2-Thiazolamine, 4-(2-naphthalenyl)-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The experimental phase of this study was performed between 30 May 2012 and 10 July 2012.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Test guideline 4

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

Meets the requirements of the Japanese Regulatory Authorities including METI, MHLW and MAFF, OECD Guidelines for Testing of Chemicals No. 471 "and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP Certificate included in Attached Background Material

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-(naphthalen-2-yl)-1,3-thiazol-2-amine

EC Number:

606-740-6

Cas Number:

21331-43-1

Molecular formula:

C13H10N2S

IUPAC Name:

4-(naphthalen-2-yl)-1,3-thiazol-2-amine

Test material form:

solid: particulate/powder

Details on test material:

Sponsor's identification : 4-(2-naphthalenyl)-2-thiazolamine
Description : Off white powder
Purity : 98.8%
Batch number : 107 (ex Pentagon)
Date received : 08 May 2012
Expiry date : Not supplied
Storage conditions : Room temperature in the dark

Method

Target gene:

Histidine for Salmonella.
Tryptophan for E.Coli

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/beta­naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

Preliminary Toxicity Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Experiment 1: Salmonella strains (absence of S9-mix): 0.5, 1.5, 5, 15, 50, 150, 500 µg/plate. Salmonella strains (presence of S9-mix)
and E.coli strain WP2uvrA (presence and absence of S9-mix): 1.5, 5, 15, 50, 150, 500, 1500 µg/plate.

Experiment 2: Salmonella strains (absence of S9-mix): 0.5, 1.5, 5, 15, 50, 150, 500 µg/plate.
Salmonella strain TA100 (presence of S9-mix only): 15, 50, 100, 150, 200, 500, 1500 µg/plate.
Salmonella strains TA1537, TA98 & TA1535 (presence of S9-mix)
E.coli strain WP2uvrA (presence and absence of S9-mix): 1.5, 5, 15, 50, 150, 500, 1500 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide.
- Justification for choice of solvent/vehicle: The test item was fully soluble in dimethyl sulphoxide at 50 mg/ml in solubility checks performed
in-house. Following solubility information provided by the Sponsor, sterile distilled water was not evaluated as a potential vehicle in this test system. Dimethyl sulphoxide was therefore selected as the vehicle.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 1 µg/plate

Remarks:

With S9 mix

Controls 2

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 2 µg/plate

Remarks:

With S9 mix

Controls 3

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 2 µg/plate

Remarks:

With S9 mix

Controls 4

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 10 µg/plate

Remarks:

With S9 mix

Controls 5

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With S9 mix

Migrated to IUCLID6: Benzo(a)pyrene: 5 µg/plate

Controls 6

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9 mix

Migrated to IUCLID6: 4-Nitroquinoline-1-oxide: 0.2 µg/plate

Controls 7

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix

Migrated to IUCLID6: 9-Aminoacridine: 80 µg/plate

Controls 8

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9 mix

Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 3 µg/plate

Controls 9

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 mix

Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 5 µg/plate

Controls 10

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 mix

Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 2 µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: Experiment 1: in agar (plate incorporation). Experiment 2: Pre-incubation

DURATION
- Preincubation period for bacterial strains: 10h
- Exposure duration: 48 - 72 hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable

NUMBER OF REPLICATIONS: Triplicate plating.

DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.

Evaluation criteria:

Acceptance Criteria:

The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.
All tester strain cultures should be in the approximate range of 1 to 9.9 x 109 bacteria per ml.
Each mean positive control value should be at least twice the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
There should be a minimum of four non-toxic test material dose levels.
There should not be an excessive loss of plates due to contamination.

Evaluation criteria:
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.

Statistics:

Standard deviation
Dunnetts method of linear regression

Results and discussion

Test results

Test results 1

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Tested up to maximum recommended dose of 5000 µg/plate and toxic limit

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

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:

cytotoxicity

Remarks:

Tested up to toxic limit

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test item was fully soluble in dimethyl sulphoxide at 50 mg/ml in solubility checks performed in-house. Following solubility information provided by the Sponsor, sterile distilled water was not evaluated as a potential vehicle in this test system. Dimethyl sulphoxide was
therefore selected as the vehicle.
- Precipitation: A test item precipitate (particulate in appearance) was noted at and above 1500 µg/plate, this observation did not prevent the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test:
The test item was initially toxic to TA100 at 150 µg/plate and from 500 µg/plate to WP2uvrA. The test item formulation and S9 mix used in this experiment were both shown to be sterile.

COMPARISON WITH HISTORICAL CONTROL DATA:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).

Results for the negative controls (spontaneous mutation rates) were considered to be acceptable.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 150 and 500 µg/plate in the absence and presence of S9-mix respectively. The sensitivity of the bacterial tester strains
to the toxicity of the test item varied slightly between strain type, exposures with or without S9 mix and experimental methodology. The test item
was tested up to the toxic limit.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

RESULTS

Preliminary ToxicityTest

The test item was initially toxic to TA100 at 150 µg/plate and from 500 µg/plate to WP2uvrA.

The test item formulation and S9-mix used in this experiment were both shown to be sterile. The numbers of revertant colonies for the toxicity assay were:

With (+) or without (-) S9-mix	Strain	Dose (µg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA100	91	87	80	88	84	91	84	56S	0V	0VP	0VP
+	TA100	103	87	90	104	76	107	113	161	65S	43SP	25SP
-	WP2uvrA	36	40	31	38	37	47	33	29	33S	20SP	24SP
+	WP2uvrA	47	41	40	37	44	38	47	53	37S	23SP	24SP

S         Sparse bacterial background lawn
V         Very weak bacterial background lawn
P         Precipitate

MutationTest

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and S9‑mix used in both experiments was shown to be sterile. The culture density for each bacterial strain was also checked and considered acceptable. These data are not given in the report.

Results for the negative controls (spontaneous mutation rates) are presented in Table 1and were considered to be acceptable. These data are for concurrent untreated control plates perford on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test item, reference item and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3 for Experiment 1 and Table 4 and Table 5 for Experiment 2 (attached background material).

A history profile of vehicle/untreated and positive control values (reference items) for 2010 and 2011 are presented in Attached background material. A certificate of analysis is also presented in attached background material.

The test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 150 and

500 µg/plate in the absence and presence of S9-mix respectively. The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9‑mix and experimental methodology. The test item was tested up to the toxic limit. A test item precipitate (particulate in appearance) was noted at and above 1500 µg/plate, this observation did not prevent the scoring of revertant colonies.

No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method. Small, statistically significant increases in TA100 revertant colony frequency were observed in the first experiment at 5, 50 and 150 µg/plate and at 100 and 150 µg/plate in the presence of S9-mix in Experiment 2. These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or appropriate reproducibility. Furthermore, the individual revertant counts at the statistically significant dose levels were within the in‑house historical untreated/vehicle control range for the tester strain and the maximum fold increase was only 1.7 times the concurrent vehicle controls.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Table1               Spontaneous Mutation Rates (Concurrent Negative Controls)

Experiment 1

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
100		19		36		12		11
103	(93)	9	(19)	21	(32)	20	(14)	8	(9)
76		29		39		9		7

Experiment 2

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
115		8		41		24		9
84	(95)	31	(21)	29	(37)	32	(26)	19	(13)
87		24		41		23		11

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test item, 4-(2-naphthalenyl)-2-thiazolamine, was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Introduction.

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Methods.

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA were treated with the test item, 4-(2-naphthalenyl)-2-thiazolamine, using both the Ames plate incorporation and pre-incubation methods at seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the first experiment was determined in a preliminary toxicity assay and ranged between 0.5 and 1500 µg/plate, depending on bacterial strain type and presence or absence of S9-mix. The experiment was repeated on a separate day (pre-incubation method) using an amended dose range (ranging between 0.5 and 1500 µg/plate), fresh cultures of the bacterial strains and fresh test item formulations.

Additional dose levels and an expanded dose range were selected in both experiments in order to achieve four non-toxic dose levels and the toxic limit of the test item. Intermediate dose levels (100 and 200 µg/plate) were included in Experiment 2 to establish a better dose-response relationship and reproducibility following the observation of small but statistically significant increases in TA100 revertant colony frequency (presence of S9-mix) in Experiment 1.

Results.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive controls used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 150 and 500 µg/plate in the absence and presence of S9-mix respectively. The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9‑mix and experimental methodology. The test item was tested up to the toxic limit. A test item precipitate (particulate in appearance) was noted at and above 1500 µg/plate, this observation did not prevent the scoring of revertant colonies.

No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method. Small, statistically significant increases in TA100 revertant colony frequency were observed in the first experiment at 5, 50 and 150 µg/plate and at 100 and 150 µg/plate in the presence of S9-mix in Experiment 2. These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or appropriate reproducibility. Furthermore, the individual revertant counts at the statistically significant dose levels were within the in‑house historical untreated/vehicle control range for the tester strain and the maximum fold increase was only 1.7 times the concurrent vehicle controls.

Conclusion.

The test item, 4-(2-naphthalenyl)-2-thiazolamine, was considered to be non-mutagenic under the conditions of this test.