Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Not genotoxic

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
other: read across from supporting substance
Adequacy of study:
key study
Study period:
From January, 11 to February, 07 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
not specified
Qualifier:
according to
Guideline:
EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Range finding test: 20.58, 61.73, 185.19, 555.56, 1666.67, 5000.00 µg/plate mutagenicity tests: 61.7, 185.19, 555.56, 1666.67, 5000.0 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO, Bidistilled water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
other: 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium.
Inoculates from frozen master copies were set up monthly. They were grown in liquid nutrient broth medium (NB-medium) overnight and then plated on NB-agar. After incubation, single colonies were taken from the plates, grown overnight in liquid NB-medium and then used for the experiment.
Statistics:
A statistical analysis of the test data was not performed. The use of statistical methods concerning this particular test system is not generally recommended
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Range finding test

Six concentrations of test item ranging from 20.6 to 5000.0 µg/plate were tested with strain Salmonella typhimurium TA 100 to determine the highest concentration to be used in the mutagenicity assay. The experiments were performed with and

without metabolic activation. Normal background growth was observed. The numbers of revenant colonies were not reduced. From the results obtained, the highest concentration suitable for the mutagenicity test was selected to be 5000.0 µg/plate with and without metabolic activation.

Mutagenicity test,

In the mutagenicity tests normal background growth was observed with all strains at all concentrations. The numbers of revertant colonies were not reduced. The test substance exerted no toxic effect on the growth of the bacteria.

Conclusions:
The substance and its metabolites did not induce gene mutations in the strains of Salmonella typhimurium used.
Executive summary:

The substance has been tested for mutagenic effects in vitro in histidine-requiring strains of Salmonella typhimurium according to the OECD Guideline 471 and in compliance to the GLP Pinciples.

The following strains of Salmonella typhimurium were used: TA 98, TA 100, TA 102, TA 1535 and TA 1537. The test was performed with and without the addition of rat-liver post mitochondrial supernatant (S9 fraction) as an extrinsic metabolic activation system. The compound was tested as a suspension in DMSO at five concentrations in the range of 61.7 to 5000.0 µg/plate in the presence and absence of a metabolic activation system (rat-liver post mitochondrial supernatant: S9-fraction). In order to confirm the results, the experiments were repeated with and without metabolic activation at five concentrations in the same concentration range. Each strain was additionally tested in the presence and in the absence of a metabolic activation system with a suitable, known mutagen as positive control. In both experiments, performed with and without metabolic activation, none of the tested concentrations of test substance led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control, therefore the substance is considered as not mutagenic

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

Additional information

Test on genetic toxicity is available on similar substance 1 and similar substance 2.

The complete hypothesis and justification for the read across approach has been attached in section 13.

The assessment was based on two studies conducted on similar substance 1. The results obtained were not conclusive, therefore in order to understand the mutagenic potential, another test was considered on similar substance 2. The complete evaluation and justification for the Read Across is attached at section 13.

During the Key study (Huntsman, 1994), similar substance 1 was tested for mutagenic effects in vitro in histidine-requinng strains of Salmonella typhimurium according to the OECD Guideline 471 and in compliance to the GLP Principles. The following strains of Salmonella typhimurium were used: TA 98, TA 100, TA 102, TA 1535 and TA 1537. The test was performed with and without the addition of rat-liver post mitochondrial supernatant (S9 fraction) as an extrinsic metabolic activation system.

In both experiments, performed with and without metabolic activation, none of the tested concentrations led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control.

The other study conducted on similar substance 2 (Huntsman, 1995), the substance was tested for mutagenic effects in vitro in histidine-requinng strains of Salmonella typhimurium according to a method comparable to the OECD Guideline 471. The following strains of Salmonella typhimurium were used: TA 98, TA 100, TA 1535 and TA 1537. The test was performed with and without the addition of rat-liver post mitochondrial supernatant (S9 fraction) as an extrinsic metabolic activation system.

In both experiments without activation carried out on strain TA 98, marginally increased numbers of back-mutants were observed at the highest concentrations. No effects occurred on the other strains.

During the study conducted according to the OECD Guideline 471 on the Similar Substance 02 (Huntsman, 1982) the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 were tested at concentrations from 0.2 to 2000 µg per Petri dish both in the presence and absence of metabolic activation. No mutagenic effect was observed.

The Key study and the supporting study made in 1995 are comparable. Are performed according to similar methods. The main difference is that the strains tested are four but they should be five strains on Salmonella typhimurium (as suggested inside the OECD and the EC guidelines).

The supporting study made in 1995 shows a marginal methabolic activity without activation on strain TA 98 at highest concentration (5000 µg/plate). This data is not confirmed by the other supporting study made in 1982 with Similar Substance 02

Therefore our expert judgment is that the substance is not genotoxic with and without metabolic activation.

Justification for classification or non-classification

According to the CLP Regulation (EC n. 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 (including specific base pair changes and chromosomal translocations). The term ‘mutagenic’ and ‘mutagen’ will be used for agents giving rise to an increased occurrence of mutations in populations of cells and/or organisms.

The test substance is not capable to induce permanent mutation inside the genetic structure, therefore according to the ECHA guidance R.7a R.7.7 -1 Flow chart of the mutagenicity testing strategy, no further testing at this level are necessary and no classification is warranted according to the CLP Regulation (EC n. 1272/2008).