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

Genetic toxicity: in vitro

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

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

Test material

Test material information

Constituent 1

Reference substance name:

2-[(2-hydroxypropyl)(C16-18 sat. C18 unsat. alkyl)amino]propan-1-ol

EC Number:

695-977-9

Cas Number:

1309955-79-0

Molecular formula:

Not applicable UVCB

IUPAC Name:

2-[(2-hydroxypropyl)(C16-18 sat. C18 unsat. alkyl)amino]propan-1-ol

Test material form:

liquid

Details on test material:

Chemical name: 2-[(2-hydroxypropyl)(C16-18 sat. C18 unsat. alkyl)amino]propan-1-ol
EC no.: 695-977-9

To the best of knowledge, the sample used is representative to the boundary composition shared and agreed by each registrant

Method

Target gene:

Not applicable.

Species / strain

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.

Controls

Controls 1

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)

Controls 2

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.

Results and discussion

Test results

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'.

Any other information on results incl. tables

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)

Applicant's summary and conclusion

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.