Reaction mass of potassium trifluoroacetate and potassium trifluoromethanesulphinate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration (frequency of micronucleated polychromatic erythrocytes in bone marrow)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

between 2012-01-17 to 2012-04-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study

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 TFSK/TFAK 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 for the Reaction mass of TFSK/TFAK a new chemical substance notification in China has been prepared under the Regulation ‘Measures on the Environmental Management of New Chemical Substances' (Decree No. 7 of the Ministry of Environmental Protection of the P.R. China, also known as ‘China REACH’). Under this regulation the in vivo mutagenicity test is part of the standard data requirements for substances that are produced or imported in volumes > 10 t/y. For this reason an OECD 474 study was performed in 2012 at CiTox Lab. The results of the study are included in the dossier and serve as the valid test related to this endpoint.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Deviations:

no

GLP compliance:

yes

Remarks:

2010-12-16

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of potassium trifluoroacetate and potassium trifluoromethanesulphinate

EC Number:

911-467-3

Molecular formula:

C2O2F3K and CO2F3KS

IUPAC Name:

Reaction mass of potassium trifluoroacetate and potassium trifluoromethanesulphinate

Constituent 2

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

water

Test material form:

liquid

Remarks:

aqueous solution

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Janvier Route des Chènes Secs B.P. 4105 53940 LE GENEST-ST-ISLE, France
- Age at study initiation: approximately 7 weeks
- Weight at study initiation:
37.7 – 39.7 g (males, preliminary experiment I.)
28.2 – 30.2 g (females, preliminary experiment I.)
38.5 - 40.3 g (males, preliminary experiment II.)
26.3 - 27.9 g (females, preliminary experiment II.)
34.7 - 39.8 g (males, main test)
- Assigned to test groups randomly: [yes: ] The animals were assigned to their respective treatment groups by randomization based on body weights. Animals were randomly allocated to the negative and positive control groups based on the most recent actual body weight; SPSS/PC+ software was used in order to verify homogeneity/variation among/within groups
- 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. Cage type:II. type polypropylene/polycarbonate (37 x 22.5 x 18 cm). Bedding: Bedding of certified wood chips especially designed to keep animals in the best natural environment was provided for animals during the study. Lignocel 3/4-FASERN Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH & Co.KG (Holzmühle 1, 73494 Rosenberg, Germany) was available to animals during the study.
- Diet (e.g. ad libitum): Animals received ssniff® SM R/M-Z+H "Autoclavable complete feed for mice and rats – breeding and maintenance" (Batch number: 683 5949, Expiry date: January 2012; and Batch number: 719 6627, Expiry date: May 2012) produced by ssniff Spezialdiäten GmbH (D-59494, Soest, Germany
- Water (e.g. ad libitum): tap water from municipal supply, as for human consumption from 500 ml bottle ad libitum
- Acclimation period: 6-14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.2 - 25.0°C
- Humidity (%): 31 – 69 %
- Air changes (per hr): 15 – 20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES: From:2012-01-17 To:2012-01-23

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Distilled water Batch No.:3450611 Source: TEVA Hungary Ltd. Expiry:June 2014 Storage conditions: Room temperature
- Justification for choice of solvent/vehicle: Based on the results of the preliminary solubility test, the test item was soluble in Distilled water
- Concentration of test material in vehicle: 2000, 1000 and 500 mg active ingredients/kg body weight/day
- Amount of vehicle (if gavage or dermal): 10 mL/kg body weight

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was dissolved in the distilled water for the treatment. The necessary amount of the test item was weighed into a calibrated volumetric flask; vehiclewas added and stirred to obtain homogenous formulations. For the purity conversion, the 36.5 % purity data of the two active ingredients (potassium trifluoromethanesulfinate and potassium trifluoroacetate) was taken into consideration.

Duration of treatment / exposure:

One occasion

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Negative (vehicule) control: 15 males (In the negative control group unusually aggressive behaviour of one male animal was observed at the 24-hour necropsy. As this behaviour could have influenced the results of other four animals in the group (marked body weight loss and biting marks were recognized), additional five negative control animals were treated on using the random spare animals of the study as agreed by the Sponsor. These additional negative control animals were individually caged, but otherwise the experimental procedure was the same. Necropsy and slide preparation from these additional animals were performed 24-hours after treatment.

test item 500 mg/kg: 5 males
Test item 1000 mg/kg: 5 males
Test item 2000 mg/kg: 10 + 2 males (Two additional male mice were dosed in highest dose group (2000 mg/kg body weight/day) of the main experiment to replace any animals which die before the scheduled sacrifice time. Bone marrow smears were not prepared from additional mice as they were not used as replacement)

Positive control (cyclophosphamide): 5 males

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide Batch No.:079K1569 Supplier: Sigma-Aldrich Co.Expiry date: July 2012 Storage condition: Refrigerated (2-8 °C)
- Justification for choice of positive control(s): No data
- Route of administration: intraperitoneal injection
- Doses / concentrations: 60mg/kg using a dose volume of 10 mL/kg

Examinations

Tissues and cell types examined:

Two thousand polychromatic erythrocytes (PCEs) were scored per animal to assess the incidence of the micronucleated cells.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In a preliminary study, 2000 and 1000 mg active ingredients/kg body weigh/day doses were tested in groups of 2 male and 2 female animals. All animals were free of clinical signs in the preliminary experiments except of one male showing piloerection.
Based on the results of the preliminary experiment, dose levels of 2000, 1000 and 500 mg active ingredients/kg body weight/day were selected for the micronucleus test. The main experiment was performed using male mice only as male mice were at least as sensitive to the test item toxic effects as female mice based on the results of the preliminary experiments.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Group Treatment mg active ingredients/kg bw/day* No. of Analysable Animals Period Between Treatment and Sampling time

1. Negative (vehicle) control -- 15 Males** 24 and 48 hours
2. Reaction mass of TFSK/TFAK, low dose 500 5 Males 24 hours
3. Reaction mass of TFSK/TFAK, mid dose 1000 5 Males 24 hours
4. Reaction mass of TFSK/TFAK, high dose 2000 10 + 2 Males*** 24 and 48 hours
5. Positive control (Cylophosphamide) 60 5 Males 24 hours

* Purity conversion was applied for the test item formulations (36.5 % purity data of the two active ingredients was taken into consideration).
** Additional five negative (vehicle) control animals were dosed in the main experiments (for further details see 6.2.).
***Two additional male mice were dosed in highest dose group (2000 mg/kg body weight/day) of the main experiment to replace any animals which die before the scheduled sacrifice time. Bone marrow smears were not prepared from additional mice as they were not used as replacement.


DETAILS OF SLIDE PREPARATION:
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.

METHOD OF ANALYSIS:
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. 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.

OTHER:

Evaluation criteria:

Criteria for a positive response:
The test item is considered to have shown genotoxic activity if statistically significant increases in the frequency of micronucleated polychromatic erythrocytes are observed in treated animals compared to the corresponding negative controls, and the increases are dose-related. Historical control data could be taken into consideration when evaluating the biological significance of small increases.

Criteria for a negative response:
The test item is concluded to have given a negative response if no reproducible, statistically significant increases are observed above the concurrent and historical control values.

Equivocal response:
Results which do not meet the criteria for a positive or negative response are considered to be equivocal. Further investigations or scoring of additional cells may be necessary in case of an equivocal result

Statistics:

Kruskal Wallis test

Results and discussion

Test results

Key result

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
- Dose range:1000 and 2000 mg/kg active ingredient
- Solubility: soluble at 200 mg/mL
- Clinical signs of toxicity in test animals: All animals were free of clinical signs in the preliminary experiments except of one male showing piloerection.
- Evidence of cytotoxicity in tissue analyzed: No
- Rationale for exposure: Based on the results of the preliminary experiment, dose levels of 2000, 1000 and 500 mg active ingredients/kg body weight/day were selected for the micronucleus test.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay):
- Ratio of PCE/NCE (for Micronucleus assay): See results table
- Appropriateness of dose levels and route: 500, 1000 and 2000 mg/kg active ingredient par oral route (gavage)

- Statistical evaluation: The group treated with 500 mg active ingredients/kg bw/day, which gave the highest individual number of micronuclei at 24h, were compared with the vehicle control group using the Kruskal Wallis test. This gave a value of H = 3.540. Comparison of the vehicle control data and the high dose of the test agent (2000 mg active ingredients/kg bw/day) at 48h gave a value of H = 0.565. Both H values are non-significant, giving a negative response.

The positive and negative control results were also compared, and gave a value of H = 7.211 (p<0.01). The positive control treatment therefore caused a large, statistically significant increase, demonstrating the sensitivity of the test system.

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	8465	12	1	307
Negative (vehicle) control	8468	15	1	396
(Distilled water)	8474	21	1	355
	8483	30	0	339
	8499	46	0	311
	8457	4	1	308
	8469	16	1	377
	8480	27	2	317
	8496	43	0	368
	8501	48	0	414
Mean:			0.7	349.2
SD:			0.675	38.91
Group 2	8456	3	7	400
Reaction mass of	8472	19	2	419
TFSK/TFAK	8478	25	1	283
(500 mg/kg bw/day)	8484	31	1	442
	8491	38	1	437
Mean:			2.4	396.2
SD:			2.608	65.40
Group 3	8460	7	3	447
Reaction mass of	8485	32	1	390
TFSK/TFAK	8488	35	2	371
(1000 mg/kg bw/day)	8500	47	5	490
	8502	49	4	436
Mean:			3.0	426.8
SD:			1.581	47.31
Group 4	8462	9	0	367
Reaction mass of	8466	13	4	453
TFSK/TFAK	8467	14	3	405
(2000 mg/kg bw/day)	8479	26	3	429
	8494	41	3	341
Mean:			2.6	399.0
SD:			1.517	45.39
Group 5	8459	6	41	450
Positive Control	8461	8	87	404
(Cyclophosphamide,	8463	10	55	429
60 mg/kg bw/day)	8470	17	35	426
	8476	23	68	440
Mean:			57.2	429.8
SD:			21.005	17.27

MNPCE: Number of Micronucleated Polychromatic Erythrocytes referring to counts of 2000 PCE.

PCE: Polychromatic Erythrocyte

NCE: Normochromatic Erythrocyte

Micronucleus Data Mice sacrificed 48 hours after dosing

Treatment	ID Number	Animal Number	MNPCE/ 2000 PCE	PCE/1000 PCE+NCE
Group 1	8455	2	1	406
Negative (vehicle) control	8458	5	0	314
(Distilled water)	8473	20	1	386
	8486	33	0	273
	8490	37	0	306
Mean:			0.4	337.0
SD:			0.548	56.45
Group 4	8471	18	0	321
Reaction mass of	8481	28	2	432
TFSK/TFAK	8482	29	0	416
(2000 mg/kg bw/day)	8497	44	1	430
	8498	45	1	353
Mean:			0.8	390.4
SD:			0.837	50.42

Applicant's summary and conclusion

Conclusions:

In conclusion, no induction of micronuclei in bone marrow erythrocytes was observed following administration of Reaction mass of TFSK/TFAK to mice at up to and including 2000 mg active ingredients/kg bw/day; thus, there was no evidence of any genotoxic activity of the test item under the conditions of this study.

Executive summary:

In a NMRI mouse micronucleus assay on erythrocytes micronucleus test (cytoxlab study, 2012), from 5 to 15 males per dose were treated orally (gavage) with TFSK/TFAK at doses of 500, 1000 and 2000 mg/kg.

There were no mortality or signs of systemic toxicity during the study. No market effect of the test item treatment on the body weight of the mice was observed in the main test.

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.

In conclusion, no induction of micronuclei in bone marrow erythrocytes was observed following administration of Reaction mass of TFSK/TFAK to mice at up to and including 2000 mg active ingredients/kg bw/day; thus, there was no evidence of any genotoxic activity of the test item under the conditions of this study

This study is classified as acceptable. This study was conducted comparably to the requirements of the Test Guideline OECD 474 for in vivo cytogenetic mutagenicity.