1-Butanone, 3-(dodecylthio)-1-[(1R,2S)-2,6,6-trimethyl-3-cyclohexen-1-yl]-, rel-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames Test (OECD 471, GLP, K, rel. 1): non mutagenic up to 5000 µg/plate in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA102.

- CHO/hprt gene mutation assay (OECD 476, GLP, K, rel. 1): non mutagenic up to limit dose (10 mM)

- Human lymphocyte chromosome aberration test (OECD 473, GLP, K, rel. 1): non clastogenic up to limit dose (10 mM)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 31 March 2004 to 13 May 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Programme (Inspected on 2002-12-02)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Storage condition of test material: room temperature under nitrogen in the dark

Target gene:

Histidine gene

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbitone/beta-naphthoflavone induced rat liver S9.

Test concentrations with justification for top dose:

Preliminary cytotoxicity / range-finding test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate. Main tests: 0, 50, 150, 500, 1500 and 5000 µg/plate (the maximum recommended dose level).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (dried on molecular sieve)
- Justification for choice of solvent/vehicle: to improve water solubility of the test substance. Well known solvent/vehicle not reacting with the test substance. Volume of vehicle/solvent in the medium: not a requirement of the test guideline.
Vehicle/solvent controls (negative control) were tested in the current study.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

See Table 7.6.1/2

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

mitomycin C

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

See Table 7.6.1/2

Positive control substance:

benzo(a)pyrene

other: 2-Aminoanthracene; 1,8-Dihydroxyanthraquinone

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: ca. 48 hours at 37°C

NUMBER OF REPLICATIONS: triplicate plate per dose level

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (Preliminary cytotoxicity test)

OTHER: ACCEPTANCE CRITERIA: The reverse mutation assay was considered valid if the following criteria were met:
1. All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (according to historical control 2003 & 2004).
2. The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.
3. All tester strain cultures should be in the approximate range of 1 to 9.9 billion bacteria per ml.
4. Each mean positive control value should be at least two times the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
5. There should be a minimum of four non-toxic test material dose levels.
6. There should be no evidence of excessive contamination.

Rationale for test conditions:

Experiment 1 and 2 - Maximum concentration was 5000 μg/plate (the maximum recommended dose level).

Evaluation criteria:

EVALUATION CRITERIA: The test material may be considered positive in this test system if the following criteria are met: the test material should have induced a reproducible, dose-related and statistically (Dunnett's method of linear regression) significant increase in the revertant count in at least one strain of bacteria.

Statistics:

Dunnett's method of linear regression if necessary

Key result

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Evaporation from medium: Test material vapour pressure (0.0000075 Pa at 25 °C) is too low to expect a significant effect of evaporation on test results.
- Water solubility: Test substance was solubilized in DMSO to improve solubility
- Precipitation: observed at 1500 & 5000 µg/plate
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: A preliminary cytotoxicity/range-finding test was carried out to determine the toxicity of the test material and to select the appropriate dose levels for use in the main test (See in "Remarks on results including tables and figures"). No Cytotoxicity was observed for TA100 strain (no reduction in the background number of revertants per plate was observed for each concentration tested).

COMPARISON WITH HISTORICAL CONTROL DATA: All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls. The comparison was made with the historical control ranges for 2002 and 2003 of the corresponding Testing Laboratory (See Tables of results - Appendix 1 in "Background attached material").

ADDITIONAL INFORMATION ON CYTOTOXICITY: The test material was non-toxic to the bacterial background lawns of the strains of bacteria used.

1.) Preliminary Cytotoxicity Test:

The test material not toxic to the strain of Salmonella used (TA100). The test material formulation and S9-mix used in this experiment were both shown to be sterile.

The number of revertant colonies for the toxicity assay were:

With (+) or without (-) Metabolic Activation (S9)	Strain	Dose (μg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA100	86	81	77	87	77	96	87	85	75	74P	87P
+	TA100	115	63	118	125	118	98	86	96	92	78P	105P
P: Precipitate

 

2.) Mutation Test:

Results for the negative controls (spontaneous mutation rates) are presented in Table 1 (See Tables of results in “Background attached material”) and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations for the test material, vehicle and positive controls both with and without metabolic activation, are presented in Table 2 to Table 5 (See Tables of results in “Background attached material”).

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. An oily precipitate was observed at and above 1500 µg/plate, this did not prevent the scoring of revertant colonies.

No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

A small statistically significant increase in revertant colony frequency was observed in tester strain TAI00 at 1500 µg/plate (without metabolic activation) in experiment 1. However, the plate counts were within the acceptable range for this particular tester strain, there was no evidence of a dose-response relationship or reproducibility and the fold increase was only 1.28 times that of the concurrent solvent control. Therefore, the response was considered to of no biological or toxicological significance.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-rnix and the sensitivity of the bacterial strains.

Conclusions:

Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium (strains TA1535, TA 1537, TA98, TA100 & TA102).

Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 473, and in compliance with GLP, S. typhimurium strains TA 1535, TA 1537, TA 98, TA100 and TA 102 were exposed to the test material diluted in DMSO both in the presence and absence of mammalian metabolic activation (Phenobarbitone/beta-naphthoflavone induced rat liver S9).

The dose range was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations.

The vehicle (acetone) control plates gave counts of revertant colonies generally within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. An oily precipitate was observed at and above 1500 µg/plate, this did not prevent the scoring of revertant colonies.

No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

A small statistically significant increase in revertant colony frequency was observed in tester strain TA100 at 1500 µg/plate (without metabolic activation) in experiment 1. However, the plate counts were within the acceptable range for this particular tester strain, there was no evidence of a dose-response relationship or reproducibility and the fold increase was only 1.28 times that of the concurrent solvent control. Therefore, the response was considered to of no biological or toxicological significance.

Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium (strains TA1535, TA1537, TA98, TA100 & TA102).

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 19 August 2011 to 8 December 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline study complying with GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP (Date of inspection: 19-21 July 2011)

Type of assay:

mammalian cell gene mutation assay

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Source: CHO-K1 cell line was obtained from ECACC, Salisbury, Wiltshire.
- Type and identity of media: Ham's F12 medium, supplemented with 5% foetal bovine serum (FBS) and antibiotics (Penicillin/Streptomycin at 100 units/100 µg per mL) at 37°C with 5% CO2 in air
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: was done upon receipt of cells, not periodically checked
- Periodically checked for karyotype stability: not required
- Periodically "cleansed" against high spontaneous background: yes. Before the stocks of cells were frozen down they were cleansed of HPRT- mutants by culturing in HAT medium for 4 days. This is Ham's F12 growth medium supplemented with Hypoxanthine (13.6 µg/ml, 100 µM), Aminopterin (0.0178 µg/ml, 0.4 µM) and Thymidine (3.85 µg/ml, 16 µM). After 4 days in medium containing HAT, the cells were passaged into HAT-free medium and grown for 4 to 7 days. Bulk frozen stocks of HAT cleansed cells were frozen down, with fresh cultures being recovered from frozen before each experiment

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from the livers of male rats treated with phenobarbitone (80 mg/kg) and beta-naphthoflavone (100 mg/kg)

Test concentrations with justification for top dose:

123.44, 246.88, 493.75, 987.5, 1975, 3950 µg/mL in experiments 1 and 2, with and without S9 (based on the results of a preliminary cytotoxicity test)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The test item had previously been tested at Harlan Laboratories Ltd in a Chromosome Aberration Study, project number 0161/0495 and the solubility data from this study was used to select Acetone as the most suitable solvent.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

500 and 750 µg/mL in Exp 1. 200 and 300 µg/mL in Exp 2.

Positive control substance:

ethylmethanesulphonate

Remarks:

In the absence of metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

0.5 and 1 µg/mL

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

In the presence of metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: Experiment 1: overnight
Experiment 2: 2 days
- Exposure duration: Experiment 1: 4 hours without S9 / 4 hours with S9 (1 %);
Experiment 2 : 24 hours without S9 / 4 hours with S9 (2 %).
- Expression time (cells in growth medium): 7 days in non-selective medium; then 14 days in selective-medium

SELECTION AGENT (mutation assays): guanine or 6-TG
STAIN: Giemsa

NUMBER OF REPLICATIONS: duplicate

NUMBER OF CELLS EVALUATED: 2 x 10E5 cells/75 cm² flask to determine mutant frequency.


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency.
In triplicate at 200 cells/25 cm² flask in 5 mL of Hams F12 with 5% serum to determine cloning efficiency. Flasks were incubated for 7 days, fixed with methanol and stained with Giemsa. Colonies were manually counted, counts were recorded for each culture and the percentage cloning efficiency for each dose group calculated.

Rationale for test conditions:

Doses used in both experiment 1 & 2 were chosen based on the results of a preliminary cytotoxicity test

Evaluation criteria:

For a test item dose level to be considered as positive the mutant frequency value must exceed the vehicle control value by 20 x 10E-6 per survivor in order to compensate for random fluctuation in the 0 to 25 x 10E-6 background mutant frequencies that are typical for this assay.
A single dose level that meets the minimum criterion for a positive response within a range of assayed concentrations is not sufficient to evaluate the test item as a mutagen. The following test results must be obtained for either with or without-metabolic activation conditions before this conclusion can be made.
i) A dose-related or toxicity-related increase in mutant frequency should be observed. If possible this relationship should be obtained for at least four dose levels, but this will depend on the concentration steps chosen for the assay and the level of toxicity at which mutagenic activity appears.
ii) Within-group replicates will be pooled for final analysis. If replicates are not similar, it will be noted in the report.
iii) An equivocal/weak mutagenic dose-response in one mutation assay should be confirmed in a second assay. Test items that are insoluble or extremely toxic (rapid increase in toxicity over a small change in concentration) along with normal assay to assay variation may lead to different dose ranges being reported in the two assays.
iv) Treatments that reduce relative clonal survival to less than ten percent will not be scored for mutant frequency in the assay.
v) A test item will be evaluated as non-mutagenic in two assays only if the minimum increase in mutant frequency is not observed and the dose range applied, should ideally, give toxicity with about 10 to 20% survival. If the test article is relatively non-toxic it should have been tested to the maximum recommended dose level of 10 mM or 5 mg/ml, providing that solubility was not a problem.

Statistics:

If a test item gives a marked and dose-related increase in the mutant frequency over the vehicle controls it will be designated as mutagenic and statistical analysis will not be required. However, if weaker responses are observed then statistical analysis will be performed using the SPSS program or a suitable alternative. All weak responses will be assessed by the Study Director for biological relevance and justified in the report.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

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 marked change in pH
- Effects of osmolality: did not increase by more than 50 mOsm at the dose levels investigated
- Evaporation from medium: the test material is not a V.O.C.
- Water solubility: dissolved in acetone to improve solubility
- Precipitation: YES
EXP1: A cloudy precipitate was noted at the end of exposure period at all dose levels of the test item in the 4-hour exposure group in the absence of S9 and a greasy/oily precipitate was noted at and above 987.5 µg/mL. In the 4-hour exposure group in the presence of S9 a greasy/oily precipitate was also noted at and above 987.5 µg/mL. Inconsistencies in the precipitate observations between the preliminary toxicity test and Experiment 1 were considered to be due to the subjective nature of performing precipitate observations, particularly when performed by different technicians. Also the hazy/cloudy appearance of the media containing S9 may have masked the cloudy precipitate.
EXP 2: Cloudy precipitate was noted at the end of exposure at all dose levels of the test item in the 24-hour exposure group in the absence of S9 and a greasy/oily precipitate was noted at and above 987.5 µg/ml. In the 4-hour exposure group in the presence of S9 a greasy/oily precipitate was also noted at and above 987.5 µg/ml. The precipitate observations are consistent with those seen in Experiment 1. This agreement between the two experiments was considered to indicate the slight difference to the observations made in the preliminary toxicity test had no impact on the integrity of the study.

RANGE-FINDING/SCREENING STUDIES:
A dose range of 15.43 to 3950 µg/mL was used in the preliminary cytotoxicity test. There was a dose- related reduction in cloning efficiency (CE) in all three exposure groups. The greatest toxicity was seen in the 24-hour exposure group where a reduction in cloning efficiency of 77% was achieved at 3950 µg/mL when compared to the vehicle control. The 4-hour exposure group in the absence of S9 demonstrated a gradual reduction in toxicity from 61.72 to 3950 µg/mL where a reduction in cloning efficiency of 54% was achieved when compared to the vehicle control group. The toxicity seen in the 4-hour exposure group in the presence of S9 was less marked with a 29% reduction in cloning efficiency at the maximum dose when compared to the vehicle control. It was considered that the presence of S9 may have been protective against the toxicity of the test item.
A cloudy precipitate was noted at the end of exposure period in both the 4-hour exposure groups at all the dose levels of the test item. The precipitate was observed as becoming a greasy/oily precipitate at and above 123.44 µg/ml in the 4-hour without S9 exposure group and at and above 493.75 µg/mL in the 4-hour exposure group in the presence of S9. In the 24-hour exposure group a cloudy precipitate was noted at the end of exposure at 30.86 to 987.5 µg/mL with a greasy/oily precipitate at and above 246.88 µg/mL.
A precipitate of the test item was observed at most dose levels in all three exposure groups, which increased by degree in a dose-related fashion. However, the presence of precipitate did not limit the maximum dose level used in the mutation experiments because a dose-related increase in toxicity was also observed in parallel with the precipitate observations. The toxic effects were also greater following a longer duration of exposure (24 hours vs 4 hours) and in the absence of S9 (4 hours –S9 vs 4 Hours +S9). This suggested that the dose-related toxic effects were caused by increased exposure to the test item rather than to some artefactual mechanism related to the physical presence of precipitate. Therefore, the maximum dose level selected for both Experiments 1 and 2 was based on toxicity and, because in no case did the toxic effects exceed the maximum recommended level, the maximum recommended 10 mM dose level of 3950 µg/mL was selected for all exposure groups.

COMPARISON WITH HISTORICAL CONTROL DATA:
It can be seen that the vehicle control values were all within the maximum upper limit of 25 x 10E-6 mutants per viable cell in both Experiment 1 and Experiment 2, and that the positive controls all gave marked increases in mutant frequency, indicating the test and the metabolic activation system were operating as expected

Mutagenicity Test - Experiment 1

There was a modest reduction in the Day 0 and Day 7 cloning efficiencies in the presence of S9 which was consistent with the toxicity seen in the preliminary toxicity test. In the absence of S9 there was an increase in the Day 0 cloning efficiency above the vehicle control value at 1975 and 3950 μg/ml which was not consistent with the toxicity seen in the preliminary toxicity test. The ‘B’ replicates of the Day 0 viability flasks were generally lower than the ‘A’ replicates but this is considered to be normal variation associated with CHO cells.

The Day 0 and vehicle control cloning efficiencies for the 4-hour exposure group in the absence of S9 did not achieve 70%, however since they achieved at least 50% this was considered to be acceptable.

There were no increases in mutation frequency per survivor that exceeded the vehicle control value by 20 x 10-6 at any dose level in either exposure group.

Mutagenicity test - Experiment 2

In the 24-hour exposure group there was dose related toxicity observed at Day 0 with a reduction in cloning efficiency of 57% at 3950 μg/ml when compared to the vehicle control group. The toxicity observed in the 24-hour exposure group of Experiment 1 was less than that seen in the preliminary toxicity test but was probably more representative of the true toxicity effect as it was based on replicate flasks. The 4-hour with S9 exposure group demonstrated increased toxicity at Day 0 when compared to the 4-hour exposure of Experiment 1 which is considered to be due to the reduced S9 concentration (1% vs 2%) used in Experiment 2 on the basis that S9 was considered to be protective against the toxicity of the test item.

The vehicle control Day 0 counts for the 4-hour exposure group in the presence of S9 did not achieve 70%, however since cloning efficiencies of at least 50% were achieved the data was considered to be acceptable.

There were no increases in mutation frequency per survivor that exceeded the vehicle control value by 20 x 10-6 in either exposure group.

Conclusions:

Under the test conditions, test substance is not considered as mutagenic at the HPRT locus of CHO cells in the presence and absence of metabolic activation.

Executive summary:

In a mammalian cell gene mutation assay performed according to the OECD test guideline No. 476 and in compliance with GLP, Chinese hamster ovary (CHO) cells were exposed to the test material diluted in acetone at six dose levels, in duplicate, together with vehicle (solvent) and positive controls. Four treatment conditions were used for the test, 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 and a 4-hour exposure in the absence of metabolic activation (S9). In Experiment 2, the 4-hour exposure in the presence 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 ranges selected for Experiment 1 and Experiment 2 were based on the results of the preliminary cytotoxicity test and were as follows:-

Exposure Group	Final concentration of test item (µg/mL)
4-hour without S9	123.44, 246.88, 493.75, 987.5, 1975, 3950
4-hour with S9 (2%)	123.44, 246.88, 493.75, 987.5, 1975, 3950
24-hour without S9	123.44, 246.88, 493.75, 987.5, 1975, 3950
4-hour with S9 (1%)	123.44, 246.88, 493.75, 987.5, 1975, 3950

 

The vehicle (solvent) controls gave mutant frequencies within the range expected of CHO cells at the HPRT locus.

The positive control treatments, both in the presence and absence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and of the metabolising system.

The test item demonstrated no significant increases in mutant frequency in the exposure groups in the presence or absence of metabolic activation, in either the first or second experiment. The test item was considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of the test.

 

Under the test conditions, test substance is not considered as mutagenic at the HPRT locus of CHO cells in the presence and absence of metabolic activation.

 

This study is considered as acceptable and satisfies the requirement for the mammalian cell gene mutation endpoint.

Reference 3

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:

From 28 September 2005 to 24 February 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Programme (inspected on 2005-08-30)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

- Storage condition of test material: Approximately 4 °C under nitrogen in the dark

Target gene:

Not Applicable

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

Cells: 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 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.

- Type and identity of media: Cells were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented "in-house" with L-glutamine, penicillin/streptomycin, amphotericin B and 15% foetal calf serum, at 37°C with 5% C02 in air. The lymphocytes of fresh heparinised whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA) at 90 µg/ml final concentration.
- Properly maintained: no (not applicable)
- Periodically checked for Mycoplasma contamination: no (not applicable)
- Periodically checked for karyotype stability: no (not applicable)
- Periodically "cleansed" against high spontaneous background: yes/no (not applicable)

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/β-napthoflavone induced rat liver S9 (2 or 1%)

Test concentrations with justification for top dose:

Without S9: 0*, 123, 247, 494*, 988*, 1975*, 3950* µg/mL (based on a preliminary cytotoxicity test).
With S9: 0*, 123, 247, 494*, 988*, 1975*, 3950* µg/mL (based on a preliminary cytotoxicity test).
* Dose levels selected for metaphase analysis

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone (The test material was accurately weighed, dissolved in acetone and serial dilutions prepared)
- Justification for choice of solvent/vehicle: test material not soluble in water (<0.1 mg/L @ 25 °C)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

0.4 or 0.2 µg/mL for cultures in Experiment 1 and 2, respectively

Positive control substance:

mitomycin C

Remarks:

In the absence of S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

5 µg/mL

Positive control substance:

cyclophosphamide

Remarks:

In the presence of S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: mytogenic stimulation of lymphocytes with 90 µg/mL phytohaemagglutinin (PHA) was performed 48 hours before lymphocyte treatment with the test substance.
- Exposure duration: Exp. 1 : 4 hours.
Exp. 2 : 4 hours (+S9), 24 hours (-S9)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours


SPINDLE INHIBITOR (cytogenetic assays): Demecolcine (Colcemid) 0.1 µg/mL for 2 hours (2 hours before cell harvest)
STAIN (for cytogenetic assays): 5% Gurrs Giemsa for 5 min

NUMBER OF CELLS EVALUATED: 200 (metaphase analysis)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: YES
- Determination of endoreplication: YES
- Other: any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing.

OTHER: none

Rationale for test conditions:

Based on a preliminary cytotoxicity test.

Evaluation criteria:

Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there were 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.

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.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

No clear dose response toxicity relationship observed in the preliminary toxicity test

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 effect
- Effects of osmolality: no significant effect, no variation greater than 50 mOsm
- Evaporation from medium: no significant effect (Vapour pressure 0.0000075 Pa @ 25°C and MW> 300g/mole)
- Water solubility: <0.1 mg/L @ 20°C
- Precipitation: a precipitation was observed from 30.86 µg/mL with S9 and from 62 µg/mL without S9, this had no significant effect on the results of the study.
- Other confounding effects: none


RANGE-FINDING/SCREENING STUDIES:
The dose range for the Preliminary Toxicity Test was 15 to 3950 µg/mL. The maximum dose level was equivalent to a 10 mM concentration. A precipitate of the test material was observed in the parallel blood-free cultures at the end of the exposure, at and above 62 µg/mL in the 4(20)-hour pulse exposure group without S9, at and above 31 µg/mL in the 4(20)-hour pulse exposure group with S9 and in the 24-hour continuous exposure group. The precipitate observations taken in the Preliminary Toxicity Test were considered to be representative for the whole study. Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present at up to 3950 µg/mL in the 4(20)-hour exposures in the presence and absence of metabolic activation (S9), and in the 24-hour continuous exposure group. The mitotic index data are presented in Table 1 (Any other information on results incl tables). The test material induced some evidence of toxicity in the 4(20)-hour pulse exposure group with S9 and the 24-hour continuous exposure group. However, there was no clear dose-response relationship.
The selection of the maximum dose level was initially based on the 3950 µg/mL (10 mM) concentration for all of the exposure groups investigated.

COMPARISON WITH HISTORICAL CONTROL DATA: results did not diverge from in-house historical aberration ranges

Dose and material tested during first and second experiment:

 

1st experiment:

Group	Final concentration of test material (µg/mL)
4(20)-hour without S9	0*, 123, 247, 494*, 988*, 3950*, MMC 0.4*
4(20)-hour with S9	0*, 123, 247, 494*, 988*, 3950*, CP 5*

2^nd experiment

Group	Final concentration of test material (µg/ml)
24- hour without S9	0*, 123, 247, 494*, 988*, 3950*, MMC 0.2*
4(20)-hour with S9	0*, 123, 247, 494*, 988*, 3950*, CP 5*

 

Results:

(For Tables, see “background attached material”).

1st experiment: 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, 3950 µg/ml, in the absence and presence of metabolic activation (S9).The mitotic index data are given in Table 2. They confirm the qualitative observations, and indicated that no dose-related inhibition of mitotic index was observed in either exposure group. A plateau of toxicity was seen in both exposure groups with 17 to 26% mitotic inhibition in the dose levels assessed in the absence of S9, and 23 to 39% mitotic inhibition in the presence of S9.

 

The maximum dose level selected for metaphase analysis was the 10mMconcentration dose level (3950 µg/mL).

 

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. 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 in either the absence or presence of metabolic activation.

The polyploid cell frequency data are given in Table 8. The test material did not induce any statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

 

2nd experiment: The qualitative assessment of the slides determined that there were metaphases suitable for scoring present at the maximum test material dose level of 3950 µg/mL in the presence of S9. In the absence of S9 the maximum test material dose level with metaphases was 3950 µg/mL. However, the quality of the metaphases at 1975 and 3950 µg/mL was adversely affected by the toxicity of the test material and they were considered unsuitable for inclusion in the study.

The mitotic index data are given in Table 3. They confirm the qualitative observations in that no clear dose-related inhibition of mitotic index was observed in either of the exposure groups.

The maximum dose level selected for metaphase analysis was the same as Experiment I in the presence of S9, and was the 10mM concentration dose level (3950 µg/mL), in the absence of S9 the maximum dose was limited by test material toxicity to 988 µg/mL.

 

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. The positive control materials induced statistically significant increases in the frequency of cells with aberrations. The response in the presence of S9 was very modest and only just exceeded the historical vehicle control value. Though it should be noted that a few chromatid exchanges were observed, and this type of aberration is very un-common in vehicle control cultures. It was considered that the poor response was due to the toxicity of the positive control affecting the quality of the metaphases and possibly inducing cell-cycle delay. Extra metaphases were scored to give greater power to the statistical analysis and, therefore, the response was considered acceptable. 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 in either the absence or presence of metabolic activation.

 

The polyploid cell frequency data are given in Table 8. The test material did not induce any statistically significant increases in the numbers of polyploid cells at any dose level in either of the exposure groups.

 

Conclusions:

Based on the results, it is concluded that test substance is not clastogenic in human lymphocytes under the experimental conditions.

Executive summary:

In an in vitro chromosome aberration test performed according to OECD guideline No 473 and in compliance with GLP, human primary lymphocyte cultures were exposed to the test material diluted in acetone. Four treatment conditions were used for the study, i.e. In Experiment 1, 4 hours in the presence of an induced rat liver homogenate metabolizing 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.

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 moderately toxic and did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments.

 

Based on the results, it is concluded that test substance is not clastogenic in human lymphocytes under the experimental conditions.

 

This study is considered as acceptable and satisfies the requirement for in vitro cytogenicity in mammalian cells endpoint.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Table 7.6/1: Summary of genotoxicity tests

Test n°	Test / Guideline Reliability	Focus	Strains tested	Metabolic activation	Test concentration (µg/plate/mL)	Statement
1   Safepharm, 2004	Ames Test (OECD 471) K, rel. 1	Gene mutation	TA 1535, TA 1537, TA 98, TA 100, TA102	-S9 +S9	Up to 5000	-S9 : non mutagenic +S9 : non mutagenic
2   Harlan, 2012	CHO/hprt test (OECD 476) K, rel. 1	Gene mutation	Chinese hamster ovary cells (CHO-K1)	-S9 +S9	Up to 3950 (10 mM)	-S9 : non mutagenic +S9 : non mutagenic
3   Safepharm, 2006	HL CAT (OECD 473) K, rel. 1	Chromosomal aberration	Human Lymphocytes	-S9 +S9	Up to 3950 (10mM)	-S9 : non clastogenic +S9 : non clastogenic

 

Gene mutation Assays (Tests n° 1 -2):

A bacterial reverse mutation assay (Ames test) was performed according to OECD 471 test guidelines with the test material (See Table 1). No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either in the presence or absence of metabolic activation. The test material does not induce gene mutations in bacteria whereas all positive control chemicals (with and without metabolic activation) induced significant increase of colonies. The test material is therefore considered as non-mutagenic according to the Ames test.

Inability to produce gene mutation was confirmed in mammals using an in vitro forward mutation assay in Chinese hamster ovary cells (CHO-K1)(Test n°2). None of the dose levels up to the limit concentration of 10 mM, either in the presence or absence of metabolic activation, induced significant mutant frequency increases in the initial or repeat tests. The test material does not induce gene mutations at the hprt locus in CHO-K1 cells under activation and non activation conditions whereas both positive control chemicals (with and without metabolic activation) induced significant mutant frequency increases. The test material is therefore considered as negative for inducing gene mutations at the hprt locus in CHO-K1 cells under activation and non-activation conditions used in this assay. This result confirms the results of both Ames tests and extends the non-mutagenic effect of the test material to mammalian cells.

 

Chromosomal aberration (Test n°3)

The clastogenic potential of the test material was determined using an in vitro chromosome aberration test in human lymphocytes, which measures the potential of a substance to increase the incidence the of structural chromosome aberrations in cultured human lymphocytes.

None of the dose levels up to the limit concentration of 10 mM, either in the presence or absence of metabolic activation, induced significant increases in the frequency of cells with aberrations in either of two experiments. The test material does not induce structural aberrations in the chromosomes of human lymphocytes under activation and non-activation conditions, whereas both positive control chemicals (with and without metabolic activation) induced significant increases in the frequency of aberrant cells. The test material is therefore considered as negative for inducing chromosomal mutations in human lymphocyte cells under activation and non-activation conditions used in this assay.

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification for human health according to the Regulation (EC) No. 1272/2008.

Self-classification:

Based on the available data, no additional classification is proposed regarding germ cell mutagenicity according to the Regulation (EC) No. 1272/2008 (CLP) and according to the GHS.