1-Octadecanamine, reaction products with 1,1'-methylenebis[4-isocyanatobenzene]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The experimental phases of the study were performed between 11 August 2009 and 01 December 2009.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

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:

2009

Report date:

2010

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

Guideline:

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

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:

P U Thickner

IUPAC Name:

P U Thickner

Test material form:

other: beige solid pieces

Details on test material:

Sponsor's identification: P U Thickener
Description : Beige coloured solid
Purity : >95%
Batch number : VPSG011
Label : PU-Thickener Batch: VPSG0011 ca.600g
Date received : 31 July 2009
Storage conditions: Room temperature in the dark

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 been exposed to high levels of radiation or hazardous chemicals and had not knowingly 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:

Final concentration of PU Thickener (µg/ml)
Experiment 1:
4(20)-hour without S9 0*, 3.91, 7.81, 15.63, 31.25*, 62.5*, 125*, MMC 0.4* (* Dose levels selected for metaphase analysis)
4(20)-hour with S9 0*, 3.91, 7.81, 15.63, 31.25*, 62.5*, 125*, CP 5* (* Dose levels selected for metaphase analysis)

Experiment 2:
24-hour without S9 0*, 7.81, 15.63, 31.25, 62.5*, 125*, 250*, MMC 0.2* (* Dose levels selected for metaphase analysis)
4(20)-hour with S9 0*, 7.81, 15.63, 31.25, 62.5*, 125*, 250*, CP 5*(* Dose levels selected for metaphase analysis)

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.
- The purity of the test material was >95% and was accounted for in the formulations

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

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

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.

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:

Preliminary Toxicity Test

The dose range for the Preliminary Toxicity Test was 3.91 to 1000 µg/ml; the maximum practical dose level. A precipitate of the test material was observed in the parallel blood-free cultures at the end of the exposure, at and above 31.25 µg/ml, in all three exposure groups. Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 1000 µg/ml in the 4(20)-hour exposures in the presence and absence of metabolic activation (S9). The maximum dose with metaphases present in the 24-hour continuous exposure was 1000 µg/ml. The mitotic index data are presented in Table 1 - see attached. The test material induced no clear evidence of toxicity in any of the exposure groups.
The selection of the maximum dose level in experiment 1 was initially based on the lowest dose level with marked precipitation in the 4(20)-hour exposure group with S9 (125 µg/ml), and was modified for exposure groups used in Experiment 2.

Chromosome Aberration Test - Experiment 1

The dose levels of the controls and the test material are given in the table below:
Group Final concentration of PU Thickener (µg/ml)
4(20)-hour without S9 0*, 3.91, 7.81, 15.63, 31.25*, 62.5*, 125*, MMC 0.4*
4(20)-hour with S9 0*, 3.91, 7.81, 15.63, 31.25*, 62.5*, 125*, 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 material, 125 µg/ml in the presence of metabolic activation (S9). In the absence of metabolic activation (S9) the maximum dose level of the test material with metaphases suitable for scoring was 125 µg/ml.
A precipitate of the test material was not observed at the end of exposure, in the 4 (20) hour exposure group in the absence and presence of S9.
The mitotic index data are given in Table 2 - see attached. They confirm the qualitative observations in that a dose-related inhibition of mitotic index was observed, and that 13% mitotic inhibition was achieved at 125 µg/ml in the absence of S9. In the presence of S9 18% mitotic inhibition was achieved at 125 µg/ml.

The maximum dose level selected for metaphase analysis was the maximum dose level tested (125 µg/ml).
The chromosome aberration data are given in Table 4 and Table 5 - see attached. All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials 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 material 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 Table 8 - see attached. The test material 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 material are given in the table below:
Group Final concentration of PU Thickener (µg/ml)
24-hour without S9 0*, 7.81, 15.63, 31.25, 62.5*, 125*, 250*, MMC 0.2*
4(20)-hour with S9 0*, 7.81, 15.63, 31.25, 62.5*, 125*, 250*, CP 5*

The qualitative assessment of the slides determined that there were metaphases suitable for scoring present at the maximum test material dose level of 250 µg/ml in the presence of S9. In the absence of S9 the maximum test material dose level with metaphases suitable for scoring was 250 µg/ml. A precipitate of the test material was not observed at the end of exposure, in either exposure group.

The mitotic index data are given in Table 3 - see attached. They confirm the qualitative observations in that a slight dose-related inhibition of mitotic index was observed, and that 13% mitotic inhibition was achieved at 250 µg/ml in the absence of S9. In the presence of S9 that a 10% mitotic inhibition was achieved at 250 µg/ml. As was seen in the preliminary toxicity test and Experiment 1 no clear evidence of toxicity was achieved, However, it was considered that the test material had been adequately tested.

The maximum dose level selected for metaphase analysis was the maximum dose level tested (250 µg/ml).

The chromosome aberration data are given in Table 6 and Table 7 - see attached. All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials 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 material 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 Table 8 - see attached. The test material did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

CONCLUSION

The test material 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 material 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

Please see attached Report of Results of Chromosome Aberration Test in Cultured Mammalian Cells Tables

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

CONCLUSION
The test material 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 material was therefore considered to be non-clastogenic to human lymphocytesin 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 followed 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. The study design also meets the requirements of the UK Department of Health Guidelines for Testing of Chemicals for Mutagenicity.

Methods. 

Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at three 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.

Results. 

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

All the positive control materials 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 material did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments.

Conclusion. 

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