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Toxicological information

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:
2011-12-08 - 2012-04-02
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 not 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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Government of the United Kingdom
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Sponsor's identification :
Description: Yellow slightly viscous liquid
Purity: 100%
Batch number :
Date received : 24 November 2011
Expiry Date : 31 December 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.
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 9.77 to 2500 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 test material (µg/mL)
4(20)-hour without S9 0*, 40, 80*, 160*, 320*, 480*, 640, MMC 0.4*
4(20)-hour with S9 (2%) 0*, 40, 80, 160*, 320*, 480*, 640* , 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 test material (µg/mL)
24-hour without S9 0*, 5, 10, 20, 40*, 50*, 60*, 80*, 120, MMC 0.2*
4(20)-hour with S9 (1%) 0*, 40, 80, 160*, 320*, 480, 640*, CP 5*

* Dose levels selected for metaphase analysis
MMC = Mitomycin C
CP = Cyclophosphamide
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: tetrahydrofuran (THF)
- Justification for choice of solvent/vehicle: THF 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:
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
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:
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:
Frequency of polyploid 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
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:
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 dissolved in THF.
- Precipitation:
Premlinary toxictiy test: A cloudy precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure period, at and above 156.25 µg/mL in the 24-hour exposure group and at between 312.5 and 1250 µg/mL inclusive, in both the 4(20)-hour exposure groups.

The precipitate increased in intensity with increasing concentration and was seen as precipitate at and above 312.5 µg/mL and greasy/oily precipitate at and above 625 µg/mL in all three exposure groups.

Experiment 1: A cloudy precipitate of the test item was observed at the end of the treatment period in both exposure groups at and above 320 µg/mL
which became precipitate at and above 480 µg/mL and a greasy/oily precipitate was also observed at 640 µg/mL.

Experiment 2: A cloudy precipitate of the test item was observed at the end of the treatment period at and above 160 µg/mL in the presence of S9 with a greasy/oily precipitate at and above 480 µg/mL. No precipitate was observed in the 24-hour exposure group at the end of the exposure period.

Preliminary Toxicity Test (Cell Growth Inhibition Test)
The mitotic index data are presented. It can be seen that the test item showed evidence of marked toxicity the 4(20)-hour exposure group in the absence of S9 and in the 24-hour exposure group. The toxicity seen in the 4(20)-hour exposure group in the absence of S9 increased with increasing dose up to 1250 µg/mL after which the toxicity was seen to decrease. This was considered to be due to the onset of greasy/oily precipitate at 625 µg/mL which effectively reduced exposure of the cells to the test item at dose levels above this concentration.
The 24-hour exposure group had metaphases suitable for scoring up to 78.13 µg/mL and demonstrated mitotic inhibition of 72% at this dose level.
The 4(20)-hour exposure group in the presence of S9 demonstrated a slight reduction in mitotic index of 21% at 625 µg/mL. At 1250 and 2500 µg/mL there was an increase in mitotic index over the vehicle control value which may be due to cell-cycle delay as a result of toxicity. Microscopic assessment of the slides prepared from the treatment cultures showed that metaphase cells were present up to 2500 µg/mL in both the 4(20)-hour exposure groups.

A cloudy precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure period, at and above 156.25 µg/mL in
the 24-hour exposure group and at between 312.5 and 1250 µg/mL inclusive, in both the 4(20)-hour exposure groups. The precipitate increased in
intensity with increasing concentration and was seen as precipitate at and above 312.5 µg/mL and greasy/oily precipitate at and above 625 µg/mL in all three exposure groups.
The maximum dose level selected in Experiment 1 and Experiment 2 was based on toxicity demonstrated in the 4(20)-hour exposure group in the
absence of S9 and in the 24-hour exposure group. Maximum exposure of the cells to the test item was considered to occur around the onset of greasy/oily precipitate and, therefore, the maximum dose level selected for the 4(20)-hour exposure group in the presence of S9 was also limited to 640 µg/mL as this is where the greatest level of toxicity was observed (21%). There was no clear demonstration of toxicity in this exposure group as the mitotic index values were observed to increase above the control value; though this could be due to test item toxicity inducing cell-cycle delay.

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 test material (µg/mL)
4(20)-hour without S9 0*, 40, 80*, 160*, 320*, 480*, 640, MMC 0.4*
4(20)-hour with S9 (2%) 0*, 40, 80, 160*, 320*, 480*, 640* , 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 test item dose level of 640 µg/mL in both the presence and absence of metabolic activation
(S9). It should be noted that in the absence of S9 there were clear qualitative signs of toxicity.
The results of the mitotic indices (MI) from the cultures after their respective treatments are reported. These data show no marked toxicity was demonstrated in the presence of S9, with a maximum value of 12% mitotic inhibition being achieved at 640 µg/mL. In the absence of S9 there was a dose related increase in toxicity with 48% mitotic inhibition being achieved at 480 µg/mL. The dose level of 640 µg/mL was not selected for mitotic index analysis due to the obvious toxicity observed during the qualitative assessment.
A cloudy precipitate of the test item was observed at the end of the treatment period in both exposure groups at and above 320 µg/mL which became precipitate at and above 480 µg/mL and a greasy/oily precipitate was also observed at 640 µg/mL.
The maximum dose level selected for metaphase analysis in the presence of S9 was the maximum dose level tested of 640 µg/mL. In the absence of S9 the maximum dose level selected for scoring was 480 µg/mL where near optimum toxicity was achieved.
The chromosome aberration data are reported. 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 reported. 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 test material (µg/mL)
24-hour without S9 0*, 5, 10, 20, 40*, 50*, 60*, 80*, 120, MMC 0.2*
4(20)-hour with S9 (1%) 0*, 40, 80, 160*, 320*, 480, 640*, CP 5*

The qualitative assessment of the slides determined that there were metaphases suitable for scoring present at the maximum test item dose level tested of 640 µg/mL in the presence of S9. In the 24-hour exposure group in the absence of S9 the maximum dose with metaphases suitable for scoring was 80 µg/mL. It should be noted in the absence of S9 at the higher dose levels the number of cells present were visibly seen to reduce even though the percentage of cells in metaphase did not appear to match this trend completely. This would indicate that some of the toxicity was not completely due to a cytotoxic mechanism but was due to physical disruption of the cells caused by the test item affecting the cell membrane resulting in reduced numbers of cells. The results of the mitotic indices (MI) from the cultures after their respective treatments are presented. These data show a dose related increase in toxicity in the presence of S9 with 51% mitotic inhibition being achieved at 640 µg/mL. The increased toxicity seen in Experiment 2 in the 4(20)-hour exposure group in the presence of S9 compared to Experiment 1 is considered to be due to the reduction in S9 concentration from 2% to 1%. In the 24-hour exposure group in the absence of S9, 34% mitotic inhibition was achieved at 80 µg/mL with no metaphases suitable for scoring at the higher dose level of 120 µg/mL. Although optimum toxicity was not achieved in the 24-hour exposure group due to the relatively steep toxicity curve it was considered that the test item had been adequately tested and the toxicity may also have been underestimated due to a low mitotic index value for the ‘B’ culture of the vehicle control group.

A cloudy precipitate of the test item was observed at the end of the treatment period at and above 160 µg/mL in the presence of S9 with a greasy/oily
precipitate at and above 480 µg/mL. No precipitate was observed in the 24-hour exposure group at the end of the exposure period.
The maximum dose level selected for metaphase analysis was 640 µg/mL in the presence of S9 and 80 µg/mL in the 24-hour exposure group in the
absence of S9. The ‘A’ replicate of the 80 µg/mL dose level in the 24-hour exposure group was relatively more toxic than the ‘B’ culture and only 89
metaphases were available for scoring, however this was considered to be acceptable as there were no aberrations present other than gaps.
The chromosome aberration data are reported. All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. As a result of a poor response initially an extra 100 metaphases were selected for scoring from the ‘A’ replicate of the positive control (MMC) in the 24-hour exposure group to verify the response. 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 reported. 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'.

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 anin 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 was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1997) No. 473 "Genetic Toxicology: Chromosome Aberration Test" and Method B.10 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. 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 took place. 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 for the 4(20)-hour groups (without S9, with S9 (2%), with S9 (1%)) 40, 80, 160, 320, 480 and 640 µg/mL. In the 24 -hour test group (without S9), concentrations of 5, 10, 20, 40, 50, 60, 80 and 120 µg/mL were tested.

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.

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