Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From July 30, 2015 to August 17, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
semi-solid (amorphous): gel
Remarks:
Paste
Details on test material:
- Name of test material (as cited in study report): Vegetable or animal oil, hydrogenated, reaction products with diethanolamine, maleic anhydride and sodium sulphite
- Lot No.: CH 192435/01
- Purity: Not applicable (UVCB)
- Organic Carbon: 30.2%
- Hydrotox No.: 15/5241
- Origin: DACC
- Stability: Stable within the durability and with appropriate storage conditions
- Solubility in water: ~1 g/L
- Appearance: Light beige liquid/paste
- Storage conditions: Ambient temperature, dark, dry
- Expiration date: July 15, 2015
- Safety directions: H315, H319. Use protective clothing: gloves and protection goggles.

Method

Target gene:
TA1537 hisC3076
TA98 hisD3052/R-factor*
TA1535 hisG46 Base-pair
TA100 hisG46/R-factor*
Escherichia coli: tryptophan (Trp+)
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9-mix, rat liver S9-mix induced by Aroclor 1254
Test concentrations with justification for top dose:
Dose range finding study: 1.7, 5.4, 17, 52, 164, 512, 1,600 and 5,000 µg/plate in triplicates
First experiment (direct plate assay) : 52, 164, 512, 1,600 and 5,000 μg/plate
Second experiment (plate incorporation assay): 17, 52, 164, 512, 1,600 and 5,000 μg/plate

Vehicle / solvent:
Water, saline and DMSO
Controls
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191 & 2-aminoanthracene
Remarks:
Without metabolic activation: TA1535: sodium azide (SA), TA1537: ICR-191, TA1537: 2-nitrofluorene (NF), TA98: 2-nitrofluorene (NF), TA100: methylmethanesulfonate (MMS), WP2uvrA: 4-nitroquinoline N-oxide, With metabolic activation: 2-aminoanthracene (2AA)
Details on test system and experimental conditions:
Salmonella typhimurium (all strains used) were obtained from Trinova Biochem GmbH, Germany [Master culture from Dr. Bruce N. Ames (TA1535: 2006, TA1537: 2009, TA98: 2006, TA100: 2006; and Master culture from The National Collections of Industrial and Marine Bacteria, Aberdeen, UK (WP2uvrA: 2008)]. Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid LTD, Hampshire, England) and incubated in a shaking incubator (37±1°C, 150 rpm), until the cultures reached an optical density of 1.0±0.1 at 700 nm (109 cells/mL). Freshly grown cultures of each strain were used for a test. All incubations were carried out in a controlled environment at a temperature of 37.0±1.0°C (actual range 35.8–38.5°C).
Evaluation criteria:
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.

A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

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:
cytotoxicity
Remarks:
In second mutation experiment, with and without S9 mix
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:
in both mutation experiments, with and without S9 mix
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:
no cytotoxicity
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:
in second mutation experiment, with and without S9 mix
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:
in second mutation experiment without S9 mix
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
A range finding study was conducted with the strains TA100 and the WP2uvrA in absence and presence of S9-mix. Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1,600 and 5,000 μg/plate were tested in triplicate.

COMPARISON WITH HISTORICAL CONTROL DATA: The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

First experiment: Direct plate assay
Based on the results of the dose range finding test, the following dose range was selected for the mutation assay with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 52, 164, 512, 1,600 and 5,000 μg/plate.
Precipitate: Precipitation of the test substance on the plates was not observed at the start or at the end of the incubation period. Toxicity: No reduction of the bacterial background lawn was observed.
Mutagenicity: No increase in the number of revertants was observed upon treatment with the test substance under all conditions tested.
Second experiment: Pre-incubation assay
A pre-incubation experiment was performed in the absence and presence of S9-mix. Based on the results of the first mutation assay, the test substance was tested up to the dose level of 5,000 μg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA.
Precipitate: Precipitation of test substance on the plates was not observed at the start or at the end of the incubation period.
Toxicity: In tester strain WP2uvrA, a reduction of the bacterial background lawn was only observed at the highest tested concentration in the absence of S9-mix, also the bacterial background lawn was absent. In the Salmonella typhimurium strains no reduction of the bacterial background lawn was observed.
Mutagenicity: In the pre-incubation test, no increase in the number of revertants was observed upon treatment with the test item under all conditions tested.

Applicant's summary and conclusion

Conclusions:
No mutagenic effect of test substance was observed either in the presence or absence of metabolic activation system under the conditions of this bacterial reverse mutation assay.No mutagenic effect of test substance was observed either in the presence or absence of metabolic activation system under the conditions of this bacterial reverse mutation assay.
Executive summary:

An in vitro bacterial reverse mutation assay was performed to evaluate the potential of test substance to cause gene mutation according to OECD Guideline 471 and EU Method B.13/14, in compliance with GLP. A total of two mutation experiments were conducted. Based on the results of the dose range finding test, a first mutation assay, a direct plate assay, was conducted with and without S9-mix in Salmonella typhimurium strains TA1535, TA1537 and TA98 at 52, 164, 512, 1,600 and 5,000 μg/plate. In the second mutation experiment, the substance was tested up to concentrations of 5,000 μg/plate in the S. typhimurium testerstrains TA1535, TA1537, TA98, TA100 and in Escherichia coli strain WP2uvrA in a pre-incubation assay. Toxicity was observed in WP2uvrA (absence of S9-mix only) and in TA1535, TA1537 and TA100 in the absence and presence of S9-mix. The negative and strain-specific positive control values were within the laboratory historical control data ranges, indicating that the test conditions were adequate and that the metabolic activation system functioned properly. The test substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four S. typhimurium strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. No mutagenic effect of the test substance was observed either in the presence or absence of metabolic activation system under the conditions of this bacterial reverse mutation assay (Verspeek-Rip CM, 2015a).