Amines, C16-18 and C18-unsatd. alkyl, ethoxylated

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

The experimental phases of the study were performed between 08 September 2009 and 26 October 2009.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of therelevant results.

Justification for type of information:

Data has been generated in a category approach. A justification for cross-reading and explanation of the category approach is available in Chapter 13.

Cross-reference

Reason / purpose for cross-reference:

read-across: supporting information

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-(octadec-9-enylimino)bisethanol

EC Number:

246-807-3

EC Name:

2,2'-(octadec-9-enylimino)bisethanol

Cas Number:

25307-17-9

Molecular formula:

C22H45NO2

IUPAC Name:

2,2'-[(9Z)-octadec-9-en-1-ylimino]diethanol

Constituent 2

Reference substance name:

Bis (2-hydroxyethyl) oleyl amine (CAS Number 25307-17-9)

IUPAC Name:

Bis (2-hydroxyethyl) oleyl amine (CAS Number 25307-17-9)

Details on test material:

Sponsor's identification: Bis (2-hydroxyethyl) oleyl amine (CAS Number 25307-17-9)
Description : Brown viscous liquid
Purity : 99.9%
Batch number : S-001018
Date received : 08 July 2009
Storage conditions: Approximately 4oC in the dark under nitrogen

Method

Target gene:

Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Type and identity of media:
RPMI 1640 (R0)

Properly maintained:
Yes

Periodically checked for Mycoplasma contamination:
Yes

Periodically checked for karyotype stability:
No

Periodically "cleansed" against high spontaneous background:
Yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and beta-naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

The maximum dose level used was limited by test material induced toxicity.

Vehicle and positive controls were used in parallel with the test material. Solvent (DMSO) treatment groups were used as the vehicle controls. Ethylmethanesulphonate (EMS) Sigma batch 1419706 15108051 at 400 µg/ml and 150 µg/ml for Experiment 1 and Experiment 2 respectively, was used as the positive control in the absence of metabolic activation. Cyclophosphamide (CP) Acros batch A0164185 at 2 µg/ml was used as the positive control in the presence of metabolic activation.

Vehicle / solvent:

Vehicle used:
Vehicle (DMSO) treatment groups were used as the vehicle controls.


Justification for choice of vehicle:
Formed a solution suitable for dosing at the required concentration.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Vehicle (DMSO) treatment groups were used as the vehicle controls.

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Vehicle (DMSO) treatment groups were used as the vehicle controls.

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation

Details on test system and experimental conditions:

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method used meets the requirements of the OECD (476), Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008.

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at eight dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test material at eight dose levels using a 4 hour exposure group in the presence of metabolic activation (1% S9) and a 24 hour exposure group in the absence of metabolic activation.

The dose range of test material was selected following the results of a preliminary toxicity test. The dose range for Experiment 1 was 0.31 to 10 µg/ml in the absence of metabolic activation and 1.25 to 20 µg/ml in the presence of metabolic activation. The dose range for Experiment 2 was 0.16 to 5 µg/ml in the absence of metabolic activation, and 1.25 to 20 µg/ml in the presence of metabolic activation.

The maximum dose level used was limited by test material induced toxicity. Precipitate of test material was not observed at any of the dose levels. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

Evaluation criteria:

Please see "Any other information on materials and methods incl. tables" section.

Statistics:

Please see "Any other information on materials and methods incl. tables" section.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

non-mutagenic

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Test

The dose range of the test material used in the repeat of the preliminary toxicity test was 0.31 to 80 µg/ml for all three of the exposure groups.
In all three of the exposure groups there were marked dose related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test material when compared to the concurrent vehicle controls. The toxicity curve was steep in all three of the exposure groups. Precipitate of the test material was not observed at any of the dose levels. In the subsequent mutagenicity experiments the maximum dose was limited by toxicity.

Mutagenicity Test
A summary of the results from the Test is presented in attached Table 1.

Experiment 1

The results of the microtitre plate counts and their analysis are presented in attached Tables 2 to 7.

There was evidence of toxicity following exposure to the test material in both the absence and presence of metabolic activation, as indicated by the %RSG and RTG values. There was no evidence of any significant reductions in viability (%V), therefore indicating that no residual toxicity had occurred. Optimum levels of toxicity were achieved in the presence of metabolic activation (Table 6). Optimum levels of toxicity were not achieved in the absence of metabolic activation due to the very sharp onset of toxicity, despite using a very narrow dose interval. However, with no evidence of a response in either the absence or presence of metabolic activation in this experiment, or in the second experiment where the usual upper limit of acceptable toxicity was exceeded, and the subsequent dose level to the upper surviving dose level exhibiting an excessive level of toxicity, the test material was considered to have been adequately tested (Tables 3 and 6). The excessive toxicity observed at 10 µg/ml in the absence of metabolic activation, resulted in this dose not being plated for viability or 5-TFT resistance. Acceptable levels of toxicity were seen with both positive control substances (Table 3 and Table 6).

Neither of the vehicle control mutant frequency values were markedly outside the acceptable range of 50 to 200 x 10-6 viable cells. The value for the without metabolic activation vehicle control was the lower limit of the range. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (Tables 3 and 6).

The test material did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell in either the absence or presence of metabolic activation (Tables 3 and 6). No precipitate of test material was observed at any of the dose levels.
The numbers of small and large colonies and their analysis are presented in Tables 4 and 7.

Experiment 2

The results of the microtitre plate counts and their analysis are presented in attached Tables 8 to 13.

As was seen previously, there was evidence of a marked dose-related reduction in % RSG and RTG values in cultures dosed with the test material in the both the absence and presence of metabolic activation. There was no evidence of any significant reductions in viability (%V), therefore indicating that no residual toxicity had occurred in either the absence or presence of metabolic activation. Near optimum levels of toxicity were achieved in the presence of metabolic activation. Optimum levels of toxicity were not achieved in either the absence or presence of metabolic activation due to the sharp onset of toxicity, despite using a very narrow dose interval. However, a dose level that marginally exceeded the usual acceptable upper limit of toxicity was plated for viability and TFT resistance for each of the exposure groups (Tables 9 and 12). It was therefore considered that with no evidence of any response, in either the first or second experiment, using a dose range where optimum levels of toxicity were achieved, approached or exceeded, the test material had been adequately tested. The excessive toxicity observed at 5 µg/ml in the absence of metabolic activation, and at and above 17.5 µg/ml in the presence of metabolic activation, resulted in these doses not being plated for viability or 5-TFT resistance. Both positive controls induced acceptable levels of toxicity (Tables 9 and 12).

The 24-hour exposure without metabolic activation demonstrated that the extended time point had an effect on the toxicity of the test material. It was also noted that the lowering of the S9 concentration to 1% S9 in the second experiment resulted in greater levels of toxicity than those observed in the presence of 2% S9 in the first experiment.

Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 10-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (Tables 9 and 12).

The test material did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell in either the absence or presence of metabolic activation (Tables 9 and 12). No precipitate of test material was observed at any of the dose levels.
The numbers of small and large colonies and their analysis are presented in Tables 10 and 13.

Remarks on result:

other: strain/cell type: Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Please see Attached "Tables 1 to 10"

Due to the nature and quantity of tables it was not possible to insert them in this section.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative Non-mutagenic

The test material did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non mutagenic under the conditions of the test.

Executive summary:

Introduction. 

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method used meets the requirements of the OECD (476), Method B17 of Commission Regulation (EC) No. 440/2008 of30 May 2008.

Methods. 

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at eight dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test material at eight dose levels using a 4‑hour exposure group in the presence of metabolic activation (1% S9) and a 24‑hour exposure group in the absence of metabolic activation.

The dose range of test material was selected following the results of a preliminary toxicity test. The dose range for Experiment 1 was 0.31 to 10 µg/ml in the absence of metabolic activation and 1.25 to 20 µg/ml in the presence of metabolic activation. The dose range for Experiment 2 was 0.16 to 5 µg/ml in the absence of metabolic activation, and 1.25 to 20 µg/ml in the presence of metabolic activation.

Results. 

The maximum dose level used was limited by test material induced toxicity. Precipitate of test material was not observed at any of the dose levels. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment.

Conclusion. 

The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.