Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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 01 May 2012 and 16 September 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
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 not effect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: UKDoH Guidelines for the Testing of Chemicals for Mutagenicity as detailed in the UKEMS Recommended Procedures for Basic Mutagenicity Tests (1990).
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
N-isopropylmethacrylamide
EC Number:
237-331-7
EC Name:
N-isopropylmethacrylamide
Cas Number:
13749-61-6
Molecular formula:
C7H13NO
IUPAC Name:
2-methyl-N-(propan-2-yl)prop-2-enamide
Test material form:
other: solid
Details on test material:
Sponsor's identification : N-Isopropylmethacrylamide (NIPMAA)
Description : Yellow crystalline solid
CAS number : 13749-61-6
Identifiers : TIS O4310, R-50621
Batch number : CI1L0212
Date received : 16 January 2012
Expiry Date : 01 November 2012
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. The cell-cycle time for the lymphocytes from the donors used in this study was determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (AGT). The average AGT for the regular donors used in this laboratory has been determined to be approximately 16 hours under typical experimental exposure conditions.

Cell Culture:
Cells were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented "in-house" with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal bovine serum (FBS), at 37°C with 5% CO2 in humidified air. The lymphocytes of fresh heparinised whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).
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 was 4.96, 9.92, 19.84, 39.69, 79.38, 158.75, 317.5, 635, 1270 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 N-Isopropylmethacrylamide (NIPMAA) (µg/ml)
4(20)-hour without S9 0*, 39.69, 79.38, 158.75, 317.5*, 635*, 1270*, MMC 0.4*
4(20)-hour with S9 (2%) 0*, 39.69, 79.38, 158.75, 317.5*, 635*, 1270*, CP 5*

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 N-Isopropylmethacrylamide (NIPMAA) (µg/ml)
24-hour without S9 0*, 39.69, 79.38, 158.75, 317.5*, 635*, 1270*, MMC 0.2*
4(20)-hour with S9 (1%) 0*, 39.69, 79.38, 158.75, 317.5*, 635*, 1270*, CP 5*

* Dose levels selected for metaphase analysis
MMC = Mitomycin C
CP = Cyclophosphamide

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.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Cyclophosphamide (CP)
Remarks:
In the presence of S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
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 were approximately 30 to 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:
In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) reported.


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.


PRELIMINARY TOXICITY TEST (CELL GROWTH INHIBITION TEST)
The mitotic index data are presented in the attached Appendix 1 (5) and (6) (see attached background material - Appendix 1). It can be seen that the test item showed marked evidence of toxicity in the 24-hour exposure group only. No precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure period in any of the three exposure groups.

Microscopic assessment of the slides prepared from the treatment cultures showed that metaphase cells were present up to 1270 µg/ml in all three
exposure groups.

Dose selection for Experiment 1 and Experiment 2 was based on the maximum recommended dose level, the 10 mM concentration which was
1270 µg/ml.

CHROMOSOME ABERRATION TEST - EXPERIMENT 1
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 test item dose level of 1270 µg/ml in both the presence and absence of metabolic
activation (S9).

The results of the mitotic indices (MI) from the cultures after their respective treatments are presented in the attached Form 1, Appendix 2 (attached background material). These data show no mitotic inhibition was demonstrated in the absence or presence of S9 at the maximum recommended dose of 1270 µg/ml.

No precipitate of the test item was observed at the end of the treatment period in either exposure group.

The maximum dose level selected for metaphase analysis was the maximum recommended dose level, the 10 mM concentration dose level
(1270 µg/ml).

The chromosome aberration data are given in the attached Form 1, Appendix 2 (attached background material). 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 (S9).

The polyploid cell frequency data are given in the attached Form 1, Appendix 2 (attached background material). 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 qualitative assessment of the slides determined that there were metaphases suitable for scoring present at the maximum test item dose level of
1270 µg/ml in both the presence and absence of S9.

The results of the mitotic indices (MI) from the cultures after their respective treatments are presented in the attached Form 2, Appendix 2 (attached backgroud material). These data show no growth inhibition was demonstrated in the 4(20)-hour exposure group in the presence of S9 as seen in the previous experiments. In the 24-hour exposure group in the absence of S9 there was a dose related increase in toxicity which achieved 42% and 62% mitotic inhibition at 635 and 1270 µg/ml respectively.

No precipitate of the test item was observed at the end of the treatment period in either exposure group.

The maximum dose level selected for metaphase analysis was the maximum recommended dose level, the 10 mM concentration (1270 µg/ml).

The chromosome aberration data are given in the attached Form 2, Appendix 2 (attached background material). 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 Form 2, Appendix 2. The test item did not induce a significant increase in the numbers of
polyploid cells at any dose level in either of the exposure groups.
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:

Appendix 1    Report of Results of Chromosomal Aberration Test in Cultured Mammalian Cells

Appendix 2    Results of Chromosome Aberration Test

Appendix 3    Dose Response Curves  

Applicant's summary and conclusion

Conclusions:
Interpretation of results: 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 (Scott et al, 1990).

The test method used was designed to be compatible with the following internationally accepted guidelines and recommendations:

i)            OECD Guidelines for Testing of Chemicals No. 473 "Genetic Toxicology: Chromosome Aberration Test", Method B10 of Commission Regulation (EC) No. 440/2008 of 30 May 2008.

ii)           UKDoH Guidelines for the Testing of Chemicals for Mutagenicity as detailed in theRecommended Procedures for Basic Mutagenicity Tests (1990).

iii)          US EPA OPPTS 870.5375 Guideline.

The test method was designed to be acceptable to the Japanese Ministry of Economy, Trade and Industry (METI), Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries.

Methods. 

Duplicate cultures of human lymphocytes, treated with the test item, 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, a 4-hour exposure 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-hour exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4-hour 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 o fTest Item (µg/ml)

4(20)-hour without S9

39.69, 79.38, 158.75, 317.5, 635, 1270

4(20)-hour with S9 (2%)

39.69, 79.38, 158.75, 317.5, 635, 1270

24-hour without S9

39.69, 79.38, 158.75, 317.5, 635, 1270

4(20)-hour with S9 (1%)

39.69, 79.38, 158.75, 317.5, 635, 1270

Results.

All vehicle (solvent) control groups 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 the maximum recommended dose level.

Conclusion.

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