N-[[3,5-dichloro-2-fluoro-4-(1,1,2,3,3,3-hexafluoropropoxy)phenyl]carbamoyl]-2,6-difluorobenzamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial Reverse Mutation Assay

Under the conditions of the test, the test material did not cause a positive increase in the mean number of revertants per plate with any of the tester strains either in the presence or absence of microsomal enzymes prepared from Aroclor™ induced rat liver (S9). The test material is therefore determined to be non-mutagenic in this assay.

Mammalian Chromosome Aberration Assay

The incidence of chromosomal abnormalities was determined from cultures treated with 0, 25, 100, and 200 µg/mL in the absence and presence of S-9. Statistical analyses of the data did not identify a significant difference in the frequencies of cells with aberrations between the negative control and any of the treated cultures with or without S-9 activation. Cultures treated with the positive control chemicals (i.e., mitomycin C without S-9 and cyclophosphamide with S-9) had significantly higher incidences of abnormal cells in all assays. Therefore the test material was considered to be negative in the in vitro chromosomal aberration assay utilizing rat lymphocytes.

Mammalian Gene Mutation Assay

Under the conditions of the test, based upon the frequency of TGr mutants recovered in cultures treated with the test material, the test material did not induce a mutagenic response.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 February 2001 - 02 March 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

Japan MAFF Revised Mutagenicity Guidelines, 2000

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine and tryptophan loci.

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Type and identity of media: The broth used to grow overnight cultures of the tester strains was Vogel- Bonner salt solution (Vogel and Bonner, 1956) supplemented with 2.5 % (w/v) Oxoid Nutrient Broth No. 2 (dry powder).
Bottom agar (25 mL per 15 x 100 mm petri dish) was Vogel- Bonner minimal medium E (Vogel and Bonner, 1956), supplemented with 1.5 % (w/v) agar and 0.2 % (w/v) glucose.
Top (overlay) agar was prepared with 0.7 % agar (w/v) and 0.5 % NaCl (w/v) and was supplemented with 10 mL of 1) 0.5 mM histidine/biotin solution per 100 mL agar for selection of histidine revertants, or 2) 0.5 mM tryptophan solution per 100 mL of agar for selection of tryptophan revertants. For an agar overlay, 2.0 mL of the supplemented top agar was used.
- Properly maintained: yes
- Periodically "cleansed" against high spontaneous background: yes
- Periodically checked for genotype: Tester strain cultures were checked for the following genetic markers on the day of their use in the mutagenicity assay; rfa Wall Mutation and pKM101 Plasmid.

Additional strain / cell type characteristics:

other: All Salmonella typhimurium strains; uvrB and rfa. Salmonella typhimurium stains TA98 and TA100; pKM101. Escherichia coli WP2; uvrA.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Dose range finding assay: 6.67, 10, 33.3, 66.7, 100, 333, 667, 1000, 3330 and 5000 µg/plate
Mutagenicity assay: 33.3, 100 333, 1000, 3330 and 5000 µg/plate

Vehicle / solvent:

- Vehicle used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-aminoanthracene, ICR-191

Details on test system and experimental conditions:

Two independent assays were performed, an initial mutagenicity assay followed by a confirmation assay.

METHOD OF APPLICATION: preincubation method. S9 mix (or phosphate buffer, where appropriate), the tester strain, and the test article were preincubated prior to the addition of molten agar. The agar and the preincubation reaction mixture were mixed and then overlaid onto a minimal agar plate.

DURATION
- Preincubation period: 20 ± 2 minutes at 37 ± 2 °C.
- Exposure duration: 52 ± 4 hours at 37 ± 2 °C.

NUMBER OF REPLICATIONS: All concentrations of the test article, the vehicle controls and the positive controls were plated in triplicate.

REVERTANT COLONY COUNTING: The number of revertant colonies per plate for the vehicle controls and all plates containing test article were counted manually. The number of revertant colonies per plate for the positive controls were counted by automated colony counter.

DETERMINATION OF CYTOTOXICITY
- Method: Evidence of cytotoxicity was scored relative to the vehicle control plate and was recorded along with the revertant counts for all plates at that concentration level. Lawns were scored as 1) normal, 2) slightly reduced, 3) moderately reduced, 4) extremely reduced, 5) absent, or 6) obscured by precipitate.

Evaluation criteria:

- Tester Strain TA100: For a test material to be considered positive, it had to produce at least a 2-fold dose related and reproducible increase in the mean revertants per plate over the mean revertants per plate of the appropriate vehicle control. A response that did not meet all three of the above criteria (magnitude, dose-responsiveness, reproducibility) was not evaluated as positive.
- Tester Strains TA98, TA1535, TA1537, and WP2uvrA: For a test material to be considered positive, it had to produce at least a 3-fold dose related and reproducible increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. A response that did not meet all three of the above criteria (magnitude, dose-responsiveness, reproducibility) was not evaluated as positive.

Key result

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:

valid

Positive controls validity:

valid

Key result

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:

valid

Positive controls validity:

valid

Key result

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:

valid

Positive controls validity:

valid

Key result

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:

not examined

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Key result

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:

valid

Positive controls validity:

valid

Additional information on results:

Dose Rangefinding Assay
Cytotoxicity was observed with tester strain TA100 in the absence of S9 mix at the maximum concentration tested (5000 µg per plate) as evidenced by a concentration-related decrease in the number of revertants per plate. No cytotoxicity was observed with either tester strain TA100 in the presence of S9 mix, as evidenced by no concentration-related decrease in the number of revertants per plate and a normal bacterial background lawn. No cytotoxicity was observed with tester strain WP2uvrA in the presence or absence of S9 mix, as evidenced by no concentration-related decrease in the number of revertants per plate and a normal bacterial background lawn.

Mutagenicity Assay
In the initial mutagenicity assay, and in the confirmatory assay, all data were acceptable and no positive increases in the mean number of revertants per plate were observed with any of the tester strains in either the presence or absence of S9 mix.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1: Mutagenicity Assay – Summary

Substance	Concentration µg/plate	Tester Strain										Background Lawn*
		TA98		TA100		TA1535		TA1537		WP2uvrA
		Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
With External Metabolic Activation System S9 mix
Vehicle Control		27	8	111	8	18	3	9	2	25	5	1
Test Material	33.3	26	7	91	4	14	2	8	4	34	5	1
	100	29	7	109	7	13	2	11	5	26	3	1
	333	32	4	98	5	16	2	9	1	27	2	1sp
	1000	31	4	106	5	13	2	8	2	23	4	1sp
	3330	28	3	104	17	12	2	9	4	22	7	1sp
	5000	27	5	97	19	12	2	7	3	24	6	1mp
Positive Control**		373	32	809	69	136	7	116	13	422	78	1
Without External Metabolic Activation
Vehicle Control		18	7	105	3	18	6	7	2	28	2	1
Test Material	33.3	21	1	101	12	13	6	7	3	23	4	1
	100	23	11	95	6	13	6	10	5	26	5	1
	333	18	5	100	11	13	0	6	4	28	6	1sp
	1000	17	7	88	8	12	4	6	1	21	6	1mp
	3330	12	3	98	3	9	1	4	2	16	4	1mp
	5000	13	0	86	16	13	1	6	1	18	4	1mp
Positive Control***		367	32	687	42	559	20	1170	63	545	28	1

 

Table 2: Confirmation Mutagenicity Assay – Summary

Substance	Concentration µg/plate	Tester Strain										Background Lawn*
		TA98		TA100		TA1535		TA1537		WP2uvrA
		Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
With External Metabolic Activation System S9 mix
Vehicle Control		33	4	108	7	14	3	11	1	23	4	1
Test Material	33.3	33	2	123	5	10	5	8	1	22	2	1
	100	31	3	115	17	13	3	9	5	22	7	1
	333	30	8	108	10	14	2	9	2	29	4	1sp
	1000	38	7	113	10	13	1	4	1	22	4	1sp
	3330	27	3	118	12	11	3	6	2	15	8	1mp
	5000	31	5	128	5	13	2	7	3	13	2	1mp
Positive Control**		371	52	1016	32	121	2	117	15	494	28	1
Without External Metabolic Activation
Vehicle Control		17	7	106	3	13	6	7	3	17	6	1
Test Material	33.3	27	4	116	14	13	2	4	1	13	6	1
	100	24	3	105	1	11	3	6	3	24	11	1
	333	21	4	101	7	14	3	5	1	21	3	1sp
	1000	22	10	103	6	10	2	6	2	17	2	1sp
	3330	23	7	107	6	14	5	5	1	11	3	1mp
	5000	17	5	111	5	11	1	6	5	13	5	1mp
Positive Control***		396	21	916	25	640	19	1828	19	351	23	1

 

Vehicle control DMSO 50 µL

** TA98, benzo[a]pyrene 2.5 µg/plate

TA100, 2-aminoanthracene 2.5 µg/plate

TA1535, 2-aminoanthracene 2.5 µg/plate

TA1537, 2-aminoanthracene 2.5 µg/plate

WP2uvrA, 2-aminoanthracene 25.0 µg/plate

 

*** TA98, 2-nitrofluorene 1.0 µg/plate

TA100, sodium azide 2.0 µg/plate

TA1535, sodium azide 2.0 µg/plate

TA1537, ICR-191 2.0 µg/plate

WP2uvrA, 4-nitroquinoline-N-oxide 0.4 µg/plate

 

* Background Lawn Evaluation Codes:

1 = normal; 2 = slightly reduced; 3 = moderately reduced; 4 = extremely reduced; 5 = absent; 6 = obscured by precipitate

sp = slight precipitate; mp = moderate precipitate (requires hand count); hp = heavy precipitate (requires hand count)

Conclusions:

Interpretation of results:
negative with and without metabolic activation

Under the conditions of the test, the test material did not cause a positive increase in the mean number of revertants per plate with any of the tester strains either in the presence or absence of S9 mix. The test material is therefore determined to be non-mutagenic in this assay.

Executive summary:

The genetic toxicity of the test material was determined in an in vitro bacterial reverse mutation assay, an ames test. The study was performed under GLP conditions and in accordance with the standardised guidelines OECD 471, EPA OPPTS 870.5100 and Japan MAFF Revised Mutagenicity Guidelines.

The concentrations tested in the mutagenicity assay were selected based on the results of a dose range finding assay using tester strains TA100 and WP2uvrA and ten concentrations of test article ranging from 6.67 to 5000 µg per plate.

The tester strain used in the mutagenicity assay were Salmonella typhimurium tester strains TA98, TA100, TA 1535, and TA1537 and Escherichia coli tester strain WP2uvrA. The assay was conducted with six concentration levels of test article in both the presence and absence of S9 mix along with concurrent vehicle and positive controls using three plates per concentration. The concentrations tested were 33.3, 100, 333, 1000, 3330, and 5000 µg per plate in both the presence and absence of S9 mix. The results of the initial mutagenicity assay were confirmed in the independent experiment.

Under the conditions of the test, the test material did not cause a positive increase in the mean number of revertants per plate with any of the tester strains either in the presence or absence of microsomal enzymes prepared from Aroclor™ induced rat liver (S9). The test material is therefore determined to be non-mutagenic in this assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiation 24 April 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: rat

Details on mammalian cell type (if applicable):

- Lymphocyte cultures: Blood samples were collected by cardiac puncture from male Sprague-Dawley rats, following euthanasia with carbon dioxide, from two rats in Assay A1, two rats in Assay A2, and three rats in Assay B1. In each assay, blood samples from individual rats were pooled and whole blood cultures were set up in RPMI 1640 medium (with 25 mM HEPES) supplemented with 10 % heat-inactivated foetal bovine serum, antibiotics and antimycotics (Fungizone 0.25 µg/mL; penicillin G, 100 ug/mL; and streptomycin sulfate, 0.1 mg/mL), 20 µg/mL PHA, and an additional 2 mM L-glutamine. Cultures were initiated by inoculating approximately 0.5 mL of whole blood/5 mL of culture medium. Cultures were set up in duplicate at each dose level in T-25 plastic tissue culture flasks and incubated at 37 °C.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

0, 3.13, 6.25, 12.5, 25, 50, 100, and 200 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium. Two independent assays were performed; these were denoted as assay A and assay B.

- Treatment procedure without metabolic activation: Approximately 48 hours after initiation of the cultures (Assay A1 and A2), the cell suspension was dispensed into 15 mL sterile centrifuge tubes (approximately 5.5 mL/tube, 2 cultures per dose level). The cells were sedimented by centrifugation and the culture medium removed and saved. The cells were exposed to medium (RPMI 1640, HEPES, and antibiotics) containing the test or positive or negative control treatments for approximately 4 hr at 37 °C and the exposure was terminated by washing the cells with culture medium. The cells were then placed in individual sterile disposable tissue culture flasks (T-25) along with approximately 4.5 mL of the original culture medium until the time of harvest. The cultures were harvested at approximately 24 hr after treatment initiation (i.e., approximately 20 hr after treatment termination).
Assay B1 was conducted with continuous treatment with the test material for 24 hr (approximately 1.5 normal cell cycle lengths). The treatments were added directly to the culture flasks at 48 hr after initiation of the cultures and these cultures were harvested 24 hr later.

- Treatment procedure with metabolic activation: Approximately 48 hr after initiation of the cultures, the cell suspension was dispensed into sterile disposable centrifuge tubes. The cells were sedimented by centrifugation and the culture medium removed and saved. The cells were exposed to medium (RPMI 1640, HEPES, antibiotics, and S-9) containing the test and positive and negative control treatments for approximately 4 hr at 37 °C and the exposure was terminated by washing the cells with culture medium (without serum and PHA).
The cells were then placed in individual sterile disposable tissue culture flasks (T-25) along with approximately 4.5 mL of the original culture media until the time of harvest. In both Assay A1 and B1, the cultures were harvested approximately 24 hr after treatment initiation (i.e., approximately 20 hr after treatment termination).

DURATION
- Exposure duration:
> Assay A with S9 mix: 4 hours
> Assay A without S9 mix: 4 hours
> Assay B with S9 mix: 4 hours
> Assay B without S9 mix: 24 hours, continuous treatment.
- Expression time (cells in growth medium):
> Assay A with S9 mix: 20 hours
> Assay A without S9 mix: 20 hours
> Assay B with S9 mix: 20 hours
> Assay B without S9 mix: none, continuous treatment.
- Fixation time (start of exposure up to fixation or harvest of cells):
> Assay A with S9 mix: 24 hours
> Assay A without S9 mix: 24 hours
> Assay B with S9 mix: 24 hours
> Assay B without S9 mix: 24 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid was added approximately 3 hr prior to harvest at a final concentration of 0.2 µg/mL.
STAIN (for cytogenetic assays): Giemsa.

NUMBER OF REPLICATIONS: Two.

NUMBER OF CELLS EVALUATED: 100 metaphases/replicate were examined, where possible, from each selected concentration of the test material and the negative controls (a total of 200 cells/treatment) for structural abnormalities.
In the positive control cultures, 50 metaphases/replicate (a total of 100 cells/treatment) were examined for abnormalities.
Based on the mitotic indices of the cultures, three concentrations (25, 100 and 200 µg/mL) were selected for the determination of chromosomal aberration frequencies and polyploidy.

DETERMINATION OF CYTOTOXICITY
- Method: Mitotic indices were determined as the number of cells in metaphase among 1000 cells/replicate and expressed as percentages.

SCORING
The microscopic coordinates of those metaphases containing aberrations were recorded. Only those metaphases that contained 42 ± 2 centromeres were scored with the exception of cells with multiple aberrations, in which case accurate counts of the chromosomes were not always possible. Those cells having five or more aberrations/cell were classified as cells with multiple aberrations. Gaps were not included in calculations of total cytogenetic aberrations.
The data was used to calculate the following parameters:
% Cells with aberrations: [Aberrant cells (excluding cells with gaps only)/ No. Metaphases evaluated] x 100
Aberrations/100 cells: [Total aberration (excluding gaps, miscellaneous and severely damaged)/ No. Metaphases evaluated] x 100

OTHER EXAMINATIONS:
- Determination of polyploidy: 100 metaphases/replicate were examined for the incidence of polyploidy.

Evaluation criteria:

- Validity: For a test to be acceptable, the chromosomal aberration frequency in the positive control cultures should be significantly higher than the negative controls. The aberration frequency in the negative control should be within reasonable limits of the laboratory historical values.
- Evaluation: A test chemical is considered positive in this assay if it induces a significant dose-related and reproducible increase in the frequency of cells with aberrations.

Statistics:

The proportion of cells with aberrations (excluding gaps) was compared by the following statistical methods. At each dose level, data from the replicates were pooled. A two-way contingency table was constructed to analyse the frequencies of cytogenetic abnormalities. An overall Chi-square statistic, based on the table, was partitioned into components of interest. Specifically, statistics were generated to test the two global hypotheses of (1) no differences in average number of cells with aberrations among the dose groups, and (2) no linear trend of increasing number of cells with aberrations with increasing dose (Armitage, 1971). An ordinal metric (0, 1, 2, etc.) was used for the doses in the statistical evaluation. If either statistic was found to be significant at α= 0.05 versus a one-sided increasing alternative, pairwise tests (i.e., control vs. treatment) were performed at each dose level and evaluated at α= 0.05 again versus a one-sided alternative.
Polyploid cells were analysed by the Fisher Exact probability test (Siegel, 1956). The number of polyploid cells was pooled across replicates for the analysis and evaluated at α= 0.05. The data was analysed separately based on the presence or absence of S-9 and based on the exposure time.

Key result

Species / strain:

lymphocytes: rat

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Assay A1
The highest concentration evaluated exceeded the limit of solubility in culture medium. Examination of negative control slides revealed that there were reduced numbers of cells present with little to no metaphase spreads available. Therefore, due to the lack of adequate numbers of mitosis available for scoring, this assay was repeated (Assay A2). Analytical verification of the stock solutions in this assay was not performed since the assay was to be repeated.

Assay A2
There was little to no toxicity in any of the treated cultures either with or without metabolic activation as evaluated by the Relative Mitotic Index (RMI). Without metabolic activation, the RMI varied from 66.9 % at the highest concentration of 200 µg/mL to 83.7 % at the lowest concentration. In the presence of S-9 activation, the RMI was 91.0 % at 200 µg/mL and 107.7 % at 3.13 µg/mL. Based upon these results, cultures treated with 25, 100 and 200 µg/mL were chosen for the determination of chromosomal aberration frequencies and incidence of polyploidy both in the presence and absence of S-9 activation.
Among the cultures treated with the positive control chemicals, 0.5 µg/mL of MMC and 6 µg/mL of CP were selected for evaluation of aberrations in the absence and presence of S-9, respectively.
There were no significant increases in the incidence of polyploid cells in the test material treated cultures as compared to the negative control values.
In the non-activation assay, the frequency of cells with aberrations in the negative control was 0.5 % and the corresponding values at treatment levels 25, 100 and 200 µg/mL were 0, 1.5 and 1.0 %, respectively. In the activation assay, cultures treated with the test material at concentrations of 25, 100 and 200 µg/mL had aberrant cell frequencies of 0, 0.5, and 1 %, respectively, as compared to the negative control value of 0 %. Statistical analyses of these data did not identify significant differences between the negative control and any of the treated cultures either with or without S-9 activation. The frequencies of aberrant cells observed in the test material treated cultures were within the laboratory historical background range.
Significant increases in the frequency of cells with aberrations were observed in cultures treated with the positive control chemicals. Aberrant cell frequencies in MMC (without S-9) and CP (with S-9) treated cultures were 27 % and 21 %, respectively.

Assay B1
Without metabolic activation, the RMI varied from 60.5 to 97.4 %. The RMI was 65.8 % at 200 µg/mL and 97.4 % at 3.13 µg/mL. In the presence of S-9 activation, the RMI was 76.2 % at 200 µg/mL and 95.2 % at 12.5 µg/mL. Based upon these results, cultures treated with 25, 100 and 200 µg/mL in the absence and presence of S-9 were selected for determining the chromosomal aberration frequencies.
Among the cultures treated with the positive control chemicals, 0.075 µg/mL of MMC and 6 µg/mL of CP were selected for the evaluation of aberrations.
There were no significant increases in the incidence of polyploid cells in test material treated cultures as compared to the negative control values.
In the non-activation assay, the frequency of cells with aberrations in the negative control was 0 % and the corresponding values at treatment levels 25, 100, and 200 µg/mL were 1.0, 1.0, and 0 %, respectively. In the activation assay, cultures treated with the test material at concentrations of 25, 100 and 200 µg/mL had aberrant cell frequencies of 0, 2.0, and 1.0 %, respectively as compared to the negative control value of 0.5 %. Statistical analyses of these data did not identify significant differences between the negative control and any of the treated cultures either with or without S-9 activation. The frequencies of aberrant cells observed in the test material treated cultures were within the laboratory historical background range.
Significant increases in the frequency of cells with aberrations were observed in cultures treated with the positive control chemicals. Aberrant cell frequencies in MMC (without S-9) and CP (with S-9) treated cultures were 23 % and 23 %, respectively.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Analytical Verification of Test Concentrations

The analytically detected concentrations of the test material in the stock solutions varied from 94 to 110 % of the target.

Table 1: Mitotic Indices and Incidence of Polyploidy in Assay A2: 4 Hours After Treatment With and Without Metabolic Activation

Substance	Dose (µg/mL)	% M.I.				Polyploidy (%)
		With Metabolic Activation		Without Metabolic Activation		With Metabolic Activation	Without Metabolic Activation
		Average of Replicates A & B	RMI (%)	Average of Replicates A & B	RMI (%)	Average of Replicates A & B	Average of Replicates A & B
Negative Control*		15.6	100.0	16.6	100.0	0.0	0.0
Test Material	3.13	16.8	107.7	13.9	83.7	-	-
	6.25	13.5	86.5	13.7	82.5	-	-
	12.5	14.4	92.3	11.9	71.7	-	-
	25	13.8	88.5	12.5	75.3	0.0	0.0
	50†	12.2	78.2	12.9	77.7	-	-
	100‡	12.0	76.9	11.7	70.5	0.0	0.5
	200‡	14.2	91.0	11.1	66.9	0.0	0.0
Positive Control	0.5**	-	-	6.9	41.6	-	0.0
	4.0***	13.6	87.2	-	-	-	-
	6.0***	9.7	62.2	-	-	0.0	-

* 1 % DMSO; ** MMC; ***CP

† Treatment medium cloudy upon addition of test material

‡ Treatment medium cloudy with precipitate upon addition of the test material

Table 2: Chromosomal Aberration Results in Assay A2: 24 Hours After Treatment Without Metabolic Activation (Replicate A + B)

Substance	Dose (µg/mL)	No. of Cells Evaluated	Total Aberrations Excluding Gaps (%)	No. of Cells with Aberrations (%)	Miscellaneous Aberrations	Cells with Multiple Aberrations, ≥ 5
Negative Control*		200	1 (0.5)	1 (0.5)	0	0
Test Material	25	200	0 (0.0)	0 (0.0)	0	0
	100	200	3 (1.5)	3 (1.5)	0	0
	200	200	2 (1.0)	2 (1.0)	0	0
Positive Control	0.5**	100	49 (49.0)	27 (27.0)†	0	0

* 1 % DMSO; ** MMC

† Significantly (α <0.05) different from the negative control

Table 3: Chromosomal Aberration Results in Assay A2: 24 Hours After Treatment With Metabolic Activation (Replicate A + B)

Substance	Dose (µg/mL)	No. of Cells Evaluated	Total Aberrations Excluding Gaps	No. of Cells with Aberrations	Miscellaneous Aberrations	Cells with Multiple Aberrations, ≥ 5
Negative Control*		200	0 (0.0 %)	0 (0.0 %)	0	0
Test Material	25	200	0 (0.0 %)	0 (0.0 %)	0	0
	100	200	1 (0.5 %)	1 (0.5 %)	0	0
	200	200	2 (1.0 %)	2 (1.0 %)	0	0
Positive Control	6.0***	100	27 (27.0 %)	21 (21.0 %)†	0	0

* 1 % DMSO; *** CP

† Significantly (α <0.05) different from the negative control

Table 4: Mitotic Indices and Incidence of Polyploidy in Assay B1: 4 Hours After Treatment With Metabolic Activation and 24 Hours After Treatment Without Metabolic Activation (Replicate A + B)

Substance	Dose (µg/mL)	% M.I.				Polyploidy (%)
		With Metabolic Activation		Without Metabolic Activation		With Metabolic Activation	Without Metabolic Activation
		Average of Replicates A & B	RMI (%)	Average of Replicates A & B	RMI (%)	Average of Replicates A & B	Average of Replicates A & B
Negative Control*		4.2	100.0	3.8	100.0	0.0	0.0
Test Material	3.13	-	-	3.7	97.4	-	-
	6.25	-	-	3.3	86.8	-	-
	12.5	4.0	95.2	3.5	92.1	-	-
	25	3.5	83.3	3.1	81.6	0.0	0.0
	50	-	-	2.9	73.6	-	-
	100	3.3	78.6	2.3	60.5	0.5	0.0
	200†	3.2	76.2	2.5	65.8	0.0	0.0
Positive Control	0.05**	-	-	1.3	34.2	-	-
	0.075**	-	-	1.2	31.6	-	0.0
	4.0***	3.0	71.4	-	-	-	-
	6.0***	2.7	64.3	-	-	0.0	-

* 1 % DMSO; ** MMC; ***CP

† Treatment medium cloudy upon addition of test material

Table 5: Chromosomal Aberration Results in Assay B1: 24 Hours After Treatment Without Metabolic Activation (Replicate A + B) 

Substance	Dose (µg/mL)	No. of Cells Evaluated	Total Aberrations Excluding Gaps (%)	No. of Cells with Aberrations (%)	Miscellaneous Aberrations	Cells with Multiple Aberrations, ≥ 5
Negative Control*		200	0 (0.0)	0 (0.0)	0	0
Test Material	25	200	2 (1.0)	2 (1.0)	0	0
	100	200	2 (1.0)	2 (1.0)	0	0
	200	200	0 (0.0)	0 (0.0)	0	0
Positive Control	0.075**	100	49 (49.0)	23 (23.0)†	0	1

* 1 % DMSO; **MMC

† Significantly (α <0.05) different from the negative control

Table 6: Chromosomal Aberration Results in Assay B1: 24 Hours After Treatment With Metabolic Activation (Replicate A + B)  

Substance	Dose (µg/mL)	No. of Cells Evaluated	Total Aberrations Excluding Gaps (%)	No. of Cells with Aberrations (%)	Miscellaneous Aberrations	Cells with Multiple Aberrations, ≥ 5
Negative Control*		200	1 (0.5)	1 (0.5)	0	0
Test Material	25	200	0 (0.0)	0 (0.0)	0	0
	100	200	4 (2.0)	4 (2.0)	0	0
	200	200	2 (1.0)	2 (1.0)	0	0
Positive Control	6.0***	100	34 (34.0)	23 (23.0)†	0	0

* 1 % DMSO; ***CP

† Significantly (α <0.05) different from the negative control

Conclusions:

Interpretation of results:
negative with and without metabolic activation

Under the conditions of the test, the test material was considered to be negative in the in vitro chromosomal aberration assay utilising rat lymphocytes.

Executive summary:

The clastogenicity of the test material was determined in an in vitro chromosomal aberration assay utilising rat lymphocytes. The study was performed under GLP conditions and in accordance with the standardised guidelines OECD 473, EPA OPPTS 870.5375, EU Method B.10 and Japan MAFF guidelines for screening mutagenicity testing of chemicals.

Approximately 48 hr after the initiation of whole blood cultures, cells in the absence and presence of S-9 activation were treated for 4 hr with targeted test concentrations of 0 (negative control) to 200 µg/mL of culture medium and harvested 20 hours later. Based upon the mitotic indices, cultures treated with targeted concentrations of 0, 3.13, 6.25, 12.5, 25, 50, 100, and 200 µg/mL in the absence and presence of S-9 activation were selected for determining the incidence of chromosomal aberrations. There were no significant increases in the frequencies of cells with aberrations in this assay. In a confirmatory assay, cultures were treated as above except that the cultures were treated continuously for 24 hr until the time of their harvest in the absence of S-9.

The incidence of chromosomal abnormalities was determined from cultures treated with 0, 25, 100, and 200 µg/mL in the absence and presence of S-9. Statistical analyses of the data did not identify a significant difference in the frequencies of cells with aberrations between the negative control and any of the treated cultures with or without S-9 activation. Cultures treated with the positive control chemicals (i.e., mitomycin C without S-9 and cyclophosphamide with S-9) had significantly higher incidences of abnormal cells in all assays. Therefore the test material was considered to be negative in the in vitro chromosomal aberration assay utilizing rat lymphocytes.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 April 2001 - 26 July 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

Hypoxanthine-guanine-phosphoribosyl transferase (HGPRT)

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Indicator cell line: CHO-K1-BH4
- Type and identity of media:
> Maintained medium: Ham's F-12 nutrient mix supplemented with 5 % (v/v) heat-inactivated (56 °C, 30 min), dialyzed foetal bovine serum, antibiotics and antimycotics (penicillin G, 100 units/mL; streptomycin sulfate, 0.1 mg/mL; fungizone, 0.25 µg/mL) and an additional 2 mM L-glutamine.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes (Selection medium: Ham's F-12 nutrient mix without hypoxanthine, supplemented with 10 µM 6-thioguanine and 5 % serum and the above-mentioned antibiotics.)

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Assay A1 (preliminary toxicity assay): 1.56, 3.13, 6.25, 12.5, 25, 50, 100 and 200 µg/mL (with and without metabolic activation)
Assay B1 (initial mutagenicity assay): 12.5, 25, 50, 100 and 200 µg/mL (with and without metabolic activation)
Assay C1 (confirmatory mutagenicity assay): 6.66, 20, 66.6 and 200 µg/mL (with and without metabolic activation)

Vehicle / solvent:

- Vehicle used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

other: 20-methylcholanthrene

Details on test system and experimental conditions:

Three assays' were performed; Assay A1 the preliminary toxicity assay, Assay B2 the initial mutagenicity assay, and Assay C1 the confirmatory mutagenicity assay.

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 7-9 days
- Fixation time (start of exposure up to fixation or harvest of cells): 6-10 days

FIXATION AND STAINING AGENT: methanol/crystal violet

METHOD: Cells in logarithmic growth phase were trypsinized and placed in medium containing 5 % serum at a standard density of 3.0 x 10^6 cells/T-75 flask approximately 24 hr prior to treatment. At the time of treatment, the culture medium was replaced with serum-free medium, S-9 mix (when applicable) and the test material, the negative control solvent or the positive control chemical. The cells were treated for approximately 4 hours at 37 °C and the exposure was terminated by washing the cells with phosphate buffered saline.
The number of cells treated at each dose level was adjusted to yield at least 1 x 10^6 surviving cells. The cultures were trypsinized at the end of the treatment and replated at a density of 1 x 10^6 cells/100 mm dish (at least two dishes/replicate) for phenotypic expression. During the phenotypic expression period (7-9 days), cells in the larger petri dishes were subcultured every 2-3 days and plated (at least two dishes/replicate) at a density of about 1 x 10^6 cells/100 mm petri dish. At each subculture, cells from various dishes within each replicate were pooled prior to replating. At the end of the expression period, the cultures were trypsinized and plated at a density of 2 x 10^5 cells/100 mm dish (a total of 10 dishes/treatment) in the selection media (Ham's F-12 without hypoxanthine and with 6-thioguanine) for the determination of HGPRT mutants and 200 cells/60 mm dish (three dishes/treatment) in Ham's F-12 medium without hypoxanthine for determination of cloning efficiency. Treatments resulting in less than approximately 10 % relative cell survival (based upon the concurrent toxicity assay results) were not used for determining either the cloning efficiency or mutation frequencies. The dishes were incubated for about 6-10 days and the colonies were fixed/stained with methanol/crystal violet.

NUMBER OF REPLICATIONS: Two

DETERMINATION OF MUTATION FREQUENCY
- Method: The mutation frequency (expressed as mutants per 10^6 clonable cells) for each replicate were calculated by the following formula (Kirkland, 1989):
MF = K x (m/c)
Where:
K = (Pc x 10^6)/Pm
Pc = the number of cells plated for the survival plates
Pm = the number of cells plated for the mutation plates
c = the mean colonies per plate for survival plates for each replicate
m = the mean colonies per plate for mutation plates for each replicate

DETERMINATION OF CYTOTOXICITY
- Method: Cloning Efficiency
The cytotoxicity of the test material was assessed by determining the ability of the treated cells to form colonies. This assay was conducted for selecting concentrations of the test material to be used in the gene mutation assay. Cells were seeded into T-25 flasks (1.0 x 10^6 cells/flask) approximately 24 hours prior to treatment. Treatment was for approximately 4 hours with various concentrations of the test material in the presence and absence of S-9 factor. After termination of treatment, the cells were trypsinized and replated at a density of 200 cells/dish into 60 mm dishes (3/dose) and the dishes incubated for 6-7 days to allow colony formation. The colonies were then fixed/stained with methanol/crystal violet. The number of colonies/dish were counted and the mean colonies/treatment were expressed relative to the negative control value.

Evaluation criteria:

- Validity: For an assay to be acceptable, the mutation frequency in positive controls should have been significantly higher than the negative controls. An additional criteria, was that the mutation frequency in the negative controls should have been within reasonable limits of the laboratory historical control values and literature values.
- Evaluation: The test chemical was considered positive if it induced a statistically significant, dose related, reproducible increase in mutation frequency. The final interpretation of the data took into consideration such factors as the mutation frequency and cloning efficiencies in the negative controls.

Statistics:

The frequencies of mutants per 10^6 clonable cells were statistically evaluated using a weighted analysis of variance (Hsie et al., 1980); weights were derived from the inverse of the mutation frequency variance. The actual plate counts were assumed to follow a Poisson distribution; therefore the mean plate count was used as an estimate of variance (Kirkland, 1989).
A linear trend test and lack of fit test were employed (α= 0.05) as omnibus tests to compare treated groups to the negative control. If there was a significant increasing trend or a significant lack of fit, a Dunnett's t-test was conducted (Winer, 1971), comparing each treated group and the positive control to the negative control (α= 0.05, one-sided). The lack of fit test is just an indicator that further analysis needed to be done (i.e., the Dunnett’s test). An additional comparison of the positive control to the negative control (α= 0.05) was conducted using a linear contrast statement.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

ASSAY A1: PRELIMINARY TOXICITY ASSAY
The treated cultures both with and without S-9 activation showed little to no toxicity with the Relative Cell Survival (RCS) values ranging from 66.9 to 122.2 % in the absence of S-9 and from 78.5 to 151.8 % in the presence of S-9. Based upon the results of this assay, dose levels of 12.5 to 200 µg/mL of the test material were selected for the gene mutation assay in both the presence and absence of metabolic activation.

ASSAY B1: INITIAL MUTAGENICITY ASSAY
Several of the concentrations formed a precipitate in the treatment medium. There was only slight toxicity observed in two concentrations (50 and 100 µg/mL) in the absence of S-9 activation. The remaining concentrations in the absence of S-9, and those treated in the presence of S-9, displayed no toxicity. The mutant frequencies observed in cultures treated with the test material in the absence and presence of S-9 at all concentration levels were not significantly different from the concurrent negative control values. All mutation frequencies were within a reasonable range of historical background values. The analytically observed concentrations of the test material in the DMSO stock solutions in Assay B1 ranged from 94-99 % of target.

ASSAY C1: CONFIRMATORY MUTAGENICITY ASSAY
In the absence of S-9 activation, there was little to no toxicity evident. The RCS values ranged from 79-119 %. In the presence of S-9, the RCS values in the treated cultures ranged from 46-83 % of the negative control values. The mutant frequencies observed in cultures treated with the test material in the absence or presence of S-9 in this assay were not significantly different from the concurrent negative control values and were within the range of the laboratory historical background. The observed concentrations of the test material in the treatment stock solutions used in Assay C1 ranged from 90-105 % of the targeted concentrations.

CONTROLS
In all assays, the positive control chemicals induced significant increases in mutation frequencies and this data confirmed the adequacy of the experimental conditions for detecting induced mutations.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1: Assay A1 - Relative Cell Survival (%) of Treated Cells (based on three replicates)

Substance	Dose (µg/mL)	Without S-9	With S-9
Negative Control*		100.0	100.0
Test Material	1.56	91.4	135.3
	3.13	85.8	91.7
	6.25	110.9	78.5
	12.5	101.9	104.1
	25	122.2	135.5
	50	117.9	101.7
	100†	87.9	151.8
	200†	66.9	133.1

*1 % DMSO

† Test material was slightly cloudy to cloudy with precipitate in the treatment medium

Relative cell survival (%) = [mean number of colonies/dish in the treated * 100)/mean number of colonies/dish in the negative control (avg. of replicates)]

 

Table 2: Assay B1 - Summary of Mutation Assay Results in the Absence of S-9

Substance	Dose (µg/mL)	RCS (%) No. of Colonies/Dish	Total TGr Colonies/Dish	Cloning Efficiency (%)	Clonable Cells per 10^6
Negative Control*		110.2	8	67.2	6.0
Negative Control*		93.2	3	75.0	2.0
Test Material	12.5	127.8	3	91.5	1.6
	12.5	112.3	10	83.0	6.0
	25	112.5	24	96.5	12.4
	25	101.8	2	74.0	1.4
	50†	56.4	18	81.3	11.1
	50†	105.9	c	c	c
	100†	83.3	4	100.0	2.0
	100†	65.0	1	89.5	0.6
	200†	94.4	16	107.5	7.4
	200†	143.3	7	56.5	6.2
Positive Control		31.8	411	31.7	648.9^f
Positive Control		40.5	312^e	32.0	487.8^f

 

Table 3: Assay B1 - Summary of Mutation Assay Results in the Presence of S-9

Substance	Dose (µg/mL)	RCS (%) No. of Colonies/Dish	Total TGr Colonies/Dish	Cloning Efficiency (%)	Clonable Cells per 10^6
Negative Control*		117.9	11	71.2	7.7
Negative Control*		82.1	5	77.8	3.2
Test Material	12.5	87.8	20^e	66.5	15.0
	12.5	103.3	13	77.2	8.4
	25	95.9	3	97.5	2.3
	25	96.1	9^e	88.2	5.0
	50†	71.8	11	82.7	6.7
	50†	98.7	c	c	c
	100†	75.1	13^e	61.0	10.9
	100†	91.3	14^e	83.5	7.5
	200†	90.4	8^e	56.7	6.9
	200†	97.6	11^e	73.8	7.7
Positive Control		101.8	390^e	59.3	328.7^f
Positive Control		69.7	680^e	68.3	497.6^f

 

Table 4: Assay C1 - Summary of Mutation Assay Results in the Absence of S-9

Substance	Dose (µg/mL)	RCS (%) No. of Colonies/Dish	Total TGr Colonies/Dish	Cloning Efficiency (%)	Clonable Cells per 10^6
Negative Control*		110.0	13	91.5	7.1
Negative Control*		90.0	19	83.3	11.4
Test Material	6.66	116.1	^21	89.3	11.8
	6.66	116.9	12	78.2	7.7
	20†	119.2	8	65.3	6.1
	20†	101.0	c	c	c
	66.6†	106.2	c	c	c
	66.6†	102.3	5	77.8	3.2
	200†	102.6	4	74.2	2.7
	200†	79.0	1	80.3	0.6
Positive Control		37.7	465	42.0	553.6^e
Positive Control		47.3	422	54.5	387.2^e

 

Table 5: Assay C1 - Summary of Mutation Assay Results in the Presence of S-9

Substance	Dose (µg/mL)	RCS (%) No. of Colonies/Dish	Total TGr Colonies/Dish	Cloning Efficiency (%)	Clonable Cells per 10^6
Negative Control*		89.3	10	82.0	6.1
Negative Control*		110.7	17	75.7	11.2
Test Material	6.66	57.1	11	58.5	9.4
	6.66	66.0	10	72.2	6.9
	20†	52.4	17	73.8	11.5
	20†	83.2	11	65.0	8.5
	66.6†	46.4	12	58.0	10.3
	66.6†	56.7	14	73.7	9.5
	200†	66.9	4	55.7	3.6
	200†	58.3	11	60.7	9.1
Positive Control		41.7	612	46.5	658.1^e
Positive Control		50.6	430	46.0	467.4^e

*1 % DMSO

† Test material was slightly cloudy to cloudy with precipitate in the treatment medium

c = lost to contamination; e = adjusted value based on 10 dishes; f = the frequency of TGr mutants is significantly higher than the concurrent negative control value (α=0.05).

Relative cell survival (%) = [(mean number of colonies/dish in the treated * 100)/(mean number of colonies/dish in the negative control (avg. of replicates))]

TGr = 6-Thioguanine resistant

CE (%) = [(Mean number of colonies/dish * 100)/(No. of cells seeded/dish)]

Conclusions:

Interpretation of results:
negative with and without metabolic activation

Under the conditions of the test, based upon the frequency of TGr mutants recovered in cultures treated with the test material, the test material did not induce a mutagenic response.

Executive summary:

The mammalian gene mutation potential of the test material was determined in an in vitro Chinese hamster ovary cell/hypoxanthine-guanine-phosphoribosyl transferase (CHO/HGPRT) forward gene mutation assay. The study was performed under GLP conditions and in accordance with the standardised guidelines OECD 476, EPA OPPTS 870.5300 and EU Method B.17.

The genotoxic potential of the test material was assessed in two independent assays in the absence and presence of an externally supplied metabolic activation (S-9) system with concentrations ranging from 6.6 to 200 µg/mL. The adequacy of the experimental conditions for detection of induced mutation was confirmed by employing positive control chemicals, ethyl methanesulfonate for assays without S-9 and 20-methylcholanthrene for assays with S-9. Negative control cultures were treated with the solvent used to dissolve the test material.

Under the conditions of the test, based upon the frequency of TGr mutants recovered in cultures treated with the test material, the test material did not induce a mutagenic response.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Mammalian Chromosome Aberration Assay

In the key study (Erexson, 2001) the clastogenicity of the test material was determined in an in vivo mouse micronucleus assay, performed under GLP conditions and in accordance with the following standardised guidelines OECD 474, EPA OPPTS 870.5395, EU Method B.12 and Japanese guidelines for screening mutagenicity testing of chemicals.

In the dose range-finding study, the test material was suspended in 0.5 % methylcellulose. Four animals per sex were dosed once daily by oral gavage for 2 consecutive days with the test article at dose levels of 500, 1000, or 2000 mg/kg.

Based on results from the dose range-finding study, dose levels of 500, 1000, or 2000 mg/kg/day were selected for testing in male animals only in the micronucleus study. The test article was suspended in 0.5 % methylcellulose. Seven animals were dosed once daily by oral gavage for 2 consecutive days with either the test article at dose levels of 500, 1000, or 2000 mg/kg, or the vehicle. Approximately 24 hours prior to harvest, seven animals were dosed one-time only by oral gavage with the positive control article. Six animals per group were euthanized approximately 24 hours after the last dose for extraction of the bone marrow. At least 2000 PCEs per animal were analysed for the frequency of micronuclei. Cytotoxicity was assessed by scoring the number of PCEs and normochromatic erythrocytes (NCEs) in at least the first 200 erythrocytes for each animal.

Under the conditions of the test, the test material induced no signs of clinical toxicity in the treated animals and was not cytotoxic to the bone marrow (i.e., no statistically significant decrease in the PCE:NCE ratio). A statistically significant increase in micronucleated PCEs was not observed at any dose level. Therefore the test material was considered negative in the mouse bone marrow micronucleus assay.

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:

29 May 2001 - 17 June 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

ICR

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Range-finding study = 8 weeks old. Definitive test = 9 weeks old.
- Weight at study initiation: Range-finding study = 32.8 - 38.8 g (males) and 26.0 - 30.9 g (females). Definitive test = 32.1 - 37.3 g (males).
- Assigned to test groups randomly: yes, using a computer program.
- Housing: in groups by sex.
- Diet: commercial rodent feed, ad libitum.
- Water: Tap water, ad libitum.
- Acclimation period: at least 7 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 64 - 79 °F
- Humidity (%): 30 - 70 %
- Air changes (per hr): at least 10 per hour
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle.

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: 0.5 % methylcellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Prior to dosing, the dosing stocks of the test material were prepared on each day of dosing by mixing the appropriate volume of the vehicle, 0.5 % methylcellulose, with pre-weighed quantities of the test article, forming homogenous suspensions.

Duration of treatment / exposure:

Two consecutive days

Frequency of treatment:

Daily

Post exposure period:

24 hours after administration of the last dose.

Dose / conc.:

500 mg/kg bw/day

Remarks:

Basis:
actual ingested

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Basis:
actual ingested

Dose / conc.:

2 000 mg/kg bw/day

Remarks:

Basis:
actual ingested

No. of animals per sex per dose:

Range finding test: 4 per sex per dose
Definitive test: 7 males per dose (6 animals/group were required for micronucleus analysis)

Control animals:

yes, concurrent vehicle

Positive control(s):

- Positive control: Cyclophosphamide
- Route of administration: the positive control was dissolved in sterile deionized water at approximately 12 mg/mL and administered once, approximately 24 hours prior to harvest, via oral gavage at approximately 10 mL/kg to achieve the final dosing concentration.
- Doses / concentrations: 120 mg/kg

Tissues and cell types examined:

The PCE:NCE cell ratio was examined in bone marrow (erythrocytes).

Details of tissue and slide preparation:

EXTRACTION OF BONE MARROW
At the appropriate harvest time point, the animals were euthanized by CO2 inhalation followed by incision of the diaphragm. The hind limb bones (tibias) were removed for marrow extraction from six surviving animals. For each animal, the marrow flushed from the bones was combined in an individual centrifuge tube containing 3 to 5 mL foetal bovine serum (one tube per animal). Animals not needed for bone marrow collection were euthanised at the completion of the assay.

PREPARATION OF SLIDES
Following centrifugation to pellet the bone marrow tissue, the supernatants were removed by aspiration and portions of the pellets were spread on slides and air dried. The slides were fixed in methanol, stained with May-Grünwald solution and Giemsa, protected by mounting with coverslips, and analysed by fluorescence microscopy. For control of scoring bias, all slides were coded prior to analysis.

SLIDE ANALYSIS
Slides prepared from the bone marrow collected from six animals per group were scored for micronuclei and the PCE:NCE cell ratio. The micronucleus frequency (expressed as percent micronucleated cells) was determined by analysing the number of micronucleated PCEs from 2000 PCEs per animal. The PCE:NCE ratio was determined by scoring the number of PCEs and NCEs observed in the optic fields while scoring the first 200 erythrocytes on a slide.
The criteria for the identification of micronuclei were those of Schmid (1976). Micronuclei were darkly stained and generally round, although almond and ring-shaped micronuclei occasionally occurred.
Micronuclei had sharp borders and were generally between one-twentieth and one-fifth the size of the PCEs. The unit of scoring was the micronucleated cell, not the micronucleus; thus, the occasional cell with more than one micronucleus was counted as one micronucleated PCE, not two (or more) micronuclei.
The staining procedure permitted the differentiation by colour of PCEs and NCEs (bluish-grey and red, respectively).

Evaluation criteria:

The criteria for a positive response was the detection of a statistically significant increase in micronucleated PCEs for at least one dose level, and a statistically significant dose-related response. A test article that did not induce both of these responses was considered negative. Statistical significance was not the only determinant of a positive response; the Study Director also considered the biological relevance of the results in the final evaluation.

Statistics:

The raw data on the counts of micronucleated PCEs for each animal were first transformed by adding 1 to each count and then taking the natural log of the adjusted number. The transformed micronucleated PCE data and the data on percent PCE were analysed by a one-way analysis of variance (Winer, 1971) when the variances were homogeneous. Ranked proportions were used for heterogeneous variances. If the analysis of variance was significant (p # 0.05), a Dunnett's t-test (Dunnett, 1955; 1964) was used to determine which dose groups, if any, were significantly different from the vehicle control. Additionally, parametric or nonparametric tests for trend may have been employed to identify any dose-related response. The alpha level at which all tests were conducted was 0.05.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

DOSE RANGE FINDING STUDY
- Toxicity:
> 500 mg/kg/day dose group: Clinical signs of toxicity were observed in one female (# 6086), approximately 5 hours after administration of the first dose, until the individual was found dead prior to administration of the second dose. Signs included: hypoactive, cold to touch, hunched posture, rough haircoat, flattened posture, squinted eyes.
> 1000 mg/kg/day: Clinical signs of toxicity were observed in one male (# 6078), approximately 5 hours after administration of the first dose, until the individual was found dead immediately prior administration of the second dose. Signs included: hypoactive, hunched posture, rough haircoat, irregular respiration, flattened posture and squinted eyes.
> 2000 mg/kg/day: Clinical signs of toxicity were observed in three individuals.
Slight hypoactivity was observed in one male (#6070), one day after administration of the first dose, before being found dead immediately prior to administration of the second dose.
A second male (# 6081) displayed slight hypoactivity with the left eye sealed shut one day after administration of the first dose. Both eyes were sealed shut one hour after administration of the second dose. One day after administration of the second dose this individual was cold to the touch, both eyes were sealed shut and the animal was recumbent. This individual was found dead four to five hours later.
One female (# 6088) displayed clinical signs of toxicity approximately 5 hours after administration of the first dose, until the individual was found dead one day after administration of the sceond dose. Signs included: slightly hypoactive, hunched posture, rough haircoat, hypoactive, squinted eyes, cold to touch and flattened posture.
- Mortality:
> 500 mg/kg/day dose group: One female died (# 6086).
> 1000 mg/kg/day dose group: One male died (# 6078).
> 2000 mg/kg/day dose group: Two males (#6070 and # 6081), and 1 female died (# 6088).
All mortalities were necropsied to ascertain whether the condition(s) were related to dosing mishap. None of the animals exhibited evidence of dosing error. However, all five mortalities in the dose range-finding study were attributed to colon/rectal perforations which could be evidence of body temperature errors and thus they are noted to be accidental deaths.
- Body temperature: No relevant variances in the body temperature were observed in any of the dose groups in the dose range-finding study.
- Conclusion: Based on these results, the maximum dose selected was 2000 mg/kg/day, the limit dose for this assay.

DEFINITIVE TEST
- Toxicity: All animals in the vehicle and positive control groups, and all animals in all dosage groups appeared normal after dosing and remained healthy until harvest.
- Mortality: No unscheduled mortality was observed during the definitive test.
- Cytotoixicty: No cytotoxicity was observed in the bone marrow, the test material did not induce a statistically significant decrease in the PCE:NCE ratio. A statistically significant increase in micronucleated PCEs was not observed at any dose level.
- Positive control: Cyclophosphamide, induced statistically significant increases in micronucleated PCEs as compared to that of the vehicle controls, with a mean and standard error of 1.52 ± 0.18 %.

Table 3: Micronucleus Data Summary

Treatment	Dose (mg/kg/day)	Harvest Time** (hours)	% Micronucleaded PCEs		Ratio PCE:NCE
			Mean	S.E.	Mean	S.E.
Vehicle control	0.5 % MC	24	0.08	0.02	0.63	0.07
Positive control	CP 120	24	1.52	0.18*	0.87	0.06
Test material	500	24	0.07	0.04	0.79	0.02
	1000	24	0.04	0.02	0.58	0.06
	2000	24	0.04	0.02	1.08	0.08

* Significantly greater that the corresponding vehicle control, p 0.01.

**Post second dose

Mean values determined from 2000 PCE’s per animal.

MC = Methyl cellulose; CP = Cyclophophamide; PCE = Polychromatic erythrocyte; NCE = Normochromactic erythrocyte

Conclusions:

Interpretation of results: negative
Under the conditions of the test, the test material induced no signs of clinical toxicity in the treated animals and was not cytotoxic to the bone marrow (i.e., no statistically significant decrease in the PCE:NCE ratio). A statistically significant increase in micronucleated PCEs was not observed at any dose level. Therefore the test material was considered to be negative in the mouse bone marrow micronucleus assay.

Executive summary:

The clastogenicity of the test material was determined in an in vivo mouse micronucleus assay, performed under GLP conditions and in accordance with the following standardised guidelines OECD 474, EPA OPPTS 870.5395 and EU Method B.12.

In the dose range-finding study, the test material was suspended in 0.5 % methylcellulose. Four animals per sex were dosed once daily by oral gavage for 2 consecutive days with the test article at dose levels of 500, 1000, or 2000 mg/kg.

Based on results from the dose range-finding study, dose levels of 500, 1000, or 2000 mg/kg/day were selected for testing in male animals only in the micronucleus study. The test article was suspended in 0.5 % methylcellulose. Seven animals were dosed once daily by oral gavage for 2 consecutive days with either the test article at dose levels of 500, 1000, or 2000 mg/kg, or the vehicle. Approximately 24 hours prior to harvest, seven animals were dosed one-time only by oral gavage with the positive control article. Six animals per group were euthanized approximately 24 hours after the last dose for extraction of the bone marrow. At least 2000 PCEs per animal were analysed for the frequency of micronuclei. Cytotoxicity was assessed by scoring the number of PCEs and normochromatic erythrocytes (NCEs) in at least the first 200 erythrocytes for each animal.

Under the conditions of the test, the test material induced no signs of clinical toxicity in the treated animals and was not cytotoxic to the bone marrow (i.e., no statistically significant decrease in the PCE:NCE ratio). A statistically significant increase in micronucleated PCEs was not observed at any dose level. Therefore the test material was considered negative in the mouse bone marrow micronucleus assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In Vitro

Bacterial Reverse Mutation Assay

In the key study (Mecchi, 2001)the genetic toxicity of the test material was determined in an in vitro bacterial reverse mutation assay, an Ames test. The study was performed under GLP conditions and in accordance with the standardised guidelines OECD 471, EPA OPPTS 870.5100 and Japan MAFF Revised Mutagenicity Guidelines.

The concentrations tested in the mutagenicity assay were selected based on the results of a dose range finding assay using tester strains TA100 and WP2uvrA and ten concentrations of test article ranging from 6.67 to 5000 µg per plate.

The tester strain used in the mutagenicity assay were Salmonella typhimurium tester strains TA98, TA100, TA 1535, and TA1537 and Escherichia coli tester strain WP2uvrA. The assay was conducted with six concentration levels of test article in both the presence and absence of S9 mix along with concurrent vehicle and positive controls using three plates per concentration. The concentrations tested were 33.3, 100, 333, 1000, 3330, and 5000 µg per plate in both the presence and absence of S9 mix. The results of the initial mutagenicity assay were confirmed in the independent experiment.

Under the conditions of the test, the test material did not cause a positive increase in the mean number of revertants per plate with any of the tester strains either in the presence or absence of microsomal enzymes prepared from Aroclor™ induced rat liver (S9). The test material is therefore determined to be non-mutagenic in this assay.

 

This result is supported by the findings reported in the second study (Lawlor, 1997), where the test material was determined to be non-mutagenic in an Ames screening study, performed according to sound scientific principles and under GLP conditions.

 

Mammalian Chromosome Aberration Assay

In the key study (Linscombe et. al., 2001) the clastogenicity of the test material was determined in an in vitro chromosomal aberration assay utilizing rat lymphocytes. The study was performed under GLP conditions and in accordance with the standardised guidelines OECD 473, EPA OPPTS 870.5375, EU Method B.10 and Japan MAFF guidelines for screening mutagenicity testing of chemicals.

Approximately 48 hour after the initiation of whole blood cultures, cells in the absence and presence of S-9 activation were treated for 4 hour with targeted test concentrations of 0 (negative control) to 200 µg/mL of culture medium and harvested 20 hours later. Based upon the mitotic indices, cultures treated with targeted concentrations of 0, 25, 100, and 200 µg/mL in the absence and presence of S-9 activation were selected for determining the incidence of chromosomal aberrations. There were no significant increases in the frequencies of cells with aberrations in this assay. In a confirmatory assay, cultures were treated as above except that the cultures were treated continuously for 24 hour until the time of their harvest in the absence of S-9.

The incidence of chromosomal abnormalities was determined from cultures treated with 0, 25, 100, and 200 µg/mL in the absence and presence of S-9. Statistical analyses of the data did not identify a significant difference in the frequencies of cells with aberrations between the negative control and any of the treated cultures with or without S-9 activation. Cultures treated with the positive control chemicals (i.e., mitomycin C without S-9 and cyclophosphamide with S-9) had significantly higher incidences of abnormal cells in all assays. Therefore the test material was considered to be negative in the in vitro chromosomal aberration assay utilizing rat lymphocytes.

 

This result is supported by the findings reported in the second study (Linscombe, 1997), where the test material was determined to be non-mutagenic in a mammalian chromosome aberration screening study, performed according to sound scientific principles.

 

Mammalian Gene Mutation Assay

In the key study (Linscombe and Beuthin, 2001), the mammalian gene mutation potential of the test material was determined in an in vitro Chinese hamster ovary cell/hypoxanthine-guanine-phosphoribosyl transferase (CHO/HGPRT) forward gene mutation assay. The study was performed under GLP conditions and in accordance with the standardised guidelines OECD 476, EPA OPPTS 870.5300 and EU Method B.17.

The genotoxic potential of the test material was assessed in two independent assays in the absence and presence of an externally supplied metabolic activation (S-9) system with concentrations ranging from 6.6 to 200 µg/mL. The adequacy of the experimental conditions for detection of induced mutation was confirmed by employing positive control chemicals, ethyl methanesulfonate for assays without S-9 and 20-methylcholanthrene for assays with S-9. Negative control cultures were treated with the solvent used to dissolve the test material.

Under the conditions of the test, based upon the frequency of TGr mutants recovered in cultures treated with the test material, the test material did not induce a mutagenic response.

 

In Vivo

Mammalian Chromosome Aberration Assay

In the key study (Erexson, 2001) the clastogenicity of the test material was determined in an in vivo mouse micronucleus assay, performed under GLP conditions and in accordance with the following standardised guidelines OECD 474, EPA OPPTS 870.5395, EU Method B.12 and Japanese guidelines for screening mutagenicity testing of chemicals.

In the dose range-finding study, the test material was suspended in 0.5 % methylcellulose. Four animals per sex were dosed once daily by oral gavage for 2 consecutive days with the test article at dose levels of 500, 1000, or 2000 mg/kg.

Based on results from the dose range-finding study, dose levels of 500, 1000, or 2000 mg/kg/day were selected for testing in male animals only in the micronucleus study. The test article was suspended in 0.5 % methylcellulose. Seven animals were dosed once daily by oral gavage for 2 consecutive days with either the test article at dose levels of 500, 1000, or 2000 mg/kg, or the vehicle. Approximately 24 hours prior to harvest, seven animals were dosed one-time only by oral gavage with the positive control article. Six animals per group were euthanized approximately 24 hours after the last dose for extraction of the bone marrow. At least 2000 PCEs per animal were analysed for the frequency of micronuclei. Cytotoxicity was assessed by scoring the number of PCEs and normochromatic erythrocytes (NCEs) in at least the first 200 erythrocytes for each animal.

Under the conditions of the test, the test material induced no signs of clinical toxicity in the treated animals and was not cytotoxic to the bone marrow (i.e., no statistically significant decrease in the PCE:NCE ratio). A statistically significant increase in micronucleated PCEs was not observed at any dose level. Therefore the test material was considered negative in the mouse bone marrow micronucleus assay.

 

Overall Reliability of the Dataset

Each key study was assigned a reliability score of 1 (reliable without restrictions) and the two supporting studies were assigned a reliability score or 2 (reliable with restrictions), in line with the principles of Klimisch (1997). The overall quality of the database is considered to be high and the results conclusive.

Justification for selection of genetic toxicity endpoint

Multiple studies have been provided to address the different endpoints of genetic toxicity, each key study assesses a different type of genetic toxicity. Since all the studies showed negative results, a single study could not be selected as key over the others.

Short description of key information:

In Vitro

Negative, bacterial reverse gene mutation assay, OECD 471, EPA OPPTS 870.5100 and Japan MAFF revised mutagenicity guidelines, Mecchi 2001.

Negative, chromosome aberration assay, OECD 473, EPA OPPTS 870.5375, EU Method B.10 and Japan MAFF revised mutagenicity guidelines, Linscombe et. al. 2001.

Negative, mammalian gene mutation assay, OECD 476, EPA OPPTS 870.5300 and EU Method B.17, Linscombe and Beuthin 2001.

In Vivo

Negative, mouse micronucleus assay, OECD 474, EU Method B.12, EPA OPPTS 870.5395 and Japan MAFF revised mutagenicity guidelines, Erexson 2001.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the substance does not require classification with respect to genetic toxicity.