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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:
11 May 2016 - 01 June 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
This information is used for Geranyl Isobutyrate MCS.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
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:
Phenobarbital/β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Experiment 1, all 5 strains:
With and without S9-mix: 3, 10, 33, 100, 333, 1000, 2500, and 5000 μg/plate
Experiment 2,
TA100 with and without S9-mix: 3, 10, 33, 100, 333, 1000, 2500, and 5000 μg/plate
TA98, TA1535, TA1537 and WP2uvrA with and without S9-mix: 33, 100, 333, 1000, 2500, and 5000 μg/plate
Vehicle / solvent:
- Vehicle used: DMSO
- Justification for choice of vehicle: The test substance was dissolved in DMSO (purity > 99 %). The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine in DMSO for TA1537 and TA98
Remarks:
Without S9-mix
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene in DMSO, 2.5 μg/plate for TA1535, TA1537, TA98 and TA100, and 10.0 μg/plate for WP2uvrA
Remarks:
With S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3 replicates in both experiments.

NUMBER OF CELLS EVALUATED: 10^8 cells

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity of the test item can be evident as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.
Evaluation criteria:
Evaluation of Results:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Acceptability of the Assay:
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data
- the positive control substances should produce an increase above the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control
- a minimum of five analysable dose levels should be present with at least three dose levels showing no signs of toxic effects, evident as a reduction in the number of revertants below the indication factor of 0.5.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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
Remarks:
at 2500, and 5000 μg/plate with S9-mix only
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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
Remarks:
at 5000 μg/plate in experiment 2 with S9-mix only.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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
Remarks:
at 333, 1000, 2500, and 5000 μg/plate with S9-mix only
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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test item precipitated in the overlay agar in the test tubes from 2500 to 5000 μg/plate in the first experiment and at 5000 μg/plate in the second experiment. No precipitation of the test item in the overlay agar on the incubated agar plates was observed.

HISTORICAL CONTROL DATA:
These data represent the laboratory´s historical control data from January 2015 until December 2015 representing approx. 450 experiments (WP2 uvrA the historical data are based on approx. 200 experiments).

Strain
without S9 mix with S9 mix
Mean SD Min Max Mean SD Min Max
Solvent control 11 2.15 7 23 12 2.14 7 21
TA 1535
Untreated control 12 2.97 6 24 12 2.71 7 26
Positive control 1090 123.80 334 1372 392 62.85 176 549

Solvent control 10 1.83 6 18 13 3.27 7 27
TA1537
Untreated control 10 2.29 6 20 14 3.72 7 25
Positive control 83 12.28 55 131 175 44.44 82 327

Solvent control 24 3.75 16 36 33 5.55 18 51
TA 98
Untreated control 26 4.72 15 43 36 5.83 17 56
Positive control 344 51.13 211 599 3822 857.83 319 5048

Solvent control 155 24.19 84 194 145 31.81 81 204
TA 100
Untreated control 174 21.92 90 206 170 23.62 93 212
Positive control 1956 279.93 658 2528 3606 676.07 722 4940

Solvent control 41 5.72 27 63 51 6.91 37 72
WP2uvrA
Untreated control 42 6.01 31 63 53 7.05 38 88
Positive control 732 161.66 322 1066 362 72.26 212 858
Mean = mean value of revertants/plate SD = standard deviation Min = minimal value/Max = maximal value

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Minor toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in strains TA 1537, TA 98 and, TA 100.

Applicant's summary and conclusion

Conclusions:
The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and Escherichia coli reverse mutation assay performed according to OECD 471 guideline and GLP principles.
Executive summary:

The mutagenic activity of the substance was evaluated in accordance with OECD 471 guideline and according to GLP principles. The test was performed in two independent experiments, experiment 1 a plate incorporation test and experiment 2 a pre-incubation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. Dose levels up to and including 5000 μg/plate were tested in the absence and presence of S9-mix. Adequate positive controls and solvent controls were included.

No precipitation of the test item in the overlay agar on the incubated agar plates was observed. The plates incubated with the test item showed reduced background growth in the strains TA 1537 and TA 100 in the presence of S9 mix in both experiments. Minor toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in strains TA 1537, TA 98 and, TA 100.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). Based on the results, the substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.