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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item did not induce genetic toxicity in vitro, proved by the results of an Ames test (RL1, GLP).

Link to relevant study records
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30.11.2009 - 09.04.2010
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Adopted: July 21, 1997
according to guideline
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
Adopted: May 31, 2008
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium, other: TA 97a, TA 98, TA 100, TA 102 and TA 1535
Details on mammalian cell type (if applicable):
Species Salmonella typhimurium LT2
Strains TA 1535, TA 97a, TA 98, TA 100 and TA 102
TA97a: hisD6610, uvrB, pKM 101, rfa
TA 98: hisD3052, uvrB, pKM 101, rfa
TA 100 : hisG46, uvrB, pKM 101, rfa
TA102: hisG428, pKM 101, rfa
TA1535 : hisG46, uvrB, rfa.
Metabolic activation:
with and without
Metabolic activation system:
microsomal fraction (S9) produced from the livers of male Sprague-Dawley rats treated i.p. with 500 mg Aroclor 1254/kg body weight
Test concentrations with justification for top dose:
1st experiment: 5004, 1501, 500, 150, and 50 µg/plate (plate incorporation)
2nd experiment: 15, 5, 1.5, 0.5, and 0.15 µg/plate (pre-incubation method)
3rd experiment: 14.8, 7.4, 3.7, 1.85, 0.93, 0.47 and 0.24 µg/plate (pre-incubation method)
Vehicle / solvent:
- Solvent: Ethanol
- Justification for choice of solvent/vehicle: high tolerance for the tester strains, complete dissolution of the test substance
Untreated negative controls:
with water
Negative solvent / vehicle controls:
with ethanol and DMSO as comparison
True negative controls:
not specified
Positive controls:
Positive control substance:
other: see below "Details on test system"
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation: 1st experiment / preincubation: 2nd and 3rd experiment

- Preincubation period: 20 min, 37 °C
- Exposure duration: 48 h, 37 °C


- Method: relative total growth ==> inhibition of growth of the background lawn

sodium azide (1 µg/plate; TA 100 and TA 1535 without metabolic activation)
4-nitro-o-phenylenediamine (20 µg per plate; TA 97a, TA 98 and TA 102 without metabolic activation)
2-aminoanthracene (1 µg/plate; TA 97a, TA 100, TA 102 and TA 1535 with metabolic activation)
benzo(a)pyrene (20 µg/plate; TA 98 with metabolic activation)

Evaluation criteria:
A test item is considered to have mutagenic potential, if a significant, reproducible increase of in the number of revertant colonies per plate (increase factor >= 2) in at least one strain can be observed. A concentration-related increase can also be taken as a sign of mutagenic activity.
calculation of mean values with standard deviations
Key result
Species / strain:
S. typhimurium, other: TA 97a, TA 98, TA 100, TA 102 and TA 1535
Metabolic activation:
with and without
reproducible in two experiments
Cytotoxicity / choice of top concentrations:
>= 15 µg/plate (in 3rd experiment)
Vehicle controls validity:
Untreated negative controls validity:
Positive controls validity:
Additional information on results:

In general, the determined values for the spontaneous reversion rates as well as the positive controls were within the normal range of the laboratory.
In isolated cases of outliers, the differences to the respective maximum or minimum of the history were marginal only (see Report Annex 6)

In the cytotoxicity pre-tests using all tester strains, no inhibition of growth was found for unexplained reasons, contrary to findings in the main tests.Therefore, a third experimental series at lower concentration had to be repeated.
Remarks on result:
other: all strains/cell types tested
Migrated from field 'Test system'.
The test item did not show mutagenic effects in both evaluable experiments. The number of revertant colonies was not increased in comparison with the spontaneous revertants (solvent only).
Therefore it can be stated, that under the test conditions, the test item C13/C15 Amin is not mutagenic in the Bacterial Reverse Mutation Test using Salmonella typhimurium, strains TA 97a, TA 98, TA 100, TA 102 and TA 1535.
Executive summary:

In an in vitro gene mutation test according to OECD 471 and GLP C13/C15 amine was tested for mutagenicity with the strains TA97a, TA98, TA100, TA102, and TA1535 of Salmonella typhimurium.

In the first toxicity control (performed before finalisation of the study plan) the test item did not show any cytotoxicity towards the bacteria. Therefore, 5000 ug/plate was chosen as highest concentration in the first experiment. But the test item showed cytotoxicity towards the bacteria at all five concentrations in the first experiment. Therefore, it must be presumed that the test item shows cytotoxicity down to a concentration of 50 ug/plate. A second toxicity control was performed with the following concentrations: 15 ug/plate, 5 ug/plate, 1.5 ug/plate and 0.5 ug/plate. In none of the treatments, toxicity was observed. Taking into account the results of the second toxicity control, a second mutagenicity experiment using the plate-incorporation method was performed with lower concentrations (ranging from 15 - 0.15 ug/plate). In this experiment, no toxicity was detected. A third experiment was performed with seven concentrations (14.8 - 0.232 ug/plate) using the preincubation-method. In both experiments, no signs of mutagenicity were observed.

The confirmation tests of the genotype did not show any irregularities. The control of the titre was above the demanded value. The number of revertant colonies of the positive controls was in the range of the historical data of the laboratory and were definitely increased in comparison with the negative controls, as well as showing mutagenic potential of the diagnostic mutagens. Spontaneous revertants were within the normal range in comparison with the historical data of the laboratory.

For these reasons, the result of the test is considered valid and the test item is considered not mutagenic.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

In an in vitro gene mutation test according to OECD 471 and GLP C13/C15 amine was tested for mutagenicity with the strains TA97a, TA98, TA100, TA102, and TA1535 of Salmonella typhimurium.

The mutagenicity studies were conducted in the absence and in the presence of a metabolizing system derived from rat liver homogenate. In two experiments (plate-incorporation method and pre-incubation method with five concentrations from 0.15 - 15 µg/plate in the first experiment and seven concentrations from 0.232 - 14.8 µg/plate in the second one) no signs of mutagenicity were observed.

Justification for classification or non-classification

Based on the available in vitro study (Ames test, RL1, GLP), the test substance has not to be classified according to Regulation (EC) No. 1272/2008.