Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance is not mutagenic in bacterias as determined in two bacterial reverse mutation assays, with the latest from 2016, both according to OECD 471.

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:
12 Jul 2016 to 21 Jul 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
30 May 2008
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
August 1998
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 001-152602
- Expiration date of the lot/batch: 01 Jul 2025
- Purity test date: 01 Jul 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: stable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The homogeneity of the test substance was ensured by mixing before preparation of the test substance solutions.
- Final preparation of a solid: The test substance was weighed and topped up with the chosen vehicle to achieve the required concentration of the stock solution. The test substance was dissolved in dimethyl sulfoxide (DMSO).
Target gene:
S. typhimurium: his
E. coli: trp
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-naphthoflavone induced rat liver S9 fraction or Uninduced hamster liver S9 fraction
Test concentrations with justification for top dose:
0; 33; 100; 333; 1000; 2500 and 5000 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used:DMSO;
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.
Untreated negative controls:
yes
Remarks:
Sterility control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
congo red
other: 2-aminoanthracene (2AA) / N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) / 4-nitro-o-phenylenediamine (NOPD) see section "Any other information on materials and methods incl. tables"
Remarks:
See 'Any other information on materials and methods incl. tables' for details on positive controls
Details on test system and experimental conditions:
EXPERIMENT 1, 2:
METHOD OF APPLICATION: in medium; in agar (plate incorporation)

DURATION
- Exposure duration: 48 - 72 hours at 37°C

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
Toxicity detected by a
• decrease in the number of revertants (factor ≤ 0.6)
• clearing or diminution of the background lawn (= reduced his- or trp- background growth) was recorded for all test groups both with and without S9 mix in all experiments and indicated in the tables. Single values with a factor ≤ 0.6 were not detected as toxicity in low dose groups.
Evaluation criteria:
ACCEPTANCE CRITERIA
Generally, the experiment was considered valid if the following criteria were met:
• The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
• The sterility controls revealed no indication of bacterial contamination.
• The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
• Fresh bacterial culture containing approximately 10E+9 cells per mL were used.

ASSESSMENT CRITERIA
The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.
Key result
Species / strain:
S. typhimurium, other: TA 98, TA 100, TA1535, TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
See any other information on results
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
See any other information on results
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Solubility - Precipitation: Precipitation of the test substance was found at 5000 μg/plate with and without S9 mix.

BACTERIOTOXIC EFFECT

Decreased revertant numbers were observed at following concentrations (μg/plate):

Experiment

S9

TA 1535

TA 100

TA 1537

TA 98

E. coli

1st-SPT

Without

-

1000 - 5000

2500 - 5000

-

5000

With

5000

-

-

5000

-

2nd-Prival

Without

5000

5000

5000

5000

5000

With

5000

-

5000

5000

5000

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In the latest reverse mutation assay (BASF, 2016), strains of Salmonella typhimurium and Escherichia coli were exposed to five concentrations of the test item in a Standard plate test (SPT) as well as Prival preincubation test (Prival) both with and without metabolic activation. The study is according to OECD 471 and US EPA OPPTS 870.5100, Aug 1998, performed in compliance with GLP criteria.

All strains were exposed to the concentrations of 0, 33, 100, 333, 1000, 2500 and 5000 µg test item per plate. Each experiment included negative controls in order to check for possible contaminants (sterility control) and to determine the spontaneous mutation rate (vehicle control). Additionally, positive controls were used to check the mutability of the bacteria and the activity of the S9 mix. Precipitation of the test substance was found at 5000 μg/plate with and without S9 mix. A bacteriotoxic effec (decrease in the number of his+ or trp+ revertants) was observed in the standard plate test depending on the strain and test conditions from about 1000 μg/plate onward. In the prival preincubation assay bacteriotoxicity (decrease in the number of his+ or trp+ revertants) was observed depending on the strain and test conditions at 5000 μg/plat.

A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the prival preincubation test either without S9 mix or after the addition of a metabolizing system. Based on the results of this study it is concluded that the test substance is not mutagenic in the S. typhimurium/E. coli reverse mutation assay in the absense and presence of metabolic activation.

In the other Ames study (Batelle, 1978), also performed according to OECD 471, the test substance was tested with the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 at concentrations from 0.2 to 2000 µg (or nL) per Petri dish both in the presence and absence of metabolic activation by microsomal enzymes from rat liver. The compounds to be tested were prepared fresh daily in DMSO or in sterile water. The appropriate dilutions were made from a 20 mg (or nL)/mL stock. The S-9 mix was prepared fresh daily from sterile stocks. The test substance was tested at five dose levels: 0.2, 2, 20, 200 and 2000 µg (or nL) per Petri dish. The test substance was dissolved in DMSO and every concentration was tested in triplicate. The criteria of mutagenicity used in this test are a doubling of the spontaneous reversion rate and a dose-effect relationship.

Under the experimental conditions defined in the protocol, and employing a doubling of the spontaneous reversion rate and dose-effect relationship as criteria of mutagenicity, the test substance was not mutagenic for S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100. The lack of evidence for mutagenicity existed both in the absence and presence of a liver microsomal enzyme preparation (S9-mix) from male rats pretreated with Aroclor 1254 and over a concentration range of 0.2 to 2000 µg (or nL) of product per Petri dish.

Justification for classification or non-classification

Based on the available information no classification is warranted according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.