Propanol, iminobis-, N-(C16-18 and C18-unsatd. alkyl) derivs.

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The experimental phases of the study were performed between 13 July 2011 and 20 October 2011.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable.

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

For each experiment, sufficient whole blood was drawn from the peripheral circulation of a volunteer who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not recently suffered from a viral infection.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone and beta-naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

Preliminary Toxicity Test The dose range of test item used initially was 15 to 3837 µg/ml. However, due to excessive toxicity being observed in the 24-hour exposure group this exposure group was repeated with a revised dose range of 0.125 to 32 µg/ml.

The dose levels used in the main experiments were selected using data from the preliminary toxicity test and were as follows:
Group Final concentration of test item (µg/ml)
4(20)-hour without S9 1, 2, 4, 8, 16, 24,
4(20)-hour with S9 (2%) 2, 4, 8, 16, 24, 32
24-hour without S9 2, 4, 8, 12, 16, 24,
4(20)-hour with S9 (1%) 4, 8, 12, 16, 24, 28, 32

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO was selected as the solvent because the test material was readily soluble in it at the required concentrations.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide (DMSO)

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

In the presence of S9

Migrated to IUCLID6: (CP)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide (DMSO)

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

In the absence of S9

Migrated to IUCLID6: (MMC)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
in medium

DURATION
- Preincubation period: 48 hrs

- Exposure duration:
Experiment 1 - 4 hrs with and without S9.
Experiment 2 - 24 hrs without S9, 4 hrs with S9.

- Expression time (cells in growth medium): 20 hrs for 4 hrs exposure.

- Selection time (if incubation with a selection agent): Not applicable.

- Fixation time (start of exposure up to fixation or harvest of cells): 24 hrs.

SELECTION AGENT (mutation assays): No selection agent.

SPINDLE INHIBITOR (cytogenetic assays): Demecolcine

STAIN (for cytogenetic assays): When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and coverslipped using mounting medium.


NUMBER OF REPLICATIONS: Duplicate cultures


NUMBER OF CELLS EVALUATED: 100/culture


DETERMINATION OF CYTOTOXICITY
- Method:
mitotic index - A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

-Scoring of Chromosome Damage:
Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there was approximately 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing. Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.

OTHER EXAMINATIONS:
- Determination of polyploidy: Frequency of polyploid cells


OTHER:
None.

Evaluation criteria:

A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

Species / strain:

lymphocytes: Human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Refer to information on results and attached tables.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no significant change in pH when the test material was dosed into media.
- Effects of osmolality: The osmalality did not increase by more than 50 mOsm.
- Evaporation from medium: Not applicable.
- Water solubility: Not applicable, test material suspended in MEM
- Precipitation: See results section below

Preliminary Toxicity Test
The dose range for the Preliminary Toxicity Test was 15 to 3837 µg/ml for all three exposure groups. The maximum dose was the maximum recommended dose level, the 10 mM concentration. However, as a result of excessive toxicity being observed in the 24-hour exposure group this was repeated with a revised dose range of 0.125 to 32 µg/ml.
A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure period, at and above 30 µg/ml and 60 µg/ml in the 4 (20)-hour exposure groups in the presence and absence of S9 respectively. In the 24-hour continuous exposure group precipitate was seen at and above 119.9 µg/ml. Haemolysis was also noted at the end of the exposure period in the blood cultures at and above 60 µg/ml in the 4(20)-hour exposure group in the absence of S9 and in the 24-hour exposure group. In the 4(20)-hour exposure group in the presence of S9 haemolysis was seen at and above 119.9 µg/ml at the end of the exposure period.
Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 15 µg/ml in the 4(20)-hour exposure group in the absence of metabolic activation and 30 µg/ml in the presence of metabolic activation. The maximum dose with metaphases present in the 24-hour continuous exposure was 16 µg/ml in the repeated toxicity test. The mitotic index data are presented in the attached Table 1. The test item induced evidence of toxicity in all three exposure groups.
The selection of the maximum dose level for the main experiments was based on toxicity and was 32 µg/ml for the both the 4(20) hour exposure groups in the presence of S9 in Experiment 1 and Experiment 2. The 4(20)-hour exposure group in the absence of S9 and the 24-hour continuous exposure groups used a maximum dose of 24 µg/ml.
 
Chromosome Aberration Test - Experiment 1
The dose levels of the controls and the test item are given in the table below:

Group Final concentration of Primary Fatty Amine propoxylated (µg/ml)
4(20)-hour without S9 0*, 1, 2*, 4*, 8*, 16*, 24, 32, MMC 0.4*
4(20)-hour with S9 (2%) 0*, 2, 4, 8*, 16*, 24*, 32*, CP 5*

The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present at the maximum dose level of test item tested, 32 µg/ml in the presence of metabolic activation (S9). In the absence of metabolic activation (S9) the maximum dose level of the test item with metaphases suitable for scoring was 16 µg/ml and although there were a few metaphases present at 24 µg/ml there were insufficient for scoring. No precipitate of the test item was observed at the end of exposure in either exposure group.
The mitotic index data are given in the attached Table 2. They confirm the qualitative observations in that a dose-related inhibition of mitotic index was observed, and that 70% mitotic inhibition was achieved at 16 µg/ml in the absence of S9 and in the presence of S9 63% mitotic inhibition was achieved at 32 µg/ml.
The maximum dose level selected for metaphase analysis was based on toxicity and was 16 µg/ml in the absence of S9 and 32 µg/ml in the presence of S9. The chromosome aberration data are given in the attached Table 4 and Table 5. All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control items induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected. The test item did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of
metabolic activation.
The polyploid cell frequency data are given in the attached Tables 4 and 5. The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Chromosome Aberration Test - Experiment 2
The dose levels of the controls and the test item are given in the table below:

Group Final concentration of Primary Fatty Amine propoxylated (µg/ml)
24-hour without S9 0*, 2, 4*, 8*, 12*, 16*, 24, MMC 0.2*
4(20)-hour with S9 (1%) 0*, 4*, 8*, 16*, 24, 28, 32, CP 5*

The qualitative assessment of the slides determined that there were metaphases suitable for scoring present at 28 µg/ml in the presence of S9; however due to the obvious toxicity observed the maximum dose selected for mitotic index analysis was 24 µg/ml. In the absence of S9 the maximum test item dose level with metaphases suitable for scoring was 16 µg/ml. No precipitate of the test item was observed at the end of exposure in either exposure group.
The mitotic index data are given in the attached Table 3. They confirm the qualitative observations in that a dose-related inhibition of mitotic index was observed, and that 52% mitotic inhibition was achieved at 16 µg/ml in the presence of S9. In the 24-hour continuous exposure group 62% mitotic inhibition was achieved at 16 µg/ml. The maximum dose level selected for metaphase analysis was 16 µg/ml for both exposure groups of Experiment 2.
The chromosome aberration data are given in the attached Table 6 and Table 7. All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control items induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected. The test item did not induce any statistically significant increases in the frequency of cells with chromosome aberrations either in the absence or presence of metabolic activation. The polyploid cell frequency data are given in the attached Tables 6 and 7. The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups. There was however an unusually high number of polyploids observed in the ‘A’ replicate of the 4(20)-hour with S9 exposure group at 16 µg/ml but since it was noted in only one culture at the point of optimum toxicity, was not dose related and was not statistically significant it was considered to be of no biological relevance.

CONCLUSION
The test item did not induce a statistically significant increase in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system in either of two separate experiments. The test item was therefore considered to be non-clastogenic to human lymphocytes in vitro.

Remarks on result:

other: strain/cell type:

Remarks:

Migrated from field 'Test system'.

For the tables and figures of resluts mentioned above, please refer to the attached background material section for the following tables:

Table 1: Mitotic Index - Preliminary Toxicity Test

Table 2: Mitotic Index - Experiment 1

Table 3: Mitotic Index - Experiment 2

Table 4: Results of Chromosome Aberration Test - Experiment 1 Without Metabolic Activation (S9)

Table 5: Results of Chromosome Aberration Test - Experiment 1 With Metabolic Activation (S9)

Table 6: Results of Chromosome Aberration Test - Experiment 2 Without Metabolic Activation (S9)

Table 7: Results of Chromosome Aberration Test - Experiment 2 With Metabolic Activation (S9)

Conclusions:

Interpretation of results (migrated information):
negative

The test item did not induce a statistically significant increase in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system in either of two separate experiments. The test item was therefore considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

Introduction

This report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scottet al, 1990). The method used was designed to be compatible with that described in the OECD Guidelines for Testing of Chemicals (1997) No. 473 "Genetic Toxicology: Chromosome Aberration Test" and Method B10 of Commission Regulation (EC) No. 440/2008 of 30 May 2008 and is acceptable to the Japanese New Chemical Substance Law (METI).

Methods

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study, i.e. In Experiment 1, 4 hours in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4 hours exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

The dose levels used in the main experiments were selected using data from the preliminary toxicity test and were as follows:

Group	Final concentration of test item (µg/ml)
4(20)-hour without S9	1, 2, 4, 8, 16, 24,
4(20)-hour with S9 (2%)	2, 4, 8, 16, 24, 32
24-hour without S9	2, 4, 8, 12, 16, 24,
4(20)-hour with S9 (1%)	4, 8, 12, 16, 24, 28, 32

 

Results

All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test item did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included dose levels that exceeded or were close to the optimum level of 50% mitotic inhibition.

Conclusion

The test item was considered to be non-clastogenic to human lymphocytes in vitro.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The experimental phases of the study were performed between 07 July 2011 and 12 September 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

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

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

RPMI 1640

- 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 in the mutagenicity test was limited by test item induced toxicity.
Vehicle and positive controls were used in parallel with the test item. Solvent (DMSO) treatment groups were used as the vehicle controls. Ethylmethanesulphonate (EMS), Sigma batch 0001423147 (Experiment 1) and 100930580 (Experiment 2) 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(s)/solvent(s) used:
Solvent (DMSO) treatment groups were used as the vehicle controls.


- Justification for choice of solvent/vehicle:
soluble

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

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

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Solvent (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 item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method used was designed to be compatible with the OECD Guidelines for Testing of Chemicals No.476 "In Vitro Mammalian Cell Gene Mutation Tests", Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and be acceptable to the Japanese EPA/METI/MHLW guidelines for testing of new chemical substances.
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 item 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 item 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 item was selected following the results of a preliminary toxicity test and for the first experiment was 0.16 to 7.5 µg/ml in the absence of metabolic activation and 0.63 to 15 µg/ml in the presence of metabolic activation. For the second experiment the dose range was 0.06 to 5 µg/ml in the absence of metabolic activation and 0.63 to 17.5 µg/ml in the presence of metabolic activation.
Results. The maximum dose level used in the mutagenicity test was limited by test item induced toxicity. 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 items 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

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:

The test item had a purity value of 97.3% and, therefore, an allowance was made when dose formulations were prepared. There were no marked changes in pH, and the osmolality did not increase by more than 50 mOsm

Preliminary Toxicity Test
The first Preliminary Toxicity Test was repeated due to excessive toxicity and therefore the revised dose range of the test item used in the repeat Preliminary Toxicity Test was 0.12 to 30 µg/ml.
Marked reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item were observed in all 3 of the exposure groups when compared to the concurrent vehicle control groups. The onset of toxicity was very steep in all 3 exposure groups and a limit of 17.5 µg/ml was selected as the maximum dose level used in the subsequent Mutagenicity Test. No precipitate of test item was observed
Mutagenicity Test
A summary of the results from the test is presented in Table 1.
Experiment 1
The results of the microtitre plate counts and their analysis are presented in Tables 2 to 7.
There was evidence of marked test item-induced toxicity following exposure to the test item in both the absence and presence of metabolic activation, as indicated by the %RSG values (Tables 3 and 6). There was no evidence of reductions in viability (%V) in both the absence and presence of metabolic activation, indicating that residual toxicity had not occurred (Tables 3 and 6).The highest levels of toxicity observed in both the absence and presence of metabolic activation were approaching the optimum levels of toxicity (Tables 3 and 6). With adequate toxicity being achieved in Experiment 2 it was considered that the test item overall had been adequately tested. Acceptable levels of toxicity were seen with both positive control substances (Tables 3 and 6).
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 3 and 6).
The test item 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 item was observed at any dose level.
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 Tables 8 to 13.
As was seen previously, there was evidence of marked toxicity in both the absence and presence of metabolic activation following exposure to the test item, as indicated by the %RSG values (Tables 9 and 12). There was no evidence of reductions in viability (%V) in both the absence and presence of metabolic activation, indicating that no residual toxicity had occurred (Tables 9 and 12). Optimum levels of toxicity were achieved in both the absence and presence of metabolic activation as indicated by the RTG values (Tables 8 and 12). It was therefore considered that the test item had been suitably tested. Acceptable levels of toxicity were seen with both positive control substances (Tables 9 and 12).
The 24-Hour exposure without metabolic activation demonstrated that the extended time point had a marked effect on the toxicity of the test item.
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 item 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). All test item mutation values were within the acceptable range for vehicle control cultures. No precipitate of test item was observed at any dose level.
The numbers of small and large colonies and their analysis are presented in Tables 10 and 13.

Remarks on result:

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

Remarks:

Migrated from field 'Test system'.

Please see attached tables 1 - 13

Due to the quantity of the tables it was not possible to inset them into this section

Conclusions:

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

CONCLUSION
The test item did not induce any statistically significant or dose related 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:

SUMMARY

Introduction. The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method used was designed to be compatible with the OECD Guidelines for Testing of Chemicals No.476 "In Vitro Mammalian Cell Gene Mutation Tests", Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and be acceptable to the Japanese EPA/METI/MHLW guidelines for testing of new chemical substances.

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 item 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 item 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 item was selected following the results of a preliminary toxicity test and for the first experiment was 0.16 to 7.5 µg/ml in the absence of metabolic activation and 0.63 to 15 µg/ml in the presence of metabolic activation. For the second experiment the dose range was 0.06 to 5 µg/ml in the absence of metabolic activation and 0.63 to 17.5 µg/ml in the presence of metabolic activation.

Results. The maximum dose level used in the mutagenicity test was limited by test item induced toxicity. 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 items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test item did not induce any statistically 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 item was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

The experimental phase of this study was performed between 09 September 2009 and 11 October 2009.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Justification for type of information:

See section 13.2 for the read-across justification.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

Meets the requirements of the Japanese Regulatory Authorities including METI, MHLW and MAFF, OECD Guidelines for Testing of Chemicals No. 471 "and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine for Salmonella.
Tryptophan for E.Coli

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/beta­naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

Preliminary Toxicity Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment one: Salmonella strains (absence of S9): 0.5, 1.5, 5, 15, 50, 150, 500 µg/plate.
Salmonella strains (presence of S9), E.coli strain WP2uvrA- (absence and presence of S9): 1.5, 5, 15, 50, 150, 500, 1500 µg/plate.
Experiment two: Salmonella strains (absence of S9): 0.15, 0.5, 1.5, 5, 15, 50, 150 µg/plate.
Salmonella strains (presence of S9), E.coli strain WP2uvrA- (absence and presence of S9): 0.5, 1.5, 5, 15, 50, 150, 500 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulphoxide.
- Justification for choice of solvent/vehicle: The test material was fully miscible in dimethyl sulphoxide at 50 mg/ml in solubility checks performed in house. Distilled water was not evaluated as a potential vehicle in this test system as information provided by the sponsor suggested it was immiscible with the test material. Dimethyl sulphoxide was therefore selected as the vehicle.

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 1 µg/plate

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 2 µg/plate

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 2 µg/plate

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 10 µg/plate

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With S9 mix Migrated to IUCLID6: Benzo(a)pyrene: 5 µg/plate

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9 mix Migrated to IUCLID6: 4-Nitroquinoline-1-oxide: 0.2 µg/plate

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix Migrated to IUCLID6: 9-Aminoacridine: 80 µg/plate

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 3 µg/plate

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 5 µg/plate

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 2 µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period for bacterial strains: 10h
- Exposure duration: 48 - 72 hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable

NUMBER OF REPLICATIONS: Triplicate plating.

DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.

Evaluation criteria:

Acceptance Criteria:

The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.
All tester strain cultures should be in the approximate range of 1 to 9.9 x 109 bacteria per ml.
Each mean positive control value should be at least twice the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
There should be a minimum of four non-toxic test material dose levels.
There should not be an excessive loss of plates due to contamination.

Evaluation criteria:
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.

Statistics:

Standard deviation
Dunnett's Linear Regression Analysis

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Solubility: The test material was fully miscible in dimethyl sulphoxide at 50 mg/ml in solubility checks performed in house. Distilled water was not evaluated as a potential vehicle in this test system as information provided by the sponsor suggested it was immiscible with the test material. Dimethyl sulphoxide was therefore selected as the vehicle.
- Precipitation: A precipitate (particulate in appearance) was initially noted at and above 500 µg/plate, this observation did not prevent the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test:
The test material initially exhibited toxicity to the strains of bacteria used (TA100 and WP2uvrA-) from 150 and 500 µg/plate, respectively. The test material formulation and S9-mix used in this experiment were both shown to be sterile.

COMPARISON WITH HISTORICAL CONTROL DATA:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).

Results for the negative controls (spontaneous mutation rates) were considered to be acceptable.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

ADDITIONAL INFORMATION ON CYTOTOXICITY: In the first experiment (plate incorporation method) the test material caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially at 150 and 500 µg/plate in the absence and presence of S9, respectively. In the second experiment (pre-incubation method) the test material induced toxicity to the bacterial background lawns of all of the tester strains initially from 50 µg/plate. The test material was, therefore tested up to the toxic limit.

 RESULTS

Preliminary Toxicity Test

The test material initially exhibited toxicity to the strains of bacteria used (TA100 and WP2uvrA^-) from 150 and 500 µg/plate, respectively.  The test material formulation and S9-mix used in this experiment were both shown to be sterile.

The numbers of revertant colonies for the toxicity assay were:

With (+) or without (-) S9-mix	Strain	Dose (µg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA100	87	69	71	71	84	84	71	0V	0T	0T	0TP
+	TA100	73	86	95	77	80	62	83	77	0V	0T	0TP
-	WP2uvrA^-	32	33	24	28	31	25	25	17	0V	0T	0TP
+	WP2uvrA^-	30	33	28	26	27	21	20	31	15S	0T	0TP

S         Sparse bacterial background lawn
V         Very weak bacterial background lawn
T         Toxic, no bacterial background lawn

P         Precipitate

 

MutationTest

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile.

 

Results for the negative controls (spontaneous mutation rates) are presented inTable 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations for the test material, vehicle and positive controls both with and without metabolic activation, are presented in Table 2 to Table 5.

In the first experiment (plate incorporation method) the test material caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially at 150 and 500 µg/plate in the absence and presence of S9, respectively. In the second experiment (pre-incubation method) the test material induced toxicity to the bacterial background lawns of all of the tester strains initially from 50 µg/plate. The test material was, therefore tested up to the toxic limit. A precipitate (particulate in appearance) was initially noted at and above 500 µg/plate, this observation did not prevent the scoring of revertant colonies.

No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. A small, statistically significant increase in TA100 revertant colony frequency was observed (presence of S9) at 5 µg/plate in Experiment 1. This response was considered not to be toxicologically significant because it was non-reproducible in three separate Experiments (including the Preliminary Toxicity Assay), the mean revertant count at 5 µg/plate was only 1.17 times the concurrent vehicle control value and individual revertant counts were within the acceptable in-house historical range for the bacterial tester strain.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

 

Table 1               Spontaneous Mutation Rates (Concurrent Negative Controls

Range-finding Test

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA^-		TA98		TA1537
123		11		22		26		10
98	(105)	15	(15)	18	(19)	16	(22)	12	(11)
95		18		18		24		11

Main Test

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA^-		TA98		TA1537
128		15		21		22		14
100	(108)	16	(16)	22	(22)	22	(22)	12	(12)
95		18		22		22		11

 

Table 2               Test Results: Range-Finding Test– Without Metabolic Activation

 

Test Period		From: 27 September 2009					To: 30 September 2009
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535		WP2uvrA^-			TA98		TA1537
-	0	109 97 89	(98) 10.1#	14 14 18	(15) 2.3	25 22 21		(23) 2.1	17 22 21	(20) 2.6	10 10 8	(9) 1.2
-	0.5	96 102 85	(94) 8.6	13 21 15	(16) 4.2	N/T			22 18 16	(19) 3.1	13 13 8	(11) 2.9
-	1.5	85 99 106	(97) 10.7	15 14 14	(14) 0.6	24 19 16		(20) 4.0	17 17 18	(17) 0.6	9 12 11	(11) 1.5
-	5	114 98 106	(106) 8.0	15 15 17	(16) 1.2	19 19 22		(20) 1.7	22 20 25	(22) 2.5	9 9 10	(9) 0.6
-	15	104 92 110	(102) 9.2	17 16 12	(15) 2.6	19 19 22		(20) 1.7	22 20 19	(20) 1.5	15 9 10	(11) 3.2
-	50	136 105 90	(110) 23.5	13 19 16	(16) 3.0	19 20 21		(20) 1.0	19 18 18	(18) 0.6	7 8 7	(7) 0.6
-	150	92 V 49 V 48 V	(63) 25.1	7 V 6 V 15 V	(9) 4.9	20 17 17		(18) 1.7	0 V 0 V 0 V	(0) 0.0	0 V 0 V 0 V	(0) 0.0
-	500	0 TP 0 TP 0 TP	(0) 0.0	0 TP 0 TP 0 TP	(0) 0.0	0 V 0 V 0 V		(0) 0.0	0 TP 0 TP 0 TP	(0) 0.0	0 TP 0 TP 0 TP	(0) 0.0
-	1500	N/T		N/T		0 TP 0 TP 0 TP		(0) 0.0	N/T		N/T
Positive controls   S9-Mix   -	Name Concentration (μg/plate) No. colonies per plate	ENNG		ENNG		ENNG			4NQO		9AA
		3		5		2			0.2		80
		383 293 381	(352) 51.4	205 276 243	(241) 35.5	719 761 668		(716) 46.6	191 106 126	(141) 44.4	1011 1003 913	(976) 54.4

 ENNG N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

N/T     Not tested at this dose level

P        Precipitate

T        Toxic, no bacterial background lawn

V        Very weak bacterial background lawn

#        Standard deviation

Table 3               Test Results: Range-Finding Test– With Metabolic Activation

Test Period		From: 27 September 2009					To: 30 September 2009
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535		WP2uvrA^-			TA98		TA1537
+	0	80 84 85	(83) 2.6#	15 11 12	(13) 2.1	22 19 20		(20) 1.5	24 24 24	(24) 0.0	8 7 11	(9) 2.1
+	1.5	82 90 91	(88) 4.9	11 10 11	(11) 0.6	21 21 20		(21) 0.6	25 24 25	(25) 0.6	13 10 13	(12) 1.7
+	5	101 101 90	$$$ (97) 6.4	10 8 12	(10) 2.0	22 19 19		(20) 1.7	21 25 18	(21) 3.5	7 12 7	(9) 2.9
+	15	82 90 81	(84) 4.9	11 7 10	(9) 2.1	20 21 24		(22) 2.1	21 17 17	(18) 2.3	10 9 9	(9) 0.6
+	50	89 88 80	(86) 4.9	8 9 8	(8) 0.6	19 19 22		(20) 1.7	24 27 22	(24) 2.5	9 8 8	(8) 0.6
+	150	80 79 80	(80) 0.6	8 9 8	(8) 0.6	17 18 20		(18) 1.5	21 20 15	(19) 3.2	12 11 10	(11) 1.0
+	500	0 V 0 V 0 V	(0) 0.0	0 T 0 T 0 T	(0) 0.0	12 S 9 S 11 S		(11) 1.5	0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T	(0) 0.0
+	1500	0 TP 0 TP 0 TP	(0) 0.0	0 TP 0 TP 0 TP	(0) 0.0	0 TP 0 TP 0 TP		(0) 0.0	0 TP 0 TP 0 TP	(0) 0.0	0 TP 0 TP 0 TP	(0) 0.0
Positive controls   S9-Mix   +	Name Concentration (μg/plate) No. colonies per plate	2AA		2AA		2AA			BP		2AA
		1		2		10			5		2
		1510 2079 2379	(1989) 441.4	112 156 183	(150) 35.8	371 340 348		(353) 16.1	215 251 208	(225) 23.1	313 292 314	(306) 12.4

2AA    2-Aminoanthracene

BP      Benzo(a)pyrene

P        Precipitate

S        Sparse bacterial background lawn

T        Toxic, no bacterial background lawn

V        Very weak bacterial background lawn

$$$     p<0.005

#        Standard deviation

PLEASE SEE OVERALL REMARKS,) Tables 4 and 5 (Experiment 2)

Conclusions:

Interpretation of results (migrated information):
negative

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

Executive summary:

Introduction.

The method was designed to conform to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It also meets the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Directive 2000/32/EC and the, EPA (TSCA) OPPTS harmonised guidelines.

Methods.

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA^-were treated with the test material using both the Ames plate incorporation and pre-incubation methods at seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the first experiment was determined in a preliminary toxicity assay and ranged between 0.5 and 1500 µg/plate, depending on bacterial strain type and presence or absence of S9. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test material formulations. The test material dose range was suitably amended to allow for toxicity and ranged between 0.15 and 500 µg/plate, depending on bacterial strain type and presence or absence of S9.

Additional dose levels and an expanded dose range were selected in both experiments in order to achieve both four non-toxic dose levels and the toxic limit of the test material.

Results.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

In the first experiment (plate incorporation method) the test material caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially at 150 and 500 µg/plate in the absence and presence of S9, respectively. In the second experiment (pre-incubation method) the test material induced toxicity to the bacterial background lawns of all of the tester strains initially from 50 µg/plate. The test material was, therefore tested up to the toxic limit. A precipitate (particulate in appearance) was initially noted at and above 500 µg/plate, this observation did not prevent the scoring of revertant colonies.

No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. A small, statistically significant increase in TA100 revertant colony frequency was observed (presence of S9) at 5 µg/plate in Experiment 1. This response was considered not to be toxicologically significant because it was non-reproducible in three separate Experiments (including the Preliminary Toxicity Assay), the mean revertant count at 5 µg/plate was only 1.17 times the concurrent vehicle control value and individual revertant counts were within the acceptable in-house historical range for the bacterial tester strain.

Conclusion.

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

di-(2-hydroxypropyl) C16-C18 (evennumbered), C18 unsaturated-alkyl amine CAS No. 1309955-79-0 was previously registered as Propanol, iminobis-, N-C16-C18 (evennumbered), C18 unsatd. alkyl) derivs. CAS No. 68951-72-4.

Propanol, iminobis-, N-C16-C18 (evennumbered), C18 unsatd. alkyl) derivs. was tested for mammalian cell gene mutation in the L5178Y TK +/- Mouse lymphoma assay and was not mutagenic. In addition it was tested in a Chromosomal aberration test in human lymphocytes in-vitro and was found to be not clastogenic. Propanol, iminobis-, N-C16-C18 (evennumbered), C18 unsatd. alkyl) derivs. was not tested in a bacterial reverse mutation assay (Ames) as there was a test on the read across substance Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derives CAS No. 61791-31-9 which was not mutagenic with or without S9 mix

 

All tests were to current OECD/EU protocols carried out to GLP and with a clearly defined and described test substance. Based on this it can be concluded thatPropanol, iminobis-, N-C16-C18 (evennumbered), C18 unsatd. alkyl) derivs . CAS No 1309955-79-0 would not be expected to have any genotoxic hazard to human health.

 

Justification for selection of genetic toxicity endpoint

All three in-vitro genetic toxicity studies are negative

 

Short description of key information:

Propanol, iminobis-, N-C16-C18 (evennumbered), C18 unsatd. alkyl) derivs . CAS No 1309955-79-0 was tested in two in-vitro genotoxicity studies and read across data are available for the third test, all to current protocol and carried out to GLP with well defined test substance. All studies were negative, therefore Propanol, iminobis-, N-C16-C18 (evennumbered), C18 unsatd. alkyl) derivs . would not be expected to have any genotoxic hazard to human health.

 

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the three negative in-vitro tests for genotoxicity, Propanol, iminobis-, N-C16-C18 (evennumbered), C18 unsatd. alkyl) derivs. CAS No 1309955-79-0 does not require classification as a mutagen according to the European Union CLP/GHS criteria.