.
The test substance was considered to cause chromosomal damage and/or damage to the mitotic apparatus, if it showed a positive response, namely: the mean number of MPE/2000 PE was statistically significantly higher compared to the negative control group.
The test substance was considered to be negative in this micronucleus test if it produced no positive response at the dose levels analysed.
The test substance or its metabolites were considered to be cytotoxic to the bone marrow via general circulation, if the test substance statistically significantly reduced the main number of PE.
Both statistical significance and biological relevance were considered together in the evaluation.
Statistics:
Data on MPE and PE were subjected to a One Way Analysis of Variance (ANOVA) with factor treatment group. Males and females were analysed separately. If the ANOVA yielded a significant effect (p<0.05), it was followed by non parametric tests (if residues were not normal distributed) or the Cochran approach (if spreading within the groups was different). These tests were applied to the negative controlgroup versus the treatment groups and the positive control group.
All statistical tests were performed using SAS V9.1 statistical software Copyright (c)2002-2003 by SAS Institute Inc., Cary, NC, USA.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
Two males and two females were dosed orally with a dose level of 500 mg/kg bw. On the next day, the animals received the same dose level for the second time. All animals showed loss of weight. Clinical signs were observed but judged as not severe. Therefore, it was decided to continue with 1000 mg/kg bw as the next higher dose level.
Two males and two females were dosed with a dose level of 1000 mg/kg bw. Due to severe clinical signs, as a result of treatment with the test substance, one male was euthanized for ethical reasons before the second administration and the remaining male and two females were found dead after the second administration. All animals showed loss of weight. Because of lethality at this dose level, it was decided to continue with 750 mg/kg bw as the next lower dose level.
After overnight fasting prior to dosing, two males and two females were dosed with a dose level of 750 mg/kg bw. On the next day, one female was found dead before the second administration. The remaining female and two males received the same dose level for the second time and survived. All animals showed loss of weight.
Based on the observed clinical signs and loss of body weights at the dose level of 500 mg/kg bw and the severe clinical signs and lethality at the two higher dose levels of 750 and 1000 mg/kg bw/day, it was decided to perform the main micronucleus test with dose levels of 500, 250 and 125 mg/kg bw/day.

RESULTS OF DEFINITIVE STUDY
Due to severe clinical signs, as a result of treatment with the highest dose level of 500 mg/kg bw/day, two females were euthanised for ethic reasons and one female was found dead before scheduled sacrifice. One female was replaced by a reserve female. Therefore, three instead of five females were available for micronuclei analysis in the high dose group. The clinical signs, observed during the performance of the main micronucleus test, were bilateral blepharospasm, hunched back, piloerection, pale, cold, slit-eyes and sluggishness. Although not the required 10 animals (5/sex) were available for analysis, the results obtained in the bone marrow of the remaining animals (5 males and 3 females) was enough for a reliable assessment.

For both male and female rats in the low dose group 2 (125 mg/kg bw/day), no loss of weight was observed. In the mid dose group 3 (250 mg/kg bw/day), four males and three females showed loss of weight. In the high dose group 4 (500 mg/kg bw/day), all males and two females showed loss of weight.

Positive control group
Statistical analysis of the test results indicated there was a statistically significant increase (p value 0.0082) in the mean number of MPE in the positive control group, when compared to the negative (vehicle) control group. This increase was within the range of means of the historical data. This indicates that the positive control substance Mitomycin C reached the bone marrow and induced damage to the chromosomes and/or to the spindle apparatus of the bone marrow cells of male rats. These results, together with the normal MPE/PE ratio in the negative control group, demonstrate the validity of the test system.

There were no statistically significant differences in the mean number of PE between the males of the positive control group and the males of the negative (vehicle) control group.

Treatment groups
Statistical analysis of the test results indicated there were no statistically significant increases in the group mean numbers of MPE/2000 PE, at any of the dose levels used, when compared to the negative (vehicle) control group. This indicates that treatment with the substance, up to 500 mg/kg bw/day, did not result in damage to the chromosomes and/or to the spindle apparatus of the bone marrow cells of male and female rats.
The mean numbers of MPE/2000 PE in the negative control group (water for injection) were within the historical range of means.

Statistical analysis of the test results indicated there were no statistically significant differences in the group mean numbers of PE/200 PE, at any of the dose levels used, when compared to the negative (vehicle) control group. This indicates that treatment with the substance, up to 500 mg/kg bw/day, did not result in cytotoxicity to the bone marrow of male and female rats.

Applicant's summary and conclusion