Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Non mutagenic in the AMES assay.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From July 13 to August 4, 2000
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Complete read-across justification is attached in section 13. Source study has reliability 1.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
1992
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
liver microsomal activation
Test concentrations with justification for top dose:
33, 100, 333, 1000, 2500 and 5000 µg/plate
No relevant toxic effects at 5000 µg/plate.
Vehicle / solvent:
- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: due to solubility properties and relative non toxicity to bacteria.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine
Remarks:
without S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation (exp. I) and preincubation (exp. II);
- Cell density at seeding: 100 µl of bacterial suspension

EXPERIMENTAL PERFORMANCE
The following materials were mixed in a test tube and poured onto the minimal agar plates:
100 µl test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control),
500 µl S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 µl bacteria suspension
2 ml overlay agar

In the pre-incubation assay 100 µl test solution, 500 µl S9 mix / S9 mix substitution buffer and 100 µl bacterial suspension were mixed in a test tube and shaken at 37 °C for 60 minutes. After pre-incubation 2.0 ml overlay agar (45 °C) was added to each tube. The mixture was poured on minimal agar plates.
After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.

NUMBER OF REPLICATIONS: 3 plates for each strain and dose level including controls

DETERMINATION OF CYTOTOXICITY
A prestudy was performed with all strains: 8 concentrations are tested for toxicity and mutation induction with 3 plates each in strains TA 98 and TA 100; 6 concentrations were tested in strains TA 1535, TA 1537, and WP2 uvrA. The experimental conditions in this pre-experiment are the same as for the experiment I (plate incorporation test).
Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.
The pre-experiment is reported as main experiment I, if the following criteria are met: valuable plates (> 0 colonies) at five concentrations or more in all strains used.

DATA RECORDING
The colonies were counted using the AUTOCOUNT (Artek Systems Corporation, BIOSYS GmbH, D-61184 Karben). The counter was connected to an IBM AT compatible PC with printer to print out the individual values and the means from the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates. Due to precipitation of the test item, revertant colonies were partly counted manually at higher concentrations.

ACCEPTABILIITY OF THE ASSAY
Bacteria reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data
- the positive control substances should produce a significant increase in mutant colony frequencies

EVALUATION OF RESULTS
A test item is considered positive if either a dose related increase in the number of revertants or a biologically relevant increase for at least one test concentration is induced.
A test item producing neither a dose related increase in the number of revertants, nor a biologically relevant positive response at any one of the test points is considered nonmutagenic in this system.
A test item is considered mutagenic if in the strains TA 98, TA 100, and WP2 uvrA the number of reversions will be at least twice as high and in the strains TA 1535 and TA 1537 at least three times higher as compared to the spontaneous reversion rate.
Also, a dose-dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test item regardless whether the highest dose induced the above described enhancement factors or not.
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Test item 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 the Escherichia coli strain WP2 uvrA.
The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate.

In experiment II, the colony counts of the negative and solvent controls of strain TA 1535 were far beyond our historical range in the presence and absence of metabolic activation. Therefore, the results were dismissed and an additional experiment with strain TA 1535 was performed. The results of this additional experiment are included in experiment II.

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 both experiments.
No toxic effects, evident as a reduction in the number of revendants, occurred in the test groups with and without metabolic activation.
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with test substance at any dose level, with or without 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.
An isolated minor increase was observed in strain TA 1535 at 5000 µg/plate in the experiment I without metabolic activation. However, this increase was judged as an artefact based upon fluctuations at such low numbers since it was not reproduced in the second assay using the more sensitive preincubation assay or in the first assay at any other concentration.

Positive controls showed distinct increase of induced revertant colonies


Conclusions:
Tets substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay
Executive summary:

Method

The potential of test substance to induce gene mutations was investigated in 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 the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate.

In experiment II, the colony counts of the negative and solvent controls of strain TA 1535 were far beyond the historical range in the presence and absence of metabolic activation. Therefore, the results were dismissed and an additional experiment with strain TA 1535 was performed. The results of this additional experiment are included in experiment II.

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 metabolic activation in both independent experiments.

Appropriate reference mutagens were used as positive controls.

Results

No toxic effects, evident as a reduction in the number of reventants, occurred in the test groups with and without metabolic activation. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with test substance 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. Positive controls showed a distinct increase of induced revertant colonies.

Overall, test substance was considered as non mutagenic in the AMES assay.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), a mutation means a permanent change in the amount or structure of the genetic material in a cell. The term ‘mutation’ applies both to heritable genetic changes that may be manifested at the phenotypic level and to the underlying DNA modifications when known.

The more general terms ‘genotoxic’ and ‘genotoxicity’ apply to agents or processes which alter the structure, information content, or segregation of DNA, including those which cause DNA damage by interfering with normal replication processes, or which in a non- physiological manner (temporarily) alter its replication.

For the purpose of classification for germ cell mutagenicity, substances are allocated to one of two categories:

 

Category 1: substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans.

Category 1A: based on positive evidence from human epidemiological studies.

Category 1B: based on:

- positive result(s) from in vivo heritable germ cell mutagenicity tests in mammals; or

- positive result(s) from in vivo somatic cell mutagenicity tests in mammals, in combination with some evidence that the substance has potential to cause mutations to germ cells.

- positive results from tests showing mutagenic effects in the germ cells of humans, without demonstration of transmission to progeny; for example, an increase in the frequency of aneuploidy in sperm cells of exposed people.

 

Category 2: substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans, based on positive evidence obtained from experiments in mammals and/or in some cases from in vitro experiments, obtained from:

- somatic cell mutagenicity tests in vivo, in mammals; or

- other in vivo somatic cell genotoxicity tests which are supported by positive results from in vitro mutagenicity assays

 

Based on negative result in the available study, relying on a read across approach, a mutagenic potential to bacteria was excluded for OB 199.

Accordingly, the substance is not classified under the CLP Regulation (EC 1272/2008).