3-amino-2-chlor-6-methylphenol

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:

13.04. - 03.05.2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 001

Cas Number:

80419-48-3

Test material form:

solid

Details on test material:

beige powder

Specific details on test material used for the study:

Analogy
CAS 80419-48-3, 2-Chlor-3-Amino-6-methylphenol x HCI

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Experiment I: 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

In the pre-experiment the concentration range of the test item was 3 - 5000 µg/plate. The pre-experiment is reported as part of experiment I since no relevant toxic effects were observed and 5000 µg/plate were chosen as maximal concentration. Based on the toxic effects observed in experiment 1, seven concentrations were tested in experiment II.

Controls

Controls 1

Untreated negative controls:

other: Concurrent untreated and solvent controls were performed.

Remarks:

concurrent untreated

Controls 2

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without metabolic activation, TA 1535, TA 100; purity: at least 99%; dissolved in: water deionised; concentration: 10 µg/plate

Controls 3

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine, 4-NOPD

Remarks:

without metabolic activation, strain TA 1537, TA 98; purity: >99.9%; dissolved in: DMSO; concentration: 10 µg/plate in TA 98, 50 µg/plate in TA 1537

Controls 4

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation, strain TA 102; purity: >99.0%; dissolved in: water deionised; concentration: 4.0 µL/plate

Controls 5

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, 2-AA

Remarks:

with metabolic activation, TA 1535, TA 1537, TA 98, TA 100, TA 102; purity: 97.5%; dissolved in: DMSO; concentration: 2.5 µg/plate (10.0 µg/plate in TA 102)

Details on test system and experimental conditions:

The histidine dependent strains are derived from S. typhimurium strain LT2 through a mutation in the histidine locus. Additionally due to the "deep rough" (rfa·) mutation they possess a faulty lipopolysaccharide envelope which enables substances to penetrate the cell wall more easily. A further mutation (deletion cf the uvrB gene) causes an inactivation cf the excision repair system. The latter alteration also includes a deletion in the nitrate reductase and biotin genes. In the strains TA 98, TA 100, and TA 102 the R-factor plasmid pKM 101 carries umu DC analogous genes that are involved in error-prone repair and the ampicillin resistance marker. The strain TA 102 does not contain the uvrf -mutation and is excision repair proficient. Additionally, TA 102 contains the multicopy plasmid pAQ1 carrying the hisG428 mutation (ochre mutation in the hisG gene ) and a tetracycline resistance gene.

Regular checking cf the properties cf the strains regarding the membrane permeability, ampicillin- and tetracycline-resistance as well as spontaneous mutation rates is performed in the laboratory cf RCC Cytotest Cell Research according to B. Ames et al. and D. Maron and B. Ames. In this way it was ensured that the experimental conditions set down by Ames were fulfilled.
The bacterial strains TA 1535 and TA 1537 were obtained from Ames (University cf California, 94720 Berkeley, U.S.A.). The bacterial strain TA 98 was obtained from E. Merck (D-64293 Darmstadt). The bacterial strains TA 100 and TA 102 were obtained from RCC Ltd (CH-4332 Stein).

Storage:
The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO (MERCK, D-64293 Darmstadt) in liquid nitrogen.

Precultures:
From the thawed ampoules of the strains 0.5 mL bacterial suspension was transferred into 250 mL Erlenmeyer flasks containing 20 mL nutrient medium. A solution of 20 µL ampicillin (25 µg/mL) was added to the strains TA 98, TA 100, and TA 102. Additionally 20 µL tetracycline (2 µg/mL) was added to strain TA 102. This nutrient medium contains per litre:
8 g Merck Nutrient Broth (MERCK, D-64293 Darmstadt)
5 g NaCI (MERCK, D-64293 Darmstadt)
The bacterial cultures were incubated in a shaking water bath for 4 hours at 37° C.

Selective Agar:
The plates with the minimal agar were obtained from E. Merck, D-64293 Darmstadt.

Overlay Agar:
The overlay agar contains per litre:
6.0 g MERCK Agar Agar*
6.0 g NaCI*
10.5mg L-Histidine X HCI X H20*
12 .2 mg Biotin*
* (MERCK, D-64293 Darmstadt)
Sterilisations were performed at 121 ° C in an autoclave.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed
.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration
.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number cf revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Test results 3

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment I

Test results 4

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Test results 5

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment II

Test results 6

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment I and II

Any other information on results incl. tables

DISCUSSION OF RESULTS

The test item A 094 was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment 1) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls, were tested in triplicate. The test item was tested at the following concentrations:

Experiment I: 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used in experiment I with the exception of strain TA 98 where reduced background growth was observed at 100 µg/plate without metabolic activation and at 333 µg/plate with metabolic activation. In experiment II, reduced background growth was observed at 5000 µg/plate in strain TA 98 without metabolic activation and in strain TA 102 with metabolic activation.

Toxic effects, evident as a reduction in the number of revertants, were observed in strains TA 1537 (exp. 1) and TA 98 (exp. II) without metabolic activation and in strain TA 102 (exp. I and 11) with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with A 094 at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowled­ged border of biological relevance.

In experiment 1, in the presence of metabolic activation, the number of colonies did not quite reach the lower limit of our historical control data in in the solvent control of strain TA 98 and in the negative and solvent control of strain TA 100. Since these deviation are rather small, these effects are judged to be based upon biologically irrelevant fluctuations and have no detrimental impact on the outcome of the study.

The historical range of positive controls was exceeded in strains TA 1535 and TA 100 (experiment I and II) without metabolic activation and with metabolic activation in strain TA 1537 (experiment I). These effects indicate the sensitivity of the strains rather than compromising the assay.

Appropriate reference mutagens were used as positive controls. They showed a distinct in­crease in induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, A 094 is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of A 094 to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Experiment I: 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used in experiment I with the exception of strain TA 98 where reduced background growth was observed at 100 µg/plate without metabolic activation and at 333 µg/plate with metabolic activation. In experiment II, reduced background growth was observed at 5000 µg/plate in strain TA 98 without metabolic activation and in strain TA 102 with metabolic activation.

Toxic effects, evident as a reduction in the number of revertants, were observed in strains TA 1537 (exp. I) and TA 98 (exp. II) without metabolic activation andin strain TA 102 (exp. I and II) with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with A 094 at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct in­crease of induced revertant colonies.