[No public or meaningful name is available]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 July - 19 August 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Standards for Mutagenicity Test using Microorganisms (JMOL)

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Guidelines for Screening Toxicity Testing of Chemicals and Guidelines for Toxicity Testing of Chemicals (JMITI)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

liquid

Method

Target gene:

tryptophan (E. coli strain) and histidine (Salmonella strains)

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital and 5,6-benzoflavone, S9 induced rat liver

Test concentrations with justification for top dose:

Preliminary test: 10, 50, 100, 500, 1000, 5000 µg/plate
Main test: 313, 625, 1250, 2500, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: test substance was found to be soluble in DMSO and acetone, but not in water.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: AF-2; 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

Remarks:

-MA: TA 100, WP2 uvrA - 0.01 µg/plate; TA 98 - 0.1 µg/plate

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

-MA: TA 1535 - 0.5 µg/plate

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

-MA: TA 1537 - 80.0 µg/plate

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-AA (2-Aminoanthracene)

Remarks:

+MA: TA 1535 - 2.0 µg/plate; WP2 uvrA - 10.0 µg/plate

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

+MA: TA 100, TA 98, TA 1537 - 5.0 µg/plate

Details on test system and experimental conditions:

ACTIVATION:
S9 mix per ml:
S9 fraction: 10 v/v%
MgCl2: 8 µmol
KCl: 33 µmol
G-6-P: 5 µmol
NADPH: 4 µmol
NADH: 4 µmol
0.5 ml S9 mix was added to a total volume of 2.7 ml, giving a final concentration of approximately 2% S9.


METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 37°C for 48 hours

SELECTION AGENT (mutation assays): minimum glucose agar

NUMBER OF REPLICATIONS: triplicate plates, experiment repeated

DETERMINATION OF CYTOTOXICITY
- increase in number of revertant colonies

Evaluation criteria:

The number of revertant colonies of each test group were compared with and analysed for a statistically significant increase (p<0.01) from that of the negative control group at each dose in each bacterial strain with and without metabolic activation.

Statistics:

Dunnett's multiple comparison (one-sided test)
Linear regression

Results and discussion

Test results

Test results 1

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Observations:
The test substance produced neither toxic effects nor a statistically significant increase in the number of revertant colonies in any conditions in any bacterial strains.

All of the positive controls produced significant increases in the number of revertant colonies in all bacterial strains with and without metabolic activation. This shows the test method employed in the test is effective for the purpose.

Remarks on result:

other: all strains/cell types tested

Any other information on results incl. tables

Table 1: Preliminary test - number of revertants per plate (mean of 2 plates)

Dose (µg/plate)	TA 100	TA 100	TA 1535	TA 1535	WP2 uvrA	WP2 uvrA	TA 98	TA 98	TA 1537	TA 1537
	- MA	+ MA	- MA	+ MA	- MA	+ MA	- MA	+ MA	- MA	+ MA
DMSO	133	124	9	11	28	31	26	29	10	10
10	133	115	9	13	24	30	26	27	8	12
50	126	120	11	11	24	29	26	34	8	9
100	123	116	8	14	30	35	20	30	7	12
500	135	133	9	14	23	32	21	36	9	9
1000	127	139	12	10	30	32	22	27	6	10
5000	126	138	14	10	29	36	27	36	5	5
Positive control	372	1227	301	413	99	733	497	277	730	53

 

Table 2: Main test 1 - number of revertants per plate (mean of 3 plates)

Dose (µg/plate)	TA 100	TA 100	TA 1535	TA 1535	WP2 uvrA	WP2 uvrA	TA 98	TA 98	TA 1537	TA 1537
	- MA	+ MA	- MA	+ MA	- MA	+ MA	- MA	+ MA	- MA	+ MA
DMSO	117	128	11	12	29	30	20	24	6	10
313	110	137	13	14	27	37	22	25	5	7
625	110	142	11	11	24	37	23	29	6	8
1250	111	128	9	12	29	31	21	32	8	8
2500	110	128	11	14	25	35	27	30	5	8
5000	116	116	10	12	27	37	23	25	5	6
Positive control	354	1178	285	444	85	818	457	273	990	55

 

Table 3: Main test 2 - number of revertants per plate (mean of 3 plates)

Dose (µg/plate)	TA 100	TA 100	TA 1535	TA 1535	WP2 uvrA	WP2 uvrA	TA 98	TA 98	TA 1537	TA 1537
	- MA	+ MA	- MA	+ MA	- MA	+ MA	- MA	+ MA	- MA	+ MA
DMSO	115	122	11	12	25	33	2.5	28	7	11
313	112	121	10	11	29	34	2.3	31	8	10
625	124	135	9	12	30	36	1.5	30	8	8
1250	107	136	9	14	22	34	3.0	29	6	8
2500	119	117	9	10	27	33	6.0	23	8	9
5000	113	122	11	9	28	32	1.5	26	9	7
Positive control	438	1334	223	437	111	1087	456	273	1077	56

 

Applicant's summary and conclusion

Conclusions:

The test substance has been tested for mutagenicity to bacteria, in a study which was conducted according to the OECD TG 471 and 472 and in compliant with GLP. No evidence of a test-substance related increase in the number of revertants was observed with or without metabolic activation in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 or E. coli WP2 uvrA in the initial or the repeat experiments up to limit concentrations. Appropriate positive and solvent controls were included and gave the expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.