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Genetic toxicity: in vitro

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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 12 May 2008 and 28 August 2008.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: 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 no effect the quality of the relevant results.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Date of GLP Inspection: 19-21 July 2011. Date of Signature: 31 August 2011
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Sponsor's identification: Diurea thickener 8
Description: Light brown solid
Purity: 95 %
Batch number: Not supplied
Date received: 12 September 2011
Expiry date: Not supplied
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 suitabilityThe 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:
Cell Growth Inhibition Test:
4(20)-hour without S9: 0, 2.0, 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250 and 500 µg/ml.
4(20)-hour with S9: 0, 2.0, 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250 and 500 µg/ml.
20-hour without S9: 0, 2.0, 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250 and 500 µg/ml.

Experiment 1
i) 4-hour exposure to the test item without S9-mix followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 0, 2, 4, 8, 16, 24 and 32 µg/ml.
ii) 4-hour exposure to the test item with S9-mix followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 0, 2, 4, 8, 16, 24 and 32 µg/ml.
Experiment 2
i) 24-hour continuous exposure to the test item without S9-mix prior to cell harvest. The dose range of test item used was 0, 2, 4, 8, 12, 16 and 24 µg/ml.
ii) 4-hour exposure to the test item with S9-mix followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 0, 2, 4, 8, 16, 24 and 32 µg/ml.
Vehicle / solvent:
Tetrahydrofuran
Justification for choice of solvent/vehicle:
The test item was accurately weighed, suspended in tetrahydrofuran (THF) and serial dilutions prepared. The molecular weight of the test item was provided by the Sponsor and was greater than 500. The test item was insoluble or non-suspendable in dimethyl sulphoxide at 100, 125, 250 and 500 mg/ml, insoluble in MEM at 50 mg/ml, insoluble at 100, 125, 250 and 500 mg/ml in acetone and insoluble in THF at 100 and 500 mg/ml. However, the test item was an emulsion in THF at 500 mg/ml, therefore, the maximum dose level was 500 µg/ml (the maximum achievable dose level). THF is toxic to human lymphocytes at and above 50 µl; therefore, the test item formulations were dosed using 50 µl aliquots. The purity of the test item was 95% and, therefore, an allowance was made when dose formulations were prepared.
Controlsopen allclose all
Untreated negative controls:
no
Remarks:
THF
Negative solvent / vehicle controls:
yes
Remarks:
THF
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
Remarks:
THF
Negative solvent / vehicle controls:
yes
Remarks:
THF
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:
METHODS OF APPLICATION:
In medium

DURATION
- Pre-incubation period:
48 hours
- Exposure duration:
Experiment 1 – 4 hours with and without S9. Experiment 2 – 24 hours without S9, 4 hours with S9.
- Expression time (cells in growth medium):
20 hours for 4 hours exposure
- Selection time (in incubation with a selective agent):
Not applicable
- Fixation time (start of exposure up to fixation or harvest of cells):
24 hours

SELECTION AGENT (MUTATION ASSAYS):
No selection agent selected

SPINDLE INHIBITOR (Cytogenetic assays):
Demecolcine

STAIN (for cytogenetic assays):
When 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 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 (Appendix 1). 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
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Preliminary Toxicity Test (Cell Growth Inhibition Test)
The dose range for the Preliminary Toxicity Test was 2.0 to 500 µg/ml. The molecular weight of the test item was greater than 500. However, the test item was not soluble in any of the acceptable vehicles to dose at 5000 µg/ml, therefore the maximum practical dose level was 500 µg/ml. A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, initially at and above 2.0 and 3.9 µg/ml, in the absence and presence of S9, in the 4(20)-hour cultures, respectively. A precipitate of the test item was observed in the parallel blood-free cultures at the end of the continuous exposure period at and above 3.9 µg/ml. It should be noted that in the absence of S9 the precipitate aggregated at and above 15.62 µg/ml, effectively reducing exposure of the test item to the cells.
Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells suitable for scoring were present up to 500 µg/ml in all of the exposure groups. There was some evidence of modest reductions in mitotic index in all of the exposure groups at the upper dose levels in the 4(20)-hour cultures, whilst no clear dose relationship was observed, it was apparent that maximum exposure was occurring at and around 31.25 µg/ml. The mitotic index data are presented in Table 1.
Therefore the selection of the maximum dose levels for the exposure groups was based on the onset of marked precipitating dose levels for all exposure groups in both experiments and the apparent maximum exposure occurring at and around 31.25 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 Diurea thickener 8 (µg/ml)
4(20)-hour without S9 0*, 2, 4*, 8, 16*, 24, 32*, MMC 0.4*
4(20)-hour with S9 0*, 2, 4*, 8, 16*, 24, 32*, CP 5*
*: dose levels selected for metaphase analysis

The qualitative assessment of the slides determined that test item induced precipitate 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, 32 µg/ml, in the absence and presence of metabolic activation (S9). A precipitate of the test item was observed at the end of exposure at and above 4 and 8 µg/ml in the absence and presence of S9 in the whole blood cultures. No media only cultures were employed for precipitate assessment.
The mitotic index data are given in Table 2. They confirm the qualitative observations in that no marked or dose-related inhibition of mitotic index was observed in the absence of S9 with 13% mitotic inhibition at 32 µg/ml in the presence of metabolic activation only. The maximum dose level of 32 µg/ml was selected for metaphase analysis for both exposure groups. Although the toxicity observed in the preliminary toxicity test was not reproduced, it was still considered that the test item had been adequately tested.
The chromosome aberration data are given in Table 4 and Table 5. All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated.
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 Table 4 and Table 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 Diurea thickener 8 (µg/ml)
24-hour without S9 0*, 2, 4, 8*, 12*, 16*, 24*, MMC 0.2*
4(20)-hour with S9 0*, 2, 4*, 8*, 16*, 24*, 32, CP 5*
* :dose levels selected for metaphase analysis

The qualitative assessment of the slides determined that there were metaphases suitable for scoring present at the maximum test item dose level of 24 µg/ml in the absence of S9 and at 32 µg/ml in the presence of S9. A precipitate of the test item was observed at the end of exposure, at and above 16 µg/ml, in the presence of S9, and at 24 µg/ml in the absence of S9. Based on the above observations at least one clear precipitating dose level was selected for mitotic index evaluation.
The mitotic index data are given in Table 3. There was a modest reduction in mitotic index (35%) in the continuous exposure group (without S9) at the upper dose of 24 µg/ml. No reduction in mitotic index was observed in the in the 4(20)-hour culture (with S9).
The maximum dose level selected for metaphase analysis was 24 µg/ml in both exposure groups.
The chromosome aberration data are given in Table 6 and Table 7. All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated.
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 Table 6 and Table 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.
Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

See overall remarks and attachments

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Thetest 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 method was designed to be compatible with 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 was also compatible with the UK Department of Health Guidelines for Testing of Chemicals for Mutagenicity.

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 Experiments 1 and 2 were selected using data from the preliminary toxicity test and were as follows:

Experiment

Group

Final concentration of test item (µg/ml)

1

4(20)-hour without S9

2, 4, 8, 16, 24 and 32

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

2, 4, 8, 16, 24 and 32

2

24-hour without S9

2, 4, 8, 12, 16 and 24

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

2, 4, 8, 16, 24 and 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 that the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item was considered to be only moderately toxic and 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 a dose level that was the lowest precipitating dose level.

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