Acetic acid, 2,2-difluoro-2-[[2,2,4,5-tetrafluoro-5-(trifluoromethoxy)-1,3-dioxolan-4-yl]oxy]-, ammonium salt (1:1)

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Remarks:

chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3 December 2009 to 13 July 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline-conform study under GLP without deviations.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.11 (Mutagenicity - In Vivo Mammalian Bone-Marrow Chromosome Aberration Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5385 (In Vivo Mammalian Cytogenetics Tests: Bone Marrow Chromosomal Analysis)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

chromosome aberration assay

Test material

Test material information

Constituent 1

Reference substance name:

cyclic C6O4 ammonium salt

IUPAC Name:

cyclic C6O4 ammonium salt

Constituent 2

Reference substance name:

Acetic acid, 2,2-difluoro-2-[[2,2,4,5- tetrafluoro-5-(trifluoromethoxy)-1,3- dioxolan-4-yl]oxy]-, ammonium salt (1:1)

IUPAC Name:

Acetic acid, 2,2-difluoro-2-[[2,2,4,5- tetrafluoro-5-(trifluoromethoxy)-1,3- dioxolan-4-yl]oxy]-, ammonium salt (1:1)

Constituent 3

Reference substance name:

ammonium 2,2-difluoro-2-{[2,2,4,5-tetrafluoro-5-(trifluoromethoxy)-1,3-dioxolan-4-yl]oxy}acetate

EC Number:

682-238-0

Cas Number:

1190931-27-1

Molecular formula:

C6H4F9NO6

IUPAC Name:

ammonium 2,2-difluoro-2-{[2,2,4,5-tetrafluoro-5-(trifluoromethoxy)-1,3-dioxolan-4-yl]oxy}acetate

Details on test material:

- Name of test material (as cited in study report): cC6O4 ammonium salt (dry salt)
- Substance type: pure substance (dry salt)
- Physical state: solid
- Analytical purity: 97.5 %
- Impurities (identity and concentrations): not reported
- Composition of test material, percentage of components: not reported
- Isomers composition: not reported
- Purity test date: not reported
- Lot/batch No.: 156/39
- Expiration date of the lot/batch: December 31, 2020
- Stability under test conditions: not reported
- Storage condition of test material: In the refrigerator (+2 to +8 °C)

Test animals

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories B.V., Postbus 6174 5960 AD Horst / The Netherlands
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 180.2 ± 5.5 g
- Assigned to test groups randomly: yes. The animals are identified by their cage number and tail tags.
- Fasting period before study: not reported
- Housing: In group, Cage Type: Makrolon Type IV, with wire mesh top; Bedding: granulated soft wood bedding.
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum (Harlan Laboratories GmbH, 33178 Borchen, Germany)
- Water (e.g. ad libitum): tap water, ad libitum (Gemeindewerke, 64380 Roßdorf, Germany)
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 2°C
- Humidity (%): 33 - 65 %
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12 hours - (artificial light: 6.00 a.m. - 6.00 p.m.)


IN-LIFE DATES: The animals were sacrifed 24 h after treatment. Animals of one of the two groups tested at high dose were sacrifed 48 h after treatment .

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: sterile water (Supplier: B. Braun Melsungen AG; Catalogue no.: 6724092.00.00)
- Justification for choice of solvent/vehicle: The vehicle was chosen to its relative non-toxicity for the animals.
- Amount of vehicle (if gavage or dermal): All animals received a single standard volume of 10 ml/kg b.w. orally

The vehicle of the test item was used as vehicle control.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: On the day of the experiment, the test item was formulated in sterile water.
All animals received a single standard volume of 10 ml/kg b.w. orally.

DIET PREPARATION:
Pelleted standard diet. Supplied by Harlan Laboratories GmbH, 33178 Borchen, Germany.

Duration of treatment / exposure:

The animals received the test item by gavage.

Frequency of treatment:

The animals received the test item once.

Post exposure period:

24 hours. For the highest dose level an additional sample was taken at 48 hours after treatment.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

312.5 mg/kg bw/day (nominal)

Remarks:

Sampled at 24 hours post treatment

Doses / concentrations 2

Dose / conc.:

625 mg/kg bw/day (nominal)

Remarks:

Sampled at 24 hours post treatment

Doses / concentrations 3

Dose / conc.:

1 250 mg/kg bw/day (nominal)

Remarks:

Sampled at 24 hours post treatment

Doses / concentrations 4

Dose / conc.:

1 250 mg/kg bw/day (nominal)

Remarks:

Sampled at 48 hours post treatment

No. of animals per sex per dose:

7 males are assigned to each test group.
One group were tested at low dose (Sampled at 24 hours post treatment).
One group were tested at medium dose (Sampled at 24 hours post treatment).
Two groups were tested at the highe dose. (Sampled at 24 and 48 hours post treatment).

Control animals:

yes, concurrent vehicle

yes, historical

Positive control(s):

Name: CPA; Cyclophosphamide (Supplier: Sigma-Aldrich Vertriebs-GmbH 82041 Deisenhofen, Germany; Catalogue no.: C 0768)
Dissolved in: sterile water
Dosing: 15 mg/kg b.w.
Route and Frequency of Administration: orally, once
Volume Administered: 10 ml/kg b.w.
Solution prepared on day of administration.

The stability of CPA at room temperature is good. At 25°C only 3.5 % of its potency is lost after 24 hours.

Examinations

Tissues and cell types examined:

Bone marrow cells from the femoral epiphyses.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: A preliminary study on acute toxicity was performed with two animals per sex and test group each under identical conditions as in the mutagenicity study concerning. (Doses: 1000, 1250, 1750, 2000 mg/kg b.w)
The animals were treated once orally with the test item and examined for acute toxic symptoms at intervals of 1 h, 2-4h, 6 h, 24h, 30 h (except for the first pre-experiment), and 48 h after each administration of the test item.
It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly or 2000 mg/kg as the upper limit for non-toxic test items.
The maximum tolerated dose levels were determined to be the doses that caused toxic reactions without having effects on survival within 48 hours after the treatment. On the basis of the preliminary study, 1250 mg/kg b.w. was estimated to be suitable as the highest dose for the main study.
In the main study three adequately spaced dose levels, spaced by a factor of 2, were applied at the central sampling interval 24 h after treatment. For the highest dose level an additional sample was taken at 48 h after treatment.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): At the beginning of the treatment the animals were weighed and the individual volume to be administered was adjusted to the animal's body weight. The animals received the test item once. Seven males were treated per dose group. The animals were examined for acute toxic symptoms around 1 h, 2-4 h, 6 h, 24 h and 48 h after treatment.
Prior (2.5 to 3 hours) to sacrifice, animals were injected intraperitoneally with the spindle inhibitor colcemid (2.0 mg/kg b.w.), to arrest cells in metaphase.

DETAILS OF SLIDE PREPARATION: The femora were removed, the epiphyses were cut off and the marrow was flushed out with approximately 5 ml hypotonic potassium chloride solution (0.56 % w/v, prewarmed to 37 °C). The hypotonic cell suspension was then incubated for 20 min at 37 °C. The cells were sedimented by a brief centrifugation (1000 rpm), the hypotonic supernatant was discarded and the cell pellet was fixed with 3+1 absolute methanol+glacial acetic acid fixative for 60 min. Then the cell pellet was gently resuspended with fixative and stored overnight at 4°C. Prior to making
slides the fixative was changed and enough fixative was added to make a relatively thin cell suspension. The fixative-cell suspension was spread by flame-drying and stained with Giemsa. Cover slips were mounted with EUKITT (KINDLER, 79110 Freiburg, Germany).
One or more slides were made from each bone marrow sample.

METHOD OF ANALYSIS: Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. Gaps, breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations. At least 100 well spread metaphases per animal were scored for cytogenetic damage on coded slides. The number of chromosome aberrations per metaphase was determined. Only metaphases with the characteristic chromosome number of 42 ± 2 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis; 1000 cells are scored) was determined.

Evaluation criteria:

A test item is classified as mutagenic if it induces either a dose-related increase in the number of structural chromosomal aberrations and a reproducible statistically significant positive response for at least one of the test points.
A test item producing neither a dose-related increase in the number of structural chromosomal aberration nor a statistically significant and reproducible positive response at any of the test points is considered non-mutagenic in this system.
This can be confirmed by means of the non-parametric Mann-Whitney test.
However, both biological and statistical significance should be considered together.

The test is acceptable since at least 5 evaluable animals per group were available, the positive control shows a statistically significant response and the aberration rate of the vehicle control (excl. gaps) is below 2 %.

Statistics:

Mann-Whitney test.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Toxic reactions in the main test: Ruffled fur at 312.5 mg/kg b.w. Ruffled fur and reduction of spontaneous activity at 625 and 1250 mg/kg b.w.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 1000. 1250. 1750, 2000 mg/kg b.w.
- Clinical signs of toxicity in test animals: Ruffled fur at 1000 mg/kg b.w. Ruffled fur, reduction of spontaneous activity and hunchback at 1250 mg/kg. Ruffled fur, reduction of spontaneous activity, hunchback, salivation and death at 1750 mg/kg. Ruffled fur, reduction of spontaneous activity, hunchback and death at 2000 mg/kg.
- Evidence of cytotoxicity in tissue analyzed: Tissue were not analyzed during the preliminary study. However the mitotic indices calculated in the main test were not relevantly reduced after treatment with cC6O4 AMMONIUM SALT, indicating that the test item did not have cytotoxic effects in the bone marrow.

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels:
reported in TABLE 2

- Appropriateness of dose levels and route:
Dose levels and route are appropriate.

- Statistical evaluation:
Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.
No statistically significant or biologically relevant enhancement of the aberration frequencies occurred (aberration rates of the test item treated animals ranged between 0.0-1.1%) as compared to the vehicle control value (0.4%). The mean aberration frequencies observed after treatment with cC6O4 AMMONIUM SALT were consistently below 2 % aberrant cells exclusive gaps (given as the upper limit of a tolerable vehicle
control value; see acceptance criteria). There were also no relevant dose dependent increases in the aberration rates of the treated animals.
The study was acceptable as the positive control showed a statistically significant response (aberration rate excluding gaps was 7.3 %) and the aberration frequency exclusive gaps of the vehicle control was below 2 %.

Any other information on results incl. tables

The mitotic indices were not relevantly reduced after treatment with cC6O4 AMMONIUM SALT, indicating that the test item did not have cytotoxic effects in the bone marrow.

Table 1: Summary of Results

Experimental group	dose mg/kg b.w.	preparation hours post administration	number of cells scored	% aberrant cells		mean mitotic index (%)
				incl. gaps	excl. gaps
1: sterile water	0	24	1000	0.4	0.4	4.67
2: cC604 ammonium salt	312.5	24	1000	0.0	0.0	5.27
3: cC604 ammonium salt	625	24	1000	0.7	0.7	3.06
4: cC604 ammonium salt	1250	24	1000	0.7	0.7	3.94
5: cyclophosphamide	15	24	1000	7.3	7.3	3.59
6: cC604 ammonium salt	1250	48	1000	1.1	1.1	5.19

 

 

Table 2: Analysis of aberration types

Group	gap	isogap	break	isobreak	fragment	iso-fragment	deletion	multiple aberration a	exchange	chrom. Disintegration b
1	0	0	0	0	2	1	0	0	0	0
2	0	0	0	0	0	0	0	0	0	0
3	0	0	2	0	2	3	0	0	0	0
4	0	0	1	0	4	1	0	0	0	0
5	0	0	39	0	9	4	0	4	18	0
6	0	0	1	0	3	4	0	0	0	0

 

a More than 5 aberrations excluding gaps in one cell; exchanges (but no other aberrations) were recorded separately

b Pulverization

Applicant's summary and conclusion

Conclusions:

During the mutagenicity test described and under the experimental conditions reported, the test item did not induce chromosome damages as determined by the chromosome aberration test with bone marrow cells of the rat.
Therefore, cC6O4 AMMONIUM SALT is considered to be non-mutagenic in this chromosome aberration assay in vivo.

Executive summary:

This study was performed to investigate the potential of cC6O4 AMMONIUM SALT to induce chromosome aberrations in bone marrow cells of the rat.

The test item was formulated in sterile water. The volume administered orally was 10 ml/kg body weight (b.w.). The animals were treated once. 24 h and 48 h (only the high dose group) after the treatment the bone marrow cells were collected for chromosome

aberration analysis. 7 males per test group were evaluated for the occurrence of cytogenetic damage. Per animal 100 well spread metaphases were scored for gaps, breaks, fragments, deletions, multiple aberrations, exchanges, and chromosomal disintegrations.

The test item was investigated in this cytogenetic assay at the following doses:

Males: 312.5, 625, and 1250 mg/kg b.w.

The maximum doses for the cytogenetic assay were determined in a pre-experiment for toxicity. 1250 mg/kg b.w. was estimated as to be close to the maximum tolerated doses. The animals treated with this dose showed clinical signs like a reduction in spontaneous activity, ruffled fur and hunchback posture indicating bioavailability of the test item.

No relevant reduction of the mitotic indices could be observed after treatment with the test item, indicating that the test item at the indicated concentrations was not cytotoxic in the bone marrow.

No statistically significant increase in the frequency of aberrant cells occurred after treatment with the test item as compared to the vehicle control.

An appropriate reference mutagen (cyclophosphamide) was used as positive control and showed a distinct and statistically significant increase of induced aberration frequency.

In conclusion, it can be stated that during the mutagenicity test described and under the experimental conditions reported, the test item did not induce chromosome mutations as determined by the chromosome aberration test with bone marrow cells of the rat.

Therefore, cC6O4 AMMONIUM SALT is considered to be non-mutagenic in this chromosome aberration assay in vivo.