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Toxicological information

Genetic toxicity: in vitro

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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:
21.08.2003 - 03.11.2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report date:
2003

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

Constituent 1
Chemical structure
Reference substance name:
4-chlororesorcinol
EC Number:
202-462-0
EC Name:
4-chlororesorcinol
Cas Number:
95-88-5
Molecular formula:
C6H5ClO2
IUPAC Name:
4-chlorobenzene-1,3-diol
Test material form:
other:
Remarks:
beige powder
Details on test material:
Molecular weight: 144.65

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:
33, 100, 333, 1000, 2500, 5000 ug/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 relevant toxic effects were only observed at the maximum concentration. Therefore, 5000 µg/plate were chosen as maximal concentration in the main experiments.
Controlsopen allclose all
Untreated negative controls:
other: Concurrent untreated and solvent controls were performed.
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without metabolic activation, TA 1535, TA 100, concentration: 10 µg/plate, purity: at leat 99%, dissolved in: water deionised
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine, 4-NOPD
Remarks:
without metabolic activation, TA 1537, TA 98, concentration: 10 µg/plate in TA 98, 50 µg/plate in TA 1537, purity: >99%, dissolved in: DMSO (purity >99 %)
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without metabolic activation, TA 102, concentration: 4.0 µ/plate, purity: >99%, dissolved in: water deionised
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, concentration: 2.5 µg/plate (10.0 µg/plate in TA 102), purity: 97.5 %, dissolved in: DMSO (purity >99 %)
Details on test system and experimental conditions:
Characterisation of the Salmonella typhimurium Strains
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 of the uvrB gene) causes an inactivation of 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 I01 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 uvrB--mutation and is excision repair proficient. Additionally, TA 102 contains the multicopy plasmid pAQl carrying the hisG428 mutation (ochre mutation in the hisG gene ) and a tetracycline resistance gene.

Regular checking of the properties of the strains regarding the membrane permeability, ampicillin- and tetracycline-resistance as well as spontaneous mutation rates is performed in the laboratory of RCC Cytotest Cell Research according to Ames et al. (1). In this way it was ensured that the experimental conditions set down by Ames were fulfilled. The bacterial strains TA 1535, TA 1537, and TA 100 were obtained from Ames (University of California, 94720 Berkeley, U.S.A.). The bacterial strain TA 98 was obtained from E. Merck (D-64293 Darmstadt). The bacterial strain TA 102 was 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 (2µg/mL) was added to strain
TA 102. This nutrient medium contains per litre:
8 g Merck Nutrient Broth (MERCK, D-64293 Darmstadt)
5 g NaCl (MERCK, 0-64293 Darmstadt)
The bacterial cultures were incubated in a shaking water bath for 4 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*
10.5 mg L-Histidine X HCI X H20*
12.2 mg Biotin*
* (MERCK, D-64293 Darrnstadt)
Sterilisation was 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 of 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
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100, TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity

Any other information on results incl. tables

DlSCUSSlON OF RESULTS

The test item A 012 was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I) 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: 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. In experiment II, reduced background growth was observed at 5000 µg/plate without metabolic activation in strains TA 1535, TA 1537, TA, 98, and TA 102 and with metabolic activation in strains TA 1535 and TA 102. In strain TA 100 reduced background growth was observed at 2500 and 5000 pglplate in the presence of metabolic activation. Relevant toxic effects, evident as a reduction of the number of revertant colonies below 0.5 times the corresponding solvent control, occurred at the following concentrations (in µg/plate):

 Strain

 Experiment I

 Experiment II

   without S9 mix  with S9 mix  without S9 mix  with S9 mix
 TA 1535  5000  5000  /  /
 TA 1537  /  5000  5000  5000
 TA 98  /  /  5000  5000
 TA 100  5000  /  5000  5000
 TA 102  5000  5000  5000  5000

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with A 012 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 I, with metabolic activation, the number of colonies did not quite reach the lower limit of our historical control data in the negative control of strains TA 1535 and TA 1537 whereas the data in the negative control of strain TA 98 was slightly above our historical control range. Since these deviations are rather small, these effect are judged to be based upon biological fluctuations and have no detrimental impact on the outcome of the study.

The historical range of positive controls was exceeded in strains TA 1535 (experiment II) without metabolic activation and in strains TA 1537 and TA 98 (experiment I) with metabolic activation. 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 012 is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay in the absence and presence of metabolic activation.
Executive summary:

This study was performed to investigate the potential of A 012 to induce gene mutations according to the plate incorporation test (experiment 1) 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: 33; 100; 333; 1000; 2500; and 5000 µg/plate Relevant toxic effects, evident as a reduction in the number of revertants, occurred in some of the strains at the maximum concentration with and without metabolic activation. 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 1. In experiment II, reduced background growth was observed at 5000 µg/plate without metabolic activation in strains TA 1535, TA 1537, TA, 98, and TA 102 and with metabolic activation in strains TA 1535 and TA 102. In strain TA 100 reduced background growth was observed at 2500 and 5000 µg/plate in the presence of metabolic activation. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with A 012 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.