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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item was considered to be non-mutagenic under the conditions of an Ames test (OECD 471, EU Method B.13/14 and USA, EPA OCSPP harmonized guideline- Bacterial Reverse Mutation Test). The test item was also found to be non-clastogenic to human lymphocytes in vitro (OECD 474, EU Method B.10, OPPTS 870.5375 and guidelines/recommendations of the Japanese Ministry of Economy, Trade and Industry (METI), Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries).

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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:
04 June 2014 to 23 June 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: There were no deviations (unplanned changes) from the study plan.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Principles of method if other than guideline:
No
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
TA1537: his C 3076; rfa-; uvrB-
TA98: his D 3052; rfa-;uvrB-; R-factor
TA1535: his G 46; rfa-; uvrB-
TA100: his G 46; rfa-; uvrB-; R-factor
WP2uvrA: trp-; uvrA-
Species / strain / cell type:
E. coli WP2 uvr A
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
The S9 Microsomal fraction was prepared in-house from male rats induced with Phenobarbitone/beta-Naphthoflavone
Test concentrations with justification for top dose:
Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Vehicle / solvent:
The test item was miscible in dimethyl sulphoxide (after an extended period) and acetone at 50 mg/mL in solubility checks performed in-house
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-Aminoanthracene
Details on test system and experimental conditions:
Without Metabolic Activation
0.1 mL of the appropriate concentration of test item, vehicle or appropriate positive control was added to 2 mL of molten trace amino-acid supplemented media containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.

With Metabolic Activation
The procedure was the same as described previously except that following theaddition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the molten trace amino-acid supplemented media instead of phosphate buffer.

All of the plates were incubated at 37 °C± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).
Evaluation criteria:
All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks.
All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
All tester strain cultures should be in the range of 0.9 to 9 x 10^9 bacteria per mL.
Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation.
There should be a minimum of four non-toxic test item dose levels.
There should be no evidence of excessive contamination.

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
initially from 150 and 500 μg/plate in the absence and presence of S9-mix respectively.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
initially from 150 and 500 μg/plate in the absence and presence of S9-mix respectively.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate. Results from the first mutation test showed that the test item induced toxicity to all of the bacterial tester strains, initially from 150 and 500 μg/plate in the absence and presence of 89-mix respectively. These results meant that the test item was tested up to the toxic limit in Experiment 2. Results from the second mutation test were identical to Experiment 1 with weakened bacterial background lawns initially noted from 150 and 500 μg/plate in the absence and presence of 89-mix respectively. The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type and exposures with or without 89-mix. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of 89-mix.
There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in the first mutation test. Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 2.
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 89-mix and the sensitivity of the bacterial strains.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
The test item was considered to be non-mutagenic under the conditions of this test.
Executive summary:

GUIDELINE

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA OCSPP harmonized guideline- Bacterial Reverse Mutation Test.

METHODS

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using the Ames plate incorporation method at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended, following the results of Experiment 1, and ranged between 0.5 and 1500 µg/plate, depending on presence or absence of S9-mix.

Up to seven test item dose levels were selected in Experiment 2 in arder to achieve both four non-toxic dose levels and the toxic limit of the test item.

RESULTS

The vehicle (acetone) control plates gave counts of revertant colonies 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 maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. Results from the first mutation test showed that the test item induced toxicity to all of the bacterial tester strains, initially from 150 and 500 µg/plate in the absence and presence of S9-mix respectively. These results meant that the test item was tested up to the taxie limit in Experiment 2. Results from the second mutation test were identical to Experiment 1 with weakened bacterial background lawns initially noted from 150 and 500 µg/plate in the absence and presence of S9-mix respectively. The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type and exposures with or without S9-mix. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in the first mutation test. Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 2.

CONCLUSION

The test item was considered to be non-mutagenic under the conditions of this test.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 July 2014 to 11 December 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
Qualifier:
according to
Guideline:
other: 40 CFR 799.9537 TSCA in vitro mammalian chromosome aberration test
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: chromosome aberration
Target gene:
Not applicable
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES
buffer (MEM), supplemented "in-house" with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal bovine serum (FBS), at approximately 37 °C with 5% C02 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).
Metabolic activation:
with and without
Metabolic activation system:
S9 Microsomal fraction prepared in-house from male rats induced with Phenobarbitone/p-Naphthoflavone at 80/100 mg/kg/day, orally, for 3 days prior to preparation on day 4
Test concentrations with justification for top dose:
PRELIMINARY TOXICITY TEST
- Dose levels of 0, 9.42, 18.85, 37.70, 75.39, 150.78. 301.56, 603.13, 1206.25 and 2412.5 µg/mL

EXPERIMENT 1
- Dose levels of 0, 5, 10, 20, 40, 60 and 80 μg/mL (4-hour exposure to the test item without S9-mix, followed by 20-hour culture in treatment-free media prior to cell harvest)
- Dose levels of 0, 10, 20, 40, 60, 80 and 160 μg/mL (4-hour exposure to the test item with S9-mix (2 %) followed by 20-hour culture in treatment-free media prior to cell harvest)

EXPERIMENT 2
- Dose levels of 0, 5, 10, 20, 30, 40 and 60 0 μg/mL (24-hour continuous exposure to the test item without S9-mix prior to cell harvest)
- Dose levels of 0, 5, 10, 20, 40, 60 and 80 0 μg/mL (4-hour exposure to the test item with S9-mix (1 %) followed by 20-hour culture in treatment-free medium prior to cell harvest)
Vehicle / solvent:
Acetone
Negative solvent / vehicle controls:
yes
Positive control substance:
other: acetone
Remarks:
Fisher Scientific UK (Batch 1488268)
Positive controls:
yes
Remarks:
Experiment 1 in the absence of S9 mix
Positive control substance:
mitomycin C
Remarks:
Sigma (Batch SLBD1982V) dissolved in Minimal Essential Medium; 0.4 µg/mL
Positive controls:
yes
Remarks:
Experiment 2 in the absence of S9 mix
Positive control substance:
mitomycin C
Remarks:
Sigma (Batch SLBD1982V) dissolved in Minimal Essential Medium; 0.2 µg/mL
Positive controls:
yes
Remarks:
Experiment 1 in the presence of S9 mix
Positive control substance:
cyclophosphamide
Remarks:
Acros (Batch A0302605) dissolved in dimethyl sulphoxide; 5 µg/mL
Positive controls:
yes
Remarks:
Experiment 2 in the presence of S9 mix
Positive control substance:
cyclophosphamide
Remarks:
Sigma (Batch SLBG4216V) dissolved in dimethyl sulphoxide; 5 µg/mL
Details on test system and experimental conditions:
CELLS
- For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer who had been previously screened for suitability.
- The volunteer had not knowingly 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 mean value of the AGT for the pool of regular donors used in the laboratory had been determined to be approximately 16 hours under typical experimental exposure conditions.

TEST ITEM PREPARATION
- The molecular weight of the test item was given as 482.5, therefore, the maximum dose level was 4825 μg/mL, which was calculated to be equivalent to 10 mM, the maximum recommended dose level. However, due to solubility issues and the sensitivity of human lymphocytes to acetone, the maximum achievable concentration was 2412.5 μg/mL (equivalent to 5mM).
- The purity of the test item was 100 % and was not accounted for in the test item formulations.
- The test item was insoluble in dimethyl sulphoxide at 482.5 mg/mL but was soluble in acetone at the same concentration in solubility checks performed in-house. Normally, the solvent vehicle is dosed at 100 μL, thereby dosing at 1% of the culture volume (10 mL). However, human lymphocytes are sensitive to acetone at 100 μL; therefore, all of the formulations were prepared at concentrations two times greater than required in the culture flasks.
- To compensate, each formulation was dosed using 50 μL (0.05 mL) aliquots. Consequently, the maximum
concentration was the highest achievable concentration (2412.5 μg/mL).
- Prior to each experiment, the test item was accurately weighed, formulated in acetone and serial dilutions
prepared.
- There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al., 1991).
- The pH and osmolality readings are presented in the table below.
- The test item was formulated within two hours of it being applied to the test system; the test item formulations were assumed to be stable.
- No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation because it is not a requirement of the guidelines. This is an exception with regard to GLP and was reflected in the GLP compliance statement.

MICROSOMAL ENZYME FRACTION
- Lot numbers PB/PNF S9 27/07/14 and PB/PNF S9 12/10/14 were used in this study.
- The S9 microsomal fraction was prepared in-house from male rats induced with phenobarbitone/p-Naphthoflavone at 80/100 mg/kg/day, orally, for 3 days prior to preparation on day 4.
- The S9 homogenate was produced by homogenizing the liver in a 0.15 M KCl solution (1 g liver to 3 mL KCl) followed by centrifugation at 9000 g.
- The protein content of the resultant supernatant was adjusted to 20 mg/mL.
- Aliquots of the supernatant were frozen and stored at approximately -196 °C.
- Prior to use, each batch of S9 was tested for its capability to activate known mutagens in the Ames test.
- The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (10 to 20% (v/v)), MgCl2 (8mM), KCl (33 mM), sodium orthophosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM) and NADP (5mM).
- The final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2% (preliminary toxicity test and Experiment 1) or 1 % (Experiment 2).
- The procedure was designed and conducted to cause the minimum suffering or distress to the animals consistent with the scientific objectives and in accordance with the Harlan Laboratories Ltd, Shardlow, UK policy on animal welfare and the requirements of the United Kingdom's Animals (Scientific Procedure) Act 1986 Amendment Regulations 2012. The conduct of the procedure may be reviewed, as part of the Harlan Laboratories Ltd, Shardlow, UK Ethical Review Process.

CULTURE CONDITIONS
- Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing MEM, 10 % FBS (9.05 mL), Li-heparin (0.1 mL), phytohaemagglutin (0.1 mL) and heparinised whole blood (0.75 mL).

WITH METABOLIC ACTIVATION (S9) TREAMENT
- After approximately 48 hours incubation at approximately 37 °C, 5% C02 in humidified air, the cultures were transferred to tubes and centrifuged.
- Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 0.05 mL of the appropriate solution of vehicle control or test item was added to each culture.
- For the positive control, the appropriate solution (0.1 mL) was added to the cultures.
- S9-mix 20 % (1 mL) was added to the cultures of the preliminary toxicity test and of Experiment 1 to give 2 % final concentration of S9 in standard co-factors.
- In Experiment 2, S9-mix 10 % (1 mL) was added to give. 1 % final concentration of S9 in standard co-factors,
- All cultures were then returned to the incubator. The nominal final volume of each culture was 10 mL.
- After 4 hours at approximately 37 °C, 5% and C02 in humidified air, the cultures were centrifuged,
the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium.
- After a further centrifugation the wash medium was removed by suction and replaced with the original culture medium. The cells were then re-incubated for a further 20 hours at approximately 37 °C in 5% C02 in humidified air.

WITHOUT METABOLIC ACTIVATION (S9) TREATMENT
- In Experiment 1, after approximately 48 hours incubation at approximately 3 7 °C with 5% C02
in humidified air, the cultures were decanted into tubes and centrifuged.
- Approximately 9 mL of the culture medium was removed and reserved. The cells were then resuspended in the required volume of fresh MEM (including serum) and dosed with 0.05 mL of the appropriate vehicle control, test item solution or 0.1 mL of positive control solution. The total volume for each culture was a nominal 10 mL.
- After 4 hours at approximately 37 °C in 5% C02 in humidified air, the cultures were centrifuged the treatment medium was removed by suction and replaced with an 8 mL wash of MEM culture medium.
- After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium. The cells were then returned to the incubator for a further 20 hours.
- In Experiment 2, in the absence of metabolic activation, the exposure was continuous for 24 hours. Therefore, when the cultures were established the culture volume was a nominal 9.9 mL.
- After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 0.05 mL of vehicle control, test item dose solution or 0.1 mL of positive control solution. The nominal final volume of each culture was 10 mL. The cultures were then incubated at approximately 37 °C in 5% C02 in humidified air for 24 hours.
- The preliminary toxicity test was performed using both of the exposure conditions as described for Experiment 1 and for Experiment 2 in the absence of metabolic activation only.

PRELIMINARY TOXICITY TEST
- Three exposure groups were used.
(i) 4 hours exposure to the test item without S9-mix, followed by a 20-hour recovery period
in treatment-free media, 4(20)-hour exposure.
(ii) 4 hours exposure to the test item with S9-mix (2%), followed by a 20-hour recovery
period in treatment-free media, 4(20)-hour exposure.
(iii) 24-hour continuous exposure to the test item without S9-mix.
- The dose range of test item used was 0, 9.42, 18.85, 37.70, 75.39, 150.78, 301.56, 603.13, 1206.25 and 2412.5 μg/mL.
- Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.
- Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for mitotic index evaluation. Mitotic index data was used to estimate test item toxicity and for selection of the dose levels for the main test.

EXPERIMENT 1
- Two exposure groups were used.
(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, 5, 10, 20, 40, 60 and 80 μg/mL.
(ii) 4-hour exposure to the test item with S9-mix (2%), followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 0, 10, 20, 40, 60, 80 and 160 μg/mL.

EXPERIMENT 2
- Two exposure groups were used.
(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, 5, 10, 20, 30, 40 and 60 μg/mL.
(ii) 4-hour exposure to the test item with S9-mix (1 %) followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 0, 5, 10, 20, 40, 60 and 80 μg/mL.

CELL HARVEST
- Mitosis was arrested by addition of demecolcine (Colcemid 0.1 μg/mL) two hours before the required harvest time.
- After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in 0.075M hypotonic KCl.
- After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded.
- The cells were re-suspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v).
- The fixative was changed at least three times and the cells stored at approximately 4 °C to ensure complete fixation prior to slide preparation.

PREPARATION OF METAPHASE SPREADS
- The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and resuspension
in a small amount of fixative.
- Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labelled with the appropriate identification data.

STAINING
- When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

QUALITATIVE SLIDE ASSESSMENT
- The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test item.
- These observations were used to select the dose levels for mitotic index evaluation.

CODING
- The slides were coded using a computerized random number generator.

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 at least 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 and the ISCN (1985) (Appendix 1).
- Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding
the slides.
- In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) reported. In addition, the incidence of cells with endoreduplicated chromosomes was also reported.
- Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors. The current historical range is shown in Appendix 1 (attached).
Evaluation criteria:
EVALUATION CRITERIA
- Negative control: The frequency of cells with chromosome aberrations (excluding gaps) in the vehicle control cultures will normally be within the laboratory historical control data range.
- Positive control: All the positive control chemicals must induce a clear positive response (p:S0.01). Acceptable positive responses demonstrate the validity of the experiment and the integrity of the S9-mix.

CRITERIA FOR DETERMINING THE STUDY CONCLUSION
- A test item can be classified as non-genotoxic if (i) the number of induced chromosome aberrations in all evaluated dose groups is within the range of laboratory historical control data and (ii) no toxicologically or statistically significant increase of the number of structural chromosome aberrations is observed following statistical analysis.
- A test item can be classified as genotoxic if (i) the number of induced structural chromosome aberrations is not in the range of laboratory historical control data and (ii) either a concentration-related or a statistically significant increase of the number of structural chromosome aberrations is observed. Marked increases only observed in one dose level will be assessed on a case by case basis.
- Biological relevance of the results will be considered first. Statistical methods will be used to analyse the increases in aberration data as recommended in the OECD 473 guideline. However, statistical significance will not be the only determining factor for a positive response.
- A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case by case basis.
Statistics:
STATISTICAL ANALYSIS
- 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 (Richardson et al. 1989).
Key result
Species / strain:
other: human lymphocytes
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
- The dose range for the Preliminary Toxicity Test was 9.42 to 2412.5 μg/mL.
- The maximum dose was the maximum practical dose level which was based on the 5 mM concentration.
- A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, at and above 301.56 μg/mL in the 4(20)-hour exposure groups and at and above 603.13 μg/mL in the continuous exposure group.
- Haemolysis was observed following exposure to the test item between 150.78 and 1206.25 μg/mL in the 4(20)-hour exposure group in the absence of S9-mix, between 75.39 and 1206.25 μg/mL in the 4(20)-hour exposure group in the presence of S9-mix and at and above 301.56 μg/mL in the 24-hour continuous exposure
group. Haemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes.
- Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 37.70 μg/mL in the 4(20)-hour exposure without S9-mix, up to 75.39 μg/mL in the 4(20)-hour exposure with S9-mix and up to 18.85 μg/mL in the 24-hour continuous exposure. The mitotic index data are presented in Table 1 and shows that the test item induced excessive toxicity in all exposure groups.
- The selection of the maximum dose level was, therefore, based on toxicity for both Experiment 1 and Experiment 2.

CHROMOSOME ABERRATION TEST – EXPERIMENT 1
- Dose levels of the controls and the test item are given in the table below.
- 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 up to 40 μg/mL and 80 μg/mL in the absence and presence of S9-mix, respectively.
- The qualitative assessment also indicated a rapid onset of toxicity resulting in no scorable metaphases present above these dose levels.
- No precipitate or haemolysis was observed in the cultures at the end of exposure in either exposure group.
- The mitotic index data are given in Table 2 (attached). They confirm the qualitative observations in that in the 4(20)-hour exposure group in the absence of S9-mix, no dose-related inhibition of mitotic index was observed and 32% and 12% mitotic inhibition was achieved at 20 and 40 μg/mL, respectively. Above this dose level, there were either too few or no metaphases suitable for analysis. In the presence of S9-mix, a dose-related inhibition of mitotic index was observed with 28%, 48% and 48% mitotic inhibition observed at 20, 40 and 60 μg/mL, respectively (although 40 μg/mL was not analysed for chromosome aberrations). Again, above this dose level, there were either too few or no metaphases suitable for analysis.
- The maximum dose level selected for metaphase analysis was, therefore, 40 μg/mL and 60 μg/mL in the absence and presence S9-mix, respectively.
- The chromosome aberration data are given in Table 4 and Table 5 (attached).
- The vehicle control cultures in the absence of metabolic activation had frequencies of cells with chromosome aberrations within the expected range. The vehicle control cultures in the presence of metabolic activation had frequencies of cells with chromosome aberrations that slightly exceeded the historical range, however, they were still considered to be acceptable.
- 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.
- The polyploid cell frequency data are given in Table 8 (attached). 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
- Dose levels of the controls and the test item are given in the table below.
- Qualitative assessment of the slides determined that the toxicity was similar to that observed in the previous experiment that there were metaphases suitable for scoring present up to 60 μg/mL and 80 μg/mL in the absence and presence of S9-mix, respectively.
- No precipitate or haemolysis was observed in the cultures at the end of exposure in either exposure group.
- The mitotic index data are given in Table 3 (attached). They confirm the qualitative observations in that in the 24-hour continuous exposure group, a moderate dose-related inhibition of mitotic index was observed where 31 % and 66% mitotic inhibition was achieved at 40 and 60 μg/mL, respectively. In the presence of S9-mix, there was excessive toxicity noted at 80 μg/mL but no dose-related inhibition of mitotic index below this concentration. Therefore, the maximum dose level selected for metaphase analysis was 60 μg/mL for both exposure groups.
- The chromosome aberration data are given in Table 6 and Table 7 (attached).
- All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range.
- The positive control items induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.
- The test item did not induce any statistically significant increases in the frequency of cells with chromosome aberrations either in the absence or presence of metabolic activation.
- The polyploid cell frequency data are given in Table 8 (attached). 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.

DOSE LEVELS OF THE CONTROLS AND TEST ITEM IN THE CHROMOSOME ABERRATION TEST – EXPERIMENT 1

Group

Final concentration of test item (µg/mL)

4(20)-hour without S9

0*, 5, 10*, 20*, 40*, 60, 80, MMC 0.4*

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

0, 10*, 20*, 40*, 60*, 80, 160, CP 5*

* =Dose levels selected for metaphase analysis

MMC=Mitomycin C

CP=Cyclophosphamide

 

DOSE LEVELS OF THE CONTROLS AND TEST ITEM IN THE CHROMOSOME ABERRATION TEST – EXPERIMENT 2

Group

Final concentration of test item (µg/mL)

24-hour without S9

O*, 5, 10, 20*, 30*, 40*, 60*, MMC 0.2*

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

O*, 5, 10, 20*, 40*, 60*, 80, CP 5*

* =Dose levels selected for metaphase analysis

MMC=Mitomycin C

CP=Cyclophosphamide

Conclusions:
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:

GUIDELINE

Structural chromosomal aberrations were investigated in cultured mammalian cells in accordance withOECD Guidelines for Testing of Chemicals No. 473"In Vitro Mammalian Chromosome Aberration Test", adopted 21st July 1997, Method B.10 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, US EPA OPPTS 870.5375 Guideline, 40 CFR 799.9537 TSCAin vitromammalian chromosome aberration test and guidelines/recommendations of the Japanese Ministry of Economy, Trade and Industry (METI), Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries.

 

METHODS

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at 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 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.The dose levels used in the main experiment were selected using data from the preliminary toxicity test. The dose levels used in the main test were0, 5, 10, 20, 40, 60, 80 µg/mL for 4(20)-hour without S9, 0, 10, 20, 40, 60, 80 and 160 µg/mL for 4(20)-hour with S9 (2%), 0, 5, 10, 20, 30, 40, 60 µg/mL for 24-hour without S9 and 0, 5, 10, 20, 40, 60, 80 µg/mL for 4(20)-hour with S9 (1%).

 

RESULTS

All vehicle (acetone) 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. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected. The test item was toxic to human lymphocytes but did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments.

 

CONCLUSION

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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The test item was considered to be non-genotoxic under the conditions of the test (OECD 474, EU Method B.12, OPPTS 870.5395 and Japanese METI/MHLW/MAFF guidelines).

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 June 2014 to 03 August 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
not applicable
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes
Type of assay:
other: damage to chromosomes and/or aneuploidy
Species:
mouse
Strain:
ICR
Remarks:
CD-1 albino
Sex:
male
Details on test animals and environmental conditions:
ANIMALS AND ANIMAL HUSBANDRY
- Sufficient albino Hsd: ICR (CD-1) strain mice were obtained from Harlan Laboratories UK Ltd, Oxon, UK.
- At the start of the main test the mice weighed 22.7 to 30.0 g and were approximately six to ten weeks old.
- After a minimum acclimatisation period of five days the animals were selected at random and given a number unique within the study by tail marking and a number written on a colour coded cage card.
- Animals were housed in groups of up to seven in solid-floor polypropylene cages with wood-flake bedding.
- Free access to mains drinking water and food (Harlan Teklad 2014C Global Certified Rodent Diet supplied by Harlan Laboratories UK Ltd, Oxon, UK) was allowed throughout the study.
- Representative analyses of food and water quality were retained in the laboratory archive.
- Temperature and relative humidity were set to achieve limits of 19 to 25 °C and 30 to 70 % respectively.
- Rate of air exchange was approximately 15 changes per hour.
- Lighting was controlled by a time switch to give twelve hours light and twelve hours darkness.
Route of administration:
oral: gavage
Vehicle:
Arachis oil
Details on exposure:
TEST ITEM PREPARATION
- The test item was freshly prepared as required as a solution of appropriate concentration in arachis oil.
- No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and was reflected in the GLP compliance statement.
- The test item was formulated within two hours of it being applied to the test system and it was assumed that the formulation was stable for that duration.

POSITIVE CONTROL ITEM
- The positive control item was freshly prepared as required as a solution at the appropriate concentration in distilled water (Laboratoire Aguettant 3010081).

RANGE-FINDING TOXICITY TEST
- A range-finding toxicity test was performed to determine a suitable dose level and route of administration for the main test. The dose level selected should ideally be the maximum tolerated dose level or that which produces some evidence of toxicity up to a maximum recommended dose of 2000 mg/kg.
- The range-finding toxicity test was also used to determine if the main test should be performed using both sexes or males only.
- Groups of mice were dosed orally as shown in the table below.
- All animals were dosed once only at the appropriate dose level by gavage using a metal cannula attached to a graduated syringe. The volume administered to each animal was calculated according to its body weight at the time of dosing.
- Where applicable, animals were observed within the time points of 1 and 4 hours after dosing and subsequently once daily for two days.

MICRONUCLEUS TEST
- Groups of seven mice were dosed once only via the oral route with the test item at 250, 500 or 1000 mg/kg.
- Once group of mice from each dose level was killed by cervical dislocation 24 hours following treatment and a second group, dosed with the test item at 1000 mg/kg, was killed after 48 hours.
- One group of seven mice was dosed via the oral route with vehicle alone (arachis oil) and killed 24 hours after dosing.
- One group of five mice was dosed orally with cyclophosphamide and killed 24 hours after dosing. Cyclophosphamide is a positive control item known to produce micronuclei under the conditions of the test.
- Experimental design is summarised in the table below.
- All animals were observed 1 and 4 hours after dosing and then once daily, as applicable, and immediately prior to termination.
Duration of treatment / exposure:
24 or 48 hours
Frequency of treatment:
Single dose
Post exposure period:
Not applicable
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Seven
Control animals:
yes, concurrent vehicle
Positive control(s):
POSITIVE CONTROL ITEM
- Identification: Cyclophosphamide
- Source: Acros Organics
- Lot number: A0302605
- Harlan serial number: R-5819
- Purity: 97 %
- Expiry date: 06 January 2016
- Storage conditions: Approximately 4 °C in the dark
Tissues and cell types examined:
Both femurs were dissected from each animal immediately following sacrifice.
Details of tissue and slide preparation:
SLIDE PREPARATION
- Immediately following termination (24 or 48 hours following dosing), both femurs were dissected from each animal, and aspirated with foetal bovine serum.
- Bone marrow smears were prepared following centrifugation and re-suspension.
- The smears were air-dried, fixed in absolute methanol, stained in May-Grünwald/Giemsa and allowed to air-dry. A cover slip was then applied using mouting medium.
Evaluation criteria:
SLIDE EVALUATION
- Stained bone marrow smears were coded and examined blind using light microscopy at x1000 magnification.
- The incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored. Micronuclei are normally circular in shape, although occasionally they may be oval or half-moon shaped, and have a sharp contour with even staining.
- In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes was counted. These cells were also scored for incidence of micronuclei.
- The ratio of polychromatic to normochromatic erythrocytes was calculated together with appropriate group mean values and standard deviations.

INTERPRETATION OF RESULTS
- A comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the test item groups and the number occurring in the vehicle control group.
- A positive mutagenic response was demonstrated when a statistically significant, dose-responsive, toxicologically relevant increase in the number of micronucleated polychromatic erythrocytes was observed for either the 24 or 48-hour kill times when compared to the vehicle control group.
- If these criteria were not fulfilled, the test item was considered to be non-genotoxic under the conditions of the test.
- A positive response for bone marrow toxicity was demonstrated when the dose group mean polychromatic to normochromatic ratio was shown to be statistically significantly lower than the vehicle control group.
Statistics:
- All data were statistically analysed using appropriate statistical methods as recommended by the UKEMS sub-committee on Guidelines for Mutagenicity Testing Report, Part III (1989).
- Data was analysed following a square route (x + 1) transformation using Student’s t-test (two tailed) and any significant results were confirmed using the one way analysis of variance.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING TOXICITY TEST
- Mortality data are summarised in the table below.
- Clinical signs observed in animals dosed with the test item at 2000 mg/kg via the oral route were hunched posture, ptosis, lethargy, ataxia, decreased respiration, laboured respiration and prostration. Due to the severity of the clinical signs, one of the animals was killed in extremis.
- In animals dosed with the test item at 1200 mg/kg via the oral route, clinical signs observed were hunched posture, ptosis, lethargy, ataxia and splayed gait.
- In animals dosed with the test item at 1000 mg/kg via the oral route, clinical signs observed were hunched posture, ptosis, lethargy and tip-toe gait.
- Confirmatory animals were dosed with the test item at 1000 mg/kg and clinical signs of hunched posture, ptosis, ataxia and tip-toe gait were observed.
- The test item showed no marked difference in its toxicity to male or female mice and it was therefore considered acceptable to use males only for the main test.
- With evidence of acceptable toxicity via the oral route, the considered maximum tolerated dose (MTD) of the test item (1000 mg/kg) was selected for use in the main test with 500 mg/kg and 250 mg/kg as the lower dose levels.

MICRONUCLEUS TEST
- There were no premature deaths seen in any of the dose groups.
- Clinical signs of hunched posture, ptosis, ataxia, lethargy and splayed gait were observed at 1000 mg/kg in the 24 and 48-hour dose groups.

EVALUATION OF BONE MARROW SLIDES
- A summary of the results of the micronucleus test is given in Table 1 (attached).
- Individual and group mean data are presented in Tables 2 to 7 (attached).
- There were no statistically significant decreases in the PCE/NCE ratio in any test item exposure group.
- There were no statistically significant increases in the frequency of micronucleated PCEs in the 48-hour 1000 mg/kg dose group or the 250 mg/kg 24-hour 250 mg/kg dose group. Modest but statistically significant increases were observed in the 24-hour 1000 mg/kg and 500 mg/kg dose groups when compared to the vehicle control group. However, the group mean for the vehicle control was very low, none of the individual animals had any marked increases in the number of micronucleated PCE, and the group mean values were well within the historical control range. The responses were therefore considered to be artefactual and of no toxicological significance.
- The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

MORTALITY DURING RANGE-FINDING TOXICITY TEST

Dose level (mg/kg)

Sex

Number of animals treated

Route

Deaths on Day 0

Deaths on Day 1

Deaths on Day 2

2000

Male

1

Oral

1*

-

-

2000

Female

1

Oral

0

0

0

1200

Male

1

Oral

0

0

0

1200

Female

1

Oral

0

0

0

1000

Male

1

Oral

0

0

0

1000

Female

1

Oral

0

0

0

1000

Male

2

Oral

0

0

0

* = Animal killed in extremis

- = No data

Conclusions:
The test item was considered to be non-genotoxic under the conditions of the test.
Executive summary:

GUIDELINE

The study was performed to assess the potential of the test item to produce damage to chromosomes or aneuploidy when administered to mice. The method was designed to be compatible with the 1997 OECD Guidelines for Testing of Chemicals No 474 “Mammalian Erythrocyte Micronucleus Test”, Method B.12 of Commission Regulation (EC) No 440/2008 of 30 May 2008, the US EPA (TSCA) OPPTS 870.5395, EPA 712-C-98-226, August 1998 guidelines, and be acceptable to the Japanese METI/MHLW/MAFF guidelines for testing of new chemical substances.

 

METHODS

A range-finding test was performed to identify suitable dose levels of the test item, route of administration and to investigate whether there was a marked difference in toxic response between the sexes. There was no marked difference in toxicity between the sexes and the main test was performed using only male mice. The micronucleus test was conducted using the oral route in groups of seven male mice at the maximum tolerated dose of 1000 mg/kg with 500 mg/kg and 250 mg/kg as the two lower dose levels. Animals were killed 24 or 48 hours later, the bone marrow extracted, and smear preparations made and stained. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei. Additional groups of mice were given a single oral dose of arachis oil (7 mice) or doses orally with cyclophosphamide (5 mice) to serve as vehicle and positive controls respectively. Vehicle and positive control animals were killed 24 hours later.

 

RESULTS

There were no premature deaths in any of the dose groups. Clinical signs of hunched posture, ptosis, ataxia, lethargy and splayed gait were observed at 1000 mg/kg in the 24 and 48-hour dose groups. No statistically significant decreases in the PCE/NCE ratio were observed in any test item dose groups when compared with the vehicle control group. There were no statistically significant increases in the frequency of micronucleated PCEs in the 48-hour 1000 mg/kg group or the 24-hour 250 mg/kg group. Modest but statistically significant increases were observed in the 24-hour 1000 mg/kg and 500 mg/kg groups when compared to the vehicle control group. However, the group mean for the vehicle control was very low, none of the individual animals had any marked increases in the number of micronucleated PCE, and the group mean values were well within the historical control range. The responses were therefore considered to be artefactual and of no toxicological significance. The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the know mutagenic activity of cyclophosphamide under the conditions of the test.

 

CONCLUSION

The test item was considered to be non-genotoxic under the conditions of the test.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Ames test

GUIDELINE

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA OCSPP harmonized guideline- Bacterial Reverse Mutation Test.

METHODS

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using the Ames plate incorporation method at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended, following the results of Experiment 1, and ranged between 0.5 and 1500 µg/plate, depending on presence or absence of S9-mix.

Up to seven test item dose levels were selected in Experiment 2 in arder to achieve both four non-toxic dose levels and the toxic limit of the test item.

RESULTS

The vehicle (acetone) control plates gave counts of revertant colonies 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 maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. Results from the first mutation test showed that the test item induced toxicity to all of the bacterial tester strains, initially from 150 and 500 µg/plate in the absence and presence of S9-mix respectively. These results meant that the test item was tested up to the taxie limit in Experiment 2. Results from the second mutation test were identical to Experiment 1 with weakened bacterial background lawns initially noted from 150 and 500 µg/plate in the absence and presence of S9-mix respectively. The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type and exposures with or without S9-mix. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in the first mutation test. Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 2.

CONCLUSION

The test item was considered to be non-mutagenic under the conditions of this test

Chromosome aberration test

GUIDELINE

Structural chromosomal aberrations were investigated in cultured mammalian cells in accordance with OECD Guidelines for Testing of Chemicals No. 473"In Vitro Mammalian Chromosome Aberration Test", adopted 21st July 1997, Method B.10 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, US EPA OPPTS 870.5375 Guideline, 40 CFR 799.9537 TSCA in vitro mammalian chromosome aberration test and guidelines/recommendations of the Japanese Ministry of Economy, Trade and Industry (METI), Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries.

 

METHODS

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at 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 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.The dose levels used in the main experiment were selected using data from the preliminary toxicity test. The dose levels used in the main test were0, 5, 10, 20, 40, 60, 80 µg/mL for 4(20)-hour without S9, 0, 10, 20, 40, 60, 80 and 160 µg/mL for 4(20)-hour with S9 (2%), 0, 5, 10, 20, 30, 40, 60 µg/mL for 24-hour without S9 and 0, 5, 10, 20, 40, 60, 80 µg/mL for 4(20)-hour with S9 (1%).

 

RESULTS

All vehicle (acetone) 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. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected. The test item was toxic to human lymphocytes but did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments.

 

CONCLUSION

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 lymphocytesin vitro.

Mouse micronucleus test

GUIDELINE

The study was performed to assess the potential of the test item to produce damage to chromosomes or aneuploidy when administered to mice. The method was designed to be compatible with the 1997 OECD Guidelines for Testing of Chemicals No 474 “Mammalian Erythrocyte Micronucleus Test”, Method B.12 of Commission Regulation (EC) No 440/2008 of 30 May 2008, the US EPA (TSCA) OPPTS 870.5395, EPA 712-C-98-226, August 1998 guidelines, and be acceptable to the Japanese METI/MHLW/MAFF guidelines for testing of new chemical substances.

 

METHODS

A range-finding test was performed to identify suitable dose levels of the test item, route of administration and to investigate whether there was a marked difference in toxic response between the sexes. There was no marked difference in toxicity between the sexes and the main test was performed using only male mice. The micronucleus test was conducted using the oral route in groups of seven male mice at the maximum tolerated dose of 1000 mg/kg with 500 mg/kg and 250 mg/kg as the two lower dose levels. Animals were killed 24 or 48 hours later, the bone marrow extracted, and smear preparations made and stained. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei. Additional groups of mice were given a single oral dose of arachis oil (7 mice) or doses orally with cyclophosphamide (5 mice) to serve as vehicle and positive controls respectively. Vehicle and positive control animals were killed 24 hours later.

RESULTS

There were no premature deaths in any of the dose groups. Clinical signs of hunched posture, ptosis, ataxia, lethargy and splayed gait were observed at 1000 mg/kg in the 24 and 48-hour dose groups. No statistically significant decreases in the PCE/NCE ratio were observed in any test item dose groups when compared with the vehicle control group. There were no statistically significant increases in the frequency of micronucleated PCEs in the 48-hour 1000 mg/kg group or the 24-hour 250 mg/kg group. Modest but statistically significant increases were observed in the 24-hour 1000 mg/kg and 500 mg/kg groups when compared to the vehicle control group. However, the group mean for the vehicle control was very low, none of the individual animals had any marked increases in the number of micronucleated PCE, and the group mean values were well within the historical control range. The responses were therefore considered to be artefactual and of no toxicological significance. The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the know mutagenic activity of cyclophosphamide under the conditions of the test.

 

CONCLUSION

The test item was considered to be non-genotoxic under the conditions of the test.

Justification for classification or non-classification

In vitro tests demonstrated that the test material was non-mutagenic (Ames test) and non-clastogenic (chromosome aberration test). An in vivo study (mouse micronucleus test) also concluded that the test item was non-genotoxic. Classification in accordance with Regulation (EC) No 1272/2008 is therefore not required.