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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In five different in vitro Ames tests, with the latest from 2017, the test substance showed no mutagenic effects with and without microsomal activation

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:
05 Oct 2016 to 21 Oct 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
- Name of test substance as used in the report: Orasol Red 363
- Source and lot/batch No.of test material: 001-151902
- Expiration date of the lot/batch: 20 Aug 2020
- Purity test date: 20 Aug 2015

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

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:
Salmonella strains: his-
E. coli strain: 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 details on positive controls under 'Any other information on materials and methods incl. tables'
Details on test system and experimental conditions:
EXPERIMENT 1:
METHOD OF APPLICATION: 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. Single values with a factor ≤ 0.6 were not detected as toxicity in low dose groups.

EXPERIMENT 2:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 30 min at 30°C
- 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. 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
- Precipitation: Test substance precipitation was found from about 333 μg/plate onward 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

-

5000

2500 - 5000

1000 - 5000

5000

With

5000

2500 - 5000

5000

5000

2500 - 5000

2nd-Prival

Without

5000

-

2500 - 5000

-

2500 - 5000

With

5000

5000

2500 - 5000

-

1000 - 5000

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In vitro assays with bacteria

In the latest reverse mutation assay (BASF, 2017), 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. 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 from about 333 μg/plate onward with and without S9 mix. A bacteriotoxic effect was observed depending on the strain and test conditions from about 1000 μg/plate onward. 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 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 / Escherichia reverse mutation assay.

In three older different reverse mutation assays, strains of Salmonella typhimurium were exposed to five concentrations of the test item in the presence and absence of mammalian metabolic activation (BASF, 1981, 801672, 810097 and 810098). The conducted studies satisfy the OECD Guideline 471 (Bacterial Reverse Mutation Assay) with one deviation: 4 strains had been tested instead of 5 (TA 98, TA 100, TA 1535, TA 1537). The same vehicle and the same positive controls were used for each strain in all studies. All tests were performed with the following concentrations of the trial substance with and without microsomal activation: 25, 75, 225, 675 and 2025 µg/0.1 mL. Acetone was used for the negative controls and a positive control was tested for each strain. In all three studies the test substance did not induce a dose-related, two-fold increase in the number of revertant (His+) colonies in each of the four tested strains (TA 98, TA 100, TA 1535, TA 1537) both in the absence and presence of S9 -metabolic activation. The spontaneous reversion in the control was within the historical control range for each strain and the positive controls induce the appropriate responses in the corresponding strains.Therefore, the test results are considered valid. Based on the results of these studies it is concluded that the test substance is not mutagenic in the S. typhimurium reverse mutation assay.


In a reverse mutation assay in bacteria (Batelle, 1979), strains of S. typhimurium were exposed to different concentrations of the test item in the presence and absence of mammalian metabolic activation. The conducted study satisfies the OECD Guideline 471 (Bacterial Reverse Mutation Assay) with one deviation: 4 strains had been tested instead of 5 (TA 98, TA100, TA 1535, TA 1537). All four strains were exposed to concentrations of 0.2, 2, 20, 200 and 2000 µg per petri dish of the test item in the absence and presence of S9-mix. Strain TA 98 was additionally exposed to the concentrations of 200, 500, 1000, 2000 and 4000 µg test item per petri dish. DMSO was used for the negative controls and a positive control was tested for each strain. In the study report a questionable mutagenic effect with strain TA 98 is described after exposure to a concentration range of 0.2 to 2000 µg/0.1 mL. Although an increase of the spontaneous reversion with increasing concentration occurred, no two fold increase in revertants compared to the control was observed. Exposure of strain TA 98 to a concentration range of 200 to 4000 µg/0.1 mL also showed no two fold increase in the number of revertant colonies both in the absence and presence of S9 -metabolic activation. Because the colony count in relation to the negative control is not double at any concentration, the test substance is considered to be nonmutagenic. The spontaneous reversion in the control was within the historical control range for each strain except for a very slight increase for TA 1537.

Justification for classification or non-classification

The Ames tests were negative and therefore no classification is warranted according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.