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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
Study period:
Jan - Feb 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Jul 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
Aug 1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: test substance was weighed and topped up with DMSO and shaken thoroughly
- Final dilution of a dissolved solid, stock liquid or gel: further concentrations were diluted from the stock solution according to the planned doses

FORM AS APPLIED IN THE TEST: solution

Method

Target gene:
his and trp
Species / strainopen allclose all
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:
Type and composition of metabolic activation system:
- source of S9 : rat liver
- method of preparation of S9 mix: At least 5 male Wistar rats [Crl:WI(Han)] received 80 mg/kg b.w. phenobarbital i.p. and β-naphthoflavone orally each on three consecutive days. Twenty-four hours after the last administration, rats were sacrificed, and livers were prepared
using sterile solvents and glassware at a temperature of +4°C. Livers were weighed and
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
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): yes
Test concentrations with justification for top dose:
- 1st and second experiment: 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 at least in the 1st Experiment. However, this maximum dose was tested even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate or > 5 μL/plate might also be tested in repeat experiments for further clarification/substantiation.
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
Controls
Untreated negative controls:
yes
Remarks:
omitting tester strains
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
other: • +S9: 2-aminoanthracene (2.5 µg/plate for S. typhimurium, 60 µg/plate for E. coli) • -S9: N-methyl-N'-nitro-N-nitrosoguanidine (5 µg/plate for TA 1535, TA 100) • -S9: 4-nitro-o-phenylenediamine (10 µg/plate for TA 98)
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : 2
- 1st experiment: Standard plate test with and without S9 mix
- 2nd experiment: Preincubation test with and without S9 mix

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): approximately 10^9 cells per mL
- Test method: preincubation and plate incorporation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20 minutes
- Exposure duration/duration of treatment: 48-72 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition; decrease in the number of revertants (factor ≤ 0.6)

METHODS FOR MEASUREMENTS OF GENOTOXICIY :
- Individual plate counts, the mean number of revertant colonies per plate and the standard deviations were given for all dose groups as well as for the positive and negative (vehicle) controls in all experiments.

OTHER:
- Precipitation
Evaluation criteria:
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 range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at and above 2500 µg/plate (slight decrease in the number of his+ revertants) at plate incorporation test only (1st experiment)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at and above 2500 µg/plate and at 5000 µg/plate with and without S9, respectively.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 1000 µg/plate and 5000 µg/plate without S9
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Solubility:
Test substance precipitation was observed at and above 2500 μg/plate with and without S9 mix.TEST-SPECIFIC CONFOUNDING FACTORS

Cytotoxicity:
A weak bacteriotoxic effect (slight decrease in the number of his+ revertants) was observed only using tester strain TA 1535 with and without S9 mix at and above 2500 μg/plate. In the preincubation assay bacteriotoxicity (slight decrease in the number of his+ revertants) was occasionally observed depending on the strain and test conditions at and above 1000 μg/plate.

Any other information on results incl. tables

Experimental Results:

Standard Plate Test:

Mean revertants per plate
Strain TA 1535 TA 100 TA 1537 TA 98 WP2uvrA
Dose (µg/plate) -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
DMSO 18.7 18.0 115.0 116.0 9.0 11.0 22.3 32.7 31.3 30.0
33 15.0 19.0 114.3 123.0 6.7 14.3 21.0 28.7 26.3 30.3
100 14.0 19.0 111.7 117.7 7.0 7.7 19.0 35.3 26.7 34.0
333 15.0 15.3 119.3 129.3 10.7 12.0 16.0 30.3 31.7 28.0
1000 21.7 12.3 125.7 120.3 6.7 12.7 19.0 33.7 24.3 30.3
2500 10.7 12.0 109.7 112.3 8.3 8.7 17.3 27.0 22.7 27.3
5000 11.7 9.7 101.7 112.3 8.0 7.7 16.7 23.3 22.3 24.0
positive control 5536.3 284.0 3517.7 2227.0 943.3 162.7 871.3 2520.3 1276.7 226.3

Preincubation test:

Mean revertants per plate
Strain TA 1535 TA 100 TA 1537 TA 98 WP2uvrA
Dose (µg/plate) -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
DMSO 13.7 12.3 114.7 115.0 9.3 8.3 27.7 28.3 30.0 29.7
33 14.0 9.3 111.7 104.0 6.3 6.7 23.0 22.7 25.7 26.3
100 15.7 8.7 109.3 109.7 11.0 8.0 22.3 29.0 29.0 25.0
333 12.7 9.3 108.3 108.3 9.7 9.7 22.7 32.0 32.0 26.0
1000 12.7 14.7 109.3 104.3 11.0 10.7 17.0 29.3 26.0 28.0
2600 15.7 10.3 102.7 104.3 5.7 6.0 18.3 26.7 26.0 24.3
5200 11.0 9.0 101.3 98.0 4.0 4.7 16.3 23.0 24.0 20.7
positive control 4773.0 126.7 3041.3 1307.7 756.0 105.0 876.0 1128.0 647.0 240.3

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.
Executive summary:

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay. Strains used were TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA. The dose range was 33 μg - 5000 μg/plate (SPT) 33 μg - 5000 μg/plate (PIT). Test conditions chosen were the standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats). Precipitation of the test substance was observed at and above 2500 μg/plate with and without S9 mix. A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions at and above 1000 μg/plate. 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 preincubation test without S9 mix or after the addition of a metabolizing system. Under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.