L-Glutamic acid, N-coco acyl derivs., monosodium salts

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test:

Test chemical did not induce mutation in Salmonella Typhimurium TA 1538, 1535, 1537, 98 and 100 with and without mammalian S-9 microsomal fraction. Hence it is not likely to classify as a gene mutant in vitro.

Chromosome aberration:

The test chemical did not induce chromosome aberration in the CHL/IU cells both in the presence and absence of S9 activation system and hence it is not likely to be gene mutant in vitro.

Mammalian cell Gene mutation toxicity:

Test chemical did not induce mutation in L5178Y TK ± mouse lymphoma with and without mammalian S-9 microsomal fraction. Hence it is not likely to classify as a gene mutant in vitro.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Justification for type of information:

Data is fom secondary source

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

In – vitro Bacterial reverse mutation assay of test chemical in S. typhimurium.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

not specified

Species / strain / cell type:

S. typhimurium, other: TA 1538, 1535, 1537, 98 and 100

Details on mammalian cell type (if applicable):

not specified

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

not specified

Metabolic activation:

with and without

Metabolic activation system:

S-9 activation

Test concentrations with justification for top dose:

1, 4, 16, 64 and 256 mg/plate (without S-9 activation)4, 16, 64, 256 and 1024 mg/plate (with S-9 activation)

Vehicle / solvent:

Double deionized water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Double deionized water

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Remarks:

not specified

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
NUMBER OF REPLICATIONS: Three plates per dose level

Rationale for test conditions:

not specified

Evaluation criteria:

reverse mutations

Statistics:

not specified

Species / strain:

S. typhimurium, other: TA 1538, 1535, 1537, 98 and 100

Metabolic activation:

with and without

Genotoxicity:

negative

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not specified

Additional information on results:

In the initial toxicity assays, the test substance without metabolic activation was cytotoxic to the tester strains TA 1535 and TA 1537 at 1000 mg/plate and cytotoxic to tester strain TA 1538 at 200 mg/plate. The test substance with metabolic activation was cytotoxic to the tester strains TA 1535 and TA 1538 at 5000 mg/plate and tester strain TA 1537 at 1000 mg/plate.

Remarks on result:

other: Non mutagenic

Conclusions:

Test chemical did not induce mutation in Salmonella Typhimurium TA 1538, 1535, 1537, 98 and 100 both in the presence and absence of mammalian S-9 microsomal fraction and hence it is not likely to classify as a gene mutant in vitro.

Executive summary:

Gene toxicity test was performed to determine the mutagenic nature of test chemical. The study was performed using Salmonella Typhimurium TA 1538, 1535, 1537, 98 and 100 both in the presence and absence of mammalian S-9 microsomal fraction. The test material did not induce reverse mutations. Therefore, Test chemical did not induce mutation in Salmonella Typhimurium TA 1538, 1535, 1537, 98 and 100 with and without mammalian S-9 microsomal fraction. Hence it is not likely to classify as a gene mutant in vitro.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

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

Version / remarks:

as below

Principles of method if other than guideline:

Gene toxicity study of test chemical in L5178Y TK ±mouse lymphoma assay.

GLP compliance:

not specified

Type of assay:

other: mammalian cell gene mutation assay

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

not specified

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

not specified

Metabolic activation:

with and without

Metabolic activation system:

not specified

Test concentrations with justification for top dose:

0.001, 0.01, 0.1, 1.0, 10 and 100 µL/mL

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Remarks:

not specified

Details on test system and experimental conditions:

not specified

Rationale for test conditions:

not specified

Evaluation criteria:

Mutation frequency were observed

Statistics:

not specified

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

Not specified

Remarks on result:

other: No mutagenic potential observed

Conclusions:

Test chemical did not induce mutation in L5178Y TK ± mouse lymphoma and hence it is not likely to classify as a gene mutant in vitro.

Executive summary:

Gene toxicity test was performed to determine the mutagenic nature of test chemical. The study was performed using L5178Y TK ± mouse lymphoma both in the presence and absence of mammalian S-9 microsomal fraction. The test material did not significantly increase mutation frequency over the average mutation frequency of solvent control. Therefore, Test chemical did not induce mutation in L5178Y TK ± mouse lymphoma with and without mammalian S-9 microsomal fraction. Hence it is not likely to classify as a gene mutant in vitro.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from secondary source

Qualifier:

according to guideline

Guideline:

other: As mentioned below

Principles of method if other than guideline:

To determine the mutagenic nature of test chemical.

GLP compliance:

not specified

Type of assay:

bacterial forward mutation assay

Target gene:

Thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Remarks:

L5178Y/TK+/-

Metabolic activation:

with and without

Metabolic activation system:

Not specified

Test concentrations with justification for top dose:

0.0038 to 0.050 μl/ml (10 concentrations) without S9
0.012 to 0.16 μl/ml (10 concentrations) with S9

Vehicle / solvent:

Not specified

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

not specified

Details on test system and experimental conditions:

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable:
- Exposure duration/duration of treatment: 2 days
- Harvest time after the end of treatment (sampling/recovery times): 24 and 48 hours

Other: At the end of the expression period, the cells were placed in cloning medium. Cell counts were made for each preparation and the appropriate number of cells were removed and plated. Total number of colonies per plate and the mutation frequency were determined .

Rationale for test conditions:

Not specified

Evaluation criteria:

The criteria for a positive test were: if there was a positive dose response and one or more of the three highest doses exhibited a mutant frequency which was two-fold greater than the background level. A two-fold increase without dose response was considered equivocal and the test was considered negative if no cultures exhibited a two-fold increase in mutant frequency.

Statistics:

Not specified

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

Not specified

Remarks on result:

other: No mutagenic potential observed

Conclusions:

The test chemical was tested in the L5178Y TK+/- Mouse Lymphoma Mutagenesis assay in the presence and absence of rat liver S-9 to determine the mutagenic nature of the test chemical. None of the treated cultures that were cloned exhibited a significant increase in mutant frequency over the average mutant frequency of the solvent controls. Thus, the test chemical was considered to be not mutagenic in nature.

Executive summary:

The test chemical was tested in the L5178Y TK+/- Mouse Lymphoma Mutagenesis assay in the presence and absence of rat liver S-9 to determine the mutagenic nature of the test chemical. The study was performed at various concentrations 0.0038 to 0.050 μl/ml (10 concentrations) without S9 and 0.012 to 0.16 μl/ml (10 concentrations) with S9 metabolic activation system. The cells were exposed to the test chemical, positive control and negative control for 4 hours. An expression time of 2 days was allowed with cell population adjustment at 24 and 48 hours. At the end of the expression period, the cells were placed in cloning medium. Cell counts were made for each preparation and the appropriate number of cells were removed and plated. Total number of colonies per plate and the mutation frequency were determined. None of the treated cultures that were cloned exhibited a significant increase in mutant frequency over the average mutant frequency of the solvent controls and no dose response was observed. Thus, the test chemical was considered to be not mutagenic in nature.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Justification for type of information:

Data is fom secondary source

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

In – vitro Bacterial reverse mutation assay of test chemical in S. typhimurium.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

not specified

Species / strain / cell type:

S. typhimurium, other:

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S-9 activation

Test concentrations with justification for top dose:

not specified

Vehicle / solvent:

not specified

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Remarks:

not specified

Details on test system and experimental conditions:

not specified

Rationale for test conditions:

not specified

Evaluation criteria:

reverse mutations

Statistics:

not specified

Species / strain:

S. typhimurium, other:

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

not specified

Remarks on result:

other: Non mutagenic

Conclusions:

Test chemical did not induce mutation in Salmonella Typhimurium other and hence it is not likely to classify as a gene mutant in vitro.

Executive summary:

Gene toxicity test was performed to determine the mutagenic nature of test chemical. The study was performed using Salmonella Typhimurium other both in the presence and absence of mammalian S-9 microsomal fraction. The test material did not induce reverse mutations. Therefore, Test chemical did not induce mutation in Salmonella Typhimurium other with and without mammalian S-9 microsomal fraction. Hence it is not likely to classify as a gene mutant in vitro.

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Justification for type of information:

Data is fom secondary source

Qualifier:

according to guideline

Guideline:

other: as below

Principles of method if other than guideline:

In – vitro Bacterial reverse mutation assay of test chemical in S. typhimurium.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

not specified

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

not specified

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix obtained from the liver of Arochlor 1254-induced rats

Test concentrations with justification for top dose:

0, 0.001, 0.005, 0.00, 0.050, 0.100, 0.125, 0.150 and 0.300 ml per plate

Vehicle / solvent:

Deionized water

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Remarks:

not specified

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS: Three plates per dose level

Evaluation criteria:

reverse mutations

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

The range finding study results indicated that the test substance was toxic towards the tester strains at 0.146 ml and higher. In the main study, no substantial increases in the revertant colony numbers of any of the five strainstested were observed following treatment with the test substance at any dose level, either in the presence or absence of S-9 activation.Cytotoxic concentration: 0.586 ml per plate and above (based on range-finding study)Genotoxic effects: 0.146 ml per plate and above (based on range-finding study)Negative with and without activation in the main study

Remarks on result:

other: Non mutagenic

Conclusions:

Test chemical did not induce mutation in Salmonella Typhimurium TA 1535, TA 1537, TA 98 and TA 100 both in the presence and absence of mammalian S-9 microsomal fraction and hence it is not likely to classify as a gene mutant in vitro.

Executive summary:

Gene toxicity test was performed to determine the mutagenic nature of test chemical. The study was performed using S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 both in the presence and absence of mammalian S-9 microsomal fraction. The test material did not induce reverse mutations. Therefore, Test chemical did not induce mutation in Salmonella Typhimurium TA 1535, TA 1537, TA 98 and TA 100 with and without mammalian S-9 microsomal fraction. Hence it is not likely to classify as a gene mutant in vitro.

Reference 6

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Justification for type of information:

Data is from secondary source

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

In – vitro Bacterial reverse mutation assay of test chemical in S. typhimurium.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

not specified

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 was prepared in-house from the livers of male Sprague-Dawley rats

Test concentrations with justification for top dose:

0, 50, 150, 500, 1500 and 5000 mg per plate

Vehicle / solvent:

Deionized water

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Details on test system and experimental conditions:

METHOD OF APPLICATION: ; in agar (plate incorporation
DURATION
- Preincubation period: approximately 10 hours.
- Exposure duration: 48 hours
NUMBER OF REPLICATIONS: triplicate

Rationale for test conditions:

not specified

Evaluation criteria:

number of induced revertants were compared

Statistics:

not specified

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

The test material caused a visible reduction in the growth of the bacterial lawn to all of the strains tested. The first evidence of toxicity was observed at 150 mg/plate. No significant increase in the frequency of revertant colonies of bacteria was recorded for any of the strains used, at any dose level with or without metabolic activation. All of the positive control chemicals used in the test produced marked increases in the frequency of revertant colonies and the activity of the S9 fraction was found to be satisfactory.

Remarks on result:

other: Non mutagenic

Conclusions:

Test chemical did not induce mutation in Salmonella Typhimurium TA 1535, TA 1537, TA 98 and TA 100 both in the presence and absence of mammalian S-9 microsomal fraction and hence it is not likely to classify as a gene mutant in vitro.

Executive summary:

Gene toxicity test was performed to determine the mutagenic nature of test chemical. The study was performed using S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 both in the presence and absence of mammalian S-9 microsomal fraction. The test material caused a visible reduction in the growth of the bacterial lawn to all of the strains tested. The first evidence of toxicity was observed at 150 mg/plate. No significant increase in the frequency of revertant colonies of bacteria was recorded for any of the strains used, at any dose level with or without metabolic activation. All of the positive control chemicals used in the test produced marked increases in the frequency of revertant colonies and the activity of the S9 fraction was found to be satisfactory. The test material did not induce reverse mutations. Therefore, Test chemical did not induce mutation in Salmonella Typhimurium TA 1535, TA 1537, TA 98 and TA 100 with and without mammalian S-9 microsomal fraction. Hence it is not likely to classify as a gene mutant in vitro.

Reference 7

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

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:

To determine the gene mutation toxicity of test chemical.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100, TA 1538 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : liver of male Sprague-Dawley rats
- method of preparation of S9 mix : The S9 fraction was prepared from the liver of male Sprague-Dawley rats, weighing about 220g (seven weeks old), pretreated with polychlorinated biphenyl (KC 500) at a dose of 500mg/kg body weight five days before sacrifice.
- concentration or volume of S9 mix and S9 in the final culture medium : 0.1 ml
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability)

Test concentrations with justification for top dose:

1, 5, 10, 50, 100, 500, 1000 and 5000 µg/plate

Vehicle / solvent:

Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2) and 9-aminoacridine

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : not specified

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): not specified
- Test substance added in medium; in agar (plate incorporation): 0.1ml of bacterial strain and 0.5ml of S9 mix or sodium phosphate buffer (pH 7.4) were added to a sterile test tube containing 0.1ml of various concentrations of the chemicals. This mixture was preincubated in a shaker water bath at 37°C for 20 minutes, and then added to 2ml of molten top agar (45°C).

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20 minutes
- Exposure duration/duration of treatment: 48 hours
- Harvest time after the end of treatment (sampling/recovery times): not specified

Rationale for test conditions:

not specified

Evaluation criteria:

Number for revertants analysed

Statistics:

not specified

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100, TA 1538 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Remarks:

DMSO

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

Not specified

Remarks on result:

other: No mutagenic potential observed

Table 1: Mutagenic activity of test chemical

Compound	Dose µg/ plate	Revertant colonies/ plate
		TA 100		TA 1535		WP2 uvrA		TA98		TA 1537		TA 1538
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
DMSO (Control)		150±16.8	154±17.5	30±5.6	15±7.1	30±9.7	34±10.9	32±7.7	42±10.1	18±8.6	22±6.3	22±7.3	28±5.7
Positive control
AF-2	0.01	501±84.7	-b	-	-	-	-	-	-	-	-	-	-
	0.05	-	-	-	-	1082±293.7	-	278±64.8	-	-	-	-	-
ENNG	0.5	-	-	1101±683.1	-	-	-	-	-	-	-	-	-
9AC	80.0	-	-	-	-	-	-	-	-	889±275.7	-	-	-
4NQO	0.25	-	-	-	-	-	-	-	-	-	-	270±66.2	-
B(a)P	5.0	-	1084±236.3	-	-	-	-	-	809±108.4	-	313±41.6	-	354±89.4
2AA	5.0	-		-	440±198.6	-	359±127.0	-	-	-	-	-	-
Test chemical	1	150	169	35	19	22	53	28	40	6	12	16	28
	5	156	162	29	20	33	42	27	36	13	13	23	22
	10	141	166	35	11	39	36	31	35	5	11	21	34
	50	152	161	32	13	31	33	23	35	8	12	19	23
	100	165	161	39	14	31	39	23	31	12	8	20	20
	500	152	163	26	25	22	34	31	37	10	13	24	30
	1000	176	168	31	20	28	39	34	56	10	15	18	27
	5000	139	185	24	23	25	41	19	28	8	10	17	16

-b: Not tested

Conclusions:

Test chemical did not induce mutation in Salmonella Typhimurium TA 1538, 1535, 1537, 98, 100 and E. coli WP2 and hence it is not likely to classify as a gene mutant in vitro.

Executive summary:

Gene mutation study was performed on Salmonella Typhimurium strains to determine the mutagenic nature of test chemical. Salmonella Typhimurium TA 1538, 1535, 1537, 98, 100 and E. coli WP2 were used during the study. The study was performed in the presence and absence of S9 metabolic activation. The S9 fraction was prepared from the liver of male Sprague-Dawley rats, weighing about 220g (seven weeks old), pretreated with polychlorinated biphenyl (KC 500) at a dose of 500mg/kg body weight five days before sacrifice. The positive controls used were 4-nitroquinoline-N-oxide, 9-aminoacridine, 2-nitrofluorene, N-ethyl-N-nitro-N-nitrosoguanidine, benzo(a)pyrene, 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2) and 9-aminoacridine. 0.1ml of bacterial strain and 0.5ml of S9 mix or sodium phosphate buffer (pH 7.4) were added to a sterile test tube containing 0.1ml of various concentrations of the chemical. The test concentrations used were 1, 5, 10, 50, 100, 500, 1000 and 5000 µg/plate and the test chemical was dissolved in DMSO. This mixture was preincubated in a shaker water bath at 37°C for 20 minutes, and then added to 2ml of molten top agar (45°C). This mixture was preincubated in a shaker water bath at 37°C for 20 minutes, and then added to 2ml of molten top agar (45°C). The contents of each tube were mixed and poured onto a minimal glucose agar plate immediately. The plates were incubated at 37°C for 48 hours, and then the number of revertant colonies on each plate was scored with an automated colony counter. The background bacterial lawn was checked routinely by dissected microscope. Negleble amount of revertant colonies were observed as compared to positive control substances. Thus the test chemical can be considered as non-mutagenic when Salmonella Typhimurium TA 1538, 1535, 1537, 98, 100 and E. coli WP2 were treated in the presence and absence of S9 activation system.

Reference 8

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

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 collection of data source.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: As mentioned below

Principles of method if other than guideline:

To determine the gene mutation toxicity of test chemical according to study 7 and 8.

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

No data

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Remarks:

7

Species / strain / cell type:

mammalian cell line, other: CHL/IU

Details on mammalian cell type (if applicable):

- Type and identity of media: Eagle MEM culture broth supplemented with 10 vol% of inactivated calf serum was used for the culture
- Properly maintained: No data
- Periodically checked for Mycoplasma contamination: No data
- Periodically checked for karyotype stability: No data
- Periodically "cleansed" against high spontaneous background: No data

Additional strain / cell type characteristics:

not specified

Remarks:

8

Metabolic activation:

with and without

Metabolic activation system:

S9 Metabolic activation system

Test concentrations with justification for top dose:

7. Without metabolic activation: 0.3, 1.0 or 3.0 µg/mL for 4 hrs
Without metabolic activation: 1.0 or 3.07 µg/mL for 7 or 18 hrs
With metabolic activation: 1.0, 3.0 or 10.0 µg/mL for 18 hrs
With metabolic activation: 10.0 µg/mL for 7 and 28 hrs

8. Main test: (without S9) : 0, 50, 75, 100, 125 or 150 µg/mL
Additional test (without S9): 0, 100, 110, 120, 130, 140 or 150 µg/mL
With and without S9: 0, 100, 125, 150, 175 or 200 µg/mL

Vehicle / solvent:

7.No data
8.- Vehicle(s)/solvent(s) used: Physiological saline
- Justification for choice of solvent/vehicle: The test chemical was soluble in physiological saline

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Remarks:

7

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

mitomycin C

Remarks:

8

Details on test system and experimental conditions:

7. METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: No data
- Exposure duration: 4 hrs
- Expression time (cells in growth medium):
Cells were washed in glucose-containing saline and cultured in normal medium for 7, 18 and 28 hours.
- Selection time (if incubation with a selection agent): Two hours (7 hour interval) or 2.5 hours (18 and 28 hours intervals)
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: Duplicate

NUMBER OF CELLS EVALUATED: 100 metaphase cells were examined

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Yes, cell proliferation was measured

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: No data
- Other:

OTHER: No data
8.METHOD OF APPLICATION: in medium
No. of cells at the start of the experiment: 20000 cells

DURATION
- Preincubation period: No data
- Exposure duration: Continuous treatment: 24 hrs
Short term treatment: 6 hrs
- Expression time (cells in growth medium):
Continuous treatment: 24 hrs
Short term treatment: 18 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: 200 groups of metaphase cells per group for structural abnormalities and ploidy cells

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Yes, cell proliferation was measured

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: No data
- Other:

OTHER: No data

Rationale for test conditions:

No data

Evaluation criteria:

7.Chromosomes were analyzed for structural chromosomal aberrations (breaks, fragments, deletions, exchanges and chromosomal disintegrations). Chromosomal gaps were recorded separately.
8.Chromosomes were analyzed based on the classification method by the Japan Society for Environmental Mutagenesis and Mammalian Testing, and structural abnormalities such as breakage and exchange of chromosome type or chromosome type, presence and absence of gap, and ploidy The presence or absence of cells (polyploid) was observed. The gap was not included in structural abnormality. The frequency of cells with chromosomal abnormality is negative, less than 5% negative, less than 10% false positive, more than 10% Positive.

Statistics:

No data

Species / strain:

Chinese hamster lung fibroblasts (V79)

Remarks:

7

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

mammalian cell line, other: CHL/IU

Remarks:

8

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

7.No data
8.TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: To determine the treatment concentration of the test substance used for the chromosomal aberration test, the influence of the test substance on cell proliferation was investigated. In the presence and absence of S9 mix in the short-time treatment method, 7 concentrations are similarly in the concentration range of 50 to 500 μg / mL, and 6 concentrations in the concentration range of 12.5 to 200 μg / mL in the 24-hour treatment in the continuous treatment method Respectively. The growth inhibitory effect of the test substance on CHL / IU cells was determined by counting the cells of each group using a hemocytometer and using the relative growth rate relative to the negative (solvent) control group as an index. The concentration showing 50% inhibition of cell proliferation was calculated from the linear equation connecting the two doses with 50% of the cell proliferation rate. As a result, the 50% cell proliferation inhibitory concentration was 242 μg / mL in the presence of S9 mix in the short-time treatment method, 146 μg / mL in the absence of S9 mix, 107 μg / mL in 24-hour treatment with the continuous treatment method

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

Remarks on result:

other: No mutagenic potential observed

Conclusions:

The test chemical did not induce chromosome aberration in the Chinese hamster V79 cells both in the presence and absence of S9 activation system and hence it is not likely to be gene mutant in vitro.

Executive summary:

Data available for the target chemical was reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

In vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical. The study was performed using Chinese hamster V79 cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in suitable solvent and used. The doses for the main study were based on the preliminary dose range finding study. Evaluated dose levels were 1.0 μg/ml without S9-mix and 10.0 μg/ml with S9-mix for 7 hours; 0.3, 1.0 and 3.0 μg/ml without S9-mix and 1.0, 3.0 and 10.0 μg/ml with S9-mix for 18 hours; and 3.0 μg/ml without S9-mix and 10.0 μg/ml with S9-mix for 28 hours.Logarithmically growing cells were incubated with the test substance in serum-free culture medium for 4 hrs. Cells were subsequently washed in glucose-containing saline and cultured in normal medium for 7, 18 and 28 hours. Ethylmethanesulfonate and cyclophosphamide were used as positive controls in 18-hour cultures without and with S9-mix, respectively. Two hours (7 hour interval) or 2.5 hours (18 and 28 hours intervals) before the end of the incubation period, colcemid was added to the cultures. The cells were put onto glass slides, treated with hypotonic potassium chloride solution, fixed in methanol and acetic acid and stained with Giemsa solution. In each experimental group two parallel cultures were set up. Per culture 100 metaphases were scored for structural chromosomal aberrations (breaks, fragments, deletions, exchanges and chromosomal disintegrations). Chromosomal gaps were recorded separately. There were no biologically relevant and statistically significant increases in cells with structural aberrations after treatment with the test substance at any fixation interval either with or without metabolic activation. Based on the observations made, the test chemical did not induce chromosome aberration in the Chinese hamster V79 cells both in the presence and absence of S9 activation system and hence it is not likely to be gene mutant in vitro.

In another experiment, vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical. The study was performed using CHL/IU cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in physiological saline and used at dose level of 0, 100, 125, 150, 175 or 200 µg/mL (short term treatment method) and 0, 50, 75, 100, 125 or 150 µg/mL (main test) and 0, 100, 110, 120, 130, 140 or 150 µg/mL (additional test) without S9 in the continuous treatment method. The doses for the main study were based on the preliminary dose range finding study. The test was performed by continuous treatment method for 24 hrs and short term treatment method for 6 hrs. The cells were allowed to express for 18 hrs in short term treatment method. Two hours before the end of the culture, colcemid was added to the culture solution to a final concentration of about 0.1 μg / ml. Chromosome specimens were prepared according to a conventional method. Two slide specimens were prepared for each dish. The prepared specimen was stained with 3% Giemsa solution for 20 mins. Chromosomes were analyzed based on the classification method by the Japan Society for Environmental Mutagenesis and Mammalian Testing, and structural abnormalities such as breakage and exchange of chromosome type or chromosome type, presence and absence of gap, and ploidy. The presence or absence of cells (polyploid) was observed. The gap was not included in structural abnormality. The frequency of cells with chromosomal abnormality is negative, less than 5% negative, less than 10% false positive, more than 10% Positive. Based on the observations made, the test chemical did not induce chromosome aberration in the CHL/IU cells both in the presence and absence of S9 activation system and hence it is not likely to be gene mutant in vitro.

Test chemical did not induce chromosomal aberration in Chinese hamster V79 cells and CHL/IU cells in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames assay:

Data available for the target chemical was reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

Study 1:

Gene toxicity test was performed to determine the mutagenic nature of test chemical. The study was performed using Salmonella Typhimurium TA 1538, 1535, 1537, 98 and 100 both in the presence and absence of mammalian S-9 microsomal fraction. The test material did not induce reverse mutations. Therefore, Test chemical did not induce mutation in Salmonella Typhimurium TA 1538, 1535, 1537, 98 and 100 with and without mammalian S-9 microsomal fraction. Hence it is not likely to classify as a gene mutant in vitro.

Study 2: 

Gene toxicity test was performed to determine the mutagenic nature of test chemical. The study was performed using Salmonella Typhimurium other both in the presence and absence of mammalian S-9 microsomal fraction. The test material did not induce reverse mutations. Therefore, Test chemical did not induce mutation in Salmonella Typhimurium other with and without mammalian S-9 microsomal fraction. Hence it is not likely to classify as a gene mutant in vitro.

Study 3: 

Gene toxicity test was performed to determine the mutagenic nature of test chemical. The study was performed using S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 both in the presence and absence of mammalian S-9 microsomal fraction. The test material did not induce reverse mutations. Therefore, Test chemical did not induce mutation in Salmonella Typhimurium TA 1535, TA 1537, TA 98 and TA 100 with and without mammalian S-9 microsomal fraction. Hence it is not likely to classify as a gene mutant in vitro.

Study 4:

Gene toxicity test was performed to determine the mutagenic nature of test chemical. The study was performed using S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 both in the presence and absence of mammalian S-9 microsomal fraction. The test material caused a visible reduction in the growth of the bacterial lawn to all of the strains tested. The first evidence of toxicity was observed at 150 mg/plate. No significant increase in the frequency of revertant colonies of bacteria was recorded for any of the strains used, at any dose level with or without metabolic activation. All of the positive control chemicals used in the test produced marked increases in the frequency of revertant colonies and the activity of the S9 fraction was found to be satisfactory. The test material did not induce reverse mutations. Therefore, Test chemical did not induce mutation in Salmonella Typhimurium TA 1535, TA 1537, TA 98 and TA 100 with and without mammalian S-9 microsomal fraction. Hence it is not likely to classify as a gene mutant in vitro.

  

Study 5:

Gene mutation study was performed on Salmonella Typhimurium strains to determine the mutagenic nature of test chemical. Salmonella Typhimurium TA 1538, 1535, 1537, 98, 100 and E. coli WP2 were used during the study. The study was performed in the presence and absence of S9 metabolic activation. The S9 fraction was prepared from the liver of male Sprague-Dawley rats, weighing about 220g (seven weeks old), pretreated with polychlorinated biphenyl (KC 500) at a dose of 500mg/kg body weight five days before sacrifice. The positive controls used were 4-nitroquinoline-N-oxide, 9-aminoacridine, 2-nitrofluorene, N-ethyl-N-nitro-N-nitrosoguanidine, benzo(a)pyrene, 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2) and 9-aminoacridine. 0.1ml of bacterial strain and 0.5ml of S9 mix or sodium phosphate buffer (pH 7.4) were added to a sterile test tube containing 0.1ml of various concentrations of the chemical. The test concentrations used were 1, 5, 10, 50, 100, 500, 1000 and 5000 µg/plate and the test chemical was dissolved in DMSO. This mixture was preincubated in a shaker water bath at 37°C for 20 minutes, and then added to 2ml of molten top agar (45°C). This mixture was preincubated in a shaker water bath at 37°C for 20 minutes, and then added to 2ml of molten top agar (45°C). The contents of each tube were mixed and poured onto a minimal glucose agar plate immediately. The plates were incubated at 37°C for 48 hours, and then the number of revertant colonies on each plate was scored with an automated colony counter. The background bacterial lawn was checked routinely by dissected microscope. Negleble amount of revertant colonies were observed as compared to positive control substances. Thus the test chemical can be considered as non-mutagenic when Salmonella Typhimurium TA 1538, 1535, 1537, 98, 100 and E. coli WP2 were treated in the presence and absence of S9 activation system.

Test chemical did not induce gene mutation in Salmonella Typhimurium strains TA 1538, 1535, 1537, 98, 100 and E. coli WP2 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

 

 

Chromosomal Abberation:

 

Data available for the target chemical was reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

 

In vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical. The study was performed using Chinese hamster V79 cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in suitable solvent and used. The doses for the main study were based on the preliminary dose range finding study. Evaluated dose levels were 1.0 μg/ml without S9-mix and 10.0 μg/ml with S9-mix for 7 hours; 0.3, 1.0 and 3.0 μg/ml without S9-mix and 1.0, 3.0 and 10.0 μg/ml with S9-mix for 18 hours; and 3.0 μg/ml without S9-mix and 10.0 μg/ml with S9-mix for 28 hours.Logarithmically growing cells were incubated with the test substance in serum-free culture medium for 4 hrs. Cells were subsequently washed in glucose-containing saline and cultured in normal medium for 7, 18 and 28 hours. Ethylmethanesulfonate and cyclophosphamide were used as positive controls in 18-hour cultures without and with S9-mix, respectively. Two hours (7 hour interval) or 2.5 hours (18 and 28 hours intervals) before the end of the incubation period, colcemid was added to the cultures. The cells were put onto glass slides, treated with hypotonic potassium chloride solution, fixed in methanol and acetic acid and stained with Giemsa solution. In each experimental group two parallel cultures were set up. Per culture 100 metaphases were scored for structural chromosomal aberrations (breaks, fragments, deletions, exchanges and chromosomal disintegrations). Chromosomal gaps were recorded separately. There were no biologically relevant and statistically significant increases in cells with structural aberrations after treatment with the test substance at any fixation interval either with or without metabolic activation. Based on the observations made, the test chemical did not induce chromosome aberration in the Chinese hamster V79 cells both in the presence and absence of S9 activation system and hence it is not likely to be gene mutant in vitro.

 

In another experiment, vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical. The study was performed using CHL/IU cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in physiological saline and used at dose level of 0, 100, 125, 150, 175 or 200 µg/mL (short term treatment method) and 0, 50, 75, 100, 125 or 150 µg/mL (main test) and 0, 100, 110, 120, 130, 140 or 150 µg/mL (additional test) without S9 in the continuous treatment method. The doses for the main study were based on the preliminary dose range finding study. The test was performed by continuous treatment method for 24 hrs and short term treatment method for 6 hrs. The cells were allowed to express for 18 hrs in short term treatment method. Two hours before the end of the culture, colcemid was added to the culture solution to a final concentration of about 0.1 μg / ml. Chromosome specimens were prepared according to a conventional method. Two slide specimens were prepared for each dish. The prepared specimen was stained with 3% Giemsa solution for 20 mins. Chromosomes were analyzed based on the classification method by the Japan Society for Environmental Mutagenesis and Mammalian Testing, and structural abnormalities such as breakage and exchange of chromosome type or chromosome type, presence and absence of gap, and ploidy. The presence or absence of cells (polyploid) was observed. The gap was not included in structural abnormality. The frequency of cells with chromosomal abnormality is negative, less than 5% negative, less than 10% false positive, more than 10% Positive. Based on the observations made, the test chemical did not induce chromosome aberration in the CHL/IU cells both in the presence and absence of S9 activation system and hence it is not likely to be gene mutant in vitro.

 

Test chemical did not induce chromosomal aberration in Chinese hamster V79 cells and CHL/IU cells in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

 

In vitro gene mutation study in mammalian cells:

Data available for the target chemical was reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

 

Gene toxicity test was performed to determine the mutagenic nature of test chemical. The study was performed using L5178Y TK ± mouse lymphoma both in the presence and absence of mammalian S-9 microsomal fraction. The test material did not significantly increase mutation frequency over the average mutation frequency of solvent control. Therefore, Test chemical did not induce mutation in L5178Y TK ± mouse lymphoma with and without mammalian S-9 microsomal fraction. Hence it is not likely to classify as a gene mutant in vitro.

 

The test chemical was tested in the L5178Y TK+/- Mouse Lymphoma Mutagenesis assay in the presence and absence of rat liver S-9 to determine the mutagenic nature of the test chemical. The study was performed at various concentrations 0.0038 to 0.050 μl/ml (10 concentrations) without S9 and 0.012 to 0.16 μl/ml (10 concentrations) with S9 metabolic activation system. The cells were exposed to the test chemical, positive control and negative control for 4 hours. An expression time of 2 days was allowed with cell population adjustment at 24 and 48 hours. At the end of the expression period, the cells were placed in cloning medium. Cell counts were made for each preparation and the appropriate number of cells were removed and plated. Total number of colonies per plate and the mutation frequency were determined. None of the treated cultures that were cloned exhibited a significant increase in mutant frequency over the average mutant frequency of the solvent controls and no dose response was observed. Thus, the test chemical was considered to be not mutagenic in nature.

Test chemical did not induce mutation in L5178Y TK ± mouse lymphoma with and without mammalian S-9 microsomal fraction. Hence it is not likely to classify as a gene mutant in vitro.

Justification for classification or non-classification

Based on the data available for test chemical, it does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.