2-Pyridinecarboxylic acid, 4-amino-3,6-dichloro-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

4 September 1998 to 10 September 1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

The tester strains used in this study were Salmonella typhimurium tester strains TA98 and TA100. The assay was conducted using seven doses of test material in the presence of S9 mix. All doses of test material and positive controls were plated using two plates per dose. The vehicle controls were plated in triplicate.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Purity: not reported

Target gene:

Histidine locus

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

- Type and identity of media:
Culturing broth = Vogel-Bonner salt solution supplemented with 2.5 % w/v Oxiod Nutrient Broth
Agar plates = Vogel-Bonner minimal medium E supplemented with 1.5 % w/v agar and 0.2 % glucose
Overlay agar = 0.7 % agar w/v and 0.5 % NaCl w/v and will be supplemented with 10 mL of 0.5 mM histidine/biotin solution per 100 mL agar.
- Properly maintained: yes

Additional strain / cell type characteristics:

other: rfa, uvrB, +R

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

- Type and identity of media:
Culturing broth = Vogel-Bonner salt solution supplemented with 2.5 % w/v Oxiod Nutrient Broth
Agar plates = Vogel-Bonner minimal medium E supplemented with 1.5 % w/v agar and 0.2 % glucose
Overlay agar = 0.7 % agar w/v and 0.5 % NaCl w/v and will be supplemented with 10 mL of 0.5 mM histidine/biotin solution per 100 mL agar.
- Properly maintained: yes

Additional strain / cell type characteristics:

other: rfa, uvrB, +R

Metabolic activation:

with

Metabolic activation system:

S9 mix (37.4 mg of protein per mL)

Test concentrations with justification for top dose:

9.99, 33.3, 99.9, 333, 1000, 3330, 5000 µg/plate

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:

benzo(a)pyrene

other: 2-aminoanthracene

Remarks:

positive control 2.5 µg/plate

Details on test system and experimental conditions:

The tester strains were exposed to the test material, positive, or vehicle controls, via the plate incorporation method, as described by Ames (1975). The test material, positive or vehicle controls, were overlaid onto a minimal agar plate. Following incubation at 37 ± 2 °C for 52 ± 4 hours, revertant colonies were counted. All doses of test material and positive controls were plated in duplicate. The vehicle controls were plated in triplicate.

PLATING PROCEDURE
To a tube containing 2.0 mL of molten selective top agar (maintained at 45 ± 2 °C), 500 µL of S9 mix was added together with 100 µL of tester strain and 50 µL of vehicle or test material dose. After the required components had been added, the mixture was vortexed and overlaid onto the surface of 25 mL of minimal bottom agar contained in a 15 x 100 mm petri dish. After the overlay had solidified, the plates were inverted and incubated for 52 ± 4 hours at 37 ± 2 °C. Positive control articles were plated using a 50 µL plating aliquot.

SCORING THE PLATES
Plates which were not evaluated immediately following the incubation period were held at 5 ± 3 °C until such time that colony counting and bacterial background lawn evaluation could take place.
- Bacterial Lawn Evaluation
The condition of the bacterial background lawn was evaluated for evidence of cytotoxicity and test material precipitate. Evidence of cytotoxicity was scored relative to the vehicle control plate and recorded along with the revertant count for that plate.
- Counting Revertant Colonies
Revertant colonies for a specific tester strain and activation condition within a given test material dilution series was counted either entirely by automated colony counter or entirely by hand. If there was sufficient test material precipitate on the plates at any dose which interfered with automated colony counting, then the plates at all doses for that specific strain and activation condition were counted by hand.

Evaluation criteria:

CRITERIA FOR A POSITIVE RESPONSE
Once the criteria for a valid assay have been met, responses observed in the assay are evaluated as follows:
For a test material to be considered positive, it must produce at least a 3-fold (TA98) or 2-fold (TA100) dose related increase in the mean revertants per plate of at least one tester strain over the mean revertants per plate of the appropriate vehicle control. A response which does not meet both of the above criteria (magnitude, dose-responsiveness) will not be evaluated as positive.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

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 applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

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 applicable

Positive controls validity:

valid

Additional information on results:

All data were acceptable and no positive increases in the number of revertants per plate were observed with either tester strain TA98 or TA100 in the presence of S9 mix.

Table 1: Results

	Dose µg/plate	Mean revertants per plate		Background Lawn*
		TA 98	TA100
Vehicle control (DMSO)		22	92	1
Test material	9.99	21	87	1
	33.3	22	93	1
	99.9	21	97	1
	333	20	94	1
	1000	28	89	1
	3330	19	90	1
	5000	20	77	1
Positive control**		432	896	1

** TA 98 = benzo[a]pyrene, 2.5 µg/plate; TA 100 = 2 -aminoanthracene, 2.5 µg/plate

* Background lawn: 1 = normal

Conclusions:

negative with metabolic activation

The results of the Salmonella/Mammalian-Microsome Reverse Mutation Screening Assay (Ames Test) indicate that under the conditions of this study, the test material did not cause a positive increase in the number of revertants per plate with either of the tester strains in the presence of S9 mix.

Executive summary:

The test material was evaluated in the Salmonella/Mammalian-Microsome Reverse Mutation Screening Assay (Ames Test); the study was conducted under GLP conditions. The tester strains used in the study were TA98 and TA100. The assay was conducted using seven dose levels of the test material in the presence of an externally supplied metabolic activation system (S-9). The concentration of the test material ranged from 9.99 to 5000 μg/plate.

The test material did not induce a positive increase in the number of revertant colonies in either strain. Hence, the test material was classified as negative in this bacterial mutagenicity screening test under the experimental conditions used.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

20 July 2000 to 1 August 2000

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Principles of method if other than guideline:

The test material was evaluated in an in vitro chromosomal aberration screening assay utilising rat lymphocytes. Approximately 48 hours after the initiation of whole blood cultures, cells in the absence of S-9 activation were treated for 24 hours with targeted doses of test material before being harvested at the end of treatment. The highest concentration tested was the limit dose of 10 mM. In the presence of S-9 activation, cultures were treated using the same dose levels of the test material for 4 hours and harvested approximately 24 hours after treatment termination. Concurrent vehicle and positive controls were included. Only one concentration level was evaluated for aberrations. Based upon the mitotic indices, cultures treated with 0 and 517.5 μg/mL of the test material in the absence of S-9 and cultures treated with 0 and 2070 μg/mL in the presence of S-9 activation were selected for determining the incidence of chromosomal aberrations.

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Purity: 95.4%

Species / strain / cell type:

lymphocytes: (cultured rat)

Details on mammalian cell type (if applicable):

Blood samples were collected and pooled from male CD rats, aged 8-20 weeks. The number of rats used was based upon the number of cultures to be set up. The animals were euthanised with carbon dioxide just prior to collecting the samples. Whole blood cultures were set up in RPMI 1640 medium supplemented with 10 % foetal calf serum, antibiotics and antimycotics (Fungizone 0.25 μg/mL; penicillin G, 100 u/mL and streptomycin sulphate, 0.1 mg/mL), 25 mM HEPES, 20 μg/mL PHA and an additional 2 mM L-glutamine. Cultures were initiated by inoculating approximately 100 μL of whole blood for each mL of complete culture medium into a sterile disposable tissue culture flask 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 (vehicle control), 16.2, 32.3, 64.7, 129.4, 258.8, 517.5, 1035, 2070 µg/mL

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

1 % DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

TREATMENT PROCEDURE WITHOUT METABOLIC ACTIVATION
The negative control, positive control, and test material were added directly to the culture flasks approximately 48 hours after initiation of the cultures. The cultures were harvested approximately 24 hours later. Prior to harvest, these cultures were rinsed at least once with RPMI 1640 (with HEPES and antibiotics).

TREATMENT PROCEDURE WITH METABOLIC ACTIVATION
Approximately 48 hours after initiation of the cultures, each culture was dispensed into 15 mL sterile disposable 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, antibiotics, and the S9 mix) containing the test or positive or negative control treatments for approximately 4 hours 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 along with approximately 4.5 mL of the original culture medium until the time of harvest. The cultures were harvested at approximately 24 hours after treatment initiation (i.e., approximately 20 hours after treatment termination).

EVALUATION
Colcemid (1 μg/culture) was added 2- 3 hours prior to harvest. The cells were swollen by hypotonic treatment (0.075 M KCl), fixed with methanol:acetic acid (3:1), dropped on microscope slides, and stained in Giemsa. Mitotic indices were determined as the number of cells in metaphase among 1000 cells/replicate and expressed as percentages.
Slides from the negative controls, positive controls, and one or more concentrations of the test material were selected for cytogenetic analysis. Whenever possible, 50 metaphases/replicate were examined from coded slides for structural abnormalities. Structural chromosomal abnormalities counted included: chromatid and chromosome gaps, chromatid breaks and exchanges, chromosome breaks and exchanges, and miscellaneous (chromosomal disintegration, chromosomal pulverization, etc.). Those cells having 5 or more aberrations/cell were classified a cell with multiple aberrations.
In addition, whenever possible, a total of 100 metaphases/replicate were examined for the incidence of polyploidy.

Evaluation criteria:

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 controls should be within reasonable limits of historical values. Historical data is maintained in the laboratory and updated continuously to reflect the most current values. 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 parameter “cells with aberrations” was analysed for statistical significance. Descriptive and comparative statistics on cytogenetic aberrations consider each of the analysed cells as an observational unit. 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 global hypotheses: 1) no difference in the 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 alpha = 0.05, versus a one-sided increasing alternative, pairwise tests (i.e., control vs. treatment) were performed at each dose level and evaluated at alpha = 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 alpha=0.05. The data were analysed separately based on the presence or absence of S9.

Key result

Species / strain:

lymphocytes: rat

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

No significant increase in the incidence of aberrant cells was noticed in the test material cultures when compared to the negative controls. The positive control cultures had significantly higher incidences of aberrant cells.

Table 1: Results of the Chromosomal Aberration Assay 24 Hours After Treatment (totals for replicate A + replicate B)

Without S9
	Negative control	Test material (517.5 µg/mL)	Positive control*
No. of cells scored	100	100	75
chromatid gaps	0	3	2
chromosome gaps	0	0	0
chromatid breaks	6	4	7
chromatid exchanges	0	0	9
chromosome breaks	0	0	1
chromosome exchanges	0	0	0
total aberrations (excl. gaps)	6	4	17
no. of cells with aberrations	3	3	17
misc. aberrations	0	0	0
cells with multiple aberrations	0	0	2
With S9
	Negative control	Test material (2070 µg/mL)	Positive control**
No. of cells scored	100	100	50
chromatid gaps	1	4	2
chromosome gaps	0	0	0
chromatid breaks	1	1	8
chromatid exchanges	0	0	11
chromosome breaks	0	0	1
chromosome exchanges	0	0	0
total aberrations (excl. gaps)	1	1	20
no. of cells with aberrations	1	1	25
misc. aberrations	0	0	2
cells with multiple aberrations	0	0	8

*Mitomycin C 0.075 µg/mL

**Cyclophosphamide 6.0 µg/mL

Conclusions:

negative with and without metabolic activation

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

Executive summary:

The clastogenic potential of the test material, to cultures rat lymphocytes, was assessed in a study which was conducted under GLP conditions.

The test material was evaluated in an in vitro chromosomal aberration screening assay. Approximately 48 hours after the initiation of whole blood cultures, cells in the absence of S9 activation were treated for 24 hours with targeted doses of 0 (negative control), 16.2, 32.3, 64.7, 129.4, 258.8, 517.5, 1035, and 2070 μg test material per mL of culture medium and harvested at the end of treatment. The highest concentration tested was the limit dose of 10 mM. In the presence of S9 activation, cultures were treated using the same dose levels of the test material for 4 hours and harvested approximately 24 hours after treatment termination. The test material was initially dissolved in dimethyl sulphoxide (DMSO) and then added to the cultures to give the desired final concentrations. Cultures treated with 0.05 and 0.075 μg/mL mitomycin C (MMC) or 4 and 6 μg/mL cyclophosphamide (CP) were used as positive controls for the non-activation and activation assays, respectively; only one concentration level was evaluated for aberrations. Based upon the mitotic indices, cultures treated with 0 and 517.5 μg/mL of the test material in the absence of S9 and cultures treated with 0 and 2070 μg/mL in the presence of S9 activation were selected for determining the incidence of chromosomal aberrations.

No significant increase in the incidence of aberrant cells was noticed in either of the treatment levels when compared to the negative controls. The positive control cultures had significantly higher incidences of aberrant cells. Hence, the test material was considered to be negative in the in vitro chromosomal aberration-screening assay utilising rat lymphocytes.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6 March 2001 to 23 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

Specific details on test material used for the study:

Purity: 94.5%

Target gene:

HGPRT locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

The cell line CHO-K1-BH4, was used in this study. The cells were grown as monolayer cultures in plastic disposable tissue culture labware under standard conditions of approximately 5 % CO2 in air at 37 °C in a humidified incubator.
- Type and identity of media: Ham's F-12 nutrient mix supplemented with 5 % (v/v) heat inactivated, dialysed foetal calf serum, antibiotics and antimycotics (penicillin G, 100 units/mL; streptomycin sulphate, 0.1 mg/mL; fungizone, 25 μg/mL) and an additional 2 mM L-glutamine.
- Properly maintained: Yes (stock cultures were stored at about -100 °C or below)
- Periodically checked for Mycoplasma contamination: Yes

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 = 0 (vehicle control), 16.2, 32.4, 64.7, 129.4, 258.8, 517.5, 1035.0, 2070.0 µg/mL
Assay B1 = 0 (vehicle control), 31.25, 62.5, 125, 250, 500, 1000, 1500, 2070 µg/mL
Assay C1 = 0 (vehicle control), 250, 500, 1000, 1500, 2070 µg/mL

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

1 % DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

other: 20-methylchloanthrene

Details on test system and experimental conditions:

TREATMENT PROCEDURE
Cells in logarithmic growth phase were trypsinised and placed in medium containing 5 % serum at a standard density of 3.0 x 10⁶ cells/ flask approximately 24 hours prior to treatment. At the time of treatment, the culture medium was replaced with serum-free medium, S9 mix (when applicable) and the test chemical, the negative control vehicle 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.

TOXICITY ASSAY
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 flasks (1.0 x 10⁶ 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 metabolic activation. After termination of treatment, the cells were trypsinised and re-plated 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 was counted and the mean colonies/treatment were expressed relative to the negative control value.

GENE MUTATION ASSAY
Each dose level was set up in duplicate from the time of treatment until the completion of the assay. The number of cells treated at each dose level was adjusted to yield at least 1 x 10⁶ surviving cells. The cultures were trypsinised at the end of the treatment and re-plated at a density of 1 x 10⁶ cells/100 mm dish (at least two dishes/replicate) for phenotypic expression. In addition, 200 cells/60 mm dish (three dishes/replicate) were also plated to determine the toxicity and incubated for approximately 6 - 8 days to permit colony formation. During the phenotypic expression period (7 - 9 days), cells in the larger petri dishes were sub-cultured every 2 - 3 days and plated (at least two dishes/replicate) at a density of about 1 x 10⁶ cells/100 mm petri dish. At each subculture, cells from various dishes within each replicate were pooled prior to re-plating. At the end of the expression period, the cultures were trypsinised and plated at a density of 2 x 10⁵ 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. The mutation frequency (expressed as mutants per 10⁶ clonable cells) for each replicate were calculated.

Evaluation criteria:

For an assay to be acceptable, the mutation frequency in positive controls should be significantly higher than the negative controls. An additional criterion is that the mutation frequency in the negative controls should be within reasonable limits of the testing laboratory’s historical control values and literature values. The test material is considered positive if it induces a statistically significant, dose related, reproducible increase in mutation frequency. The final interpretation of the data will also take into consideration such factors as the mutation frequency and cloning efficiencies in the negative controls.

Statistics:

The frequencies of mutants per 10⁶ 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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

ASSAY A1 - PRELIMINARY TOXICITY ASSAY
The treated cultures both with and without S9 activation exhibited little to no toxicity with the relative cell survival (RCS) values ranging from 80.1 to 188.1 % in the absence of S9 and 22.0 to 194.3 % in the presence of S9. Based upon the results of this assay, dose levels of 31.25 to 2070 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
There was little to no toxicity observed at any of the concentrations selected in the absence of S9. The RCS values in the assay with S9 ranged from 52 to 150 %. The mutant frequencies observed in the treated cultures were not significantly different from the concurrent negative control values and were within reasonable limits of the laboratory historical control values. The analytically observed concentrations of the test material in the DMSO stock solutions in Assay B1 ranged from 82 to 89 % of target.

ASSAY C1 - CONFIRMATORY MUTAGENICITY ASSAY
There was little to no toxicity observed, as indicated by the RCS values, both with and without S9 activation (76 to 124 % and 86 to 122 %, respectively). The mutation frequencies observed in cultures treated with the test material in the absence and presence of S9 in this assay were not significantly different from the concurrent negative control values and were within the range of the testing laboratory’s historical background. The observed concentrations of the test material in the treatment stock solutions used in Assay C1 ranged from 102 to 107 % of the targeted concentrations.

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.

Table 1: Results of the Gene Mutation Assay in CHO Cells Treated with Test Material - Assay B1

Without S9
Treatment (µg/mL)	Toxicity assay	Mutation assay	Cloning efficiency (%)	TGr mutants per 10⁶ clonable cells
	RCS (%)	total TGr colonies
negative control (1 % DMSO)	121.4	22	73.3	15.0
	78.6	11	74.2	7.4
31.25	95.7	17	81.5	10.4
	103.7	9	88.2	5.1
62.5	106.5	4	73.5	2.7
	96.3	13.3	73.0	9.1
125	96.5	11	94.7	5.8
	110.3	12	86.3	7.0
250	92.1	6	118.7	2.5
	74.4	17	83.8	10.1
500	99.3	23	120.8	9.5
	79.2	7	119.3	2.9
1000	105.7	10	62.3	8.0
	86.7	16	69.8	11.5
1500	77.1	14	111.5	6.3
	103.1	10	72.5	6.9
2070	125.6	19	60.5	15.7
	96.7	21	76.8	13.7
positive control (621 µg/mL EMS)	50.6	585	43.2	677.6
	40.1	395	28.0	705.4
With S9
Treatment (µg/mL)	Toxicity assay	Mutation assay	Cloning efficiency (%)	TGr mutants per 10⁶ clonable cells
	RCS (%)	total TGr colonies
negative control (1 % DMSO)	101.7	39	112.0	17.4
	98.3	20	81.0	12.3
31.25	89.5	12	122.0	4.9
	79.5	8	71.0	5.6
62.5	150.0	12	86.5	6.9
	60.0	1.4	83.3	0.9
125	63.7	23	74.2	15.5
	76.7	12	81.8	7.3
250	88.4	9	94.8	4.7
	78.1	24	82.0	14.6
500	78.4	9	81.2	5.5
	76.7	4	77.0	2.6
1000	74.6	2	76.0	1.3
	89.7	10	72.0	6.9
1500	64.8	15	55.8	13.4
	68.7	4	72.8	2.7
2070	60.5	36	86.7	20.8
	51.9	12	64.7	9.3
positive control (4.0 µg/mL MCA)	63.5	479	53.2	450.5
	42.7	533	65.2	409.0

TGr: 6 -thioguanine resistant

RSC: relative cell survival (%) = (Mean no. of colonies/dish in the treated x 100) / Mean no of colonies in the negative control (avg. of replicates)

Table 2: Results of the Gene Mutation Assay in CHO Cells Treated with Test Material - Assay C1

Without S9
Treatment (µg/mL)	Toxicity assay	Mutation assay	Cloning efficiency (%)	TGr mutants per 10⁶ clonable cells
	RCS (%)	total TGr colonies
negative control (1 % DMSO)	103.4	3	85.0	1.8
	96.6	11	63.5	8.7
250	87.7	7	98.5	3.6
	124.2	7	97.0	3.6
500	96.1	28	134.0	10.4
	88.1	7	112.0	3.0
1000	97.8	15	85.7	8.8
	95.7	13	108.0	6.0
1500	87.2	6	81.7	3.7
	86.2	5	69.0	3.6
2070	92.7	15	106.2	7.1
	75.6	4	74.5	2.7
positive control (621 µg/mL EMS)	38.4	508	50.2	506.3
	31.8	585	56.0	522.3
With S9
Treatment (µg/mL)	Toxicity assay	Mutation assay	Cloning efficiency (%)	TGr mutants per 10⁶ clonable cells
	RCS (%)	total TGr colonies
negative control (1 % DMSO)	106.1	7	106.0	3.3
	93.9	17	96.0	8.9
250	85.5	7	88.3	4.0
	91.5	17	92.7	9.2
500	121.6	12	86.0	7.0
	115.8	7	69.8	5.0
1000	96.0	8	61.7	6.5
	110.6	9	55.8	8.1
1500	86.4	8	58.0	6.9
	89.4	10	80.2	6.2
2070	98.6	5	67.3	3.7
	91.1	25	56.5	22.1
positive control (4.0 µg/mL MCA)	92.2	977	68.3	714.9
	75.4	632	59.2	534.1

TGr: 6 -thioguanine resistant

RSC: relative cell survival (%) = (Mean no. of colonies/dish in the treated x 100) / Mean no of colonies in the negative control (avg. of replicates)

Conclusions:

negative with and without metabolic activation

It was concluded that the test material did not induce a mutagenic response in the CHO/HGPRT gene mutation assay.

Executive summary:

The mutagenic potential of the test material was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 476, EU Method B.17 and EPA OPPTS 870.5300.

The test material was evaluated in an in vitro Chinese hamster ovary cell/hypoxanthine-guanine-phosphoribosyl transferase (CHO/HGPRT) forward gene mutation assay. The genotoxic potential of the test material was assessed in two independent assays in the absence and presence of an externally supplied metabolic activation (S9) system with concentrations ranging from 31.25 to 2070 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 S9 and 20-methylcholanthrene for assays with S9. Negative control cultures were treated with the vehicle used to dissolve the test material.

Based upon the frequency of TGr mutants recovered in cultures treated with the test material, it was concluded that the test material did not induce a mutagenic response in the assay system employed.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5 April 2001 to 25 April 2002

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

Specific details on test material used for the study:

Purity: 94.5%

Species / strain / cell type:

lymphocytes: rat

Details on mammalian cell type (if applicable):

Blood samples were collected by cardiac puncture, following euthanasia with carbon dioxide, from three rats in both Assay A1 and Assay B1 and from two rats in Assay C1. 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 mg/mL; penicillin G, 100 u/mL; and streptomycin sulphate, 0.1 mg/mL), 20 mg/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:

Assay A1 = 0 (vehicle control), 32.3, 64.7, 129.4, 258.8, 517.5, 1035, 2070 µg/mL (treatment duration: 4 hours, with and without metabolic activation).
Assay B1 = 0 (vehicle control), 125, 250, 500, 750, 1000, 1400, 1700, 2070 µg/mL (treatment duration: 24 hours, without metabolic activation).
Assay B1 = 0 (vehicle control), 62.5, 125, 500, 1000, 2070 µg/mL (treatment duration: 4 hours, with metabolic activation).
Assay C1 = 0 (vehicle control), 400, 600, 800, 1000, 1200, 1400, 1600, 1700, 1800, 2070 µg/mL (treatment duration: 24 hours, without metabolic activation).

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

1 % DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

TREATMENT PROCEDURE WITHOUT METABOLIC ACTIVATION
Approximately 48 hours after initiation of the cultures (Assay A1), the cell suspension was dispensed into 15 mL sterile centrifuge tubes (approximately 5.5 mL/tube, 2 cultures per concentration). 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 hours 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 along with approximately 4.5 mL of the original culture medium until the time of harvest. The cultures were harvested at approximately 24 hours after treatment initiation (i.e., approximately 20 hours after treatment termination). Assays B1 and C1 were conducted with continuous treatment with the test material for 24 hours (approximately 1.5 normal cell cycle length). The treatments were added directly to the culture flasks at 48 hours after initiation of the cultures and these cultures were harvested 24 hours later.

TREATMENT PROCEDURE WITH METABOLIC ACTIVATION
Approximately 48 hours 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 S9) containing the test and positive and negative control treatments for approximately 4 hours 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 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 hours after treatment initiation (i.e., approximately 20 hours after treatment termination).

HARVESTING OF CULTURES AND SLIDE EVALUATION
Colcemid was added approximately 3 hours prior to harvest at a final concentration of 0.2 mg/mL. The cells were swollen by hypotonic treatment (0.075 M KCl), fixed with methanol:acetic acid (3:1), dropped on microscope slides, and stained in Giemsa. Mitotic indices were determined as the number of cells in metaphase among 1000 cells/replicate and expressed as percentages. One hundred metaphases/replicate were examined, where possible, from coded slides at each selected concentration of the test chemical and the negative controls (a total of 200 cells/treatment) for structural abnormalities. In the positive control cultures, 50-100 metaphases/replicate (a total of 100-200 cells/treatment) were examined for abnormalities, where possible. Structural chromosomal abnormalities that were counted included: chromatid and chromosome gaps, chromatid breaks and exchanges, chromosome breaks and exchanges, and miscellaneous (chromosomal disintegration, chromosomal pulverisation, etc.). 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. In addition, one hundred metaphases/replicate were examined for the incidence of polyploidy.

Evaluation criteria:

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. A test material 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 S9 and based on the exposure time.

Key result

Species / strain:

lymphocytes: rat

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

in rat lymphocyte cultures treated continuously for 24 hours without metabolic activation (assay B1).

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in the absence of S-9 (assay B1)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

ASSAY A1
There was little to no toxicity in any of the treated cultures either with or without metabolic activation.
Without metabolic activation, the Relative Mitotic Index (RMI) varied from 63.9 % at the highest concentration of 2070 µg/mL to 124.1 % at the lowest concentration of 32.3 µg/mL. In the presence of S9 activation, the RMI was 63.1 % at 2070 µg/mL and 81.0 % at 32.3 µg/mL. Based upon these results, cultures treated with 517.5, 1035, and 2070 µg/mL in the absence of S9 activation and cultures treated with 64.7, 1035, and 2070 µg/mL in the presence of S9 were chosen for the determination of chromosomal aberration frequencies and incidence of polyploidy.
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 1 % and the corresponding values at treatment levels 517.5, 1035, and 2070 µg/mL were 1.5, 1.0, and 1.5 %, respectively. In the activation assay, cultures treated with the test material at concentrations of 64.7, 1035, and 2070 µg/mL had aberrant cell frequencies of 0.5, 0.5, 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 S9 activation. The frequencies of aberrant cells observed in the test material treated cultures were within the testing laboratory’s 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 S9) and CP (with S9) treated cultures were 20.8 % and 18.9 %, respectively.

ASSAY B1
The cultures treated in the absence of S9 showed increasing toxicity with increasing concentration. The RMIs ranged from 97.7 % at 125 µg/mL to 18.0 % at 2070 µg/mL. In the presence of S9 activation, there was little to no toxicity present. Based upon these results, cultures treated with 125, 750 and 1400 µg/mL in the absence of S9 activation and cultures treated with 62.5, 1000, and 2070 µg/mL in the presence of S9 were initially 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 0.5 % and the corresponding values at treatment levels 125, 750, and 1400 μg/mL were 1.0, 1.0, and 5.0 %, respectively. In the activation assay, cultures treated with the test material at concentrations of 62.5, 1000, and 2070 μg/mL had aberrant cell frequencies of 2.0, 1.0, and 2.5 %, respectively, as compared to the negative control value of 0.5 %. Statistical analyses of the data did not identify a significant difference between the negative control and any of the treated cultures with or without S9 activation, with the exception of the highest concentration (1400 µg/mL) in the non-activation series. Due to this increase, cultures treated with 1000 and 1700 µg/mL of test material were also analysed for the incidence of chromosomal aberrations. Statistical analysis of the data at these concentrations indicated a significant increase in the aberrant cell frequency (4.5 and 7.5 %, respectively) as compared to the negative control value.
With the exception of the cultures treated with 1700 µg/mL test material in the absence of S 9, the frequency of aberrant cells observed in the test material treated cultures in either the presence or absence of S9 were within the testing laboratory’s 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 S9) and CP (with S9) treated cultures were 34.0 % and 17.3 %, respectively.

ASSAY C1
There were no significant increases in the incidence of polyploid cells in test material treated cultures as compared to the negative control values.
Statistical analysis of the data indicated significant increases in aberrant cell frequencies in cultures treated with 1400 and 1700 µg/mL (7.0, and 7.5 %, respectively) as compared to the negative control value of 2.5 %.

Table 1: Results of the Chromosome Aberration Assay 4 Hours After Treatment (Replicate A + Replicate B) - Assay A1

Without S9
	Negative control (1 % DMSO)	Test material (517.5 µg/mL)	Test material (1035 µg/mL)	Test material (2070 µg/mL)	Positive control (MMC 0.5 mg/mL)
No. of cells scored	200	200	200	200	125
Chromatid gaps	5	1	1	3	3
Chromosome gaps	0	0	0	0	0
chromatid breaks	2	3	1	3	12
Chromatid exchanges	0	0	0	0	18
Chromosome breaks	0	0	1	0	4
Chromosome exchanges	0	0	0	0	0
Total aberrations (excl. gaps)	2	3	2	3	34
No. of cells with aberrations	2	3	2	3	26
Misc. aberrations	0	0	0	0	0
Cells with multiple aberrations	0	0	0	0	2
With S9
	Negative control (1 % DMSO)	Test material (64.7 µg/mL)	Test material (1035 µg/mL)	Test material (2070 µg/mL)	Positive control (CP 6.0 mg/mL)
No. of cells scored	200	200	200	200	175
Chromatid gaps	0	1	0	2	4
Chromosome gaps	0	0	0	0	0
Chromatid breaks	1	0	1	1	13
Chromatid exchanges	0	0	0	0	25
Chromosome breaks	0	1	0	1	3
Chromosome exchanges	0	0	0	0	0
Total aberrations (excl. gaps)	1	1	1	2	41
No. of cells with aberrations	1	1	1	2	33
Misc. aberrations	0	0	0	0	1
C ells with multiple aberrations	0	0	0	0	1

 

Table 2: Results of the Chromosome Aberration Assay 24 Hours After Treatment (Replicate A + Replicate B) - Assay B1

Without S9
	Negative control (1 % DMSO)	Test material (125 µg/mL)	Test material (750 µg/mL)	Test material (1000 µg/mL)	Test material (1400 µg/mL)	Test material (1700 µg/mL)	Positive control (MMC 0.075 µg/mL)
No. of cells scored	200	200	200	200	200	200	100
Chromatid gaps	3	2	1	3	3	8	3
Chromosome gaps	0	0	0	0	0	0	0
chromatid breaks	1	1	1	9	11	18	19
Chromatid exchanges	0	1	1	0	0	1	23
Chromosome breaks	0	0	0	0	0	1	0
Chromosome exchanges	0	0	0	0	0	0	0
Total aberrations (excl. gaps)	1	2	2	9	11	20	42
No. of cells with aberrations	1	2	2	9	10	15	34
Misc. aberrations	0	0	0	0	0	0	0
Cells with multiple aberrations	0	0	0	0	0	0	4
With S9
	Negative control (1 % DMSO)	Test material (62.5 µg/mL)	Test material (1000 µg/mL)	Test material (2070 µg/mL)			Positive control (CP 6.0 µg/mL)
No. of cells scored	200	200	200	200			150
Chromatid gaps	2	1	1	6			0
Chromosome gaps	0	0	0	0			0
chromatid breaks	1	4	2	4			14
Chromatid exchanges	0	0	0	1			20
Chromosome breaks	0	0	0	0			0
Chromosome exchanges	0	0	0	0			0
Total aberrations (excl. gaps)	1	4	2	5			34
No. of cells with aberrations	1	4	2	5			26
Misc. aberrations	0	0	0	0			0
Cells with multiple aberrations	0	0	0	0			3

 

Table 3: Results of the Chromosome Aberration Assay 24 Hours After Treatment (Replicate A + Replicate B) - Assay C1

Without S9
	Negative control (1 % DMSO)	Test material (1000 µg/mL)	Test material (1400 µg/mL)	Test material (1700 µg/mL)	Positive control (MMC 0.05 µg/mL)
No. of cells scored	200	200	200	200	200
Chromatid gaps	5	6	0	9	8
Chromosome gaps	0	0	0	1	0
chromatid breaks	3	9	13	16	21
Chromatid exchanges	0	0	3	0	6
Chromosome breaks	2	0	1	0	0
Chromosome exchanges	0	0	0	1	0
Total aberrations (excl. gaps)	5	9	17	17	27
No. of cells with aberrations	5	9	14	15	22
Misc. aberrations	0	0	0	0	0
Cells with multiple aberrations	0	0	0	0	1

 

Conclusions:

positive without metabolic activation

The test material induced a clastogenic response in rat lymphocyte cultures treated continuously for 24 hours. However, the magnitude of the response was relatively weak with the frequencies of aberrant cells in test material treated cultures being only slightly greater than the upper end of the laboratory historical negative control data. Hence, under the experimental conditions used, the test material was considered to be a weak clastogen in this in vitro chromosomal aberration assay in the absence of S9.

Executive summary:

The clastogenic potential of the test material was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 473, EU Method B.10, EPA OPPTS 870.5375 and JMAFF (1988).

The test material was evaluated in an in vitro chromosomal aberration assay utilising rat lymphocytes. Approximately 48 hours after the initiation of whole blood cultures, cells were treated for 4 hours with targeted concentrations of 0 (negative control) to 2070 µg test material per mL of culture medium in the absence or presence of an external metabolic activation system (S9). The cultures were harvested 20 hours after the termination of treatments (Assay A1). Based upon the mitotic indices, cultures treated with 0, 517.5, 1035, and 2070 µg/mL without S-9 and 64.7, 1035, and 2070 µg/mL with S9 were selected for evaluating the incidence of chromosomal aberrations. There were no significant increases in the frequencies of cells with aberrations in this assay. In a confirmatory assay (Assay B1), cultures were treated as above except that the cultures without S9 were treated continuously for 24 hours until the time of their harvest. The concentrations tested in Assay B1 were 0, 125, 250, 500, 750, 1000, 1400, 1700 and 2070 µg/mL without S-9 and 0, 62.5, 125, 500, 1000 and 2070 µg/mL with S9. Based upon the mitotic indices, the following cultures were initially selected for estimating the incidence of chromosomal aberrations: 0, 125, 750, and 1400 µg/mL without S9 and 0, 62.5, 1000, and 2070 µg/mL with S9. Statistical analyses of the data identified a significant difference in the frequency of aberrant cells between the negative control and the highest concentration without S9 (0.5 vs. 5.0 % aberrant cells). Because of this finding, cultures treated with 1000 and 1700 µg/mL were also evaluated to determine whether the increase at 1400 µg/mL was related to treatment or a chance occurrence. The extended analysis identified significant increases in aberrant cell frequency at both 1000 and 1700 µg/mL (4.5 and 7.5 %, respectively, vs. 0.5 % in the negative control). No significant differences were observed in cultures treated in the presence of S9. A repeat assay (C1) using 0, 400, 600, 800, 1000, 1200, 1400, 1600, 1700, 1800, and 2070 µg/mL was conducted to evaluate the reproducibility of the response seen in cultures treated for 24 hours without S9. In this assay, the incidence of chromosomal abnormalities was determined from cultures treated with 0, 1000, 1400 and 1700 µg/mL of treatment. The frequencies of aberrant cells at these concentrations were 4.5, 7.0 and 7.5 %, respectively, and these values were significantly different from the negative control value of 2.5 % and were slightly outside of the laboratory historical negative control range. These results confirmed the weak clastogenic activity observed in Assay B1. In all the experiments, cultures treated with the positive control chemicals (i.e., mitomycin C without S9 and cyclophosphamide with S9) had significantly higher incidences of abnormal cells. Hence, under the experimental conditions used, the test material was considered to be a weak clastogen in the in vitro chromosomal aberration assay utilising rat lymphocytes in the absence of S9.

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 April 2001 to 21 May 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:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Purity: not reported

Target gene:

Histidine locus of Salmonella typhimurium and the tryptophan locus of Escherichia coli.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

- Type and identity of media:
Culturing broth = Vogel-Bonner salt solution, supplemented with 2.5 % w/v Oxiod Nutrient Broth
Agar plates = Vogel-Bonner minimal medium E, supplemented with 1.5 % w/v agar and 0.2 % glucose
Overlay agar = 0.7 % agar w/v and 0.5 % NaCl w/v and will be supplemented with 10 mL of 0.5 mM histidine/biotin solution per 100 mL of agar for selection of histidine revertants.
- Properly maintained: yes
- Periodically checked for karyotype stability: yes

Additional strain / cell type characteristics:

other: All strains have uvrB and rfa wall mutations. Strains TA98 and TA100 also contain the pKM101 plasmid.

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

- Type and identity of media:
Culturing broth = Vogel-Bonner salt solution, supplemented with 2.5 % w/v Oxiod Nutrient Broth
Agar plates = Vogel-Bonner minimal medium E, supplemented with 1.5 % w/v agar and 0.2 % glucose
Overlay agar = 0.7 % agar w/v and 0.5 % NaCl w/v and will be supplemented with 0.5 mM tryptophan solution per 100 mL of agar for selection of tryptophan revertants.
- Properly maintained: yes
- Periodically checked for karyotype stability: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

> Range-finding study: 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3330, 5000 µg/plate (with TA100 and WP2uvrA, with and without metabolic activation).
> Mutagenicity assay; 100, 333, 1000, 3330, 5000 µg/plate (all strains, with and without metabolic activation).

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:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-aminoanthracene, ICR-191

Details on test system and experimental conditions:

The tester strains were exposed to the test material via the pre-incubation method. The S9 mix and dilutions of the test material were prepared immediately prior to their use.

PLATING PROCEDURES
When S9 mix was required, 500 μL of S9 mix was added to 13 x 100 mm glass culture tubes, which had been pre-heated to 37 ± 2 °C. To these tubes 100 μL of the tester strain and 50 μL of vehicle or test material concentration was added. When S9 mix was not required, 500 μL of 0.1M phosphate buffer was substituted for the S9 mix. After the required components had been added, the mixture was vortexed and allowed to incubate for 20 ± 2 minutes at 37 ± 2 °C. Two mL of molten selective top agar was then added to each tube, and the mixture was vortexed and overlaid onto the surface of 25 mL of minimal bottom agar contained in a 15 x 100 mm petri dish. After the overlay solidified, the plates were inverted and incubated for 52 ± 4 hours at 37 ± 2 °C. Positive control articles were plated using a 50 μL plating aliquot.

SCORING THE PLATES
Plates which were not evaluated immediately following the incubation period were held at 5 ± 3 °C until such time that colony counting and bacterial background lawn evaluation could take place.

BACKGROUND LAWN EVALUATIONS
The condition of the bacterial background lawn was evaluated both macroscopically and microscopically (using a dissecting microscope) for indications of cytotoxicity and test material precipitate. 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. If present on the plates, macroscopic precipitate was scored as slight, moderate or heavy.

COUNTING REVERTANT COLONIES
Revertant colonies were counted either by automated colony counter or by hand. If there was sufficient test material precipitate on the plates at any concentration that interferes with automated colony counting, then the plates at that concentration were counted manually.

Evaluation criteria:

CRITERIA FOR A POSITIVE RESPONSE
Once the criteria for a valid assay have been met, responses observed in the assay are evaluated as follows:
For a test material to be considered positive, it must produce at least a 3-fold (TA98, TA1535, TA1537 and WP2uvrA) or 2-fold (TA100) dose related increase in the mean revertants per plate of at least one tester strain over the mean revertants per plate of the appropriate vehicle control. A response which does not meet both of the above criteria (magnitude, dose-responsiveness) was not be evaluated as positive.

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and 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 applicable

Positive controls validity:

valid

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 applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING ASSAY
No cytotoxicity was observed with either tester strain in the presence or absence of S9 mix as evidenced by no reduction in the number of revertants per plate and a normal bacterial background lawn. No test material precipitate was observed on any of the plates in the presence or absence of S9 mix.

MUTAGENICITY ASSAY
Concentrations tested in the initial mutagenicity assay were selected based on the results of the range-finding study. The concentrations tested were 100, 333, 1000, 3330, and 5000 μg per plate in both the presence and absence of S9 mix.
In the initial mutagenicity 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.
Concentrations tested in the confirmatory assay were selected based on the results of the initial mutagenicity assay. The concentrations tested were 100, 333, 1000, 3330, and 5000 μg per plate in both the presence and absence of S9 mix. 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.
All criteria for a valid study were met.

Table 1: Mutagenicity Assay Results - Summary

With S9 mix
Concentration (µg/plate)	Mean revertants per plate					Background lawn*
	TA98	TA100	TA1535	TA1537	WP2uvrA
0 (vehicle control)	31	98	13	8	23	1
100	30	111	9	9	19	1
333	26	100	13	8	24	1
1000	28	114	12	10	25	1
3330	29	98	10	9	26	1
5000	26	106	12	8	23	1
Positive control**	353	1000	112	139	625	1
Without S9 mix
Concentration (µg/plate)	Mean revertants per plate					Background lawn*
	TA98	TA100	TA1535	TA1537	WP2uvrA
0 (vehicle control)	20	93	12	9	19	1
100	15	109	14	9	28	1
333	14	101	11	7	20	1
1000	21	106	13	6	21	1
3330	14	92	12	6	22	1
5000	17	94	16	4	23	1
Positive control**	355	1228	893	3661	451	1

**Positive control with / without S9 mix

TA98: benzo[a]pyrene 2.5 µg/plate / 2 -nitrofluorene 1.0 µg/plate

TA100: 2-aminoanthracene 2.5 µg/plate / sodium azide 2.0 µg/plate

TA1535: 2-aminoanthracene 2.5 µg/plate / sodium azide 2.0 µg/plate

TA1537: 2-aminoanthracene 2.5 µg/plate / ICR-191 2.0 µg/plate

WP2uvrA: 2-aminoanthracene 25.0 µg/plate / 4-nitroquinoline-N-oxide 0.4 µg/plate

*Background lawn evaluation code: 1 = normal

Table 2: Confirmatory Mutagenicity Assay Results - Summary

With S9 mix
Concentration (µg/plate)	Mean revertants per plate					Background lawn*
	TA98	TA100	TA1535	TA1537	WP2uvrA
0 (vehicle control)	29	113	17	10	17	1
100	29	98	14	8	16	1
333	27	101	10	9	18	1
1000	36	110	11	11	21	1
3330	48	102	12	11	20	1
5000	32	101	13	12	17	1
Positive control**	390	1218	136	163	657	1
Without S9 mix
Concentration (µg/plate)	Mean revertants per plate					Background lawn*
	TA98	TA100	TA1535	TA1537	WP2uvrA
0 (vehicle control)	20	100	10	9	17	1
100	17	103	15	9	14	1
333	29	89	12	10	16	1
1000	20	98	11	8	16	1
3330	20	92	13	11	16	1
5000	17	93	16	10	17	1
Positive control**	365	1270	848	4203	461	1

**Positive control with / without S9 mix

TA98: benzo[a]pyrene 2.5 µg/plate / 2 -nitrofluorene 1.0 µg/plate

TA100: 2-aminoanthracene 2.5 µg/plate / sodium azide 2.0 µg/plate

TA1535: 2-aminoanthracene 2.5 µg/plate / sodium azide 2.0 µg/plate

TA1537: 2-aminoanthracene 2.5 µg/plate / ICR-191 2.0 µg/plate

WP2uvrA: 2-aminoanthracene 25.0 µg/plate / 4-nitroquinoline-N-oxide 0.4 µg/plate

*Background lawn evaluation code: 1 = normal

Conclusions:

negative with and without metabolic activation

The results indicate that, under the conditions of this study, 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 metabolic activation.

Executive summary:

The mutagenic potential of the test material was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 471, EU Method B13/14, EPA OPPTS 870.5100 and JMAFF (2000).

The objective of this study was to evaluate the ability of the test material to induce reverse mutations either in the presence or absence of mammalian microsomal enzymes at 1) the histidine locus in the genome of several strains of Salmonella typhimurium and at 2) the tryptophan locus of Escherichia coli strain WP2uvrA.

The concentrations tested in the mutagenicity assay were selected based on the results of a range-finding assay using tester strains TA100 and WP2uvrA and ten concentrations of test material ranging from 6.67 to 5000 μg per plate, one plate per concentration, both in the presence and absence of S9 mix. The tester strains used in the mutagenicity assay were Salmonella typhimurium tester strains TA98, TA100, TA1535, and TA1537 and Escherichia coli tester strain WP2uvrA. The assay was conducted with five concentration levels of test material in both the presence and absence of S9 mix along with concurrent vehicle and positive controls using three plates per concentration. The concentrations tested in the mutagenicity assay were 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 an independent experiment.

The results indicate that, under the conditions of this study, 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 metabolic activation.

Reference 6

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 October 2012 to 17 December 2012

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:

EU Method B.17 (Mutagenicity - 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

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

Purity: 93.5%

Target gene:

hgprt locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

The CHO-K1 cell line was used in this study. The cells were grown as monolayer in disposable tissue culture flasks. Cultures were free from any contamination during the conduct of the study.
- Type and identity of media: α-MEM (Minimum Essential Medium, Eagle α-Modification with nucleosides) with nucleosides with 10 % heat inactivated, sterile, foetal bovine serum was used as the culture medium to grow the CHO-K1 cell line. Culture medium was also supplemented with Penicillin (50 IU/mL of the medium) and Streptomycin (50 μg/mL). At the time of selection Minimum Essential Medium Eagle α-modification without nucleosides (α-MEM w/o NS) with 10 % dialysed foetal bovine serum was used.
The medium to eliminate the existing mutants in the culture for treatment was prepared by addition of 2 mL of reconstituted HAT supplement to 98 mL of α-MEM w/o NS with 5 % foetal bovine serum [50X vial of HAT media supplement was reconstituted using 10 mL of sterile α-MEM w/o NS. The reconstituted supplement contains 5 x 10^-3 Hypoxanthine, 2 x 10^-5 M Aminopterine and 8 x 10^-4 M Thymidine].
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (2 % v/v)

Test concentrations with justification for top dose:

Preliminary cytotoxicity test = 156.25, 312.5, 625, 1250, 2500, 5000 µg/mL (with and without 2 % v/v S9)
Trial I = 0 (vehicle control), 156.25, 312.5, 625, 1250, 2500, 5000 µg/mL (with and without 2 % v/v S9, treatment period: 4 hours)
Trial II = 0 (vehicle control), 250, 500, 1000, 2000, 3000, 5000 µg/mL (with and without 2 % v/v S9, treatment period: 4 hours)

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:

benzo(a)pyrene

ethylmethanesulphonate

Details on test system and experimental conditions:

CYTOTOXICITY TEST
Volumes of 120 μL of relevant stock solutions were added into respective tubes containing 11.88 mL of medium to obtain the required test concentrations for the treatment in the absence and presence of metabolic activation system. To each culture flask 5 mL of the treatment medium was added. Based on the results of cytotoxicity test, the test concentration of 5000 μg test material /mL was selected as the highest concentrations for main study experiment both in the absence and presence of the metabolic activation system.

MUTAGENICITY EXPERIMENT
Cultures were maintained in duplicate for each test concentration, negative and positive controls. Cultures were exposed at the concentrations of 156.25, 312.5, 625, 1250, 2500 and 5000 μg/mL in the absence and presence of metabolic activation (2 % v/v S9) for a period of 4 hours (for Trial I).
Volumes of 250 μL of relevant stock solutions were added into 24.75 mL of culture medium to prepare master mix (treatment medium). Each replicate flask was received 10 mL of this respective treatment medium (75 cm² culture flask). Serum free culture medium was used in the absence of metabolic activation while medium with S9 mix (2 % v/v S9) was used in the presence of metabolic activation.
A confirmatory experiment (Trial II) was performed using the same experimental conditions with modification in the concentration spacing. Cultures were exposed at the concentrations of 250, 500, 1000, 2000, 3000 and 5000 μg/mL both in the absence and presence of metabolic activation (2 % v/v S9) for a period of 4 hours.

CULTURE PREPARATION (DAY 0)
HAT (Hypoxanthine Aminopterine Thymidine media supplement) treated cells showing normal growth were used for preparing cultures for treatment. Approximately 24 hours prior to treatment, the required numbers of culture flasks were prepared by inoculating 2000750 cells per flask for Trial I and 2009000 cells per flask for Trial II. The culture flasks prepared for treatment were incubated at 37 ± 1 °C and 5 % CO₂ in humid air using a CO₂ incubator (standard conditions). The day of preparation of culture was recorded as ‘day 0’.

TREATMENT (DAY 1)
Thirty two (16 each for treatment in absence and presence of metabolic activation) culture flasks prepared on ‘day 0’ were observed under an inverted microscope for their growth and culture conditions. Cultures free from contamination were used during both the trials.
The media in culture flasks was removed prior to treatment. Serum free medium was used for the treatment. For treatment in presence of metabolic activation, media containing S9 was used at a final concentration 2 % v/v S9 for Trial I and II.
Duplicate culture flasks were maintained for each test concentration, negative and positive controls. The cultures were incubated at 37 ± 1 °C and 5 % CO₂ in humidified air for 4 hours in Trials I and II. At the end of the exposure period, the treatment medium was removed from the flasks and the cell surface was washed using Dulbecco’s Phosphate Buffered Saline (DPBS). The cells were trypsinised and suspended in complete medium to obtain single cells. The cell concentration was determined by using a haemocytometer and adjusted accordingly with complete medium. The cell concentration in the flasks was adjusted to 1 - 2 x 10⁵ cells/mL. A sample taken from the cell suspension was serially diluted with complete medium to approximately 1000 - 2000 cells/mL. An aliquot of 100 μL was then dispensed on the centre of 60 mm tissue culture dishes and 5 mL of complete medium was added. The plates were incubated for 8 days to determine relative survival and also to demonstrate the cytotoxic effect of selected test concentrations.
At the end of 8 day incubation period the cytotoxicity plates were removed and the medium was decanted. The colonies were fixed, stained and washed. The colonies formed on each plate were counted and recorded for calculation of relative survival/cloning efficiency following treatment.

SUBCULTURE FOR MUTATION EXPRESSION (DAY 3 AND 5)
The cultures for mutation expression were sub-cultured on day 3 and 5, following the culture processing on day 1. During the expression period (7 - 9 days) the cell concentration in the flasks was adjusted by sub-culturing on day 3 and 5. The cells in each flask were trypsinised and the cell concentration was adjusted to approximately 1 x 10⁶ cells/culture, provided with fresh complete medium and incubated under standard conditions.

SELECTION OF MUTANTS (DAY 8)
On day 8, the cells in duplicate flasks (expression flasks) were trypsinised for each test concentration, negative control and positive controls and were counted and plated for determination of survival and mutant frequency. The cell concentration was adjusted to around 1 - 2 x 10⁵ cells/mL for survival plating and 2 x 10⁵ cells/mL for selection of mutants. A sample taken from the cell suspension was diluted to approximately 1000 - 2000 cells/mL by serial dilution. An aliquot of 100 μL was then dispensed on the centre of 60 mm culture dish and 5 mL of complete medium was added. Duplicate plates were maintained for each test concentration negative and positive controls. The plates were then incubated for 8 days for the determination of survival, at this stage, to calculate the mutant frequency. One mL of the suspension (2 x 10⁵ cells/mL) was added into 100 mm culture dish with 10 mL of selective medium (α-MEM without nucleoside - complete medium containing 5 μg/mL of 6-thioguanine). For each treatment 10 dishes were maintained per replicate i.e. 20 dishes/concentration. The plates were incubated at 37 ± 1 °C and 5 % CO₂ in humidified air using a CO₂ incubator for 8 days.

ENUMERATION OF COLONIES
On day 16 the plates were removed from the incubator and the medium was decanted off and colonies were fixed using 3.7 % formaldehyde for 10 minutes. The fixative was removed and the colonies were stained using 0.4 % methylene blue for 15 minutes. The number of colonies formed on each replicate plate or flask was counted and recorded. The number of colonies was used to calculate the absolute cloning efficiency at the time of selection, number of clonable cells and the mutant frequency per 1 x 10⁶ clonable cells.

Evaluation criteria:

ASSAY ACCEPTANCE CRITERIA
A mutation assay is considered acceptable if it meets the following criteria:
a. The criteria for acceptability are a minimum 60 % absolute cloning efficiency in negative controls (solvent used) and a spontaneous Mutant frequency less than 20 per 10⁶ clonable cells (Nestmann, E.R. et al,. 1991).
b. Positive controls induce a significant increase in the mutant frequency above the concurrent negative control.

ASSAY EVALUATION CRITERIA
A test material is considered positive in the mutation assay if:
i. A positive result is defined as a concentration-related biologically significant increase in mutant frequency in comparison with concurrent negative control and the test material causes a three-fold increase (Nestmann, E.R. et al., 1991) in the number of 6-thioguanine resistant colonies relative to concurrent negative control and such increases are statistically significant and outside of the tsting laboratory’s historical negative (solvent used) control range.
ii. A net increase in mutant colonies of treated above the concurrent control is observed in at least two of the concentrations tested.
Negative results were confirmed by a repeat test (short duration), using a modification in test concentrations.

Statistics:

Weighted regression analysis was performed to evaluate the dose response relationship (Li, A.P. et al., 1987; Hsie, A.W. et al., 1981) on test material treatment groups against negative control (excluding positive controls).

Key result

Species / strain:

Chinese hamster Ovary (CHO)

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 applicable

Positive controls validity:

valid

Additional information on results:

A significant change in pH or osmolality was not observed at 0 and 4 hours in any of the tested concentrations. Furthermore, precipitation was not observed in any of the test concentrations tested up to 5000 μg/mL of culture medium. Therefore, the guideline limit dose of 5000 μg/mL was selected as the highest concentration for the cytotoxicity experiment.

CYTOTOXICITY TEST
A significant reduction in the cell count was not observed at the end of the treatment in the cultures treated at the dose level of 5000 μg/mL, both in the absence and presence of the metabolic activation system. The percent relative cloning efficiency observed was 100.92, 90.62, 83.53, 81.46, 73.38 and 52.29 % in the absence of metabolic activation and 97.89, 89.27, 91.08, 90.70, 86.93 and 66.43 % in the presence of metabolic activation (2 % v/v S9), at the test concentrations of 156.25, 312.5, 625, 1250, 2500 and 5000 μg/mL of culture medium, respectively. Therefore, a significant level of cytotoxicity (relative survival of 10 - 20 %) was not observed both in the absence and presence of metabolic activation up to the guideline limit dose of 5000 μg/mL. Based on the observed results, 5000 μg/mL was selected as the highest concentration both in the absence and presence of the metabolic activation system for the main study experiment.

MUTAGENICITY TEST
The absolute cloning efficiency in the negative control was above 60 % during both the trials. The mutant frequency in the negative control group was less than 20 per 10⁶ clonable cells during both the trials, validating the acceptability of the test system. A significant dose related increase in the mutation frequency was not observed in any of the treated concentrations and the mutation frequency was comparable to that from the negative control group. The increased mutant frequency observed in the positive controls in Trials I and II demonstrated the efficiency of the test system and suitability of the test procedures and conditions employed in the study.

Table 1: Absolute and Relative Cloning Efficiency/Survival Following Treatment

Group (µg/mL)	TRIAL I				Group (µg/mL)	TRIAL II
	-S9		+S9			-S9		+S9
	ACE (%)	RCE (%)	ACE (%)	RCE (%)		ACE (%)	RCE (%)	ACE (%)	RCE (%)
NC (DMSO)	76.60	100.00	77.13	100.00	NC (DMSO)	76.69	100.00	75.09	100.00
156.25	72.70	94.91	74.73	96.89	250	74.11	96.63	76.79	102.26
312.5	72.52	94.68	71.17	92.28	500	72.97	95.15	75.09	100.01
625	69.68	90.97	74.11	96.09	1000	70.99	92.56	68.21	90.85
1250	67.55	88.19	67.20	87.13	2000	68.26	89.01	73.13	97.39
2500	62.82	82.01	63.00	81.68	3000	64.22	83.74	67.43	89.81
5000	45.54	59.46	46.10	59.77	5000	50.89	66.36	50.88	67.77
PC	38.25	49.93	53.20	68.98	PC	43.39	56.58	47.32	63.02

NC = negative control

PC = positive control: -S9: Ethyl methanesulfonate 0.4 µl/mL; +S9: Benzo(a)pyrene: 6 µg/mL

Table 2: Absolute Cloning Efficiency at Selection and Mutant Frequency

Group (µg/mL)	TRIAL I				Group (µg/mL)	TRIAL II
	-S9		+S9			-S9		+S9
	Mean ACE	Mean MF	Mean ACE	Mean MF		Mean ACE	Mean MF	Mean ACE	Mean MF
NC (DMSO)	0.7852	16.08	0.7587	14.46	NC (DMSO)	0.7431	14.68	0.7650	11.46
156.25	0.7622	12.43	0.7535	12.23	250	0.7458	9.99	0.7080	10.62
312.5	0.7001	15.58	0.7340	13.83	500	0.7133	12.78	0.7342	11.44
625	0.7185	14.46	0.7148	15.61	1000	0.6903	10.52	0.7350	13.20
1250	0.7060	14.44	0.7035	12.01	2000	0.7139	12.22	0.7570	11.44
2500	0.7279	15.41	0.6907	14.53	3000	0.6576	10.20	0.6743	11.43
5000	0.6391	15.78	0.6250	12.68	5000	0.6210	10.81	0.5684	12.65
PC	0.5035	288.43	0.5531	176.41	PC	0.5466	253.80	0.5267	268.81

NC = negative control

PC = positive control: -S9: Ethyl methanesulfonate 0.4 µl/mL; +S9: Benzo(a)pyrene: 6 µg/mL

Conclusions:

negative with and without metabolic activation

The test material does not have potential to induce gene mutations at the hgprt locus of CHO-K1 cells, both in the absence and presence of metabolic
activation under the experimental conditions.

Executive summary:

The mutagenic potential of the test material was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 476, EU Method B.17 and EPA OPPTS 870.5300.

Dose selection for the gene mutation experiments was based on pre-tests, considering the cytotoxicity, the occurrence of precipitation and changes in the pH and osmolality.

The test material was tested in two independent experiments, with and without metabolic activation (2 % v/v S9). The cultures treated with solvent (DMSO) were kept both in the absence and presence (2 % v/v S9) of metabolic activation and served as negative controls. Ethyl methanesulfonate (0.4 μL/mL) and Benzo(a)pyrene (6 μg/mL) were used as the positive control in the absence and presence of metabolic activation, respectively.

In the first mutagenicity experiment (Trial I), the CHO-K1 cells were exposed to test material at the test concentrations of 156.25, 312.5, 625, 1250, 2500 and 5000 μg/mL of culture media both in the absence and presence of metabolic activation (2 % v/v S9) for a period of 4 hours. No significant dose-related effect was observed in any of the treated concentrations both in the absence and presence of metabolic activation (2 % v/v S9) and the induced mutation frequency was comparable to that from the negative control group.

A second trial was conducted to confirm the negative results obtained in Trial I in the absence and presence of metabolic activation (2 % v/v S9). The proliferating cells were exposed to test material at the test concentrations of 250, 500, 1000, 2000, 3000 and 5000 μg/mL of culture media both in the absence and presence of metabolic activation system (2 % v/v S9) for a period of 4 hours. No significant dose-related effect was observed in any of the treated concentrations both in the absence and presence of metabolic activation (2 % v/v S9) and the induced mutation frequency was comparable to that from the negative control group.

No significant dose-related effect was observed at any of the treated concentrations during either trial of the experiments.

No relevant influence of the test item on pH value or osmolality was observed both in the absence and presence of metabolic activation during both the trials.

The absolute cloning efficiency of the CHO-K1 cell line (negative control) was above 60 % in both the trials. The spontaneous mutation level was (negative control) within the acceptable limit [less than 20 per 10⁶ clonable cells] in both the trials, validating the acceptability of the test system. The increased mutant frequency observed in positive controls in Trials I and II demonstrated the efficiency of the test system and suitability of the test procedures and conditions employed in the study.

From these results, it is concluded that the test material does not have potential to induce gene mutations at the hgprt locus of CHO-K1 cells both in the absence and presence of metabolic activation under the experimental conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 August 2012 to 27 September 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This study was required by ICAMA to obtain aminopyralid registration in China.

Qualifier:

according to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

Deviations:

no

GLP compliance:

yes

Type of assay:

unscheduled DNA synthesis

Specific details on test material used for the study:

Purity: 94.5%

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 8 weeks
- Weight at study initiation: 264.3 - 284.6 g
- Assigned to test groups randomly: Yes. Animals were assigned to 10 treatment groups of 4 male rats each. Animals were assigned to these groups using a randomisation procedure based on equalisation of group mean body weights (MiniTab based program).
- Housing: Rats of the same sex were housed up to 5 per rodent Micro-Barrier cage. Heat-treated hardwood chips were used for bedding to absorb liquids.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 72 ± 3 F
- Humidity: 50 ± 20 % (relative)
- Air changes: at least 10 air changes per hour
- Photoperiod: 12 hours of darkness / 12 hours of light

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 0.5 % (w/v) methylcellulose (400 cps) in water

Details on exposure:

The test material-vehicle mixture, vehicle control (0.5 % (w/v) methylcellulose) and positive control (35 mg/kg dimethylnitrosamine) were administered via oral gavage at a volume of 10 mL/kg as a single administration.
Animals were weighed at least once on each day of dosing. They were observed daily for signs of illness or poor health. Animal were observed for clinical signs of toxicity prior to and following each dose administration. All rats in the experimental and control groups dose volume was based on individual body weights.

Duration of treatment / exposure:

Animals received a single treatment.

Frequency of treatment:

Animals received a single treatment.

Post exposure period:

One set of animals was dosed at 2-4 hours prior to sacrifice and second set was dosed at 12-16 hours prior to sacrifice.

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Eight animals per sex per dose (split equally between the two time points).

Control animals:

yes, concurrent vehicle

Positive control(s):

Dimethylnitrosamine (DMN)
- Route of administration: oral (gavage)
- Doses / concentrations: 35 mg/kg

Tissues and cell types examined:

The animals (3/group) were euthanised 2 to 4 hours post-dose and 12 to 16 hours post-dose to harvest hepatocytes. Hepatocytes were exposed to medium containing tritiated thymidine for 4 hours, washed and incubated another 17 to 20 hours and processed for autoradiography; examination then took place using a microscope and software to quantify the amount of tritiated thymidine incorporated into the cells, as measured by the mean net nuclear grain count (MNNGC). These data were compared between groups to determine if the test material increased incorporation of tritiated thymidine, which would be considered an indication that it caused DNA damage that induced DNA synthesis/repair.

Details of tissue and slide preparation:

PREPARATION OF HEPATOCYTE CULTURES AND SCORING
For preparation of hepatocyte cultures, each rat was anaesthetised by inhalation of isoflurane and a midventral incision was made to expose the liver. The liver was perfused with 0.5 mM ethylene glycol-bis(β-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) solution followed by collagenase solution (80 - 100 units Type I collagenase/mL culture medium). The liver was removed, transected, and shaken in a dilute collagenase solution to release the hepatocytes. The cells were pelleted by centrifugation (50 x g), re-suspended in complete Williams' Medium E (WME; buffered with 0.01 M HEPES, supplemented with 2 mM L-glutamine, 50 μg/mL gentamicin and 10 % foetal bovine serum).
Approximately 5 x 10⁵ cells were seeded into each of six 35 mm tissue culture dishes containing 25 mm coverslips and preconditioned complete WME (i.e., complete WME medium in 35 mm tissue culture dishes incubated overnight in a humidified atmosphere of 5 ± 1 % CO₂ and 37 ± 1 °C). A minimum of 6 cultures were set up for each rat. The hepatocyte cultures were maintained in a humidified atmosphere of 5 ± 1 % CO₂ and 37 ± 1 °C. The vehicle control hepatocyte viability was at least 50 % CO₂ at the time of viable cell count prior to seeding for the assay.
90 to 180 minutes after plating, the cells were washed once with complete WME and re-fed with 2.0 mL serum-free WME containing 10 μCi 3H-thymidine/mL. Four hours later, the radioactive medium was removed; the cultures were washed 3 times in serum-free WME containing 0.25 mM thymidine, and then re-fed with serum-free WME containing 0.25 mM non titrated thymidine and incubated for 17 - 20 hours.
Seventeen to 20 hours after completion of the exposure to thymidine, the coverslips bearing cultures were washed once in serum-free WME. The nuclei were swelled in 1 % sodium citrate solution and the cultures fixed in 3 changes of ethanol-glacial acetic acid fixative (3:1, v/v). The coverslips were allowed to air dry for at least 1.5 hours before mounting cell side up on glass slides.
At least 3 of the 6 slides for each rat were dipped in photographic emulsion at 43.6 °C, allowed to drain and dry for at least 1.5 hours at room temperature and were stored for 7 - 14 days at 2- 8 °C in light tight boxes with desiccant. Slides were developed in Kodak D-19 developer (diluted 1:1 in deionized water), fixed in Kodak fixer, and stained with haematoxylin-eosin stain.
The slides were viewed microscopically under a 100x oil immersion lens. An automated colony counter was interfaced with the microscope so that silver grains within each nuclei and the surrounding cytoplasm can be counted. If possible, 50 nuclei were scored from each of 3 replicate cultures for a total of 150 nuclei from each rat. A minimum of three animals per group were evaluated for UDS. Replicative DNA synthesis is evidenced by nuclei completely blackened with grains, and such cells were not counted. Cells exhibiting toxic effects of treatments, such as irregularly shaped or very darkly stained nuclei, were not counted.

Evaluation criteria:

EVALUATION OF TEST RESULTS
A test material was considered to be positive if:
- Any mean net nuclear grain count (MNNGC) which was increased by at least five counts over the vehicle control was considered significant (Butterworth et al., 1987).
- The test material induced a dose-related increase with no less than one dose significantly elevated above the vehicle control.
- There was a significant increase in the MNNGC in at least two successive doses in the absence of a dose response.

A test material was considered to be equivocal if:
- There was a significant increase in net nuclear grain counts at the high dose group only with no evidence of a dose response.
- There was a significant increase in net nuclear grain counts at one dose with no evidence of a dose response.

A test material was considered to be negative if:
- There was no significant increase in the net nuclear grain counts observed.

> Criteria for a Valid Test
The proportion of cells in repair in the vehicle control group must be less than 15 % and the MNNGC must be less than one. The MNNGC of the positive control group must be at least 5 counts over that of the vehicle control group.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

(no adverse effects noted)

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

CLINICAL SIGNS OF TOXICITY
No mortality or clinical signs were observed in any of the vehicle control-treated animals or the test material-treated animals at 500 and 1000 mg/kg prior to dosing, immediately following dosing or prior to harvest. Test material-treated animals at 2000 mg/kg had increased faecal output observed for the entire group at the time of sacrifice for the 12 to 16 hour exposure. In the positive control treated animals there were signs of diarrhoea at the time of sacrifice.

IN VIVO UDS ASSAY
2 - 4 hour exposure
The mean net nuclear grain count for the vehicle control group was -1.0 with 2 % of cells in repair. The means of the net nuclear grain counts for the 500, 1000 and 2000 mg/kg bw treatment groups were -1.0, -0.9 and -0.5 with 2, 2 and 4 % of cells in repair, respectively. The mean net nuclear grain count for the positive control group was 17.0 with 96 % of cells in repair. The mean net nuclear grain counts from the positive control or test material-treatment groups were compared to the mean net nuclear grain counts from the vehicle control group. None of the test material doses caused a significant increase in the mean net nuclear counts. The positive control compound, DMN at 35 mg/kg bw, induced an increase in the average mean net nuclear grain counts of +18.0 over that of the vehicle control. According to the protocol criteria set for evaluating the test results, the induced increase was considered to be significant in the positive control (DMN) treated animals.

12 - 16 hour exposure
The mean net nuclear grain count for the vehicle control group was -0.6 with 2 % of cells in repair. The means of the net nuclear grain counts for the 500, 1000 and 2000 mg/kg bw treatment groups were -1.6, -1.2 and 0.2 with 0, 2 and 4 % of cells in repair, respectively. The mean net nuclear grain count for the positive control group was 18.4 with 96 % of cells in repair. The mean net nuclear grain counts from the positive control or test material-treatment groups were compared to the mean net nuclear grain counts from the vehicle control group. None of the test material doses caused a significant increase in the mean net nuclear counts. The positive control compound, DMN at 35 mg/kg bw, induced an increase in the average mean net nuclear grain counts of +19.0 over that of the vehicle control. According to the protocol criteria set for evaluating the test results, the induced increase was considered to be significant in the positive control (DMN) treated animals.

DOSING FORMULATION ANALYSIS RESULTS
Analysis of the dosing formulations was performed by HPLC/UV. The concentration found from the top, middle and bottom samples for each concentration of the formulation were within 80 to 120 % of target, which indicated the samples were prepared accurately. The overall % relative standard deviation (% RSD) for all samples for each concentration was 10 %, therefore the formulations were considered homogeneous.

All criteria for a valid study were met. Positive and vehicle controls were within the expected range.

Table 1: Summary of Results (2 to 4 hour exposure)

Group	Cells Scored	Per Animal				Per Treatment Group
		Mean grain counts				Mean Net	Cells in Repair (%)
		Nuclear	Cytoplasmic	Net per nucleus	Cells in repair (%)
0.5 % (w/v) methylcellulose (10 mL/kg)
Vehicle	150	5.8	6.8	-1.0	2	-1.0	2
	150	5.5	7.1	-1.6	1
	150	5.9	6.1	-0.3	2
Test material (mg/kg)
500	150	5.1	6.4	-1.3	1	-1.0	2
	150	6.6	7.1	-0.5	4
	150	6.1	7.3	-1.2	0
1000	150	6.1	6.6	-0.6	1	-0.9	2
	150	5.9	6.9	-1.0	4
	150	5.3	6.4	-1.1	1
2000	150	7.7	9.1	-1.4	2	-0.5	4
	150	6.2	8.0	-1.8	1
	150	4.9	3.3	1.6	9
Positive control: Dimethylnitrosamine (mg/kg)
35	150	25.4	4.8	20.6	99	17.0	96
	150	21.3	6.2	15.1	97
	150	19.2	3.9	15.3	92

 

Table 2: Summary of Results (12 to 16 hour exposure)

Group	Cells Scored	Per Animal				Per Treatment Group
		Mean grain counts				Mean Net	Cells in Repair (%)
		Nuclear	Cytoplasmic	Net per nucleus	Cells in Repair (%)
0.5 % (w/v) methylcellulose (10 mL/kg)
Vehicle	150	5.5	6.8	-1.3	3	-0.6	2
	150	4.3	4.8	-0.5	1
	150	5.0	5.0	-0.1	3
Test material (mg/kg)
500	150	5.0	6.6	-1.6	1	-1.6	0
	150	5.9	8.1	-2.2	0
	150	4.3	5.3	-1.0	0
1000	150	5.3	7.2	-2.0	0	-1.2	2
	150	5.1	5.9	-0.7	3
	150	5.1	6.0	-0.9	3
2000	150	3.3	2.9	0.4	1	0.2	4
	150	6.8	6.7	0.1	7
	150	6.5	6.5	0.1	5
Positive control: Dimethylnitrosamine (mg/kg)
35	150	21.5	3.9	17.7	96	18.4	96
	150	21.7	4.0	17.7	96
	150	24.7	4.8	20.0	97

 

Conclusions:

negative
Under the test conditions, the test material did not induce a significant increase in the mean number of net nuclear grain counts in hepatocytes isolated from treated animals, and was concluded to be negative in the Unscheduled DNA Synthesis (UDS) Assay with mammalian cells in vivo.

Executive summary:

The genotoxic potential of the test material was tested in an Unscheduled DNA Synthesis (UDS) Assay with mammalian cells prepared from rats dosed in vivo. The study was conducted under GLP conditions and in accordance with the standardised guideline OECD 486. Specifically, the UDS assay was used to evaluate the potential of the test material to induce unscheduled DNA synthesis in primary hepatocyte cultures obtained from test material-treated Sprague-Dawley rats.

During the study two sets of animals were dosed orally with test material. One set of animals was dosed at 2-4 hours prior to sacrifice and second set was dosed at 12-16 hours prior to sacrifice. Each time point included five groups of four animals in each group dosed at 500, 1000, 2000 mg/kg test material along with oral dose of vehicle 0.5 % (w/v) methylcellulose and positive control 35 mg/kg dimethylnitrosamine (DMN), respectively. The test and control articles were administered at a constant volume of 10 mL/kg by a single oral gavage.

No mortality or clinical signs were observed in any of the vehicle control-treated animals and the test material-treated animals prior to dosing, immediately following dosing or prior to harvest. Clinical signs were observed in positive control-treated animals prior to harvest.

The animals (3/group) were euthanised 2 to 4 hours post-dose and 12 to 16 hours post-dose to harvest hepatocytes. Hepatocytes were exposed to medium containing tritiated thymidine for 4 hours, washed and incubated another 17 to 20 hours and processed for autoradiography; and then examined using a microscope and software to quantify the amount of tritiated thymidine incorporated into the cells, as measured by the mean net nuclear grain count (MNNGC). These data were compared between groups to determine if test material increased incorporation of tritiated thymidine, which would be considered an indication that it caused DNA damage that induced DNA synthesis/repair.

Under the conditions of the study, the test material did not cause a significant increase in average mean MNNGC at any dose level or harvest time. At both harvest times, the proportion of cells in repair in the vehicle control group was less than 15 %, the average mean MNNGC of the vehicle control group was less than 1, and the average mean MNNGC of the positive control group was at least 5 counts over that of the vehicle control group. Based on these results, all criteria for a valid study were met, and the test material was concluded to be negative for genotoxic potential in hepatocytes from male Sprague-Dawley rats given single oral doses up to 2000 mg/kg.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

IN VITRO

In vitro gene mutation in bacterial cells (Ames)

Two studies investigating in vitro gene mutation in bacterial cells are available.

In the first (Lawlor, 1998), the test material was evaluated in the Salmonella/Mammalian-Microsome Reverse Mutation Screening Assay (Ames Test); the study was conducted under GLP conditions. Since the study was not a guideline study, but was regarded as a screening test, it was assigned a reliability score of 2 in line with the criteria of Klimisch et al. (1997) and is included as supporting information.

The Salmonella typhimurium tester strains used in the study were TA98 and TA100. The assay was conducted using seven dose levels of the test material in the presence of an externally supplied metabolic activation system (S9). The concentration of the test material ranged from 9.99 to 5000 µg/plate.

The test material did not induce a positive increase in the number of revertant colonies in either strain. Hence, the test material was classified as negative in this bacterial mutagenicity screening test under the experimental conditions used.

In the second (Mecchi, 2004), the mutagenic potential of the test material was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 471, EU Method B13/14, EPA OPPTS 870.5100 and JMAFF (2000). The study was assigned a reliability score of 1 in line with the criteria of Klimisch et al. (1997) and is regarded as key data.

The objective of this study was to evaluate the ability of the test material to induce reverse mutations either in the presence or absence of mammalian microsomal enzymes at 1) the histidine locus in the genome of several strains of Salmonella typhimurium and at 2) the tryptophan locus of Escherichia coli strain WP2uvrA. The concentrations tested in the mutagenicity assay were selected based on the results of a range-finding assay using tester strains TA100 and WP2uvrA and ten concentrations of test material ranging from 6.67 to 5000 µg per plate, one plate per concentration, both in the presence and absence of S9 mix. The tester strains used in the mutagenicity assay were Salmonella typhimurium tester strains TA98, TA100, TA1535, and TA1537 and Escherichia coli tester strain WP2uvrA. The assay was conducted with five concentration levels of test material in both the presence and absence of S9 mix along with concurrent vehicle and positive controls using three plates per concentration. The concentrations tested in the mutagenicity assay were 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 an independent experiment.

The results indicate that, under the conditions of this study, 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 metabolic activation.

In vitro mammalian chromosome aberration test

Two in vitro mammalian chromosome aberration tests are available.

In the first (Linscombe & Jackson, 2000), the clastogenic potential of the test material to cultures of rat lymphocytes, was assessed in a study which was conducted under GLP conditions. The test material was evaluated in an in vitro chromosomal aberration screening assay. Since the study was not a guideline study, but was regarded as a screening test, it was assigned a reliability score of 2 in line with the criteria of Klimisch et al. (1997) and is included as supporting information.

Approximately 48 hours after the initiation of whole blood cultures, cells in the absence of S9 activation were treated for 24 hours with targeted doses of 0 (negative control), 16.2, 32.3, 64.7, 129.4, 258.8, 517.5, 1035, and 2070 µg test material per mL of culture medium and harvested at the end of treatment. The highest concentration tested was the limit dose of 10 mM. In the presence of S9 activation, cultures were treated using the same dose levels of the test material for 4 hours and harvested approximately 24 hours after treatment termination. The test material was initially dissolved in dimethyl sulphoxide (DMSO) and then added to the cultures to give the desired final concentrations. Cultures treated with 0.05 and 0.075 µg/mL mitomycin C (MMC) or 4 and 6 µg/mL cyclophosphamide (CP) were used as positive controls for the non-activation and activation assays, respectively; only one concentration level was evaluated for aberrations. Based upon the mitotic indices, cultures treated with 0 and 517.5 µg/mL of the test material in the absence of S9 and cultures treated with 0 and 2070 µg/mL in the presence of S9 activation were selected for determining the incidence of chromosomal aberrations.

No significant increase in the incidence of aberrant cells was noticed in either of the treatment levels when compared to the negative controls. The positive control cultures had significantly higher incidences of aberrant cells. Hence, the test material was considered to be negative in the in vitro chromosomal aberration-screening assay utilising rat lymphocytes.

In the second study (Linscombe et al., 2002), the clastogenic potential of the test material was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 473, EU Method B.10, EPA OPPTS 870.5375 and JMAFF (1988). The study was assigned a reliability score of 1 in line with the criteria of Klimisch et al. (1997) and is regarded as key data.

The test material was evaluated in an in vitro chromosomal aberration assay utilising rat lymphocytes. Approximately 48 hours after the initiation of whole blood cultures, cells were treated for 4 hours with targeted concentrations of 0 (negative control) to 2070 µg test material per mL of culture medium in the absence or presence of an external metabolic activation system (S9). The cultures were harvested 20 hours after the termination of treatments (Assay A1). Based upon the mitotic indices, cultures treated with 0, 517.5, 1035, and 2070 µg/mL without S9 and 64.7, 1035, and 2070 µg/mL with S9 were selected for evaluating the incidence of chromosomal aberrations. There were no significant increases in the frequencies of cells with aberrations in this assay.

In a confirmatory assay (Assay B1), cultures were treated as above except that the cultures without S9 were treated continuously for 24 hours until the time of their harvest. The concentrations tested in Assay B1 were 0, 125, 250, 500, 750, 1000, 1400, 1700 and 2070 µg/mL without S9 and 0, 62.5, 125, 500, 1000 and 2070 µg/mL with S9. Based upon the mitotic indices, the following cultures were initially selected for estimating the incidence of chromosomal aberrations: 0, 125, 750, and 1400 µg/mL without S9 and 0, 62.5, 1000, and 2070 µg/mL with S9. Statistical analyses of the data identified a significant difference in the frequency of aberrant cells between the negative control and the highest concentration without S9 (0.5 vs. 5.0 % aberrant cells). Because of this finding, cultures treated with 1000 and 1700 µg/mL were also evaluated to determine whether the increase at 1400 µg/mL was related to treatment or a chance occurrence. The extended analysis identified significant increases in aberrant cell frequency at both 1000 and 1700 µg/mL (4.5 and 7.5 %, respectively, vs. 0.5 % in the negative control). No significant differences were observed in cultures treated in the presence of S9. A repeat assay (C1) using 0, 400, 600, 800, 1000, 1200, 1400, 1600, 1700, 1800, and 2070 µg/mL was conducted to evaluate the reproducibility of the response seen in cultures treated for 24 hours without S9. In this assay, the incidence of chromosomal abnormalities was determined from cultures treated with 0, 1000, 1400 and 1700 µg/mL of treatment. The frequencies of aberrant cells at these concentrations were 4.5, 7.0 and 7.5 %, respectively, and these values were significantly different from the negative control value of 2.5 % and were slightly outside of the laboratory historical negative control range. These results confirmed the weak clastogenic activity observed in Assay B1. In all the experiments, cultures treated with the positive control chemicals (i.e., mitomycin C without S9 and cyclophosphamide with S9) had significantly higher incidences of abnormal cells. Hence, under the experimental conditions used, the test material was considered to be a weak clastogen in the in vitro chromosomal aberration assay utilising rat lymphocytes in the absence of S9.

In vitro mammalian cell gene mutation assay

Two in vitro gene mutation assays in mammalian cells are available. Both were performed under GLP conditions and followed standardised guidelines. Both studies were awarded a reliability score of 1 in line with the criteria of Klimisch et al. (1997) and are regarded as key data.

In the first (Linscombe et al., 2001), the mutagenic potential of the test material was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 476, EU Method B.17 and EPA OPPTS 870.5300. The test material was evaluated in an in vitro Chinese hamster ovary cell/hypoxanthine-guanine-phosphoribosyl transferase (CHO/HGPRT) forward gene mutation assay.

The genotoxic potential of the test material was assessed in two independent assays in the absence and presence of an externally supplied metabolic activation (S9) system with concentrations ranging from 31.25 to 2070 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 S9 and 20-methylcholanthrene for assays with S9. Negative control cultures were treated with the vehicle used to dissolve the test material.

Based upon the frequency of TGr mutants recovered in cultures treated with the test material, it was concluded that the test material did not induce a mutagenic response in the assay system employed.

In the second study (Nagane, 2012), the mutagenic potential of the test material was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 476, EU Method B.17 and EPA OPPTS 870.5300.

Dose selection for the gene mutation experiments was based on pre-tests, considering the cytotoxicity, the occurrence of precipitation and changes in the pH and osmolality. The test material was tested in two independent experiments, with and without metabolic activation (2 % v/v S9). The cultures treated with solvent (DMSO) were kept both in the absence and presence (2 % v/v S9) of metabolic activation and served as negative controls. Ethyl methanesulfonate (0.4 μL/mL) and Benzo(a)pyrene (6 μg/mL) were used as the positive control in the absence and presence of metabolic activation, respectively. In the first mutagenicity experiment (Trial I), the CHO-K1 cells were exposed to test material at the test concentrations of 156.25, 312.5, 625, 1250, 2500 and 5000 μg/mL of culture media both in the absence and presence of metabolic activation (2 % v/v S9) for a period of 4 hours. No significant dose-related effect was observed in any of the treated concentrations both in the absence and presence of metabolic activation (2 % v/v S9) and the induced mutation frequency was comparable to that from the negative control group. A second trial was conducted to confirm the negative results obtained in Trial I in the absence and presence of metabolic activation (2 % v/v S9). The proliferating cells were exposed to test material at the test concentrations of 250, 500, 1000, 2000, 3000 and 5000 μg/mL of culture media both in the absence and presence of metabolic activation system (2 % v/v S9) for a period of 4 hours. No significant dose-related effect was observed in any of the treated concentrations both in the absence and presence of metabolic activation (2 % v/v S9) and the induced mutation frequency was comparable to that from the negative control group.

No significant dose-related effect was observed at any of the treated concentrations during either trial of the experiments. No relevant influence of the test material on pH value or osmolality was observed both in the absence and presence of metabolic activation during both the trials. The absolute cloning efficiency of the CHO-K1 cell line (negative control) was above 60 % in both the trials. The spontaneous mutation level was (negative control) within the acceptable limit [less than 20 per 10⁶ clonable cells] in both the trials, validating the acceptability of the test system. The increased mutant frequency observed in positive controls in Trials I and II demonstrated the efficiency of the test system and suitability of the test procedures and conditions employed in the study. From these results, it is concluded that the test material does not have potential to induce gene mutations at the hgprt locus of CHO-K1 cells both in the absence and presence of metabolic activation under the experimental conditions.

IN VIVO

In vivo Unscheduled DNA Synthesis

The genotoxic potential of the test material was tested in an Unscheduled DNA Synthesis (UDS) Assay with mammalian cells prepared from rats dosed in vivo. The study was conducted under GLP conditions and in accordance with the standardised guideline OECD 486. The study was assigned a reliability score of 1 in line with the criteria of Klimisch et al. (1997) and is regarded as key data.

Specifically, the UDS assay was used to evaluate the potential of the test material to induce unscheduled DNA synthesis in primary hepatocyte cultures obtained from test material-treated Sprague-Dawley rats.

During the study two sets of animals were dosed orally with test material. One set of animals was dosed at 2 - 4 hours prior to sacrifice and second set was dosed at 12 - 16 hours prior to sacrifice. Each time point included five groups of four animals in each group dosed at 500, 1000, 2000 mg/kg test material along with oral dose of vehicle 0.5 % (w/v) methylcellulose and positive control 35 mg/kg dimethylnitrosamine (DMN), respectively. The test and control articles were administered at a constant volume of 10 mL/kg by a single oral gavage. No mortality or clinical signs were observed in any of the vehicle control-treated animals and the test material-treated animals prior to dosing, immediately following dosing or prior to harvest. Clinical signs were observed in positive control-treated animals prior to harvest. The animals (3/group) were euthanised 2 to 4 hours post-dose and 12 to 16 hours post-dose to harvest hepatocytes. Hepatocytes were exposed to medium containing tritiated thymidine for 4 hours, washed and incubated another 17 to 20 hours and processed for autoradiography; and then examined using a microscope and software to quantify the amount of tritiated thymidine incorporated into the cells, as measured by the mean net nuclear grain count (MNNGC). These data were compared between groups to determine if test material increased incorporation of tritiated thymidine, which would be considered an indication that it caused DNA damage that induced DNA synthesis/repair.

Under the conditions of the study, the test material did not cause a significant increase in average mean MNNGC at any dose level or harvest time. At both harvest times, the proportion of cells in repair in the vehicle control group was less than 15 %, the average mean MNNGC of the vehicle control group was less than 1, and the average mean MNNGC of the positive control group was at least 5 counts over that of the vehicle control group. Based on these results, all criteria for a valid study were met, and the test material was concluded to be negative for genotoxic potential in hepatocytes from male Sprague-Dawley rats given single oral doses up to 2000 mg/kg.

Justification for selection of genetic toxicity endpoint
Multiple studies have been provided to address the different endpoints of genetic toxicity, each key study assessing a different type of genetic toxicity. Therefore a single study could not be selected as key over the others. All key studies were conducted to standardised guidelines under GLP conditions.

Short description of key information:
In Vitro
Negative, bacterial reverse gene mutation assay, OECD 471, EU Method B.13/14, EPA OPPTS 870.5100 and Japan MAFF (2000), Mecchi (2004)
Weak clastogen (without metabolic activation), chromosome aberration assay, OECD 473, EPA OPPTS 870.5375, EU Method B.10 and Japan MAFF (1988), Linscombe et. al. (2002)
Negative, mammalian gene mutation assay, OECD 476, EPA OPPTS 870.5300 and EU Method B.17, Linscombe et al. (2001)
Negative, mammalian gene mutation assay, OECD 476, EPA OPPTS 870.5300 and EU Method B.17, Nagane (2012)

In Vivo
Negative, unscheduled DNA synthesis, OECD 486, Pant & Celestin (2012)

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.

In accordance with the criteria for classification as defined in Annex VI, Directive 67/548/EEC (DSD), the substance does not require classification with respect to genetic toxicity.