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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997-07-21
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008-05-30
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
1998-08
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch number of test material: V03 NEOVA 9-Dest. Fraktion 3-25
- Expiration date of the batch: Sept 2021

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

Method

Target gene:
his, trp
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Cytokinesis block (if used):
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:

- Source of S9: Phenobarbital- and ß-naphthoflavone-induced rat liver S9 mix

- Method of preparation of S9 mix: Male Wistar rats received 80 mg/kg b.w. phenobarbital i.p. and β-naphthoflavone orally each on three consecutive days. 24 hours after the last administration, the rats were sacrificed, and the livers were prepared washed in a weight-equivalent volume of a 150 mM KCl solution and homogenized in three volumes of KCl solution. After centrifugation of the homogenate at 9000 x g for 10 minutes at +4°C, 5 mL portions of the supernatant (S9 fraction) were stored at -70°C to -80°C.

- Concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL S9 mix (containing 50 µL S9) in a total volume of 2.7 mL

- Quality controls of S9
Sterility: Additional plates were treated with soft agar, S9 mix, buffer, vehicle and the test substance but without the addition of tester strains.
Efficacy: The S9 batch was characterized with benzo[a]pyrene.
Test concentrations with justification for top dose:
1st experiment (plate incorporation): 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate
2nd experiment (pre-incubation): 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate;
In agreement with the recommendations of current guidelines, 5 mg/plate or 5 μL/plate were generally selected as maximum test dose.
Vehicle / solvent:
- Vehicle/solvent used: DMSO (test item and all positive controls)

- 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.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
- S9 mix, tested with Escherichia coli WP2 uvrA (5 µg/plate)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
- S9 mix, tested with TA 1537 (100 µg/plate)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylenediamine
Remarks:
- S9 mix, tested with TA 98 (10 µg/plate)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-methyl-N'-nitro-N-nitrosoguanidine
Remarks:
- S9 mix, tested with TA 1535, TA 100 (5 µg/plate)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
+ S9 mix, tested with TA 1535, TA 100, TA 1537, TA 98 (2.5 µg/plate) and with Escherichia coli WP2 uvrA (60 µg/plate)
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicates (both in the 1st and 2nd experiment)

- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Plate incorporation (1st experiment), pre-incubation (2nd experiment)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 20 minutes
- Exposure duration/duration of treatment: 48 – 72 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Background growth inhibition and decrease in the number of revertants (factor ≤ 0.6)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The bacterial colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hindered the counting using the Image Analysis System.
Evaluation 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 range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other.
Statistics:
Not performed as not mandatory for this test system.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
Solubility: Test substance precipitation was observed at 5000 μg/plate with and without S9 mix.
Sterility: There was no bacterial growth in the sterility controls.

TOXICITY
A bacteriotoxic effect (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants) was observed in the standard plate test depending on the strain and test conditions at and above 1000 μg/plate.
In the preincubation assay bacteriotoxicity was observed depending on the strain and test conditions at and above 333 μg/plate.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: See section "Attached background material".

Ames test:
- Signs of toxicity: See section "Attached background material".
- Individual plate counts: See section "Attached background material".
- Mean number of revertant colonies per plate and standard deviation: See section "Attached background material".

Applicant's summary and conclusion

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