Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

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 19 June 2006 and 06 September 2006.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: 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 relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report date:
2006

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:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of Inspection: 30th August 2005. Date of signature: 21/11/05.
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
478-130-6
EC Name:
-
Cas Number:
50940-49-3
Molecular formula:
C9H12O6
IUPAC Name:
2-(acryloyloxy)ethyl hydrogen succinate
Details on test material:
Sponsor's identification: MAES
Description: Colourless turbid liquid
Batch number: J4l7708-A
Date received: 29 March 2006
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):
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 17 hours under typical experimental exposure conditions.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/β-naphthoflavone induced rat liver, S9
Test concentrations with justification for top dose:
Preliminary Toxicity Test:
The dose range of test material used was 8.4 to 2160 µg/ml.

Experiment 1:
Without S9 mix: 0*, 8.8, 17.5, 35*, 70*, 105*, 140 µg/ml
With S9 mix: 0*, 17.5, 35, 70*, 105*, 140*, 280 µg/ml

Experiment 2:
Without S9 mix: 0*, 4.38, 8.75*, 17.5*, 35*, 52.5, 70 µg/ml
With S9 mix: 0*, 35, 70*, 105*, 140*, 210, 280 µg/ml

*Dose levels selected for metaphase analysis.

Vehicle / solvent:
The test material was accurately weighed, dissolved in MEM* and serial dilutions prepared. The molecular weight of the test material was given as 216.09, and therefore the maximum dose level was limited to 2160 µg/ml, which was calculated to be equivalent to 10 mM (the maximum recommended dose level). The purity of the test material was 95% and was accounted for in the formulations. There was no significant change in pH when the test material was dosed into media and the osmolality did not increase by more than 50 mOsm. Chemical analysis of the test material formulations was not performed because it is not a requirement of the test method.

MEM*: Eagle's minimal essential medium with HEPES buffer
Controlsopen allclose all
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, mitomycin C (MMC) was used at 0.4 and 0.2 µg/ml for cultures in Experiment 1 and 2 respectively. It was dissolved in Minimal Essential Medium.
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, cyclophosphamide (CP) was used at 4.0 µg/ml in both experiments. It was dissolved in dimethyl sulphoxide.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hours.
- Exposure duration and Expression time (cells in growth medium):
Experiment 1:
i) 4-hour exposure to the test material without S9-mix followed by 20-hour culture in treatment free media prior to cell harvest.
ii) 4-hour exposure to the test material with S9-mix followed by 20-hour culture in treatment free media prior to cell harvest.
Experiment 2:
i) 24-hour continous exposure to the test material without S9-mix prior to cell harvest.
ii) 4-hour exposure to the test material with S9-mix followed by 20-hour culture in treatment free media prior to cell harvest.
- Selection time (if incubation with a selection agent): Not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours

SELECTION AGENT (mutation assays): No selection agent applicable.
SPINDLE INHIBITOR (cytogenetic assays): Mitosis was arrested by addition of demecolcine (Colcemid 0.1 µg/ml ) two hours before the required harvest time.
STAIN (for cytogenetic assays): 5% Gurrs Giemsa

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 poylploid cells.

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
Key result
Species / strain:
lymphocytes: Human
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
: Small but statistically signficant increases in the frequency of cells with chromosome aberrations
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
:(Refer to any other information on results incl. tables section).
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: no significant change in pH when the test material was dosed into media.
- Effects of osmolality: osmolality did not increase by more than 50 mOsm
- Precipitation: Preliminary Toxicity Test - No precipitate of the test material was observed in the parallel blood-free cultures at the end of the exposure period in any of the exposure groups.

RANGE-FINDING/SCREENING STUDIES:
The dose range for the Preliminary Toxicity Test was 8.4 to 2160 µg/ml. The maximum dose was a 10 mM concentration. No precipitate of the test material was observed in the parallel blood-free cultures at the end of the exposure period in any of the exposure groups. Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present at up to 540 and 270 µg/ml in the 4(20)-hour exposures in the absence and presence of metabolic activation (S9) respectively. The maximum dose with metaphases present in the 24-hour continuous exposure was 33.8 µg/ml. The mitotic index data are presented in Table 1 (see atached background information). The test material induced evidence of toxicity in all three of the exposure groups.
The selection of the maximum dose level was based on toxicity for all exposure groups.

COMPARISON WITH HISTORICAL CONTROL DATA:
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.


Remarks on result:
other: strain/cell type: Human lymphocytes
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Additional information on cytotoxicity:

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 at 105 µg/ml in the absence of metabolic activation (S9). In the presence of metabolic activation (S9) the maximum dose level ofthe test material with metaphases suitable for scoring was 140 µg/ml, with no metaphases being observed at 280 µg/ml.

The mitotic index data are given in Table 2 (see attached background material). They confirm the qualitative observations in that a dose-related inhibition of mitotic index was observed and that 63% mitotic inhibition was achieved at 105 µg/ml in the absence of S9. In the presence of S9 a dosed-related inhibition of the mitotic index was also observed, with 18% inhibition being seen at 140 µg/ml. The maximum dose level selected for metaphase analysis was based on toxicity in both the absence and presence of metabolic activation.

The chromosome aberration data are given in Table 4 and Table 5 (see attached background material).

The test material induced small but statistically significant increases in the frequency of cells with aberrations in both the absence and presence of metabolic activation. The responses in both cases only marginally exceeded the historical maxima for vehicle controls. However, the presence of cells with multiple aberrations and a small number of chromatid exchange type aberrations would indicate the responses were biologically relevant, particularly if reproducible.

 

The polyploid cell frequency data are given in Table 8 (see attached background material). 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 qualitative assessment of the slides determined that there were metaphases suitable for scoring at 140 µg/ml in the presence of S9. In the absence of S9 the maximum test material dose level with metaphases suitable for scoring was 35 µg/ml. Some metaphases were present at 52.5 µg/ml but the quality was poor and they were only observed in the 'B' culture.

The mitotic index data are given in Table 3 (see attached background information). They confirm the qualitative observations in that a dose-related inhibition of mitotic index was observed and that 50% mitotic inhibition was achieved at 140 µg/ml in the presence of S9. In the absence of S9 28% mitotic inhibition was observed at 35 µg/ml, which was the maximum dose selected for metaphase analysis; the 52.5 µg/ml dose level was excluded because of the excessive toxicity seen in the 'A' culture.

The chromosome aberration data are given in Table 6 and Table 7 (see attached background information).

The test material induced small but statistically significant dose-related increases in the frequency of cells with chromosome aberrations in both the absence and presence of metabolic activation. As was seen in Experiment 1 the responses, whilst modest, included chromatid exchange type aberrations and cells with multiple aberrations. These responses confirmed that the responses observed in the first experiment were reproducible and, therefore, to be of toxicological significance. It should be noted that the test material was supplied as being 97% pure and the material safety data sheet listed the main impurity as 2-Hydroxyethyl acrylate at approximately 2%. 2-Hydroxyethyl acrylate has been reported (Dearfield, K.L. et al 1989) as a potent clastogen, mutagen and inducer of micronuclei at dose levels in the region of 15 to 20 µg/ml. It is therefore feasible that the responses observed may have been due to the genotoxic action of this impurity rather than the main component of the test material.

The polyploid cell frequency data are given in Table 8. The test material induced a small but statistically significant increase in the numbers of polyploid cells at 140 µg/ml in the presence of metabolic activation.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive

The test material induced small but statistically significant increases in the frequency of cells with chromosome aberrations in both the absence and presence of a liver enzyme metabolising system in two separate experiments. The test material was therefore considered to be weakly 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 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 Directive 2000/32/EC. 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 up to three dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study, ie. 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 was toxic and induced small but statistically significant increases in the frequency of cells with aberrations, in two separate experiments, using a dose range that included a dose level that induced approximately 50% mitotic inhibition.

Conclusion.

The test material was considered to be weakly clastogeriic to human lymphocytes in vitro.