Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to GLP and valid testing guidelines, therefore it is considered relevant, adequate and reliable for classification.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

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 (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Butanedioic acid, sulfo-, 1-C12-18-alkyl esters, disodium salts; Disodium Lauryl Sulfosuccinate; Plantapon®SUS
- Physical state: White powder
- Analytical purity: > 95% (correction factor: 1.05)
- Impurities (identity and concentrations): See confidential details
- Composition of test material, percentage of components: See confidential details
- Purity test date: CoA 2012-07-03
- Lot/batch No.: 0008249628
- Expiration date of the lot/batch: March 20, 2014
- Stability under test conditions: stable
- Storage condition of test material: At+10°to+25°C
- Other: manufacturer/supplier: BASF Personal Care and Nutrition GmbH

Method

Target gene:
histidine
Species / strain
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
cytotoxicity test: 0.316, 1.0, 3.16, 10.0, 31.6, 100, 316, 1000, 3160 and 5000 µg test item/plate
mutagenicity test: 1.0, 3.16, 10.0, 31.6, 100 and 316 µg test item/plate (plate incorporation and preincubation)
Vehicle:
- Vehicle(s)/solvent(s) used: aqua ad iniectabilia
Controlsopen allclose all
Negative controls:
yes
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
other: Sodium azide in aqua ad iniectabilia
Remarks:
(10 µg/plate):TA 1535, TA 100, without S9-mix
Positive controls:
yes
Positive control substance:
other: 2-Nitro-fluorene in DMSO
Remarks:
(10 µg/plate): TA 98, without S9-mix
Positive controls:
yes
Positive control substance:
other: 9-Amino-acridine in ethanol, abs.
Remarks:
(100 µg/plate): TA 1537, without S9-mix
Positive controls:
yes
Positive control substance:
other: Mitomycin C in DMSO
Remarks:
( 10 µg/plate): TA 102, without S9-mix
Positive controls:
yes
Positive control substance:
other: Benzo(a)pyrene in DMSO
Remarks:
( 10 µg/plate): TA 98, TA 102, TA 1537, with S9-mix
Positive controls:
yes
Positive control substance:
other: 2-amino-anthracene in DMSO
Remarks:
(2-4 µg/plate): TA 100, TA 1535, with S9-mix
Details on test system and conditions:
METHOD OF APPLICATION:
1st independent experiment : in agar (plate incorporation);
2nd independent experiment : preincubation.

DURATION
1st independent experiment :
- Plate incorporation
- Exposure duration: 48 h to 72 h
2nd independent experiment :
- Preincubation period: 20 min
- Exposure duration: 48 h to 72 h

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS: Triplicate

DETERMINATION OF CYTOTOXICITY
- Method: other: Cytotoxicity is evidenced by a reduction in the number of revertant colonies, a clearing or diminution of the background lawn, or by the degree of survival of treatment cultures.
Cytotoxicity is defined as reduction in the number of colonies by more than 50% compared to the solvent control and/or a sparse background lawn.
Evaluation criteria:
The statistical evaluation of the results of the AMES test is still under discussion. In our laboratory, a test item is considered to show a positive response if
- the number of revertants is significantly increased (p ≤ 0.05, U-test according to MANN and WHITNEY) compared with the vehicle control to at least 2-fold of the vehicle control for TA 98, TA 100 and TA 102 and 3-fold of the vehicle control for TA 1535 and TA 1537 in both independent experiments;
Or
- a concentration-related increase of the revertants is observed (Spearman’s rank correlation coefficient).
Positive results have to be reproducible and the histidine independence of the revertants has to be confirmed by streaking random samples on histidine-free agar plates.
A test item for which the results do not meet the above mentioned criteria is considerd as non-mutagenic in the AMES test.
Statistics:
number of revertants compared with the vehicle control (p ≤ 0.05, U-test according to MANN and WHITNEY)
concentration-related increase of the revertants (Spearman’s rank correlation coefficient)

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
at concentrations of 316 µg/plate and higher
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other:
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Negative with metabolic activation
Negative without metabolic activation

Under the present test conditions the test item tested up to a cytotoxic concentration of 316 µg/plate, caused no mutagenic effect in the Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.

Executive summary:

The test item was examined in the 5 Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 in two independent experiments, each carried out without and with metabolic activation (a microsomal preparation derived from Aroclor 1254-induced rat liver). The first experiment was carried out as a plate incorporation test and the second as a preincubation test.

The test item was completely dissolved in aqua ad iniectabilia. A correction factor of 1.05 was used to correct for the purity of the test item. Aqua ad iniectabilia was used as vehicle control.

The test item was examined in a preliminary cytotoxicity test without metabolic activation in test strain TA 100 employing a plate incorporation test. Ten concentrations of 0.316, 1.0, 3.16, 10.0, 31.6, 100, 316, 1000, 3160 and 5000 µg test item/plate were tested. Cytotoxicity (scarce background lawn and reduction of the number of revertants)was noted at concentrations of 316 µg/plate and higher. Hence, 316 µg test item/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.

Six concentrations of 1.0, 3.16, 10.0, 31.6, 100 and 316 µg test item/plate were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation.
In the plate incorporation test and in the preincubation test, each carried out without and with metabolic activationcytotoxicity (scarce background lawn and reduction of the number of revertants) was noted at the top concentration of 316 µgtest item/plate in all test strains.
No increase in revertant colony numbers as compared with control counts was observed for test item, tested up to a cytotoxic concentration of 316 µg/plate, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation and preincubation test).
The results for the vehicle controls were within the range of historical control data of the laboratory. The positive control items showed a significant increase in the number of revertant colonies compared to the vehicle controls of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system.
In conclusion, under the present test conditions the test item tested up to a cytotoxic concentration of 316 µg/plate, caused no mutagenic effect in the Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.