Disodium 2-(2,4,5,7-tetraiodo-6-oxido-3-oxoxanthen-9-yl)benzoate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro bacterial cell gene mutation study

Mutagenicity testing was performed by the standard plate-incorporation assay for the test chemical on Salmonella typhimurium by using TA1535, TA1537, TA1538, TA98 and TA100 strains. The test chemical was negative in the Salmonella mutagenicity assays conducted.

In vitro mammalian chromosomal aberration study

An in vitro mammalian chromosome aberration study was conducted for the test chemical in Chinese hamster lung-derived fibroblasts (CHL) in the absence of metabolic activation system. The test chemical tested negative for mutachromosomal effects in Chinese hamster lung-derived fibroblasts (CHL).

In vitro mammalian gene mutation study

L5178Y TK ± mouse lymphoma assay was conducted to evaluate the mutagenic potential of the test chemical. The mouse lymphoma cell line, L5178Y TK± (Clone 3.7.2C), used was derived from the Fischer L5178Y line by Dr Donald Clive.None of the assayed treatments induced a mutant frequency that exceeded the minimum criterion of 69.3 x 10^-6. Hence, the test chemical can be considered to be non-mutagenic at concentrations that approached excessive toxicity under both the conditions of metabolic activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from peer-reviewed scientific journal

Qualifier:

according to guideline

Guideline:

other: AMES ASSAY

Principles of method if other than guideline:

Mutagenicity testing was performed by the standard plate-incorporation assay for the test chemical on Salmonella typhimurium by using TA1535, TA1537, TA1538, TA98 and TA100 strains.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium, other: TA1535, TA1537, TA1538, TA100 and TA98

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: Each chemical was tested without metabolic activation and with S9 mix from Aroclor 1254-induced male Fischer 344 rats and Syrian golden hamsters.
- source of S9
- method of preparation of S9 mix : The S9 mix was prepared according to the procedure described by Ames et al. (1975) and contained 0.1 ml S9 per ml of S9 mix; an aliquot of 0.5 ml of S9 mix was added per plate.
- concentration or volume of S9 mix and S9 in the final culture medium
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability)

Test concentrations with justification for top dose:

33 .0µg/plate,100 .0µg/plate,333.0 µg/plate,1000.0µg/plate,3333.0µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: [none; no data; acetone; arachis oil; beeswax; carbowaxe; castor oil; cetosteryl alcohol; cetyl alcohol; CMC (carboxymethyl cellulose); coconut oil; corn oil; cotton seed oil; DMSO; ethanol; glycerol ester; glycolester; hydrogenated vegetable oil; lecithin; macrogel ester; maize oil; olive oil; paraffin oil; peanut oil; petrolatum; physiol. saline; poloxamer; polyethylene glycol; propylene glycol; silicone oil; sorbitan derivative; soya oil; theobroma oil; vegetable oil; aqueous solvents (water or saline or culture medium)]: The solvents used were water, dimethyl sulfoxide, and acetone (exact solvent was not mention)

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: The positive control for all strains with metabolic activation was 2-aminoanthracene (2.5µg/plate for TA1535 and TA1537, 1.0µg/plate for TA1538 and TA98, and 5.0 µg/plate for TA100).

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : TRIPLICATES
- Number of independent experiments

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk : PLATE INCORPORATION

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable:
- Exposure duration/duration of treatment:
- Harvest time after the end of treatment (sampling/recovery times):

Evaluation criteria:

A response was considered positive if there was a dose-related increase in the number of revertants above spontaneous solvent controls, with a 2-fold increase for strains TA1535, TA1538, TA98 and TA100, and a 3-fold increase for TA1537.

Statistics:

No data available

Species / strain:

S. typhimurium, other: TA1535, TA1537, TA1538, TA100 and TA98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH:
- Data on osmolality:
- Possibility of evaporation from medium:
- Water solubility:
- Precipitation and time of the determination:
- Definition of acceptable cells for analysis:
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES: The range of concentrations for testing was based on preliminary toxicity tests in which the viability of the bacterial cells on complete medium was measured at concentrations up to 10 mg/plate or to the limit of solubility. When solubility and toxicity were not limiting factors, the maximum concentration tested was 10 mg/plate.

STUDY RESULTS
- Concurrent vehicle negative and positive control data

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible
- Statistical analysis; p-value if any
- Any other criteria: e.g. GEF for MLA

Ames test: The test chemical was negative in the Salmonella mutagenicity assays conducted.

Remarks on result:

other: negative

Results of the Salmonella Mutagenicity study using the Plate incorporation assay.

Dose(µg/plate)	Revertants per plate
	TA1535
	(-S)S9                                   (R)S9                            (H)S9
Solvent Cont.	30±7	16±6	20±6
Positive Cont.^b	380±6	198±17^2	119±7
33.00	21±3	19±3	18±2
100.00	28±3	21±6	21±4
333.00	19±4	15±2	16±2
1000.00	15±4	9±1	10±1
3333.00	14±2	13±3	11±3

Dose(µg/plate)	Revertants per plate
	TA1537
	(-S)S9                                   (R)S9                            (H)S9
Solvent Cont.	5±2	9±5	9±3
Positive Cont.^b	199±30	36±28^2	79±6
33.00	5±4	7±2	9±8
100.00	8±3	10±3	5±2
333.00	6±3	6±3	9±1
1000.00	5±1	5±2	7±6
3333.00	5±1	4±4	7±2

Dose(µg/plate)	Revertants per plate
	TA98
	(-S)S9                                   (R)S9                            (H)S9
Solvent Cont.	21±3	34±5	34±3
Positive Cont.^b	196±34^2	1086±74^2	991±25
33.00	18±4	25±11	24±2
100.00	17±5	27±5	26±6
333.00	19±4	31±2	25±9
1000.00	15±5	23±6	20±6
3333.00	13±4	12±2	20±7

Dose(µg/plate)	Revertants per plate
	TA1538
	(-S)S9                                   (R)S9                            (H)S9
Solvent Cont.	15±3	19±4	22±3
Positive Cont.^b	191±16^1	1040±33^2	525±15
33.00	16±4	22±5	14±9
100.00	12±5	16±4	14±9
333.00	12±2	21±7	15±4
1000.00	9±2	14±6	19±4
3333.00	11±1	14±1	10±5

Dose(µg/plate)	Revertants per plate
	TA100
	(-S)S9                                   (R)S9                            (H)S9
Solvent Cont.	171±14	156±13	191±26
Positive Cont.^b	571±7	1630±63^2	1214±61
33.00	179±21	138±2	160±21
100.00	168±11	136±10	189±21
333.00	106±10	163±20	179±34
1000.00	117±35	164±14	175±7
3333.00	102±33	138±5	171±9
^aMean and standard deviation. ^bin assays without metabofic activation, the positive controls were sodium azide for TA1535 and TA100, 1.0 µg/plate, 9-aminoacridine for TA1537, 50.0 µg/plate; 2-nitrofluorene for TA1538, 1.0 or 5.0 µg/plate, and for TA98, 2.0 or 5.0 µg/plate. In those assays in which the lower dose of 2-nitrofluorene was used, the positive control value is followed by a superscript indicating the dose level used (either^1or^2). In assays with rat-liver S9, the positive control for all strains was 2-aminoanthracene, 2.5 µg/plate except for those positive control values, followed by a numerical superscript. This number is the dose used as a positive control. In assays with hamster-fiver S9, the positive control for all strains was 2-aminoanthracene, 1.0 µg/plate, except for those positive controls followed by the superscript "c". In these cases 2.5µg/plate was used. 1 Dose level was 1.0 µg/plate. 2 Dose level was 2.0 µg/plate. 5 Dose level was 5.0 µg/plate. c Dose level was 2.5 µg/plate. d Only 2 plates

Conclusions:

The test chemical was negative in the Salmonella mutagenicity assays conducted.

Executive summary:

Mutagenicity testing was performed by the standard plate-incorporation assay for the test chemical on Salmonella typhimurium by using TA1535, TA1537, TA1538, TA98 and TA100 strains. These strains were kindly supplied by Dr. Bruce Ames, University of California at Berkeley. Strain checks were performed immediately after receipt of the cultures and on a routine basis thereafter. The test chemical was tested without metabolic activation and with S9 mix from Aroclor 1254-induced male Fischer 344 rats and Syrian golden hamsters. The S9 mix was prepared according to the procedure described by Ames et al. (1975) and contained 0.1 ml S9 per ml of S9 mix; an aliquot of 0.5 ml of S9 mix was added per plate. The test chemical was either dissolved in water, DMSO or acetone and tested at concentrations of 33.0µg/plate, 100.0µg/plate, 333.0µg/plate, 1000.0µg/plate, 3333.0µg/plate in triplicates. The range of concentrations for testing was based on preliminary toxicity tests in which the viability of the bacterial cells on complete medium was measured at concentrations up to 10 mg/plate or to the limit of solubility. When solubility and toxicity were not limiting factors, the maximum concentration tested was 10 mg/plate.In tests without metabolic activation the positive controls were sodium azide for strains TA1535 and TA100 (1.0 µg/plate), 9-aminoacridine for TA1537 (50.0 µg/plate), and 2-nitrofluorene for TA1538 and TA98 (1.0, 2.0 or 5.0 µg/plate). The positive control for all strains with metabolic activation was 2-aminoanthracene (2.5µg/plate for TA1535 and TA1537, 1.0µg/plate for TA1538 and TA98, and 5.0 µg/plate for TA100). A response was considered positive if there was a dose-related increase in the number of revertants above spontaneous solvent controls, with a 2-fold increase for strains TA1535, TA1538, TA98 and TA100, and a 3-fold increase for TA1537. The test chemical was negative in the Salmonella mutagenicity assays conducted.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from publication.

Qualifier:

according to guideline

Guideline:

other: as mentioned below

Principles of method if other than guideline:

in vitro mammalian chromosome aberration study was conducted for the given test chemical.

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

No data

Species / strain / cell type:

other: Chinese hamster lung-derived fibroblasts (CHL)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: The cell line was originally established from the lung of a newborn female at the Cancer Research Institute, Tokyo (Koyama, Utakoji & Ono, 1970),

For cell lines:
- Absence of Mycoplasma contamination: No data
- Number of passages if applicable: 4-day passages
- Methods for maintenance in cell culture: The cell line was maintained in Minimum Essential Medium (MEM; GIBCO) supplemented by 10% calf serum.
- Cell cycle length, doubling time or proliferation index : The doubling time was approximately 15 hr.
- Modal number of chromosomes: The modal chromosome number is 25

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

Colcemid (final conc 0.2 microgm/ml) was added to the culture 2 hr before cell harvesting.

Metabolic activation:

without

Metabolic activation system:

no metabolic activation systems were applied

Test concentrations with justification for top dose:

0.001 mg/ml, Top dose was expected to produce a 50% inhibition of cell growth based on data from a pre-experiment.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used [none; no data; acetone; arachis oil; beeswax; carbowaxe; castor oil; cetosteryl alcohol; cetyl alcohol; CMC (carboxymethyl cellulose); coconut oil; corn oil; cotton seed oil; DMSO; ethanol; glycerol ester; glycolester; hydrogenated vegetable oil; lecithin; macrogel ester; maize oil; olive oil; paraffin oil; peanut oil; petrolatum; physiol. saline; poloxamer; polyethylene glycol; propylene glycol; silicone oil; sorbitan derivative; soya oil; theobroma oil; vegetable oil; aqueous solvents (water or saline or culture medium)] : Physiological saline

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Details on test system and experimental conditions:

DURATION
- Exposure duration: 24 & 48 hours
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
NUMBER OF CELLS EVALUATED: 100
DETERMINATION OF CYTOTOXICITY
- Method: 50% cell growth inhibition
OTHER EXAMINATIONS:
- Determination of polyploidy: yes

Evaluation criteria:

Results were considered negative if incidence of aberrations was less than 4.9%, equivocal if it was between 5 and 9.9% and positive if it was more than 10%

Statistics:

No data

Key result

Species / strain:

other: Chinese hamster lung-derived fibroblasts (CHL)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

Chromosome aberration test (CA) in mammalian cells:

o Changes in ploidy (polyploidy cells and cells with endoreduplicated chromosomes) if seen: 0% polypliody was observed.

Remarks on result:

other: No mutagenic potential

Conclusions:

The test chemical tested negative for mutachromosomal effects in Chinese hamster lung-derived fibroblasts (CHL).

Executive summary:

In vitro mammalian chromosome aberration study was conducted for the test chemical in Chinese hamster lung-derived fibroblasts (CHL) in the absence of metabolic activation system. The cell line was originally established from the lung of a newborn female at the Cancer Research Institute, Tokyo (Koyama, Utakoji & Ono, 1970), and was maintained by 4-day passages in Minimum Essential Medium (MEM; GIBCO) supplemented by 10% calf serum. The modal chromosome number is 25 and the doubling time was approximately 15 hr. The cells were exposed to chemical at three concentrations up to 0.001 mg/ml for 24 and 48 hr. Physiological saline was used as solvent. The top dose was expected to produce a 50% inhibition on cell growth based on data from a pre-experiment. Colcemid (final conc 0.2 µg/ml) was added to the culture 2 hr before cell harvesting. The cells were then trypsinized and suspended in a hypotonic KCI solution (0.075 M) for 13 min at room temperature. After centrifugation the cells were fixed with acetic acid-methanol (1:3, v/v) and spread on clean glass slides. After air-drying, the slides were stained with Giemsa solution (1.5%, at pH 6.8; E. Merck) for 12-15 min. A hundred well-spread metaphases were observed under the microscope (x 600 with a nocover objective lens). The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. Results were considered negative if incidence of aberrations was less than 4.9%, equivocal if it was between 5 and 9.9% and positive if it was more than 10%. The test chemical tested negative for mutachromosomal effects in Chinese hamster lung-derived fibroblasts (CHL).

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

data is from peer reviewed journals

Qualifier:

according to guideline

Guideline:

other: L5178Y TK ± mouse lymphoma assay

Principles of method if other than guideline:

L5178Y TK ± mouse lymphoma assay was conducted to evaluate the mutagenic potential of the test chemical

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

.CELLS USED
- Type and source of cells: The mouse lymphoma cell line, L5178Y TK± (Clone 3.7.2C), used was derived from the Fischer L5178Y line by Dr Donald Clive, Burroughs Welcome, Research Triangle Park, NC
- Suitability of cells:
- Normal cell cycle time (negative control):

For cell lines:
- Absence of Mycoplasma contamination: Stocks were maintained in liquid nitrogen
and laboratory cultures were periodically checked for the absence of mycoplasma
contamination.
- Number of passages if applicable:
- Methods for maintenance in cell culture: Cell stock cultures were routinely exposed to medium containing 3 µg/ml thymidine, 5 µg/ml hypoxanthine, 0.1 µg/ml methotrexate and 7.5 µg/ml glycine (THMG). The cultures were then returned to THG medium for 1 day and then to normal growth medium for 2 days before use in mutagenesis experiments.
- Cell cycle length, doubling time or proliferation index :
- Modal number of chromosomes:
- Periodically checked for karyotype stability: [yes/no]
- Periodically ‘cleansed’ of spontaneous mutants: [yes/no]

For lymphocytes:
- Sex, age and number of blood donors:
- Whether whole blood or separated lymphocytes were used:
- Whether blood from different donors were pooled or not:
- Mitogen used for lymphocytes:

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: The cells were maintained in Fischer's mouse leukemia medium supplemented
with 2 mM L-glutamine, 110 µg/ml sodium pyruvate, 96 units/ml penicillin,
96µg/ml streptomycin, 0.05% Pluronic F68 and horse serum (10% by volume).
Cloning medium consisted of the preceding growth medium (minus Pluronic F68)
with the addition of agar to a final concentration of 0.35% to achieve a semisolid
state. The selection medium was the cloning medium containing 100µg/ml
of 5-bromodeoxyuridine (BrdUrd).

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: The S9 was prepared from the livers of Aroclor 1254-induced Fischer 344 male rats (Ames et al., 1975)
- source of S9
- method of preparation of S9 mix
- concentration or volume of S9 mix and S9 in the final culture medium
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability)

Test concentrations with justification for top dose:

Non- activated phase - 0, 100,200, 300, 400, 500, 600, 800 µg/ml
Activated phase- 0, 50, 150, 300, 350, 400 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: [none; no data; acetone; arachis oil; beeswax; carbowaxe; castor oil; cetosteryl alcohol; cetyl alcohol; CMC (carboxymethyl cellulose); coconut oil; corn oil; cotton seed oil; DMSO; ethanol; glycerol ester; glycolester; hydrogenated vegetable oil; lecithin; macrogel ester; maize oil; olive oil; paraffin oil; peanut oil; petrolatum; physiol. saline; poloxamer; polyethylene glycol; propylene glycol; silicone oil; sorbitan derivative; soya oil; theobroma oil; vegetable oil; aqueous solvents (water or saline or culture medium)] : deionized water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

The solvent control was prepared by adding 0.1 ml H2O per 10-ml culture.

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) :
- Number of independent experiments

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): Each treated culture consisted of 3 x 106 cells suspended in 10-ml final
volumes in 15-ml centrifuge tubes
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable:
- Exposure duration/duration of treatment: The cell cultures were exposed to the test material or control chemical for 4 h,
- Harvest time after the end of treatment (sampling/recovery times):

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): The cell cultures were exposed to the test material or control chemical for 4 h, washed and allowed an expression time of 2 days in growth medium.
- Selection time (if incubation with a selective agent): At the end of the expression period, 3 x 106 cells from each treated culture were seeded in selection medium at 1 X 106 cells per 100-mm dish.
- Fixation time (start of exposure up to fixation or harvest of cells):
- Method used: agar or microwell plates for the mouse lymphoma assay. : microwell
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure. :
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: To determine the efficiency of cloning, a portion of each cell suspension was serially diluted and 300 cells were seeded in cloning medium (non-selective) at 100 cells per 100-mm dish. BrdUrd-resistant colonies (TK~'~ mutants) and viable colonies
(non-selective medium) were counted after 10 days of incubation at 37°C in a humidified atmosphere containing 5% CO2.A Biotran II automatic counter was
used at settings allowing the scoring of colonies with diameters of - 0 . 3 mm and larger.
- Criteria for small (slow growing) and large (fast growing) colonies:

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition; mitotic index (MI); relative population doubling (RPD); relative increase in cell count (RICC); replication index; cytokinesis-block proliferation index; cloning efficiency; relative total growth (RTG); relative survival (RS); other:
- Any supplementary information relevant to cytotoxicity:

METHODS FOR MEASUREMENTS OF GENOTOXICIY

- OTHER:

Evaluation criteria:

The average of the solvent and untreated negative control mutant frequencies was used as the background or spontaneous mutant frequency for each trial. A treated culture was considered to have a significant elevated mutant frequency if the frequency exceeded 10-5 plus 1.5x the background frequency. Additional criteria, such as the presence of a dose-related or toxicity-related response and repeatability between trials were used to help assess the presence of any mutagenic activity.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

excessive toxicity was observed at 400 µg/ml in the activated phase and 800 µg/ml in the non-activated phase

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

STUDY RESULTS
- Concurrent vehicle negative and positive control data : The negative control mutant frequencies were all in the normal ranges and the positive control compounds yielded normal mutant frequencies

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible
- Statistical analysis; p-value if any
- Any other criteria: e.g. GEF for MLA

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements: Under nonactivation conditions, the test material was assayed from 100 to 600 µg/ml and non-detectable to moderate toxicities were induced (134 — 24.5% relative growths). Higher toxicities were not assayed because the toxicity curve was excessively steep. In the presence of metabolic activation, the test material was assayed from 50 to 350 µg/ml and non-detectableto moderate toxicities were induced (117.7—40.5% relative growths). Two highest concentrations (300 and 350 µg/ml) were assayed in duplicate to determine the reproducibility of the results.
o Relative total growth (RTG) or relative survival (RS) and cloning efficiency

- Genotoxicity results: Under the non-activation conditions, all of the assayed treatments produced mutant frequencies that were similar to the background mutant frequencies (solvent control). Since there was no evidence for mutagenic activity up to concentrations that approached excessive toxicity and no dose-related response was observed, the test material was considered non-mutagenic without activation in this assay.Small increases in concentration > 350 µg/ml were excessively toxic. None of the assayed treatments induced a mutant frequency that exceeded the minimum criterion of 69.3 x 10-6.

Remarks on result:

other: not mutagenic

Conclusions:

Under the non-activation conditions, all of the assayed treatments produced mutant frequencies that were similar to the background mutant frequencies (solvent control). Since there was no evidence for mutagenic activity up to concentrations that approached excessive toxicity and no dose-related response was observed, the test material was considered non-mutagenic without activation in this assay.Small increases in concentration > 350 µg/ml were excessively toxic. None of the assayed treatments induced a mutant frequency that exceeded the minimum criterion of 69.3 x 10-6. Hence, the test chemical can be considered to be non-mutagenic at concentrations that approached excessive toxicity under both the conditions of metabolic activation.

Executive summary:

L5178Y TK ± mouse lymphoma assay was conducted to evaluate the mutagenic potential of the test chemical. The mouse lymphoma cell line, L5178Y TK± (Clone 3.7.2C), used was derived from the Fischer L5178Y line by Dr Donald Clive.Stocks were maintained in liquid nitrogen and laboratory cultures were periodically checked for the absence of mycoplasma contamination. Cell stock cultures were routinely exposed to medium containing 3 µg/ml thymidine, 5 µg/ml hypoxanthine, 0.1 µg/ml methotrexate and 7.5 µg/ml glycine (THMG). The cultures were then returned to THG medium for 1 day and then to normal growth medium for 2 days before use in mutagenesis experiments. The test chemical was dissolved in deionized water just prior to use, and the aqueous solution was diluted 1:100 into the cell cultures to initiate the treatments.Negative, solvent and positive controls were used. The solvent control was prepared by adding 0.1 ml H₂O per 10-ml culture. The positive control for the non-activation portion of the assay was 0.5 µl/ml ethylmethane sulfate (EMS). This treatment was initiated by adding 0.5 ml of freshly prepared aqueous stock (10 µl/ml) to a 9.5-ml cell culture. Dimethylnitrosamine (DMN) at 0.3µg/ml was the positive control for the activation assays. A 0.3-ml aliquot of a freshly prepared aqueous stock (10 µl/ml) was added to a 9.7-ml cell culture containing the S9 activation system. The procedure was based on the method as described by Clive and Spector.Each treated culture consisted of 3 x 10⁶ cells suspended in 10-ml final volumes in 15-ml centrifuge tubes. The nonactivation and activation assays were conducted in the same manner, except the cell cultures in the activation assay contained 50 µl/ml of S9 supernatant and co-factors 2.7 mg/ml NADP and 4.5 mg/ml isocitric acid. The S9 was prepared from the livers of Aroclor 1254-induced Fischer 344 male rats (Ames et al., 1975). The cell cultures were exposed to the test material or control chemical for 4 h, washed and allowedan expression time of 2 days in growth medium. Cell counts were determined by hemacytometer and the cell cultures were diluted to 3 x 10⁵ cells/ml each day if the density had increased beyond 4 x 105 cells/ml. The average of the solvent and untreated negative control mutant frequencies was used as the background or spontaneous mutant frequency for each trial. A treated culture was considered to have a significant elevated mutant frequency if the frequency exceeded 10^-5 plus 1.5x the background frequency. Additional criteria, such as the presence of a dose-related or toxicity-related response and repeatability between trials were used to help assess the presence of any mutagenic activity.The negative control mutant frequencies were all in the normal ranges and the positive control compounds yielded normal mutant frequencies. Under non-activation conditions, the test material was assayed from 100 to 600 µg/ml and non-detectable to moderate toxicities were induced (134 — 24.5% relative growths). Higher toxicities were not assayed because the toxicity curve was excessively steep. In the presence of metabolic activation, the test material was assayed from 50 to 350 µg/ml and non-detectableto moderate toxicities were induced (117.7—40.5% relative growths). Two highest concentrations (300 and 350 µg/ml) were assayed in duplicate to determine the reproducibility of the results.Under the non-activation conditions, all of the assayed treatments produced mutant frequencies that were similar to the background mutant frequencies (solvent control). Since there was no evidence for mutagenic activity up to concentrations that approached excessive toxicity and no dose-related response was observed, the test material was considered non-mutagenic without activation in this assay.Small increases in concentration > 350 µg/ml were excessively toxic. None of the assayed treatments induced a mutant frequency that exceeded the minimum criterion of 69.3 x 10 ^-6. Hence, the test chemical can be considered to be non-mutagenic at concentrations that approached excessive toxicity under both the conditions of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In vivo HPC/DR genetic toxicity in vivo assay was performed for the test chemical by study on hepatocytes isolated from rat. Six to eight rats were perfused on a given day (ie, experiment), including one control (ie, usually corn oil-treated) from which hepatocytes were obtained for in vivo assays. One or more known positive chemicals (usually SY3) were tested .DNA repair was quantified by the autoradiographic determination of incorporated [3H]-thymidine, similar to the method of Williams [1977] and Bermudez et al [1979], as detailed in Kornbrust and Barfknecht [1984a,b]. Net nuclear grains (NNG) were determined by counting the number of grains in each nuclei and subtracting the average number of grains present in three equal-size adjacent cytoplasmic areas. A strict procedure for random selection of cells was used [Mirsalis and Butterworth,1980]. The average NNG for 60 cells (±SD) as well as the percentage of cells with 25 NNG was determined for the test chemical. single dose was given that was equal to approximately. Average net nuclear grain counts of 5 or greater were assumed to constitute a positive response, since these differed from the control net nuclear counts by greater than 2 SD. The NNG count for the test chemical was less than 5 and the test chemical was negative i.e gave no induction of DNA repair when tested at 200 mg/plate in Wistar rats.

Link to relevant study records

Reference

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviwed journal

Qualifier:

according to guideline

Guideline:

other: in vivo rat hepatocyte primary culture/DNA repair (HPC/DR) assay

Principles of method if other than guideline:

The test chemical was tested for the in vivo rat hepatocyte primary culture/DNA repair (HPC/DR) assay.

GLP compliance:

not specified

Type of assay:

other: In Vivo/ In Vitro Rat Hepatocyte Primary Culture/ DNA Repair Assays

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Kingston, NY
- Age at study initiation: No data available
- Weight at study initiation: 200-300 g
- Assigned to test groups randomly: [no/yes, under following basis: ] No data available
- Fasting period before study: No data available
- Housing: The animals were housed in humidity- and temperature-controlled rooms.

- Diet (e.g. ad libitum): Diet (ad libitum)
- Water (e.g. ad libitum): Water (ad libitum)
- Acclimation period: No data available

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Controlled
- Humidity (%):Controlled
- Air changes (per hr): No data available
- Photoperiod (hrs dark / hrs light): 12-hr light/dark cycles

IN-LIFE DATES: From: To: No data available

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: aqueous solutions or corn oil suspensions (for the non-water-soluble dyes)

Duration of treatment / exposure:

2 hour and 15 hour time point

Frequency of treatment:

Onces daily

Post exposure period:

No data available

Remarks:

Doses / Concentrations:
200 mg/kg
Basis:
no data

No. of animals per sex per dose:

six to eight rats

Control animals:

yes, concurrent vehicle

Positive control(s):

o-aminoazotoluene

Tissues and cell types examined:

Rat hepatocytes

Details of tissue and slide preparation:

OTHER: Six to eight rats were perfused on a given day (ie, experiment), including one control (ie, usually corn oil-treated) from which hepatocytes were obtained for in vitro assays. One or more known positive chemicals (usually SY3) were tested .DNA repair was quantified by the autoradiographic determination of incorporated [3H]-thymidine, similar to the method of Williams [1977] and Bermudez et al [1979], as detailed in Kornbrust and Barfknecht [1984a,b]. Net nuclear grains (NNG) were determined by counting the number of grains in each nuclei and subtracting the average number of grains present in three equal-size adjacent cytoplasmic areas. A strict procedure for random selection of cells was used [Mirsalis and Butterworth,1980]. The average NNG for 60 cells (±SD) as well as the percentage of cells with 25 NNG was determined for the test chemical. single dose was given that was equal to approximately 50% of the single dose LD50 (derived from reference toxicity data) or 500 mg/k body weight, whichever was smaller.
 

Evaluation criteria:

Average net nuclear grain counts of 5 or greater were assumed to constitute a positive response, since these differed from the control net nuclear counts by greater than 2 SD. Net nuclear grain counts below zero were considered negative responses.

For those dyes that produced responses between zero and 5 average net nuclear grains, it was generally not possible to demonstrate a statistically significant difference from the control value within a given experiment. Therefore, these responses were judged to be equivocal, unless, in addition to an average net nuclear grain count between zero and 5, at least 25% of the cells examined contained ≥ 5 net nuclear grains, in which case the response was considered weakly positive.

Concentrations of the dyes that produced approximately 90% or greater detachment of the hepatocytes from the coverslips (as assessed visually by comparing to control slides) were assumed to be toxic and were not counted.

Statistics:

Average net nuclear grains was reported as Mean ± standard deviation

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: non mutagenic

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range:
- Solubility:
- Clinical signs of toxicity in test animals:
- Evidence of cytotoxicity in tissue analysed:
- Rationale for exposure:
- Harvest times:
- High dose with and without activation:
- Other:

RESULTS OF DEFINITIVE STUDY : The test chemical was negative i.e gave no induction of DNA repair when tested at 200 mg/plate in Wistar rats
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay):
- Induction of micronuclei (for Micronucleus assay):
- Ratio of PCE/NCE (for Micronucleus assay):
- Appropriateness of dose levels and route:
- Statistical evaluation:

Responses Produced by Various Dyes in the In Vivo/In Vitro HPC/DR Assay

Dye	Dose	Time PTP^a (hr)	Avg. NNG^b	Percent cells<5 NNG	Res- ponse
Acid red 51	200	2 15	-1.0 (± 3.3) -1.1 (± 3.1)	5 5	N
^aTime that dye was administered prior to start of liver perfusion and isolation of hepatocytes. ^bAverage net nuclear grains (as defined in the Methods section); mean + standard deviation from 60 cells. ^CPercent of cells with > S net nuclear grains. ^dP. positive; WP, weak positive; E, equivocal; N, negative (as per the criteria defined in the Methods section).

Conclusions:

The test chemical was negative i.e gave no induction of DNA repair when tested at 200 mg/plate in Wistar rats.

Executive summary:

In vivo HPC/DR genetic toxicity in vivo assay was performed for the test chemical by study on hepatocytes isolated from rat. Six to eight rats were perfused on a given day (ie, experiment), including one control (ie, usually corn oil-treated) from which hepatocytes were obtained for in vivo assays. One or more known positive chemicals (usually SY3) were tested .DNA repair was quantified by the autoradiographic determination of incorporated [3H]-thymidine, similar to the method of Williams [1977] and Bermudez et al [1979], as detailed in Kornbrust and Barfknecht [1984a,b]. Net nuclear grains (NNG) were determined by counting the number of grains in each nuclei and subtracting the average number of grains present in three equal-size adjacent cytoplasmic areas. A strict procedure for random selection of cells was used [Mirsalis and Butterworth,1980]. The average NNG for 60 cells (±SD) as well as the percentage of cells with 25 NNG was determined for the test chemical. single dose was given that was equal to approximately. Average net nuclear grain counts of 5 or greater were assumed to constitute a positive response, since these differed from the control net nuclear counts by greater than 2 SD. The NNG count for the test chemical was less than 5 and the test chemical was negative i.e gave no induction of DNA repair when tested at 200 mg/plate in Wistar rats.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic toxicity in vitro

In vitro bacterial cell gene mutation study

Various studies have been evaluated to determine the mutagenic potential of the test chemical in bacterial tester strains. The results are mentioned below:

Mutagenicity testing was performed by the standard plate-incorporation assay for the test chemical on Salmonella typhimurium by using TA1535, TA1537, TA1538, TA98 and TA100 strains.

These strains were kindly supplied by Dr. Bruce Ames, University of California at Berkeley. Strain checks were performed immediately after receipt of the cultures and on a routine basis thereafter. The test chemical was tested without metabolic activation and with S9 mix from Aroclor 1254-induced male Fischer 344 rats and Syrian golden hamsters. The S9 mix was prepared according to the procedure described by Ames et al. (1975) and contained 0.1 ml S9 per ml of S9 mix; an aliquot of 0.5 ml of S9 mix was added per plate. The test chemical was either dissolved in water, DMSO or acetone and tested at concentrations of 33.0µg/plate, 100.0µg/plate, 333.0µg/plate, 1000.0µg/plate, 3333.0µg/plate in triplicates. The range of concentrations for testing was based on preliminary toxicity tests in which the viability of the bacterial cells on complete medium was measured at concentrations up to 10 mg/plate or to the limit of solubility. When solubility and toxicity were not limiting factors, the maximum concentration tested was 10 mg/plate.In tests without metabolic activation the positive controls were sodium azide for strains TA1535 and TA100 (1.0 µg/plate), 9-aminoacridine for TA1537 (50.0 µg/plate), and 2-nitrofluorene for TA1538 and TA98 (1.0, 2.0 or 5.0 µg/plate). The positive control for all strains with metabolic activation was 2-aminoanthracene (2.5µg/plate for TA1535 and TA1537, 1.0µg/plate for TA1538 and TA98, and 5.0 µg/plate for TA100). A response was considered positive if there was a dose-related increase in the number of revertants above spontaneous solvent controls, with a 2-fold increase for strains TA1535, TA1538, TA98 and TA100, and a 3-fold increase for TA1537. The test chemical was negative in the Salmonella mutagenicity assays conducted.

This is supported by a study where the FMN pre-incubation modification of the Salmonella assay was performed to evaluate the mutagenic potential of the test chemical. Salmonella Typhimurium strains TA98 and TA100 were used. The bacteria, uninduced hamster liver S9 (30% V/V), plus cofactors (FMN, NADH, G6PD, G6P), and test chemical were added to tubes, mixed, and incubated at 30°C for 30 minutes without shaking. Nitrogen was blown over the pre-incubation tube before capping to keep the atmosphere reduced. At the end of the incubation period, 2 ml of molten top agar (Ames et al., 1975) were added to each tube, and the mixture was poured on a minimal agar plate containing 0.5% glucose rather than the 2% glucosespecified by Ames et al. (1975). Plates were incubated at 37°C for 48 h. The positive control in all FMN experiments was trypan blue (228µg/ plate). All plates were counted on an Artek ® automated counter, which was calibrated before use. The test chemical was negative in the Salmonella mutagenicity assays conducted.

In a similar FMN-modified assay, the test chemical was tested under reductive conditions using Salmonella typhimurium strains TA98, TA100. Salmonella typhimurium histidine auxotrophs TA98, TA100 were obtained from Dr. Bruce Ames, University of California (Berkeley, CA). Cultures were grown overnight in Oxoid nutrient broth no. 2 and were removed from incubation when they reached a density of (1-2)x10⁹cells/mL. On the day of use, all tester strain cultures were checked for genetic integrity as recommended by Ames et al. The test chemical was dissolved in water and the final concentrations used for the FMN MODIFIED PRE-INCUBATION ASSAY were 0, solvent control, 33, 100, 333, 1000, 3333 µg/plate. The bacteria, uninduced hamster liver S9 (30% v/v), cofactors (FMN, NADH, glucose-6-phosphate dehydrogenase, and glucose-6-phosphate), and test chemical were added, mixed, and incubated at 30 °C for 30 min without shaking. Nitrogen was blown over the preincubation tube to keep the atmosphere reduced. At the end of the incubation period, 2 mL of molten top agar was added to each sample tube and the mixture was poured on a minimal agar plate containing 0.5% glucose rather than the 2% glucose specified by Ames et al. The plates were then incubated at 37°C for 48 h. The positive control in all FMN experiments was Congo red. The test chemical failed to induce mutagenic response to Salmonella Typhimurium tester strains TA 98, TA100 under reductive conductions

in the presence of uninduced hamster liver S9 metabolic activation system. Hence. the test chemical can be considered to be non mutagenic to bacterial tester strains.

In another study, Plate incorporation method was used to evaluate genetic effect of the test chemical in Salmonella typhimurium TA98, TA100, TA1535, TA1537, and TA1538.Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535, TA1537, and TA1538 were obtained from Dr. Bruce Ames, University of California (Berkeley, CA). Cultures were grown overnight in Oxoid nutrient broth no. 2 and were removed from incubation when they reached a density of (1-2)x109 cells/mL. On the day of use, all tester strain cultures were checked for genetic integrity as recommended by Ames et al. Liver S9 homogenate was prepared from male Sprague-Dawley rats and Syrian golden hamsters that had been injected with Aroclor 1254 at 500 mg/kg body weight. The post-mitochondrial (microsomal) enzyme fractions were prepared as described by Ames et al. The components of the S9 mix were 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 4 mM NADP, 100 mM sodium phosphate (pH 7.4), and the appropriate S9 homogenate at a concentration of 0.1 mL/mL of mix. For each plate receiving microsomal enzymes, 0.5 mL of S9 mix was added. The doses that were tested in the mutagenicity assay were selected based on the levels of cytotoxicity observed in a preliminary dose range-finding study using strain TA100. Ten dose levels of the chemical, one plate per dose, were tested in both the presence and the absence of induced hamster S9. If no toxicity was observed, a total maximum dose of 10 mg of test chemical per plate was used. The test chemical was dissolved in water and the concentrations used for the main study were 33, 100, 333, 1000, 3333 µg/plate. For testing in the absence of S9 mix, 100 µL of the tester strain and 50 µL of the solvent or test chemical were added to 2.5 mL of molten selective top agar at 45± 2 °C. When S9 was used, 0.5 mL of S9 mix, 50µL of tester strain, and 50 µL of solvent or test chemical were added to 2.0 mL of molten selective top agar at 45 ±2 °C. After it was vortexed, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. After the overlay had solidified, the plates were incubated for 48 h at 37± 2 °C. Appropriate concurrent solvent and positive controls, along with the test chemical concentrations were tested in triplicate on each tester strain without metabolic activation and also with activation by induced rat and hamster liver S9 preparations. The criteria used to evaluate a test were as follows: for a test article to be considered positive, it had to induce at least a doubling (TA98, TA100, and TA1535) in the mean number of revertants per plate of at least one tester strain. This increase in the mean revertants per plate had to be accompanied by a dose response to increasing concentrations of the test chemical. If the study showed a dose response with a less than 3-fold increase on TA1537 or TA1538, the response had to be confirmed in a repeat experiment. The test chemical failed to induce mutagenic response to Salmonella Typhimurium tester strains TA 98, TA100, TA1535, TA 1537, TA 1538 both in the presence and absence of rat and hamster liver S9 metabolic activation system. Hence. the test chemical can be considered to be non mutagenic to bacterial tester strains.

These results are further supported by a gene mutation toxicity study performed by the pre-incubation method to evaluate the mutagenic potency of the test chemical. Reverse mutation assays using S. typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 were carried out according to the method of Ames, McCann & Yamasaki (1975). The liver microsome fraction (S-9) was prepared from the liver of Fischer rats (Charles River Japan Co .) pre-treated 5 days before with polychlorinated biphenyls (500mg/kg body weight of Kanechlor KC-400 in olive oil, ip) . The reaction mixture (S-9 mix) contained 5 mM-glucose 6-phosphate, 4 mM-NADPH, 4 mM-NADH, 33 mN KCl, 8mM-MgCl2, 100 mM-phosphate buffer (pH 7 .4) and 3 .75 ml S-9 (129 mg protein) in a total volume of 12 .5 ml. The test chemical was dissolved in DMSO and tested upto the maximum dose of 10 mg/plate. Cells cultured overnight were pre-incubated with both the test sample and the S-9 mix for 20 min at 37°C before plating. Duplicate plates were used for each of six different concentrations of the sample. The result was considered positive if the number of colonies found was twice the number in the control (exposed to the appropriate solvent or untreated). If no reasonable dose response was detected, additional experiments using different doses or induced mutation frequency assays (Yoshikawa, Nakadate, Watabe er al. 1980) were performed. The test chemical failed to induce mutagenic activity in Salmonella Typhimurium tester strains TA92, TA1535, TA100, TA1537, TA94 and TA98 under both conditions of metabolic activation system. Hence, the test chemical can be considered to be non mutagenic.

The induction of mutations was also studied in modified liquid fluctuation tests using a tryptophan-requiring E. coli strain (sensitive to base substitutions) for the test chemical. Fluctuation test was performed for the test chemical used at a concentration of 5 mg/mL in liquid medium for 72-96 hrs.The food colour was made up in deionized water and membrane-sterilized prior to use. The test chemical was tested at its maximum sublethal concentration. The tester strain used was Escherichia coliWP2uvrA both in the presence and absence of metabolic activation system. The assay was performed for each bacterium in three separate experiments. Ethidium bromide (167 microgram), furacin (10 microgram) and mitomycin C (1 microgram) were used as positive controls. The test chemical was considered positive only if it resulted in significant more turbid tubes in a treated series when compared with an untreated set of tubes.The test chemical failed to induce genotoxicity in the bacteria Escherichia coliWP2uvrA and hence was negative for gene mutation in vitro.

Additionally, a Salmonella/microsome assay was performed to determine the mutagenic potential of the test chemical. Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538, TA98 were obtained from Dr. Bruce N. Ames (University of California, Berkeley). Frozen permanents of each tester strain and the S-9 fractions were stored in sterile plastic vials under liquid nitrogen (Linde LR-30). Broth cultures of the tester strains were prepared by inoculating 10 ml nutrient broth - 0.5% NaCI contained in each of five 25-ml Delong culture flasks from the respective frozen permanents, and incubating overnight (14-16 h) at 37 ° in a New Brunswick G24 gyrotory incubator shaker at 350 r.p.m. The test chemical was dissolved in DMSO and the concentrations of the test chemical used for the study were 50, 250, 1000 µg/plate. The mammalian liver homogenate fraction "S-9" was prepared essentially as described by Ames et al.. Female Sprague-Dawley rats (Simonsen Laboratories, Gilroy, Calif.) were maintained on Simonsen white laboratory diet. Five days prior to sacrifice the animals were injected i.p. with about 0.5 ml of a corn oil solution of Aroclor 1254 (400 mg/ml) to give a dose of 500 mg/kg body weight. Removal of livers and subsequent preparation of the S-9 fraction and "S-9 mix" have been described in detail previously in Brown, J.P. et.al, 1976. Liver microsome preparation from rats injected with Aroclor 1254, 0.25 ml "S-9 mix"/plate with 0.2 ml S9/ml "S9-mix"; - quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability). Plates containing various concentrations of the test chemical incorporated with the top agar and in some cases 100 -200 µg of the test agent contained in a 0.25-inch sterile concentration disk applied to the top agar were incubated 3 days at 35 °C. Ethylmethanesulfonate (5 µl); Methylmethanesulfonate (µl); 9 -Aminoacridine (50 µg); Nitroquinoline-N-oxide (0.22 µg); Anthragallol ( 50 µg ); 2-Anthramine (1 µg) were used as positive controls. The criteria adopted for scoring a mutagenic response in routine plate tests is that the observed number of revertants exceed twice the background value for that given assay and exceed the 99.9% confidence limit based on the historical controls. The negative and positive control values were comparable to the historical control values. The test chemical failed to induce mutagenic response in Salmonella tester strains TA1535, TA100, TA1537, TA1538, TA98 both in the presence and absence of metabolic activation system.

Furthermore, in an identical salmonella/microsome assay, the mutagenic potential of the test chemical was evaluated using Salmonella tester strains. Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 were obtained from Dr Bruce Ames, University of California at Berkeley. All indicator strains were kept at 4°C on minimal medium plates supplemented with a trace of biotin and an excess of histidine (Ames, 1980). In addition, the plates with the plasmid-carrying Salmonella strains (TA98 and TA100) were supplemented with 25 µg/ml of ampicillin to ensure stable maintenance of plasmid pKM101. The bacteria strains were cultured at 37°C in Oxoid Media no. 2 (nutrient broth), and Vogel - Bonner Medium E with 2 % glucose was used as the selective medium. S9 liver homogenates, which were prepared from Aroclor 1254-induced and non-induced adult Sprague- Dawley male rats as described by Ames el al. (1975), were purchased from Hazleton Biotechnologies Corporation (formerly Bionetics Laboratory Product, Litton Bionetics, Inc.). The overlay agar was prepared according to the method of Ames el al. (1975). The test chemical was dissolved in deionized water.The maximum concentration of 10 mg/plate was selected on the basis of slight toxicity observed and that 10 mg/plate represents a generally accepted high dose. The concentration of the test chemical used in the main study were 0, 1, 10, 100, 500, 1000, 2500, 5000, 10000 µg/plate. Criteria which were used to determine whether a chemical was mutagenic were: (i) an increase in revertants in strains TA1535, TA1537 and TA1538 of three times the solvent control; (ii) an increase in revertants in strains TA98 and TA100 of twice the solvent control; (iii) reproducibility and (iv) a dose-related response, and a consistent pattern of response between strains derived from the same parental strain. Eight dose levels ranging from 1 to 10 000µg/plate were evaluated for the test chemical. Some evidence of toxicity was observed at the high dose levels. None of the dose levels of the test chemical increased the number of revertants by a factor of two or more in any of the S. typhimurium strains either in the presence or absence of S9 rat liver homogenate. Hence the test chemical can be considered to be not mutagenic to Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538, TA98.

These results are substantiated by another bacterial mutagenicity study was performed by the method of Ames with S. typhimurium strains TA1535, TA100, TA1538, TA98 and TA1537 to evaluate the mutagenic potential of the test chemical. One-tenth ml of the appropriate food coloring in distilled water and 0.1 ml of bacterial culture were added to a soft agar overlay supplemented with 0.5 mM biotin and 0.5 mM histidine. The agar was mixed and poured over a base plate of Spizzizen's minimal medium. Revertant colonies were scored after 48 h at 37°C. The livers of male Fischer rats (F344/f Mai) pre-treated with Aroclor 1254 for the non-specific induction of enzyme activity served as the sources of enzymes for metabolic activation. In the assay, 0.5 ml S-9 Mix, consisting of liver post-mitochondrial supernatant and cofactors, was added to the soft agar overlay along with test substance and bacteria. Plates were poured and incubated as described above. The appearance of colored colonies on the agar plates was taken as an indication that the coloring had penetrated the bacteria and therefore was in close proximity to the DNA. MNNG , BaP and 4-NQO were used as positive controls. BaP with S-9 was used at 10µg/plate for strain TA98. Because of the increased sensitivity of TA100 to the action of this agent, it was used at 5 µg/plate with this strain. Each determination was the mean of 3 plates and is shown with the standard deviation around the mean. Known positive mutagens used as controls resulted in an increase in the number of revertants per plate. All strains gave negative results with test chemical. There was no indication of mutagenic activity either with or without metabolic activation with strains TA 98, TA 100, TA1535, TA1537 and TA1538. An examination of the background lawn failed to show any overt indication of toxicity. Hence, the test chemical can be considered to be non-mutagenic in nature.

These results are ably lent support by a study in which the genotoxicity of the test chemical was evaluated before and after UV irradiation. Aqueous solutions of the test chemical The lamp used was Black Light Blue Flourescent light; FL15-BLB from Matsushita Electric Industrial Co., Ltd (Osaka,Japan). The concentration of the food colours was 0.15 g/50 ml, with distilled water as solvent. The distance from the UV lamp to the surface of the solution was 12 cm. The test chemical was constantly stirred and irradiated for 14 days. The mutagenicity of the test chemical was assessed in Salmonella typhimurium TA98 and TA100 with and without S-9 mix (Maron and Ames, 1983), by the pre-incubation method. The strains were kindly provided by Professor B.N. Ames. The test chemical was applied at the maximum concentration of 3 mg/plate. The positive controls were AF-2 at the maximum concentration of 0.1 mg/plate for TA98 andTA100 in the absence of S-9 mix, and 2AA at the maximum concentration of 1.0 mg/plate for TA98 and TA100 in the presence of S-9 mix. Distilled water was used as the solvent control.Triplicate plates were run and results were shown as mean values. Samples expressing mutagenicity higher than twice spontaneous revertants with a dose±response relationship were concluded as positive. The relationship between the number of His+ revertant colonies and the dose of the test chemical was used for linear regression. The statistical test described by Furusawa et al. (1996) was used for the analysis. The test chemical was negative to Salmonella strains TA100, TA98 before and after 14 days of UV irradiation.The test chemical showed toxicity before and after UV irradiation at 3.0 mg/plate in Salmonella strains TA100 with and without S-9 mix. Hence, it can be considered that the test chemical was non-mutagenic before and after UV irradiation.

All of the above results are supported by a study in which with the help of the AMES assay the genointeractive role of the test chemical was evaluated. Salmonella typhimurium strains TA97a, TA98, TA100, TA102, and TA104 were generously provided by Prof. Bruce N. Ames. University of California, Berkeley, CA, U.S.A. Strain characteristics were checked by the procedures of Maron and Ames (1983). Working cultures were stored on nutrient agar slants at 4-10°C. Rat liver S9 OR Caecal extracts were used as metabolic activation systems.Male Wistar rats weighing 200-250 g were injected intraperitoneally with Aroclor 1254 (500 mg/kg body wt.) dissolved in DMSO (200 mg/ml) 5 days before sacrifice. On fourth day after injection, feed was removed. Liver homogenate, S9 fraction and S9 mixture were prepared according to the method of Maron and Ames (1983) and contained 25-35 mg protein/ ml as determined by the procedure of Lowry et al. (1951) with bovine serum albumin as the standard. The caecum (= 5 g wet wt.) of male Wistar rats (= 200 g body wt.) were removed, suspended in cold Kreb-Ringer's phosphate buffer, pH 7.4 (NaC1, 9 g/l; KCl, 460 mg/l; KH2PO4, 210 mg/l; MgSO 4, 380 mg/l; and Na 2 HPO4, 4.45 g/l), minced, homogenized in a Potter-Elvehjern apparatus and sonicated with 5 bursts of 30 sec in a cell disruptor (Model W-220 F, Heat Systems-Ultrasonics Inc., U.S.A.). The post 13000 × g (15 min) supernatant was filtered through a millipore filter (pore size, 1 µm) and stored at -80°C. Its protein content was equal to 5 mg/ml. Dithiothreitol (Clelands reagent) was added to the caecal extract before use (2.5 mg/ml). Double-strength nutrient broth and liquid minimal medium were added to equal volumes of pre-sterilized erythrosine solution in distilled water. The Salmonella strains were inoculated and incubated at 37°C for 16 h in the dark or in the presence of fluorescence lighting (40 W x 3, at a distance of 30 cm). Cells collected by centrifugation from 1.0 ml culture were washed twice with PBS (phosphate-buffered saline, pH 7.4, 0.01 M NaPO4) and resuspended in 2.5 ml PBS. Cell density was determined by nephelometry in a Hitachi 203 fluorescence spectrometer (excitation and analyser wavelengths set at 650 nm). The pre-incubation procedure of Yahagi et al. (1975) was followed. To 1.0 ml of 16-h-old Salmonella cultures in nutrient broth, were added the following as per requirements: comutagens (harman, 5µg in 0.1 ml DMSO; or, norharman, 7.5 µg in 0.1 ml DMSO), mutagens (sodium azide, 5 µg in 0.1 ml distilled water; 4-nitroquinoline N-oxide, 3.75µg in 0.1 ml DMSO; benzo[a]pyrene, 5µg in 0.1 ml DMSO; mitomycin C, 4µg in 0.1 ml of 10% NaCl; methyl methanesulphonate, 5µl; and ethidium bromide, 25 µg in 0.1 ml distilled water), and/or 1.0 ml of rat liver S9 mixture or rat caecal cell-free extract or PBS (when metabolic activation was not required). The suspensions were mixed rapidly and 1.0 ml of the test chemical at concentrations of 0.1, 1.0 and 10.0 mg/ml in distilled water were added. The 3-ml mixture was incubated at 37°C for 30 min in the presence or absence of fluorescent lighting, and 0.9 ml of the suspension was added to 2 ml soft agar (histidine, 0.05 mM; biotin, 0.05 mM; glucose, 4 g/l; NaCl, 5 g/l; and agar, 7 g/l) and plated out on M-9 agar plates. Revertants were scored after incubation at 37 °C for 48 h. The values for the mutagenic responses were the mean of 2 experiments, each with 5 replicates. All values were mean± S.D. for N > 12. The test chemical was toxic to the Salmonella tester strains (TA97a, TA98, TA100, TA102, TA104);D50 levels in minimal medium and in nutrient broth were about 1.0 mg/ml and 2.3 mg/ml of the test chemical. The test chemical was non-mutagenic to the Ames/Salmonella typhimurium strains TA97a, TA98, TA100, TA102, and TA104, to a concentration of 2 mg/plate. No mutative intermediates were detected on metabolism by rat caecal cell-free extracts or rat liver S9 mixture. Hence, the test chemical can be considered to be non-mutagenic to Salmonella tester strains under both conditions of metabolic activation.

Based on the available results, the test chemical can be considered to be non-mutagenic to Salmonella tester strains under both conditions of metabolic activation.

In vitro mammalian chromosomal aberration study

Various studies have been evaluated to determine the chromosomal aberration potential of the test chemical in mammalian cell lines. The results are mentioned below:

In vitro mammalian chromosome aberration study was conducted for the test chemical in Chinese hamster lung-derived fibroblasts (CHL) in the absence of metabolic activation system. The cell line was originally established from the lung of a newborn female at the Cancer Research Institute, Tokyo (Koyama, Utakoji & Ono, 1970), and was maintained by 4-day passages in Minimum Essential Medium (MEM; GIBCO) supplemented by 10% calf serum. The modal chromosome number is 25 and the doubling time was approximately 15 hr. The cells were exposed to chemical at three concentrations up to 0.001 mg/ml for 24 and 48 hr. Physiological saline was used as solvent. The top dose was expected to produce a 50% inhibition on cell growth based on data from a pre-experiment. Colcemid (final conc 0.2 µg/ml) was added to the culture 2 hr before cell harvesting. The cells were then trypsinized and suspended in a hypotonic KCI solution (0.075 M) for 13 min at room temperature. After centrifugation the cells were fixed with acetic acid-methanol (1:3, v/v) and spread on clean glass slides. After air-drying, the slides were stained with Giemsa solution (1.5%, at pH 6.8; E. Merck) for 12-15 min. A hundred well-spread metaphases were observed under the microscope (x 600 with a no cover objective lens). The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. Results were considered negative if incidence of aberrations was less than 4.9%, equivocal if it was between 5 and 9.9% and positive if it was more than 10%. The test chemical tested negative for mutachromosomal effects in Chinese hamster lung-derived fibroblasts (CHL).

This result is supported by a study where the cytogenetic effects of the test chemical were investigated on human peripheral leucocytes. Human peripheral leucocytes from 10 healthy male donors were cultured according to modifications of procedures first described by Moorhead et al..Fibroblast cultures (passage 10) were obtained from the skin of a male fetus of 10 weeks gestation. Eagle's MEM supplemented with 10% fetal calf serum was used as a culture medium. The dose concentrations were decided based on the results of preliminary dose range finding study. These dosages applied as preliminary test were ranging in concentration from 1μg/ml to 100μg/ml which are generally used in foods. The test chemical was tested at concentrations of 10 or 50μg/ml. The blood cultures were incubated for 72hr at 37℃with Colcemid (0.06-0.10μg/ml) for the final 2hr. As hypotonic treatment 0.075M KCl was used, and as fixative methanol: acetic acid (3:1). Chromosome preparations were made by an air-drying method and Giemsa staining. The test dyes were added 24hr before harvesting. The frequencies of cells with chromosome aberrations in untreated control groups were in the range of 0-5%, in agreement with the reported frequency of spontaneous aberrations. The test chemical gave no significant increase in the frequency of cells with chromosome aberrations, as compared with controls, within the dosage levels. Hence, the test chemical can be considered to be non- mutagenic to human peripheral leucocytes.

All of the above studies are also supported by another study where the genotoxicity of the test chemical was assessed using Syrian Hamster embryo cells. SHE cell cultures were grown as described previously by T. Tsutsui et.al, 1991. The test chemical was dissolved in culture medium at 2mM and filter sterilized. SHE cells (5×10⁵) in tertiary culture were plated into 75 cm2 flasks, incubated overnight, and treated with each of the 14 chemical agents at varying concentrations for 24 h. The treatment time was 1.5-fold the normal doubling time of SHE cells, which allows for optimal induction of chromosome aberrations by diverse chemical carcinogens. Three hours before harvest, Colcemid (GIBCO, Grand Island, NY, USA) was administered at 0.2µg/ml and metaphase chromosomes were prepared. After treatment, the cells were harvested with 0.1% trypsin for chromosome preparation. For determination of structural chromosome aberrations, 100 metaphases were scored per experimental group. The aberrations scored were chromatid gaps, isochromatid gaps, chromatid breaks, isochromatid breaks, exchanges, ring chromosomes, dicentric chromosomes, and fragmentations. Achromatic lesions greater than the width of the chromatid were scored as gaps unless there was displacement of the broken piece of chromatid. If there was displacement, they were scored as breaks.One hundred metaphases per experimental group were also scored for examining the induction of polyploidy and endoreduplication, but these numerical aberrations were not taken into account as chromosome aberrations in the present study. Cytotoxicity of the chemical agents tested was determined by the colony-forming efficiencies of SHE cells treated with the test chemical. SHE cells (5×105) in tertiary culture were plated into 75 cm2 flasks (Costar, Cambridge, MA, USA), incubated overnight, and treated with each of the 14 chemical agents at varying concentrations for 24 h. After harvesting with 0.1% trypsin, the cells were replated in triplicate onto 100mm dishes (Costar) at 2000 cells/dish and incubated for 7 days for colony formation. The relative colony-forming efficiency was expressed as the number of colonies in the treated dishes divided by the number in the control dishes×100. Actual colony-forming efficiency of control cells was 11.5±0.9% (S.D.).For determination of a mitotic index, more than 1000 cells were scored for each group, and the percentage of mitotic cells was calculated by dividing the number of mitotic cells by the total number of cells and multiplying by 100. Statistical analysis was performed by χ2-test to assess the significance of the difference in the incidences of chromosome aberrations between control cultures and cultures treated with chemical agents. The level of significance in the statistical analysis was determined at p < 0.05. Cytotoxicities determined by the colony-forming efficiencies of SHE cells treated with the test chemical were increased with increasing concentrations. Treatment of SHE cells with the test chemical decreased mitotic indices in a concentration-dependent manner. The test chemical did not induce chromosome aberrations at all concentrations tested.

Based on the available results, it can be considered that the test chemical did not induce chromosome aberrations at all concentrations tested.

In vitro mammalian gene mutation study

Various studies have been evaluated to determine the mutagenicity potential of the test chemical in mammalian cell lines. The results are mentioned below:

 

L5178Y TK ± mouse lymphoma assay was conducted to evaluate the mutagenic potential of the test chemical. The mouse lymphoma cell line, L5178Y TK± (Clone 3.7.2C), used was derived from the Fischer L5178Y line by Dr Donald Clive.Stocks were maintained in liquid nitrogen and laboratory cultures were periodically checked for the absence of mycoplasma contamination. Cell stock cultures were routinely exposed to medium containing 3 µg/ml thymidine, 5 µg/ml hypoxanthine, 0.1 µg/ml methotrexate and 7.5 µg/ml glycine (THMG). The cultures were then returned to THG medium for 1 day and then to normal growth medium for 2 days before use in mutagenesis experiments. The test chemical was dissolved in deionized water just prior to use, and the aqueous solution was diluted 1:100 into the cell cultures to initiate the treatments.Negative, solvent and positive controls were used. The solvent control was prepared by adding 0.1 ml H2O per 10-ml culture. The positive control for the non-activation portion of the assay was 0.5 µl/ml ethylmethane sulfate (EMS). This treatment was initiated by adding 0.5 ml of freshly prepared aqueous stock (10 µl/ml) to a 9.5-ml cell culture. Dimethylnitrosamine (DMN) at 0.3µg/ml was the positive control for the activation assays. A 0.3-ml aliquot of a freshly prepared aqueous stock (10 µl/ml) was added to a 9.7-ml cell culture containing the S9 activation system. The procedure was based on the method as described by Clive and Spector.Each treated culture consisted of 3 x 106 cells suspended in 10-ml final volumes in 15-ml centrifuge tubes. The nonactivation and activation assays were conducted in the same manner, except the cell cultures in the activation assay contained 50 µl/ml of S9 supernatant and co-factors 2.7 mg/ml NADP and 4.5 mg/ml isocitric acid. The S9 was prepared from the livers of Aroclor 1254-induced Fischer 344 male rats (Ames et al., 1975). The cell cultures were exposed to the test material or control chemical for 4 h, washed and allowed an expression time of 2 days in growth medium. Cell counts were determined by hemacytometer and the cell cultures were diluted to 3 x 10⁵cells/ml each day if the density had increased beyond 4 x 10⁵cells/ml. The average of the solvent and untreated negative control mutant frequencies was used as the background or spontaneous mutant frequency for each trial. A treated culture was considered to have a significant elevated mutant frequency if the frequency exceeded 10^-5plus 1.5x the background frequency. Additional criteria, such as the presence of a dose-related or toxicity-related response and repeatability between trials were used to help assess the presence of any mutagenic activity.The negative control mutant frequencies were all in the normal ranges and the positive control compounds yielded normal mutant frequencies. Under nonactivation conditions, the test material was assayed from 100 to 600 µg/ml and non-detectable to moderate toxicities were induced (134 — 24.5% relative growths). Higher toxicities were not assayed because the toxicity curve was excessively steep. In the presence of metabolic activation, the test material was assayed from 50 to 350 µg/ml and non-detectable to moderate toxicities were induced (117.7—40.5% relative growths). Two highest concentrations (300 and 350 µg/ml) were assayed in duplicate to determine the reproducibility of the results.Under the non-activation conditions, all of the assayed treatments produced mutant frequencies that were similar to the background mutant frequencies (solvent control). Since there was no evidence for mutagenic activity up to concentrations that approached excessive toxicity and no dose-related response was observed, the test material was considered non-mutagenic without activation in this assay.Small increases in concentration > 350 µg/ml were excessively toxic. None of the assayed treatments induced a mutant frequency that exceeded the minimum criterion of 69.3 x 10^-6. Hence, the test chemical can be considered to be non-mutagenic at concentrations that approached excessive toxicity under both the conditions of metabolic activation.

Similarly, the mutagenicity and cytotoxicity of the test chemical were evaluated in V79 hepatocyte-mediated mutation assay. The V79 Chinese hamster lung cells used throughout this study were kindly provided by Dr. E. Elmore, Northrop Services Inc., Research Triangle Park, NC. The cells were grown and maintained in Williams' medium E (WE) (Gibco, Burlington, Ont.) containing 10% fetal bovine serum (Dextran Products, Toronto, Ont.), L-glutamine (2 mM) (Flow Laboratories, Mississauga, Ont.) and gentamicin (10 µg/ml) (Schering Corp., Kenilworth, N J). EMS, ethyl methanesulfonate (direct-acting mutagen); DMBA, 7,12-dimethylbenz[a]anthracene (indirect-acting mutagen) were used as positive controls. Primary hepatocytes were freshly prepared by in vivo collagenase perfusion of the liver of a 200-300 g male Wistar rat using the method of Williams et al. (1977) with modifications as described by Rogers and Heroux-Metcalf (1983) and by Rogers et al. (1985). Cytotoxicity and mutagenicity of the test chemical were evaluated in a hepatocyte-mediated mutation assay according to protocols of Langenbach et al. (1978, 1981) but with modifications as reported by Rogers et al. (1985). The period of exposure to chemicals, with or without rat hepatocyte activation, was 48 h. Modifications to the protocol for the selection of ouabain-resistant (Ou R) mutants at the Na +, K+-ATPase gene locus included (1) a 24-h period of recovery after 48 h of chemical treatment and (2) the addition of ouabain (final concentration 1.0 mM or 0.73 mg/ml) at the time of seeding. Improvement to the selection of Ou R mutants was evident from the clarity of colony formation in stained dishes (absence of background) and by the frequencies of Ou R mutations in both control and test chemical- treated cultures. The dose range over which cytotoxicity was determined, was 0-600 µg/ml. At marginally cytotoxic dose levels of 100, 200 and 300µg/ml of medium, the test chemical failed to induce measurable changes in osmolality, as determined with the aid of a freezing point depression osmometer.The dose range over which cytotoxicity was determined, was 0-600 µg/ml. A qualitative reduction in colony size occurred at 200µg/ml, whereas a significant reduction in the number of colonies occurred at 300µg/ml and higher. The test chemical was lethal to about 90% of the cells at 400µg/ml, where surviving colonies were pinpoint in size, and to 100% of the cells at the 500µg/ml dose level. Hence, the test chemical was tested at concentrations up to 300 µg/ml in the main study. The potent, direct- acting mutagen EMS, at 1.25 ×10³/zM (155 µg/ml), was strongly mutagenic without hepatocyte- mediated activation. When activated by rat hepatocytes, the indirect-acting mutagen/carcinogen DMBA was significantly mutagenic to V79 cells at both the HGPRT and Na +, K+-ATPase gene loci. The positive controls used demonstrated that the V79 cells and hepatocytes were capable of reacting to both direct- and indirect-acting genotoxins. The test chemical was non-mutagenic to V79 ceils at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and sodium, potassium-ATPase (Na ÷, K+-ATPase) gene loci, with or without rat hepatocyte-mediated activation.

Based on the available results the test chemical can be considered to non-mutagenic to mammalian cell lines when tested in vitro under both conditions of metabolic activation.

The test chemical can be considered to be non-genotoxic when tested in vitro under both conditions of metabolic activation when tested in vitro on mammalian cell lines as well as bacterial tester strains.

The test chemical can be considered to be non-genotoxic when tested in vitro under both conditions of metabolic activation when tested in vitro on mammalian cell lines as well as bacterial tester strains.

Genetic toxicity in vivo

The non-mutagenic potential of the test chemical is further strengthened by the results of various in vivo genetic toxicity studies conducted on rats as well mice. The results are mentioned below:

In vivo/in vitro HPC/DR genetic toxicity in vivo assay was performed for the test chemical by study on hepatocytes isolated from rat. Six to eight rats were perfused on a given day (ie, experiment), including one control (ie, usually corn oil-treated) from which hepatocytes were obtained for in vitro assays. One or more known positive chemicals (usually SY3) were tested .DNA repair was quantified by the autoradiographic determination of incorporated [3H]-thymidine, similar to the method of Williams [1977] and Bermudez et al [1979], as detailed in Kornbrust and Barfknecht [1984a,b]. Net nuclear grains (NNG) were determined by counting the number of grains in each nuclei and subtracting the average number of grains present in three equal-size adjacent cytoplasmic areas. A strict procedure for random selection of cells was used [Mirsalis and Butterworth,1980]. The average NNG for 60 cells (±SD) as well as the percentage of cells with 25 NNG was determined for the test chemical. single dose was given that was equal to approximately. Average net nuclear grain counts of 5 or greater were assumed to constitute a positive response, since these differed from the control net nuclear counts by greater than 2 SD. The NNG count for the test chemical was less than 5 and the test chemical was negative i.e gave no induction of DNA repair when tested at 200 mg/plate in Wistar rats.

The results of the in vivo DNA repair study are supported by an in vivo mouse micronucleus test performed to evaluate the mutagenic properties of the test chemical. The test chemical and controls were administered acutely via i.p. injection. Adult CD-1 mice were purchased from Harlan Industries, Indianapolis, IN.Triethylenemelamine (TEM) at 3.0 mg/kg was dissolved in saline and used as the positive control, the negative control was deionized water. The test chemical was dissolved in deionized water. Eighty percent of the LD50 value was chosen as the high dose (240 mg/kg) followed by 33 and 10% of this dose for the intermediate and low doses. So the doses used in the study were 24, 80 and 240 mg/kg dose levels in distilled water.

The test chemical and controls were administered acutely via i.p. injection.Harvests were at 24 and 48 h after the administration of the test article, and at 24 h after the administration of the controls.Animals were killed with CO2 and the adhering soft tissue and epiphyses of both tibiae were removed. The marrow was flushed from the bone and transferred to centrifuge tubes containing 3 ml fetal calf serum (one tube for each animal). Following centrifugalion to pellet the tissue, the supernatant was drawn off, the cells re-suspended in 0.25 ml of serum and the suspension spread on slides and air-dried. The slides were then fixed in methanol, stained in May — Gruenwald solution followed by Giemsa and rinsed in deionized water (Schmid, 1975). Five hundred polychromatic erythrocytes (PCEs) per animal were scored. The frequency of micronucleated cells was expressed as percent micronucleated cells based on the total PCEs present in the scored optic field. The normal frequency of micronuclei in this mouse strain is around 0—0.4 %. The frequency of PCEs versus red blood cells (RBCs) was determined by scoring the number of RBCs observed in the optic fields while scoring the first 200 PCEs for micronuclei. The criteria for the identification of micronuclei were those of Maier and Schmid (1976).The results were evaluated using a statistical Student's t-test.The percent micronucleated PCEs from the negative control were within the normal range of negative controls. The positive control chemical induced a statistically significant increase in frequencies of micronuclei.The test chemical did not induce significant increases in micronucleus frequencies in bone marrow PCEs in the mouse.Hence, it was concluded that the test chemical was non-mutagenic in nature.

This result is ably supported by another study where the clastogenic potential of the test chemical was evaluated in mice by analyzing the cytogenetic endpoint micronuclei in bone marrow polychromatic erythrocytes (PCEs). Groups of male male B6C3F1 mice weighing 25-28 g were used throughout the study. The test chemical was administered twice by ip injection to groups of five mice at 50, 100 and 200 mg/kg, at intervals of 24 hr. The highest dose was selected according to data in the literature, which indicated 320 mg/kg as the LD50 in the mouse by the ip route (Lin and Brusick, 1986). Three mice, used as positive controls, received 1 mg/kg MMC once by the same route. Five mice, injected twice with distilled water, served as negative controls. Vehicle and test compounds were given in the same volume (10 ml/kg). The animals were killed 24 hr after the last administration.The animals were killed by cervical dislocation after collection of peripheral blood for B lymphocyte cultures. Femoral bone marrow cells were flushed out in 1.5 ml FCS and centrifuged for 5 min at 1000g. The pellet was resuspended in some drops of FCS, smeared on clean slides and dried overnight; marrow cells were fixed for 5 min in absolute methanol :rod stained for 20 min with 5% Giemsa in 0.01 m Sorensen buffer (pH 6.8). 200 PCEs per mouse were scored blind to determine the frequency of micronucleated PCEs (MnPCEs). Bone marrow toxicity was evaluted by the ratio polychromatic/total erythrocytes [PCEs/ PCEs + normochromatic erythrocytes (NCEs)]. The average PCE ratios in control and treated animals were compared by the Student's t-test; micronucleus frequencies were compared by the chi-square test. No increase in MnPCEs was observed in test chemical-treated mice. MMC produced a large increase of micronuclei in both PCEs and NCEs. The percentage of PCEs on total erythrocytes, an index of bone marrow toxicity, was decreased in mice treated with the highest test chemical dose, as well as with the positive control MMC. Hence, the test chemical can be considered to be non-mutagenic in nature.

In a similar study, the clastogenic potential of the test chemical was evaluated in mice by analyzing the cytogenetic endpoint sister chromatid exchanges (SCEs) in peripheral blood lymphocytes (PBLs).Groups of male male B6C3F1 mice weighing 25-28 g were used throughout the study. The test chemical was administered twice by ip injection to groups of five mice at 50, 100 and 200 mg/kg, at intervals of 24 hr. The highest dose was selected according to data in the literature, which indicated 320 mg/kg as the LD50 in the mouse by the ip route (Lin and Brusick, 1986). Three mice, used as positive controls, received 1 mg/kg MMC once by the same route. Five mice, injected twice with distilled water, served as negative controls. Vehicle and test compounds were given in the same volume (10 ml/kg). The animals were killed 24 hr after the last administration.Whole blood (0.8-1.0 ml) was taken by cardiac puncture from mice anaesthetized with Nembutal (sodium pentobarbital; Abbott Laboratories, North Chicago, IL, USA) and was cultured by a slight modification of the protocol described by Erexon et al. (1983). Briefly, blood was centrifuged, the serum collected and filter-sterilized. The blood cell pellet was washed three times with phosphate buffered saline (pH 7.4) supplemented with 2% heat-inactivated foetal calf serum (HI-FCS; Flow Laboratories, McLean, VA, USA) and incubated for 24hr in culture medium consisting of RPMI 1640 plus 25 mM HEPES buffer with L-glutamine (Gibco, Paisley, UK), 4% mouse plasma, 15% HI-FCS, 100 U penicillin/ml, 100 streptomycin µg/ml (Gibco), an additional 292/ag L-glutamine/ml (Gibco), 10U preservative-free sodium heparin/ml and 90µg lipopolysaccharide ml (Sigma).Four parallel cultures were established from each mouse, in order to ensure a sufficient number of well-differentiated metaphases. Cultures were maintained in the dark at 37"C in a 5% CO2, atmosphere for 24 hr, after which the original medium was replaced with a complete medium (without mouse plasma) containing 5µM 5-bromo-2'-deoxyuridine (Sigma). Colcemid (0.5mg/ml, Gibco) was added 3 hr before harvest. After a total culture time of 54 hr, cells were harvested by standard procedures and slides stained by the FPG technique (Perry and Wolff. 1974). When possible, 25 second division metaphases per animal were scored to estimate the SCE frequency in Blood lymphocytes. Owing to the small interindividual variability of SCE rates in PBLs, three animals were used for each experimental point. The mitotic index (MI) was calculated from 500 stimulated lymphocytes. All slides were coded before scoring. The average SCE's in control and treated animals were compared by the Student's t-test.A treatment- related toxic effect on blood lymphocytes highlighted by the severe reduction of the MI observed at he highest test chemical dose. Well stimulated blood lymphocytes were obtained from all cultures set up; however, for two of the three mice treated with the highest dose the amount of blood available was not sufficient to establish lymphocyte cultures. 25 well-differentiated metaphases were blind scored for each animal. The spontaneous SCE rates were highly uniform among animals and agreed with literature data. The frequency of SCEs was not increased in test chemical -treated mice with respect to untreated controls. Conversely, MMC clearly increased SCEs in all mice treated. These results indicate that the test chemical was inactive as a clastogen in mouse blood lymphocytes.

Based on the available results, the test chemical can be considered to be non-mutagenic when tested in vivo in mouse micronucleus assay and also failed to induce DNA damage in rat hepatocytes. Hence, the test chemical can be considered to be non-mutagenic when tested in vivo.

Justification for classification or non-classification

The test chemical can be considered to be non-genotoxic when tested in vitro under both conditions of metabolic activation when tested in vitro on mammalian cell lines as well as bacterial tester strains.

Based on the available results, the test chemical can be considered to be non-mutagenic when tested in vivo in mouse micronucleus assay and also failed to induce DNA damage in rat hepatocytes. Hence, the test chemical can be considered to be non-mutagenic when tested in vivo.

Therefore the test chemical can be concluded to be not mutagenic and classified under category "Not Classified " as per CLP Regulation.