2-Butene, 1,1,1,4,4,4-hexafluoro-, (2Z)-

Genetic toxicity: in vivo

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: 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 labelling and/or risk assessment. Although data provided have a report year after 2009, the study was performed to fulfill needs required by other governmental registrations and/or product stewardship purposes. This study was not performed to specifically fulfill an information requirement under REACH, but since the test data were already available they were provided as part of the REACH submission.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

The study was conducted in conjunction with a 90-day repeated dose study. Blood samples taken for analysis on Day 3, 28 and 92.

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day)

Deviations:

no

Remarks:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EU Method B.29 (Sub-Chronic Inhalation Toxicity Test:90-Day Repeated Inhalation Dose Study Using Rodent Species)

Deviations:

no

Remarks:

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

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.5395 (Mammalian Erythrocyte Micronucleus Test)

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

Test material information

Constituent 1

Reference substance name:

(2Z)-1,1,1,4,4,4-hexafluorobut-2-ene

EC Number:

700-651-7

Cas Number:

692-49-9

Molecular formula:

C4H2F6

IUPAC Name:

(2Z)-1,1,1,4,4,4-hexafluorobut-2-ene

Details on test material:

- Purity: >99.9%

Test animals

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: 6 weeks
- Weight at study initiation: Mean group body weight range: 149.1 - 152.1 g (males); 143.2 - 144.9 g (females)
- Housing: group housed in solid bottom caging (2-3 rats per cage) with bedding and enrichment. Each cage rack contained only animals of one sex.
- Diet (e.g. ad libitum): ad libitum, except during exposures
- Water (e.g. ad libitum): ad libitum, except during exposures
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26ºC
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

Administration / exposure

Route of administration:

inhalation: vapour

Vehicle:

clean air

Details on exposure:

TYPE OF INHALATION EXPOSURE: whole body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: All exposure chambers were constructed of stainless steel and glass (NYU style) with a nominal internal volume of 350 L. A tangential feed inside the chambers promoted uniform chamber distribution of the test atmosphere. The chamber volume was chosen so that the total body volume of the test animals did not exceed 5% of the chamber volume.
- Method of holding animals in test chamber: animals were placed in a wire-mesh module and exposed, whole-body
- Source and rate of air: Houseline high-pressure air, metered to the flask by a mass flow controller, carried the resulting atmosphere into a filtered dilutional air stream leading to the exposure chamber. The dilution air supply was metered by a mass flow controller.
- System of generating particulates/aerosols: Chamber atmospheres were generated by flash evaporation of the test substance in air with a heated, round-bottom, flash evaporation flask. The test substance was metered into the flask with a pump equipped with pistons. The flask was heated to approximately 175°C to vaporize the test substance. Houseline high-pressure air, metered to the flask by a mass flow controller, carried the resulting atmosphere into a filtered dilutional air stream leading to the exposure chamber. The dilution air supply was metered by a mass flow controller. General and dilutional air flows and the heating mantles were all monitored by the Camile Inhalation Toxicology Automated Data System (CITADS). Chamber concentrations of test substance were controlled by varying either the test substance feed rate to the flask or the amount of generational or dilutional air supply.
- Temperature, humidity, flow in air chamber: 20-24°C, 22–50% humidity, 60 L/min which provided 10 air changes per hour
- Treatment of exhaust air: exhausted into a fume hood

TEST ATMOSPHERE
- Brief description of analytical method used: During each exposure the atmospheric vapour concentration of the test substance was determined by gas chromatography at approximately 30 minute intervals. The air-control chamber was sampled once a week. Samples of the chamber atmosphere were continually drawn from the exposure chamber and directly injected into a gas chromatograph (GC) equipped with a gas sample valve and a flame-ionization detector. Samples were chromatographed isothermally at 80°C on a fused silica glass column.

Duration of treatment / exposure:

90 days

Frequency of treatment:

6 hours/day; 5 days/week

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 3000, 4000, 5000 and 7500 ppm
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 3000 ± 8.4, 4000 ± 12 , 5000 ± 10 and 7500 ± 8.3 ppm
Basis:
analytical conc.

No. of animals per sex per dose:

5 animals/sex/concentration were designated for micronucleus testing

Control animals:

yes, concurrent no treatment

Examinations

Tissues and cell types examined:

At least 20000 reticulocytes were analysed, when available, per blood sample for induction of micronuclei, and toxicity as indicated by the frequency of immature erythrocytes (%RETs) among the total (RETs plus normochromic erythrocytes [NCEs]).

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: A previous subchronic inhalation toxicity study with the test substance reported a NOAEL of 1500 ppm where the next highest exposure concentration was 10000 ppm. Since there was a large gap between the NOAEL and the next highest concentration in the previous study, the objective of this study was to assess the potential subchronic toxicity of the test substance in rats to provide a more precise no-observed-adverse-effect-level (NOAEL) as well as evaluate the ability of the test substance to cause DNA damage as assessed with the micronucleus assay. The micronucleus assay evaluated the ability of the test substance to induce an increase in the frequency of micronucleated polychromatic erythrocytes (MNPCEs) in rat peripheral blood cells following inhalation exposure. An increase in this frequency relative to negative controls indicates that a test substance induced chromosome and/or spindle damage in erythroblasts.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Each group of animals was exposed for 6 hours/day over an approximate 90-day period (weekends and holidays excluded). The micronucleus evaluation was conducted by flow cytometry, following published guidance. Peripheral blood samples were collected by tail vein venipuncture or orbital sinus (test day 92) from 5 animals per sex per test substance-exposed group. There were 3 blood collections, a bleed after the fourth exposure (test day 3), after approximately 4 weeks of exposure (test day 28), and at the time of sacrifice with the clinical pathology blood sample collection (test day 92).

DETAILS OF SLIDE PREPARATION: Approximately 2 to 3 drops (i.e., 60-120 μL) of blood were collected from the tail vein of each animal directly into a labelled microcentrifuge tube containing 350 μL Solution B found in the In Vitro MicroFlow Plus® Rat Micronucleus assay kit. The tubes were capped and inverted several times to mix the blood with Solution B. The blood/Solution B mixture was stored for up to 7 hours at room temperature prior to fixing. The blood samples (approximately 180 μL of blood/Solution B mixture) were fixed in 2 mL ultra-cold reagent grade methanol and stored below -75ºC until processed. All subsequent steps to prepare the samples for flow cytometric analysis were documented in the study records.

METHOD OF ANALYSIS: At least 20000 reticulocytes were analysed, when available, per blood sample for induction of micronuclei, and toxicity as indicated by the frequency of immature erythrocytes (%RETs) among the total (RETs plus normochromic erythrocytes [NCEs]). The samples were analysed and evaluated using the In Vitro 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. The micronucleus assay was performed on all samples collected from animals from the test days 3 and 28; the assay was performed on only the control group and rats exposed to 7500 ppm of the test substance from test day 92. The positive control biological standard, provided in the assay kit, was used as a positive methods control.

Evaluation criteria:

Micronucleus data were evaluated using scientific judgment taking into account both statistical and biological significance.

Statistics:

For each treatment group, the mean and standard deviation for %RETs and %MN-RETs were calculated. 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 were 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%.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

There were test substance-related reductions in body weights and food consumption observed in male rats exposed to 7500 ppm (50322 mg/m3)

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Additional information on results:

See study DI.K1.90Day.InhVap.R.D-17453-785-1.KD in Section 7.5.2 for additional details on toxicity results

No statistically significant increases in MN-RET frequency were observed in any evaluated test substance treated group of male or female animals exposed to any concentration at any time point. A statistically significant decrease in the %RET was observed in male animals exposed to 3000, 4000, and 7500 ppm at the test day 3 time point. These changes were considered test substance-related. However, there were no changes in red cell mass parameters (red blood cell count, haemoglobin or haematocrit at the test day 92 time point) and the absolute reticulocyte count at test day 92 was increased in male rats exposed to 5000 or 7500 ppm. Therefore, the decrease in %RET at test day 3 was considered test substance-related but non-adverse. No other test substance-related statistically significant decreases in the %RET could be observed in males at 5000 ppm, or in females at any time point. The decrease in %RE observed at the test day 28 and 92 time points were expected and are attributed to the maturation of the animals. Therefore, the observed decreases in %RET in the micronucleus evaluation are not considered adverse. 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
This study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
The test substance did not have an effect in the micronucleus assay in any animals exposed during this study as assessed by the number of micronucleated polychromatic erythrocytes

Executive summary:

Five groups of 10 male and 10 female Crl:CD(SD) rats each were exposed whole body, 6 hours per day, 5 days per week to 0, 3000, 4000, 5000 or 7500 ppm of the test substance over a 90 day period for a total of 65 exposures. Animals were weighed, observed for clinical signs of toxicity and had body weight and food consumption evaluations weekly. Five animals per sex per exposure concentration had blood collected following the fourth exposure, after approximately 4 weeks of exposure and at the time of final sacrifice for micronucleus evaluation.

Test substance-related adverse effects on body weight and food consumption were observed in male rats exposed to 7500 ppm. The test substance did not have an effect in the micronucleus assay in any animals exposed during this study as assessed by the number of micronucleated polychromatic erythrocytes.