Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Not mutagenic

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From September 26 to November 2, 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine locus
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:
liver homogenate fraction (S9)
Test concentrations with justification for top dose:
Test Group - plate incorporation test:
a: without metabolic activation:
50, 160, 500, 1600 and 5000 µg /plate
b: with metabolic activation:
50, 160, 500, 1600 and 5000 µg /plate

Test Group - preincubation test (1):
a: without metabolic activation:
50, 160, 500, 1600 and 5000 µg /plate
b: with metabolic activation:
50, 160, 500, 1600 and 5000 µg /plate

Test Group - preincubation test (2):
a: without metabolic activation:
500, 1600, 2000, 3000, 4000 (with all the strains) and 5000 µg/plate (only with the strain TA 102)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: deionized water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
With metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
Remarks:
Without metabolic activation (S9-mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Plate incorporation test: in agar;
Preincubation test period only for the second test, if done.

DURATION
- Preincubation period (only for the second test, if done): 20 - 30 minutes at 30 °C

NUMBER OF REPLICATIONS:3

OTHER EXAMINATIONS:
- Sterility check: yes
- Solubility: yes
- Toxicity: Toxicity was assessed after microscopic thinning of the bacterial lawn and at least halving of the number of spontaneously occurring mutants compared to the corresponding solvent control value.
Evaluation criteria:
Criteria for a valid assay
The assay is considered valid if the following criteria are met:
- the solvent control data are within the laboratory's normal control range for the
spontaneous mutant frequency
- the positive controls induce increases in the mutation frequency which are significant
and within the laboratory's normal range

Criteria for a positive response
A test compound is classified as mutagenic if it has either of the following effects:
a) it produces at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn
b) it induces a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test compound at complete bacterial background lawn.
If the test substance does not achieve either of the above criteria, it is considered to show no evidence of mutagenic activity in this system.
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:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: > 10 g/l
- Precipitation: Visible precipitation of the test compound on the plates was observed at 500 pg/plate and above in first preincubation test.

OTHER:
- In the preincubation test toxicity was only observed with the tester strain TA 1537 without metabolic activation at a concentration of 5000 pg/plate.
- Only in the preincubation test a slight increased number of revertants was obtained in the tester strain TA 102 in the absence of S9-mix. But in a following preincubation test this result was not confirmed.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Not mutagenic
Executive summary:

The substance to be registered has been tested according to OECD 471, EU Method B.13/14, EPA OPPTS 870.5100, and in compliance with the Good Laboratory Practice regulation. The strains used during the test were TA100, TA1535, TA 1537, TA98 and TA102. Two independent studies were conducted (one plate incorporation test and one preincubation test), each in absence and in presence of metabolic activation (S9 -mix). In the plate incorporation test the compound proved to be not toxic to the bacterial strains. In the preincubation test toxicity was only observed with the tester strain T1537 without metabolic activation at the concentration plate of 5000 µg/plate.

In the absence and in presence of the metabolic activation system the substance did not result in relevant increases in the number of revertants in any of the bacterial strain.

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

Additional information

The assessment for genetic toxicity was conducted with two In Vitro studies (Ames test) according to internationally accepted guidelines and in compliance with GLP Regulation, the key study was conducted with all the strains required by the last version of the OECD Guideline n. 471. All the available studies have negative results for all the tested strains. The information obtained from the studies are conclusive but not sufficient for the classification.

Justification for classification or non-classification

According to the CLP Regulation amutation 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. Substances known to 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

All the available test show negative results therefore the information obtained from the studies are conclusive but not sufficient for the classification.

Categories Display