Reaction mass of chlorodifluoroacetic acid and trifluoroacetic acid

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:

2012

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

Test material information

Constituent 1

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

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 Number	Animal Number	MNPCE/ 2000 PCE	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 Number	Animal Number	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.