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
Study period:
from 29 November 2007 to 14 January 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study in compliance with international guidelines

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
Perfluoroalkyl acrylate (C6-SFA)
CAS No. 17527-29-6
Lot No. 6X002
Storage: Room Temperature
Purity: 99.7%
Expiration date: 30 Oct 2008

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male
Details on test animals and environmental conditions:
Micronucleus Assay Young adult male CD-1 (ICR) BR mice were received on 13 December 2007 from Harlan, Frederick, Maryland. This is an outbred strain that maximizes genetic heterogeneity and therefore tends to eliminate strain-specific response to test articles. The mouse has been routinely utilized as an animal model of choice for the mammalian bone marrow erythrocyte micronucleus assay.
Identification and Acclimation:
Animals were randomized into groups according to Covance Standard Operating Procedures. Following randomization, each study animal was uniquely identified by ear tag. Animals were acclimated to laboratory conditions for at least 5 days, and released for study use by a staff examiner. Animals were considered acceptable for study use based upon data collected during acclimation.
Husbandry:
Housing The animals were housed in sanitary polycarbonate cages containing Sani-Chips ® Hardwood Chip Laboratory bedding. The animals were housed up to five animals per cage during acclimation, and by full dose group/harvest timepoint after randomization. Each batch of wood chips was analyzed by the manufacturer for specific microorganisms and contaminants.
Environmental Conditions:
Environmental controls were set to maintain the following animal room conditions: temperature range of 64 to 79 F, relative humidity range of 30 to 70%, 10 or greater air changes/hour, and a 12-hour light/12-hour dark cycle. The light/dark cycle was interrupted for non-study related activities. Actual temperature and humidity readings were monitored continuously and averaged twice daily. Any variations to these conditions are maintained in the raw data and had no effect on the outcome of the study.
Diet, Water, and Contaminants:
PMI Certified Rodent Diet was available ad libitum. The manufacturer analyzed the diet for nutritional components and environmental contaminants. Tap water was available ad libitum. Water samples are routinely analyzed for specified microorganisms and environmental contaminants. Results of the diet and water analyses are reviewed for acceptability and are on file at Covance-Vienna. No contaminants were known to be present in the diet or water at levels that might interfere with this study.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
The vehicle control article was olive oil with 0.5% Tween 80.
Olive Oil CAS No. 8001-25-0
Tween 80 CAS No. 9005-65-6
Details on exposure:
In the dose range-finding study, Perfluoroalkyl acrylate was formulated in olive oil (see Protocol Deviation) and administered once by oral gavage to 3 males and 3 females per dose level. The animals were dosed at 500, 1000 and 2000 mg/kg and observed for up to 2 days after dosing for toxic signs and/or mortality. In a follow-up dose range-finding study, Perfluoroalkyl acrylate was formulated in olive oil with 0.5% Tween 80 and administered once by oral gavage to 2 or 3 males and 3 females per dose level (see Protocol Deviation). The animals were dosed at 250, 500, and 1000 mg/kg and observed for up to 2 days after dosing for toxic signs and/or mortality. Based on the results of the dose range-finding study, the maximum tolerated dose was estimated to be 400 mg/kg. In the micronucleus assay, the test article was formulated in olive oil with 0.5% Tween 80.

Duration of treatment / exposure:
Single exposure.
Frequency of treatment:
Single exposure.
Post exposure period:
All animals were examined immediately after dosing, approximately 1 hour after dosing, and at least daily for the duration of this assay for signs of clinical toxicity and/or mortality.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
100
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
200
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
400
Basis:
nominal conc.
No. of animals per sex per dose:
5 male
Control animals:
no
Positive control(s):
Positive control: cyclophosphamide

Examinations

Details of tissue and slide preparation:
Micronucleus Assay
Extraction of Bone Marrow :
The hind limb bones (tibias) were removed for marrow extraction from five surviving animals in the positive control, low and mid dose groups, and from ten surviving animals in the control and high dose groups. For each animal, the marrow flushed from the bones was combined in an individual centrifuge tube containing 3 to 5 mL fetal bovine serum (one tube per animal).
Preparation of Slides:
Following centrifugation to pellet the marrow, the supernatant was removed by aspiration and portions of the pellet were spread on slides and air-dried. The slides were fixed in methanol, stained in May-Grünwald solution and Giemsa, and protected by mounting with coverslips. For control of bias, all slides were coded prior to analysis.
Evaluation criteria:
The criteria for a positive response is the detection of a statistically significant increase in micronucleated PCEs for at least one dose level, and a statistically significant dose- related response. A test article that does not induce both of these responses is considered negative. Statistical significance is not the only determinant of a positive response; the Study Director also considers the biological relevance of the results in the final evaluation.
Statistics:
The following statistical methods were used to analyze the micronucleus data.
Assay data analysis was performed using an analysis of variance (Winer, 1971) on untransformed proportions of cells with micronuclei per animal and on untransformed PCE:NCE ratios when the variances were homogeneous. Ranked proportions were used for heterogeneous variances.
If the analysis of variance was statistically significant (p 0.05), Dunnett's t-test (Dunnett, 1955; 1964) was used to determine which dose groups, if any, were statistically significantly different from the vehicle control. Analyses were performed separately for each sampling time.
The 100, 200, and 400 mg/kg dose groups, as well as the positive control group, were compared with the vehicle control group at the 5% probability level. Statistical significance is designated throughout the text of this report by the term significant.
Statistical analysis programs are referenced accordingly in the appropriate section of this report.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
The test article, Perfluoroalkyl acrylate, induced signs of clinical toxicity in the treated animals at 400 mg/kg, which included hypoactivity, squinted eyes, rough haircoat, hunched posture, wet haircoat-perineal, and/or body tremors.
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
The test article, Perfluoroalkyl acrylate, induced signs of clinical toxicity in the treated animals at 400 mg/kg, which included hypoactivity, squinted eyes, rough haircoat, hunched posture, wet haircoat-perineal, and/or body tremors. Perfluoroalkyl acrylate did not induce statistically significant increases in micronucleated PCEs at any test article dose examined (100, 200, and 400 mg/kg). In addition, Perfluoroalkyl acrylate was not cytotoxic to the bone marrow (i.e., no statistically significant decrease in the PCE:NCE ratios) at any dose of the test article. A statistically significant lower level of micronucleated PCEs and higher PCE:NCE ratio were found at 400 mg/kg without biological significance. The vehicle control group had approximately 0.04% micronucleated PCEs and the group mean was within the historical control range. The positive control, cyclophosphamide, induced a statistically significant increase in micronucleated PCEs as compared to that of the vehicle control, with a mean and standard deviation of 2.22 0.73%.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The test article, Perfluoroalkyl acrylate, was evaluated as negative in the mouse bone marrow micronucleus assay under the conditions of this assay.
Executive summary:

The objective of this study was to evaluate the test article, Perfluoroalkyl acrylate, for in vivo clastogenic activity and/or disruption of the mitotic apparatus by detecting micronuclei in polychromatic erythrocytes (PCE) in CD-1 (ICR) BR mouse bone marrow. Bone marrow was extracted and at least 2000 PCEs per animal were analyzed for the frequency of micronuclei. Cytotoxicity was assessed by scoring the number of PCEs and normochromatic erythrocytes (NCEs) in at least the first 500 total erythrocytes for each animal. The test article, Perfluoroalkyl acrylate, induced signs of clinical toxicity in the treated animals at 400 mg/kg, which included hypoactivity, squinted eyes, rough haircoat, hunched posture, wet haircoat-perineal, and/or body tremors. Perfluoroalkyl acrylate did not induce statistically significant increases in micronucleated PCEs at any test article dose examined (100, 200, and 400 mg/kg). In addition,

Perfluoroalkyl acrylate was not cytotoxic to the bone marrow (i.e., no statistically significant decrease in the PCE:NCE ratios) at any dose of the test article suggesting that the substance does not reach bone marrow.

A statistically significant lower level of micronucleated PCEs and higher PCE:NCE ratio were found at 400 mg/kg without biological significance. The test article, Perfluoroalkyl acrylate, was evaluated as negative in the mouse bone marrow micronucleus assay under the conditions of this assay.