1,1,2,2,3,3,4-heptafluorocyclopentane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Two in vitro tests are available.

Ames study:

The substance was examined for mutagenic activity in four histidine dependent auxotrophs of Salmonella typhimurium, strains TA98, TA100, TA1535 and TA1537, and one tryptophan dependent auxotroph of Escherichia coli, strain WP2uvrA, using pour-plate tests in compliance with OECD 471. The test material did not exhibit any mutagenic activity under the conditions of test.

In vitro mammalian chromosome aberration test:

The test item was tested to determine whether it would cause chromosomal aberrations or polyploidy in human lymphocytes cultured in vitro according to OECD 473. The test item has shown evidence of clastogenic but not polyploidy-inducing activity in this in vitro cytogenetic test system.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 1997-09-03 to 1997-10-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Batch No.: 9706-1A
Purity: 98.65 %

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Human lymphocytes
- Suitability of cells: The cells have been used for this type of study for a number of years. They were easy to culture in vitro but do not divide unless stimulated to do so. This is achieved by adding phytohaemagglutinin (PHA) to the culture which results in a high mitotic yield.
- Normal cell cycle time (negative control): 16 hours

For lymphocytes:
Human blood was collected aseptically from healthy male donors, pooled and diluted with RPMI 1640 tissue culture medium (Imperial) containing 10% foetal calf serum (Gibco). Aliquots (0.4 mL blood 4.5 mL medium : 0.1 mL phytohaemagglutinin (Gibco)) of the cell suspension were placed in sterile universal containers and incubated at 37°C in a slanted position for approximately 48 hours. The cultures were gently shaken once daily to resuspend the cells.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Harlan Olac Ltd
- method of preparation of S9 mix: S9 mix contained: S9 fraction (25% v/v), MgCl2 (8mM), KCl (33 mM), sodium orthophosphate buffer pH 7.4 (100 mM), glucóse-6-phosphate (5 mM), NADP (2 mM) and NADPH (2 mM). All the cofactors were filter-sterilised before use.
- concentration or volume of S9 mix and S9 in the final culture medium: 1.25 mL

Test concentrations with justification for top dose:

FIRST TEST
Set 1: With S9 mix - 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 μg/mL
Set 2: Without S9 mix - First: 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 μg/mL; Repeated: 250, 500, 750, 1000, 1200, 1400, 1600, 1800 and 2000 μg/mL.
SECOND TEST
Set 3: Without S9 mix - 100, 200, 400, 600, 800, 1000 and 1200 μg/mL.
Set 4: Without S9 mix - 50, 100, 200, 400, 600, 800, 1000 and 1200 μg/mL.
Set 5: With S9 mix -31.3, 62.5, 125, 250, 375, 500, 750 and 1000 μg/mL.

On dosing at 1 % v/v into aqueous tissue culture medium, giving a final concentration of 4090 μg/mL small oily globules were observed. Concentrations with high ionic strength and osmolality may cause chromosomal aberrations. Therefore, concentrations greater than 5000 μg/mL or 10 mM are not used in this test. In this case, the highest final concentration used for subsequent testing was 2000 μg/mL (10 mM) as this is the concentration recommended for use according to Japanese guidelines.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulphoxide

- Justification for choice of solvent/vehicle: The test item was found to be soluble in dimethyl sulphoxide at 409 mg/mL..

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- FIRST TEST:
For set 1, aliquots of S9 mix (1.25 mL) were added to one set of duplicate cultures followed by 62.5 μL aliquots of test item.
For set 2, 50 μL aliquots of the various dilutions of test item, were added to one set of duplicate cultures.
- SECOND TEST:
Three sets of cultures were initiated and maintained as previously described.
Three hours after dosing, the cultures containing S9 mix were centrifuged and the cell pellets resuspended in fresh medium. They were then incubated for a further 21 hours. Cultures treated in the absence of S9 mix were incubated for 24 and 48 hours.


TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: FIRST TEST: 3 hours treatment and 21 hours recovery; SECOND TEST: continuous treatment of 24 and 48 hours
- Harvest time after the end of treatment: FIRST TEST: 21 hours recovery; SECOND TEST: At the end of treatment


FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Harvesting and fixation: Two hours before the end of the 24 hour incubation period, mitotic activity was arrested by the addition of Colcemid (Sigma) to each culture at a final concentration of 0.1 μg/mL. After two hours incubation, each cell suspension was transferred to a conical centrifuge tube and centrifuged for 5 minutes at 500 g. The cell pellets were treated with a hypotonic solution (0.075M KCI prewarmed at 37°C). After a 10 minute period of hypotonic incubation at 37°C, the suspensions were centrifuged at 500 g for 5 minutes and the cell pellets fixed by addition of freshly prepared cold fixative (3 parts methanol : 1 part glacial acetic acid). The fixative was replaced several times. The pellets were allowed to fix for at least two hours.
- Methods of slide preparation: The pellets were resuspended using a whirlimixer, then centrifuged at 200 g for 10 minutes and finally resuspended in a small volume of fresh fixative. Two or three drops of the cell suspensions were dropped onto pre-cleaned microscope slides which were then allowed to air-dry. The slides were then stained in 10% Giemsa, prepared in buffered water (pH 6.8). After rinsing in buffered water the slides were left to air-dry and then mounted in DPX.
- Microscopic Examination:
The prepared slides were examined by light microscopy at a magnification of x160. The proportion of mitotic cells per 1000 cells in each culture was recorded except for positive control treated cultures. From these results the dose level causing a decrease in mitotic index of approximately 50% of the solvent control value or, if there was no decrease, the maximum concentration, 10 mM, was used as the highest dose level for the metaphase analysis. Intermediate and low dose levels usually approximated to 50% and 25% of the highest dose level.
The concentration of each positive control compound selected for analysis was the lowest concentration dosed unless a preliminary scan of metaphase figures indicated an insufficient level of aberrant cells
The slides were then coded. Metaphase figures were identified, using a magnification of x160 and examined at a magnification of x1000 using an oil immersion objective. Approximately 100 metaphase figures were examined, where possible, from each culture. Only cells with 44 - 46 chromosomes were analysed for structural aberrations. The vernier readings of all aberrant metaphase figures were recorded. Additionally, the proportion of polyploid cells in 100 metaphase figures, where possible, was recorded.

Statistics:

The number of aberrant metaphase figures, or polyploid cells, in each treatment group was compared with the solvent control value using Fisher's test (Fisher 1973).

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

STUDY RESULTS
- Positive control data : Both positive control compounds caused large statistically significant increases (P<0.001) in the proportion of aberrant cells.

Chromosome aberration test (CA) in mammalian cells:
FIRST TEST
- Toxicity data:
In set 1, test item caused a reduction in the mitotic index to 53% of the solvent control value at 500 μg/mL. This dose level, together with lower dose levels of 250 and 125 μg/mL, were selected for metaphase analysis. To clarify the results from the metaphase analysis an additional dose level, 1000 μg/mL, was subsequently analysed. This gave a mitotic index of 27% of the solvent control value.
In set 2, test item caused a reduction in the mitotic index to 75% of the solvent control value at 1000 μg/mL. However, because of a sudden decrease in the mitotic index to 7% of the solvent control value at 2000 μg/mL, the test was repeated. In the repeat test, test item reduced the mitotic index to 39% of the solvent control value at 1400 μg/mL. The higher concentrations (1600, 1800 and 2000 μg/mL) were excessively toxic. Therefore, 1400μg/mL was selected as the highest concentration for metaphase analysis. The lower dose levels selected were 1000 μg/mL, which reduced the relative mitotic index to 42%, and 750 μg/mL, which was the highest non-toxic dose level. To clarify the results from the metaphase analysis an additional dose level, 1200 μg/mL, was subsequently analyzed. This gave a mitotic index of 25% of the solvent control value.
- Metaphase analysis:
In set 1, test item caused a statistically significant increase (P<0.001) in the number of aberrant cells at the highest doss level analysed, 1000 μg/mL. This increase, to 6.0% with and without gaps, lies outside the upper 95% limit of the historical control range of (4.5%).
In set 2, test item caused statistically significant increases in the number of aberrant cells at 1000, 1200 and 1400 μg/mL. The increases seen at 1000 and 1200 μg/mL, to 5.0% and 4.6%, respectively, lie outside the upper 95% limit of the historical control range (4.0%). However, at all dose levels the damage was predominantly seen in one culture.
No statistically significant increases in the proportion of polyploid cells were observed in either treatment set.

SECOND TEST
- Toxicity data:
In set 3, test item caused a reduction in the mitotic index to 45% of the solvent control value at 800 μg/mL. This dose level, together with 400 and 200 μg/mL, were selected for metaphase analysis. To clarify the results from the metaphase analysis, additional dose levels were analyzed. These were 600 and 1000 μg/mL, with mitotic indices of 52% and 28%, respectively, of the solvent control value.
In set 4, test item reduced the mitotic index to 45% of the solvent control value at 1000 μg/mL. This dose level was the highest dose level selected for the metaphase analysis. The lower dose levels selected were 800 and 400 μg/mL, which were moderately toxic and non-toxic, respectively. To clarify the results from the metaphase analysis an additional dose level was analysed. This was 1200 μg/mL, with a mitotic index of 30% of the solvent control value.
In set 5, test item, reduced the mitotic index to 49% of the solvent control value at the highest dose level, 1000 μg/mL. This dose level, together with 500 and 250 μg/mL, were selected for metaphase analysis. To clarify the results from the metaphase analysis an additional dose level was analysed. This was 750 μg/mL, with a mitotic index of 54% of the solvent control value.
- Metaphase analysi:
In set 3, test item caused a statistically significant increase (P<0.001) in the number of aberrant cells at the highest dose level, 1000 μg/mL, to 6.0% and 7.0%, without and with gaps, respectively. These increases lie outside the upper 95% limit of the historical control range (4.0% and 4.25%, respectively). However, this dose level was excessively toxic in one of the replicate cultures and only 20 cells could be scored.
In set 4, test item caused a statistically significant increase (P<0.01) in the number of aberrant cells at 800 μg/mL, but only when gap damage was included. However, this increase, to 4.0%, lies just inside the upper 95% limit of the historical control range (4.25%).
In set 5, test item caused a statistically significant increase (P<0.01) in the number of aberrant cells at the highest dose level, 1000 μg/mL, but only excluding gap damage. This increase, to 3.0%, lies inside the upper 95% limit of the historical control range of (4.5%).
No statistically significant increases in the proportion of polyploid cells were observed in either treatment set.


HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
See “Appendix 1 Historical control data” in attached background material

Conclusions:

The test item has shown evidence of clastogenic but not polyploidy-inducing activity in this in vitro cytogenetic test system.

Executive summary:

 The test item was tested to determine whether it would cause chromosomal aberrations or polyploidy in human lymphocytes cultured in vitro according to OECD 473. Two sets of tests were carried out. The first test used a three hour culture treatment, with and without S9 mix, with a harvest at 24 hours (sets 1 and 2, respectively). The second test used 24 and 48 hour continuous treatments without S9 mix, and the test with S9 mix was repeated (set 3, 4 and 5, respectively). Cultures were treated with a range of dose levels, the solvent control and positive control compounds.

Cultures in all treatment sets were analysed for structural chromosomal aberrations and for the incidence of polyploidy.

The test item caused statistically significant increases in the proportion of aberrant cells in all five sets. Some of these increases lay outside the upper 95% limit ofthe historical control range. No statistically significant increases in the proportion of polyploid cells were observed in either treatment set.

It is concluded that the test item has shown evidence of clastogenic but not polyploidy-inducing activity in this in vitro cytogenetic test system.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 1997-07-31 to 1997-08-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Batch No.: 9706-1A
Purity: 98.65 %

Target gene:

histidine dependent and tryptophan dependent

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S9.

Test concentrations with justification for top dose:

First main test with metabolic activation: 313 - 5.000 micrograms/plate
First main test without metabolic activation: 313 - 5.000 micrograms/plate
Second main test with metabolic activation: 50 - 5,000 micrograms/plate
Second main test without metabolic activation: 50 - 5,000 micrograms/plate

Vehicle / solvent:

Dimethyl sulphoxide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate for Preliminary (range-finding) test, triplicate for Main mutation tests
- Number of independent experiments : Preliminary (range-finding) test, Main mutation tests (First test ans second test)

METHOD OF TREATMENT/ EXPOSURE:
- Preliminary (range-finding) test:
Aliquots (0.1 mL) of each concentration of test item were placed in sterile glass tubes. Where appropriate, 0.5 mL rat liver microsomal preparation (S9 mix) or 0.1 M sodium phosphate buffer was added to each tube. Molten top-agar (2 mL) containing 0.5mM histidine/0.5mM biotin or 0.5mM tryptophan, maintained at 45°C, and bacterial culture (0.1 mL) were then added, the tubes were inverted to mix thoroughly and the contents poured onto plates containing solidified minimal medium (25 mL).
Further plates were prepared without the inclusion of the test organisms to verify the sterility of the S9 mix and the test material. A control series of plates was prepared to confirm the inability of DMSO (0.1 mL) to induce reversion in the bacterial strains, and to provide a measure of the spontaneous mutation rates.
- First test:
Same as Preliminary (range-finding) test
- Second test:
Aliquots (0.1 mL) of each concentration of test item were placed in sterile glass tubes, followed by 0.5 mL rat liver microsomal preparation (S9 mix) or 0.1 M sodium phosphate buffer where appropriate. Bacterial culture (0.1 mL) was then added and the tubes were mixed by inversion. The mixtures were pre-incubated for 30 minutes at 37°C with shaking prior to addition of molten top-agar (2 mL) containing 0.5mM histidine/0.5mM biotin or O.5mM tryptophan, maintained at 45 °C. The tubes were again inverted to mix thoroughly and the contents poured onto plates containing solidified minimal medium (25 mL).
Further plates were prepared without the inclusion of the test organisms to verify the sterility of the S9 mix and the test material. A control series of plates was prepared to confirm the inability of DMSO (0.1 mL) to induce reversion in the bacterial strains, and to provide a measure of the spontaneous mutation rates.


TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3 days


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- After incubation, numbers of revertant colonies were counted with an automated colony counter. Total colonies on nutrient plates were counted in the same way. The minimal plates were also examined for the presence of a background lawn of non-revertant colonies.

Evaluation criteria:

For a test to be considered valid the mean of the solvent/vehicle control revertant colony numbers for each strain should lie in the range stated in the appropriate Standard Operating Procedure. These ranges are based on the laboratory's historical control values. Also, the positive control compounds must cause at least a doubling of mean revertant colony numbers over the negative control.
The mean number of revertant colonies for all treatment groups were compared with those obtained for the solvent/vehicle control groups. The mutagenic activity of a test substance was assessed by applying the following criteria:
a) If treatment with a test substance produces an increase in revertant colony numbers of at least twice the concurrent solvent/vehicle controls, with some evidence of a positive dose-relationship, in two separate experiments, with any bacterial strain either in the presence or absence of S9 mix, it is considered to show evidence of mutagenic activity in this test system. No statistical analysis is performed.
b) If treatment with a test substance does not produce reproducible increases of at least 1.5 times the concurrent solvent/vehicle controls in either mutation test it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.
c) If the results obtained fail to satisfy the criteria for a clear “positive" or "negative" response given in paragraphs a) and b), even after the additional testing outlined in the mutation test procedure, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers.

Statistics:

The statistical procedures used are those described by Mahon er al (1989) and are usually analysis of variance followed by Dunnett's test.

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

- Sterility checks, spontaneous reversion rate and viability checks:
The absence of colonies on test item and S9 mix sterility check plates indicates that these preparations were free of microbial contamination.
The total colony counts on plates numbered 17 confirmed the viability and high cell density of the cultures of the individual organisms. The counts recorded on appropriate negative control plates confirmed the characteristically low spontaneous reversion rates of the tester strains and the absence of effects on these rates of DMSO inclusion.
- Mutagenic activity of positive control chemicals:
Appropriate positive control chemicals (with S9 mix where required) induced marked increases in revertant colony numbers with all strains, confirming sensitivity of the cultures and activity of the S9 mix.

PRELIMINARY (RANGE-FINDING) TEST
No increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to test item at concentrations from 20 to 5000 μg/plate in either the presence or absence of S9 mix.
No visible thinning ofthe background lawn of non-revertant cells was obtained following exposure to test item. A top exposure concentration of 5000 μg/plate was therefore selected for use in the main mutation tests.

MAIN MUTATION TESTS
No increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to test item at concentrations from 313 to 5000 μg/plate (first main test) or 50 to 5000 μg/plate (second main test) in either the presence or absence of S9 mix.

Conclusions:

The test material did not exhibit any mutagenic activity under the conditions of test.

Executive summary:

The substance was examined for mutagenic activity in four histidine dependent auxotrophs of Salmonella typhimurium, strains TA98, TA100, TA1535 and TA1537, and one tryptophan dependent auxotroph of Escherichia coli, strain WP2uvrA, using pour-plate tests in compliance with OECD 471. The tests, which were conducted in the presence and absence of an activating system derived from rat liver (S9 mix), employed a range of concentrations of test item up to 5000μg/plate. All tests included solvent (dimethyl sulphoxide) controls with and without S9 mix.

No increases in reversion to prototrophy were obtained with any of the five bacterial strains at the concentrations tested, either in the presence or absence of S9 mix.

It was concluded that the test material did not exhibit any mutagenic activity under the conditions of test.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

One in vivo mammalian somatic cell study is available.

The effect of test item on chromosome structure in bone marrow cells was investigated following acute oral administration to mice based on OECD474. Under the conditions of test, there was no evidence of induced chromosomal or other damage leading to micronucleus formation in immature erythrocytes of treated mice 24 or 48 hours after oral administration of test item.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 1997-12-03 to 1998-01-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

Batch No.: 9706-1A
Purity: 98.65 %

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories (UK)
- Age at study initiation:41 days
- Weight at study initiation: 22.3-33.5 g
- Assigned to test groups randomly: yes, allocated non-selectively
- Housing: housed in clear plastic cages with stainless steel tops in a limited-access animal holding room.
- Diet: No. 1 maintenance diet (SQC grade obtained from Special Diets Services Ltd.), ad libitum
- Water: ad libitum
- Acclimation period: at least four days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 45-60
- Air changes: The animals were held in a room kept at positive pressure with respect to the outside. The room has its own supply of filtered，fresh air which is passed to atmosphere and not recirculated.
- Photoperiod: 12 hrs dark / 12 hrs light

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: The test material can form a doseable and apparently miscible suspension in corn oil at a concentration of approximately 100 mg/mL.
- Concentration of test material in vehicle: 20, 50, 100 mg/mL
- Amount of vehicle (if gavage or dermal): 20 mL/kg

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
`Doses were formulated in glass vessels, by injection of the test material through a septum cap using a syringe and needle and the addition of the appropriate volume of com oil, also by injection. Consequently, dosing suspensions were freshly prepared in com oil on the day of dosing, each concentration being individually formulated, and mixed prior to use.

Duration of treatment / exposure:

24/48 hours

Frequency of treatment:

one occasion

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 males and 10 females for control and 2000 mg/kg group.
5 males and 5 females for positive control and 500, 1000 mg/kg group.

Control animals:

yes, concurrent vehicle

Positive control(s):

Mitomycin C
- Route of administration: oral gavage
- Doses / concentrations: 12 mg/kg in sterile water

Tissues and cell types examined:

femurs, Marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Based on the preliminary toxicity test results, the highest dosage selected for the main micronucleus test was 2000 mg/kg.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Five male and five female mice per group were killed 24 hours after treatment; further lots of five males and five females from groups 1 and 4 were scheduled to be killed 48
hours after treatment.

DETAILS OF SLIDE PREPARATION:
Animals were killed by cervical dislocation following carbon dioxide inhalation. For the main micronucleus test, femurs from each animal were rapidly dissected out and cleaned of adherent tissue. The proximal head was cut off to obtain access to the marrow canal. Marrow cells were flushed out with 2 mL pre-filtered foetal calf serum using a syringe and needle. The recovered cells were centrifuged at 1000 rpm for five minutes. The supernatant fluid was discarded and the cell pellet resuspended in a small volume of fresh foetal calf serum. Single drops of the cell suspension were transferred to clean, dry slides, three smears were prepared for each animal, and the slides left to air-dry. Following fixation in methanol for ten minutes, they were stained manually, using 5% Giemsa stain (prepared in Sorensen's buffer: pH 6.8) for 20 minutes. After staining, slides were washed in buffer, allowed to air-dry and coverslips were applied using DPX mountant.

METHOD OF ANALYSIS:
Slides were examined under the light microscope, and regions judged to be of adequate quality to permit scoring were selected under low magnification. At high magnification (x 100, oil immersion objective), a total of at least 1000 erythrocytes per animal was examined. Each erythrocyte scored was classed as immature (polychromatic) or mature: immature cells stain blue/pink and the older (mature) cells stain red/pink. Scoring was continued until a total of at least 2000 immature erythrocytes had been examined for the presence or absence of micronuclei. When examination had been completed, the data were decoded.
The frequencies of micronucleated cells per 1000 immature erythrocytes were then calculated as an index of induced genetic damage. The incidence of micronuclei in the mature cell population 24 hours after treatment reflects the pretreatment situation, since most of these cells were produced before treatment. The proportion of immature to mature cells was also determined; a decrease in this ratio may indicate inhibition of cell division following treatment.

Evaluation criteria:

The test material will be considered to have caused clastogenic, or other damage leading to micronucleus production in this study if the following conditions are met:
- statistically significant increases in the frequency of micronucleated polychromatic erythrocytes are observed at one or more dose levels in one or both sexes
- the increases are reproducible between animals of the same sex in the treatment group(s) - the increases exceed the historical negative control range at this laboratory
- evidence of a dose-response relationship, increases in both sexes, or increases at more than one sacrifice time will be considered to support the conclusion.

Statistics:

For incidences of micronucleated immature erythrocytes, exact one sided p-values are calculated by permutation (StatXact, CYTEL Software Corporation, Cambridge, Massachusetts). Comparison of several dose levels are made with the concurrent control using the Linear by Linear Association test for trend in a step-down fashion if significance is detected; for individual inter-group comparisons (ie the positive control group) this procedure simplifies to a straightforward permutation test. For assessment of effects on the proportion of immature erythrocytes, equivalent permutation tests based on rank scores are used, ie exact versions of Wilcoxon's sum of ranks test and Jonckheere's test for trend.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 250 - 2000 mg/kg
- Solubility: In all cases the test item formed a miscible suspension in corn oil
- Clinical signs of toxicity in test animals: All animals survived to scheduled termination. No adverse reactions to treatment were recorded for any group of animals dosed with test item. Incidences of weight loss were noted but these were small and not dose-related.

RESULTS OF DEFINITIVE STUDY
- Reactions to treatment:
Incidences of weight loss were noted throughout the study but these were small and were not dose-related.
Three mice treated with test item at 500 mg/kg showed signs including piloerection, underactivity, hunched posture and partially closed eyelids approximately 2 hours after dosing and these continued until scheduled termination. At 1000 mg/kg all five males showed signs of hunched posture, immediately after dosing but had recovered 18 hours later. Two females dosed at this concentration showed signs of piloerection, underactivity and hunched posture two hours after dosing which continued until scheduled termination.
All ten males dosed with test item at 2000 mg/kg showed signs including piloerection, underactivity and hunched posture immediately after dosing and continued until scheduled termination. Two females dosed at this concentration showed signs of piloerection, underactivity and hunched posture post-dose which continued until scheduled termination 24 hours later. One of these female mice also had swelling around the left front forelimb approximately 18 hours after dosing until termination.
One other female treated at 2000 mg/kg and scheduled for termination at the 48 hour timepoint showed signs of piloerection, hunched posture and swelling around the right front forelimb and was found dead approximately 42 hours after dosing. Macroscopic post mortem of this animal showed no signs of misdosing and the data from this animal was excluded from the statistical analysis.
- Incidence of micronucleated cells:
No large differences in the frequencies of micronucleated immature cells were seen between sexes, in any group.
There were no statistically significant increases in the frequency of micronucleated immature erythrocytes in groups treated with test item, compared to the vehicle control values, at any termination time (p>0.01 in each case).
The proportion of immature erythrocytes for all groups treated with test item were similar to corresponding vehicle control group values at each sacrifice time.
Treatment with Mitomycin C produced a large, statistically significant increase in the frequency of micronucleated immature erythrocytes (p<0.001). This increase after treatment with a known mutagen demonstrates the sensitivity of the test system.

Conclusions:

Under the conditions of test, there was no evidence of induced chromosomal or other damage leading to micronucleus formation in immature erythrocytes of treated mice 24 or 48 hours after oral administration of test item

Executive summary:

The effect of test item on chromosome structure in bone marrow cells was investigated following acute oral administration to mice based on OECD474. Chromosome damage was measured indirectly by counting micronuclei.

Male and female mice were given a single dose of test item at 500, 1000 or 2000 mg/kg. In all cases the test item formed a miscible suspension in corn oil and was dosed by oral gavage.

Incidences of weight loss were noted throughout the study but these were small and were not dose-related.

Three mice treated with test item at 500 mg/kg showed signs including piloerection, underactivity, hunched posture and partially closed eyelids approximately 2 hours after dosing and these continued until scheduled termination. At 1000 mg/kg all five males showed signs of hunched posture, immediately after dosing but had recovered 18 hours later. Two females dosed at this concentration showed signs of piloerection, underactivity and hunched posture two hours after dosing which continued until scheduled termination.

All ten males dosed with test item at 2000 mg/kg showed signs including piloerection, underactivity and hunched posture immediately after dosing and continued until scheduled termination. Two females dosed at this concentration showed signs of piloerection, underactivity and hunched posture post-dose which continued until scheduled termination 24 hours later. One of these female mice also had swelling around the left front forelimb approximately 18 hours after dosing until termination.

One other female treated at 2000 mg/kg and scheduled for termination at the 48 hour timepoint showed signs of piloerection, hunched posture and swelling around the right front forelimb and was found dead approximately 42 hours after dosing. Macroscopic post mortem of this animal showed no signs of misdosing and the data from this animal was excluded from the statistical analysis.

No large differences in the frequencies of micronucleated immature cells were seen between sexes, in any group.

There were no statistically significant increases in the frequency of micronucleated immature erythrocytes in groups treated with test item, compared to the vehicle control values, at any termination time (p>0.01 in each case).

The proportion of immature erythrocytes for all groups treated with test item were similar to corresponding vehicle control group values at each sacrifice time.

It is concluded that, under the conditions of test, there was no evidence of induced chromosomal or other damage leading to micronucleus formation in immature erythrocytes of treated mice 24 or 48 hours after oral administration of test item.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Data:

Ames study: OECD 471, negative

In Vitro Mammalian Chromosome Aberration Test: OECD 473, positive

In vivo mammalian somatic cell study: OECD 474, negative

Therefore in accordance with Regulation (EC) No. 1272/2008 Table 3.5.1, the test substance should not be classified as germ cell mutagens.