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EC number: 903-945-5 | CAS number: -
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
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- Short-term toxicity to fish
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Genetic toxicity: in vivo
Administrative data
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From 09 nov 2011 to 17 may 2012
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP study, OECD 474 compliant
- Justification for type of information:
- As indicated in the EU REACH Regulation (Annex VIII), an in vivo mutagenicity test shall be considered in case of a positive result in any of the in-vitro genotoxicity studies in Annex VII or VIII. For the reaction mass of chlorodifluoroacetic acid and trifluoroacetic acid the in-vitro mutagenicity tests show negative results and do not trigger the performance of an in vivo study. It should however be noted that this in vivo mutagenicity study with chlorodifluoroacetic acid was not performed as part of the EU REACH registration. For this reason no testing proposal for the in-vivo mutagenicity study was submitted.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 012
- Report date:
- 2012
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- no
- GLP compliance:
- yes
- Remarks:
- 4-8 october 2010
- Type of assay:
- micronucleus assay
Test material
- Reference substance name:
- Chlorodifluoroacetic acid
- EC Number:
- 200-928-8
- EC Name:
- Chlorodifluoroacetic acid
- Cas Number:
- 76-04-0
- Molecular formula:
- C2HClF2O2
- IUPAC Name:
- chloro(difluoro)acetic acid
Constituent 1
Test animals
- Species:
- mouse
- Strain:
- NMRI
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Elevage Janvier (Route des Chènes Secs B.P. 4105 - 53940 LE GENEST-ST-ISLE, France)
- Age at study initiation: Approximately 7 weeks at the treatment
- Weight at study initiation:
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Fasting period before study:
- Housing: Group caging (5 animals/cage or 2 animals/cage) to allow social interaction, and with deep wood sawdust bedding, to allow digging and other normal rodent activities. Cages type II, polypropylene/polycarbonate (37 x 22.5 x 18 cm)
- Diet (e.g. ad libitum): Animals received ssniff® SM R/M-Z+H H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH (D-59494, Soest, Germany), ad libitum.
- Water (e.g. ad libitum): tap water from municipal supply, as for human consumption from 500 ml bottle ad libitum.
- Acclimation period: At least 5 days under the same conditions as for the test
ENVIRONMENTAL CONDITIONS
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 21.3 – 24.9°C (preliminary experiment)
21.7 – 24.9°C (main test)
Relative humidity: 32 – 69 % (preliminary experiment)
36 – 67 % (main test)
Ventilation: 15 – 20 air exchanges/hour
IN-LIFE DATES: From 30 nov 2011 To 01 dec 2011
Administration / exposure
- Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle(s)/solvent(s) used: Phosphate buffer saline (x10)
- Justification for choice of solvent/vehicle: the most appropriate for the test and based on the solubility of CDFA - Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
Due to the highly acidic property (pH=2.1 in 5 % aqueous solution) and known corrosive effect of the test item, formulations were prepared in a PBS buffer solution, diluted such that the pH of the administered solution was not below pH 3.0. This pH adjustment had been considered not to adversely affect the determination of the test item ability to cause genotoxic effect in the experimental animals since the systemic form of the chemical in the blood was not changed by the gavage formulation.
A preliminary toxicity test was performed to identify the appropriate maximum dose level for the main test. The preliminary toxicity test also determined whether there are large differences in toxicity between the sexes or not. Groups of two male and female mice were treated by oral gavage at two occasions with an approximately 3-hour interval in between at the dose levels of 400, 200, 100 and 50 mg/kg body weight/day. The concentration of the test item formulation was chosen to assure the same dosing volumes in mice for all dose levels (20 mL/kg body weight).
Animals were examined regularly for toxic signs and mortalities. The surviving mice were euthanized 48 hours after treatment. No bone marrow smears were prepared in the preliminary experiment. - Duration of treatment / exposure:
- 48 hours
- Frequency of treatment:
- twice
Doses / concentrations
- Remarks:
- Doses / Concentrations:
0, 100, 200 and 400 mg/kg bw
Basis:
nominal conc.
- No. of animals per sex per dose:
- 5 males per dose at 100 and 200 mg/kg bw
10 + 2 males at 400 mg/kg bw - Control animals:
- yes, concurrent vehicle
- Positive control(s):
- - Positive control: cyclophosphamide
- Justification for choice of positive control(s): usually used by the lab, and recommended by the guideline
- Route of administration: gavage
- Doses / concentrations: 60 mg/kg bw (6 mg/mL)
Examinations
- Tissues and cell types examined:
- Two thousand polychromatic erythrocytes (PCEs) were scored per animal to assess the incidence of the micronucleated cells. The frequency of micronucleated cells was expressed as percent of micronucleated cells based on the first 2000 PCEs counted in the optic field. Fewer than 2000 PCEs may be counted in the event of a clear positive effect.
The proportion of immature among total (immature + mature) erythrocytes was determined for each animal by counting a total of at least 1000 cells (immature erythrocytes, PCEs plus mature normochromatic erythrocytes, NCEs), in which the number of micronuclei were recorded in both types of erythrocytes. - Details of tissue and slide preparation:
- PREPARATION OF BONE MARROW
asphyxiation with ascending doses of carbon dioxide. Deep anaesthesia was confirmed before making incision; death was confirmed by cervical dislocation or by cutting through major cervical blood vessels before discarding carcasses. Bone marrow was obtained from two exposed femurs of mice immediately after sacrifice.
The bone marrow was flushed out of each pair of femurs with foetal bovine serum (5 mL) using a syringe and needle into a sterile centrifuge tube. After mixing, the cell suspension was concentrated by a gentle centrifugation and the supernatant was discarded. Smears of the cell pellet were made on standard microscope slides (2 slides / animal). Slides were air-dried at room temperature for approximately 24 hours.
Dried slides were fixed in methanol for a minimum of 5 minutes and allowed to air-dry. Two slides per animal were stained with 10 % Giemsa solution for 20 minutes then thoroughly rinsed with distilled water, and then air-dried at room temperature for at least 12 hours. After staining, coverslips were mounted on them.
TO BE COMPLETED - Evaluation criteria:
- EVALUATION OF THE RESULT:
The frequencies of micronucleated polychromatic erythrocytes in animals in the test and positive control groups will be compared to the values found in the corresponding negative control group. Statistically analysis will be performed using Kruskall Wallis Non Parametric ANOVA test. Cytotoxicity will be expressed by the PCE/NCE ratio.
The test item will be considered to have shown genotoxic activity in this study if the following criteria are met:
- increases in the frequency of micronucleated polychromatic erythrocytes are observed in treated animals compared to the corresponding negative control
- the increases are dose-related
- the increases are statistically significant.
The historical control range for this laboratory will also be considered when evaluating the biological significance of small increases.
The test item will be concluded to have given a negative response if no reproducible, statistically significant increases are observed above the concurrent and historic control values.
The Micronucleus Test is considered acceptable if it meets the following criteria:
- the frequencies of micronucleated polychromatic erythrocytes found in the negative and /or solvent controls falls within the range of historical laboratory control data.
- the positive control item should produce biologically relevant increases in the number of micronucleated polychromatic erythrocytes.
- Each treated and control group should include at least 5 analysable animals. - Statistics:
- Kruskal-Wallis test
Results and discussion
Test results
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS OF RANGE-FINDING STUDY
The solubility limit of the test item in the selected vehicle was examined in a Preliminary Compatibility Test. Based on the observed results, 10 mg/mL formulation using 10X PBS as vehicle was used as the highest feasible concentration formulation in the preliminary toxicity test (higher concentration test item solutions had a pH value lower than 3.0). Taking the highest applicable treatment volume (20 mL/bw kg) and two treatments/day into account, the highest feasible concentration of the test item was 400 mg/body weight/day.
Therefore, groups of two male and female mice were treated at two occasions by oral gavage at the dose levels of 400, 200, 100 and 50 mg/kg bw/day in the Preliminary Toxicity Test.
Based on the results of the preliminary experiment, dose levels of 400, 200 and 100 mg/kg body weight/day were selected for the micronucleus test. Because the toxic effect of the test item was similar in both sexes in the preliminary toxicity test, the main experiment was performed using male mice only.
RESULTS OF DEFINITIVE STUDY
Groups of five male mice were treated with the test item at 400, 200 and 100 mg/kg body weight/day or with the vehicle (10X PBS) in the main experiment. All mice in the vehicle and test item groups were dosed by oral gavage at two occasions using a dose volume of 20 mL/kg body weight. Animals of the positive control group were treated by intraperitoneal injection with Cyclophosphamide at 60 mg/kg body weight/day at one occasion using a dose volume of 10 mL/kg body weight.
No marked effect of test item treatment on the body weight of the mice was observed in the main test.
No mortality or signs of systemic toxicity were observed during the study. The animals in each dose group, furthermore in the negative (vehicle) control and positive control groups were symptom-free during the whole observation period.
2000 polychromatic erythrocytes (PCEs) were scored per animal to assess the micronucleated cells. The frequency of micronucleated cells was expressed as percent of micronucleated cells based on the first 2000 PCEs counted in the optic field. The proportion of immature among total erythrocytes was determined for each animal by also counting mature erythrocytes (NCEs) until a total of 1000 PCEs+NCEs had been observed. Results are shown in Appendix 6.
Comparison of the vehicle control data and the high dose of the test item (400 mg/kg bw/day) at 24h using the Kruskal-Wallis test gave a value of H = 0.918. This is non-significant, giving a negative response. The average number of micronuclei in the high dose group at 48h was less than the negative control group; therefore statistical analysis was not necessary at that time point.
No indication of an increase in the number of micronucleated normochromatic erythrocytes was observed in the study.
- Statistical evaluation: Kruskal-Wallis test
Any other information on results incl. tables
Micronucleus Data
Mice sacrificed 24 hours after dosing
Treatment |
ID |
Animal |
MNPCE/ |
PCE/1000 PCE+NCE |
Group 1 |
8190 |
19 |
0 |
270 |
Negative (vehicle) control |
8202 |
31 |
1 |
205 |
(Phosphate buffered saline) |
8203 |
32 |
1 |
260 |
|
8207 |
36 |
1 |
291 |
|
8210 |
39 |
0 |
220 |
Mean: |
|
|
0.6 |
249.2 |
SD: |
|
|
0.548 |
35.72 |
Group 2 |
8173 |
2 |
1 |
350 |
Chlorodifluoroacetic acid |
8191 |
20 |
1 |
682 |
(100 mg/kg bw/day) |
8198 |
27 |
0 |
320 |
|
8204 |
33 |
1 |
430 |
|
8205 |
34 |
1 |
446 |
Mean: |
|
|
1.2 |
399.4 |
SD: |
|
|
1.095 |
38.33 |
Group 3 |
8174 |
3 |
0 |
300 |
Chlorodifluoroacetic acid |
8186 |
15 |
0 |
155 |
(200 mg/kg bw/day) |
8187 |
16 |
1 |
402 |
|
8189 |
18 |
1 |
393 |
|
8200 |
29 |
1 |
507 |
Mean: |
|
|
0.6 |
351.4 |
SD: |
|
|
0.548 |
132.02 |
Group 4 |
8175 |
4 |
3 |
363 |
Chlorodifluoroacetic acid |
8195 |
24 |
1 |
391 |
(400 mg/kg bw/day) |
8212 |
41 |
1 |
460 |
|
8213 |
42 |
0 |
410 |
|
8216 |
45 |
1 |
373 |
Mean: |
|
|
0.8 |
445.6 |
SD: |
|
|
0.447 |
142.34 |
Group 5 |
8188 |
17 |
31 |
330 |
Positive Control |
8194 |
23 |
40 |
442 |
(Cyclophosphamide, |
8196 |
25 |
27 |
381 |
60 mg/kg bw/day) |
8206 |
35 |
40 |
409 |
|
8215 |
44 |
37 |
310 |
Mean: |
|
|
35.0 |
374.4 |
SD: |
|
|
5.788 |
54.61 |
Mice sacrificed 48 hours after dosing
Treatment |
ID |
Animal |
MNPCE/ 2000 PCE |
PCE/1000 PCE+NCE |
Group 1 |
8177 |
6 |
2 |
310 |
Negative (vehicle) control |
8192 |
21 |
0 |
156 |
(Phosphate buffered saline) |
8185 |
14 |
0 |
232 |
|
8181 |
10 |
1 |
258 |
|
8208 |
37 |
0 |
210 |
Mean: |
|
|
0.6 |
233.2 |
SD: |
|
|
0.894 |
57.04 |
Group 4 |
8180 |
9 |
0 |
283 |
Chlorodifluoroacetic acid |
8182 |
11 |
0 |
278 |
(400 mg/kg bw/day) |
8184 |
13 |
0 |
211 |
|
8201 |
30 |
0 |
282 |
|
8211 |
40 |
0 |
322 |
Mean: |
|
|
0.0 |
275.2 |
SD: |
|
|
0.000 |
40.08 |
MNPCE: Number of Micronucleated Polychromatic Erythrocytes referring to counts of 2000 PCE.
PCE: Polychromatic Erythrocyte
NCE: Normochromatic Erythrocyte
Applicant's summary and conclusion
- Conclusions:
- No induction of micronuclei in bone marrow erythrocytes was observed following administration of Chlorodifluoroacetic acid to mice at up to and including 400 mg/kg bw/day (highest feasible concentration); thus, there was no evidence of any in vivo genotoxic activity of the test item under the conditions of this study.
- Executive summary:
The objective of the study (2012) was to determine whether Chlorodifluoroacetic acid test item caused genotoxic effects resulting in the formation of micronuclei in erythrocytes of treated mice in accordance with the OECD guideline “Mammalian Erythrocyte Micronucleus Test”, No. 474 (1997).
In the main test, groups of male mice were treated with the vehicle (10X PBS) or the test item at 400, 200 and 100 mg/kg body weight/day by oral gavage or the positive control item (Cyclophosphamide dissolved in physiological saline) at 60 mg/kg body weight/day administered by intraperitoneal injection. Five mice from each group were examined 24 hours after dosing, and a further five mice dosed with the vehicle or test item at 400 mg/kg body weight/day were examined 48 hours after dosing. Bone marrow smears were prepared on glass slides for each of the mice, stained, and scored. Two thousand polychromatic erythrocytes (PCEs) were scored per animal to assess the micronucleated cells.
No marked effect on body weight was observed in the main test. No mortality or signs of systemic toxicity were observed during the study. The animals in each group were symptom-free during the whole observation period.
No biologically or statistically significant increases in the frequency of micronucleated polychromatic erythrocytes were seen in mice treated with the test item, compared to the negative control values at any of the sampling time points. The positive control treatment caused a large, clearly positive response demonstrating the sensitivity of the test system.
In conclusion, no induction of micronuclei in bone marrow erythrocytes was observed following administration of Chlorodifluoroacetic acid to mice at up to and including 400 mg/kg bw/day (highest feasible concentration); thus, there was no evidence of any genotoxic activity of the test item under the conditions of this study.
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