mecoprop-P (ISO); (R)-2-(4-chloro-2-methylphenoxy)propionic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Several genetic toxicity studies are available on the substance. All of the available studies were conducted to standardised guidelines which were relevant at the time of conduct. In one case, the method followed a draft guideline.

All of the studies were conducted in reputable laboratories and under GLP conditions. As such, every available study is regarded as a key study and contributes to the overall conclusion that the substance does not require classification with respect to genetic toxicity.

Ames: Thompson (2014)

Under the conditions of the study the test material was considered to be non-mutagenic.

Ames: May (2011)

Under the conditions of the study it is concluded that the test material showed no evidence of mutagenic activity in this bacterial system.

Ames: May (2010)

Under the conditions of the study it is concluded that the test material showed no evidence of mutagenic activity in this bacterial system.

Ames: May (2009)

Under the conditions of the study it is concluded that the test material showed no evidence of mutagenic activity in this bacterial system.

Ames: May (1990)

Under the conditions of the study, the test material was devoid of mutagenic activity.

Ames: Jones et al. (1993)

Under the conditions of the study it was concluded that, when tested in ethanol, the test material was not mutagenic in this bacterial system.

Chromosome Aberration: Heidemann (1994)

Under the conditions of the study and in presence of S9 mix, the test material was not clastogenic. In absence of S9 mix a final assessment of the clastogenic potential of the test material is difficult due to divergent results at cytotoxic concentrations.

Chromosome Aberration: Edwards (1990)

Under the conditions of study, the test material showed some evidence of clastogenic activity in the presence of S-9 mix, but only at a concentration which induced marked toxicity. 

In Vitro Mammalian Cell Gene Mutation Test: Adams et al. (1993)

Under the conditions of the study the test material acid did not demonstrate mutagenic potential in this in vitro test system.

In Vitro Mammalian Cell Gene Mutation Test: Lloyd (1990)

Under the conditions of the test, the test material induced no increases in mutation frequency at the HGPRT gene locus when cells were treated in the absence or presence of S-9 mix.

QSAR: Lye (2011)

All substances examined (i.e. the test material and 4 impurities) are predicted to be non-mutagenic with the restraints of the models.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 February 1990 to 23 February 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA FIFRA Guidelines

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9 : Young male CD rats, ca. 200 g bodyweight, were obtained from Charles River Breeding Laboratories (U.K.), Margate, Kent. Aroclor 1254 (500 mg/kg bodyweight in corn oil) was administered as a single intraperitoneal injection to induce microsomal enzyme activity. Four days after treatment, the animals were fasted overnight and then killed by cervical dislocation.
- Method of preparation of S9 mix: The livers were removed, washed in cold 0.15M KCl, then homogenised with more of the same medium (approximately 3 mL per g wet liver) in a Potter-Elvehjem homogeniser. Homogenates were centrifuged at 9 000 g for 10 minutes and supernatants collected and stored at -80 °C until required for preparation of the S-9 mix. Supernatant was used within 3 months of preparation.

S9-mix was prepared using:
0.4 mL 0.1 M NADP, sodium salt in aqueous solution
0.5 mL 0.1 M glucose-6-phosphate, sodium salt in aqueous solution
0.2 mL 0.4 M MgCl2·6H2O/1.65 M KCl aqueous solution
1.5 mL supernatant from liver homogenate
10 mL 0.1 M KH2PO4-Na2HPO4 buffer (pH7.4)

Test concentrations with justification for top dose:

Main Experiment: 10, 32, 100, 316 and 1 000 µg/plate (following preliminary toxicity test).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

benzo(a)pyrene

other: 2-Aminoanthracene 5 µg/plate in DMSO, with and without S9 mix, strain TA1535; 2-nitrofluorene 5 µg/plate in DMSO, without S9 mix, strain TA98.

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments : Each test, in each strain, was conducted on two separate occasions.

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added: Plate incorporation
- Pour-plate assay
An aliquot (0.1 mL) of each concentration of the test material was placed in a sterile tube. Molten, histidine-deficient top-agar (2 mL) and bacterial suspension (0.1 mL), maintained at 45 °C, was then added. The tubes were mixed by inversion and 0.5 mL rat liver microsomal preparation (S-9 mix) was added where appropriate. The tubes were again inverted to mix thoroughly and the contents poured onto plates containing solidified minimal medium (20 mL). Further plates were prepared without the inclusion of the test organisms to verify the sterility of the S-9 mix and the test material. A control series of plates was prepared to confirm the inability of DMSO (0.1 mL) to induce reversion in the bacterial strains, and to provide a measure of the spontaneous mutation rates.
Aliquots (0.1 mL) of a 10^-6 dilution of culture were spread on the surface of plates of complete medium to measure the viability and cell-density of each culture.
All plates were prepared in triplicate, allowed to solidify and incubated at 37 °C for 2 days. After incubation, numbers of revertant colonies were counted, either manually or with a Biotran II automatic colony counter. Total colonies on nutrient plates were counted in the same way. Growth of the background lawn of non-revertant cells on minimal plates was verified.


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Background growth inhibition
- Preliminary Toxicity Test: A solution of the test material was prepared in DMSO at 25 mg/mL, and an aliquot of this solution (0.2 mL) was transferred to a sterile tube containing molten, histidine-deficient top-agar (2.0 mL) maintained at 45 °C. An additional aliquot (0.1 mL) of the test material solution was similarly transferred to another tube of molten top-agar (2.0 mL).
Three serial ten-fold dilutions in molten top-agar were prepared from each of these preparations, giving a series of eight different concentrations of test material from 2.5 µg to 5 mg per plate. All tubes were inoculated with an overnight culture of strain TA98 (0.1 mL) and overlaid onto minimal medium plates. Control plates were prepared containing top-agar and culture alone, top-agar, DMS0 (0.2 mL) and culture, and top-agar and test material (0.1 mL) without bacterial culture.
The plates were incubated at 37 °C for 2 days and were then examined for the presence of a background lawn of non-revertant colonies; toxicity of the test material is shown by absence or thinning of the background lawn. The level of test material chosen as the top level for pour-plate tests is normally the lowest level causing visible thinning of the lawn. (In the absence of such thinning, a top level of 5 mg/plate would be selected):
The control plates were checked for the absence of growth on sterility checks or normal counts on negative controls in the presence and absence of the solvent.

Rationale for test conditions:

Based on results of preliminary toxicity test.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

- Preliminary toxicity test: The background lawn of non-revertant cells was absent following exposure to the test material at 2 500 µg per plate. A normal background lawn and revertant colony count was obtained with the test material at 500 µg per plate. A top exposure level of 1 000 µg per plate was therefore selected for use in the main assays.
- Sterility checks, spontaneous reversion rate and viability checks: The absence of colonies on the test material and S-9 mix sterility check plates indicates that these preparations were free of significant microbial contamination.
The total colony counts on plates number 18 confirmed the viability and high cell density of the cultures of the individual organisms. The counts recorded on appropriate negative control plates confirmed the characteristically low spontaneous reversion rates of the tester strains and the absence of effects on these rates of DMSO inclusion.
- Mutagenic activity of positive control chemicals: Appropriate positive control chemicals (with S-9 mix where required) induced marked increases in revertant colony numbers with all strains, confirming sensitivity of the cultures and activity of the S-9 mix.
- Action of the test material: No increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to the test material at levels from 10 to 1 000 µg per plate. Inhibition of growth, observed as thinning of the background lawn of non-revertant cells, occurred in all strains following exposure to the test material at 1 000 µg per plate.

Conclusions:

Under the conditions of the study, the test material was devoid of mutagenic activity.

Executive summary:

The test material was examined for mutagenic activity in four histidine-dependent auxotrophs of Salmonella typhimurium, strains TA98, TA100, TA1535 and TA1537, using pour-plate assays according to OECD Test Guideline 471 and in compliance with GLP. Each test, in each strain, was conducted on two separate occasions.

The studies, which were conducted in the absence and presence of an activating system derived from rat liver (S-9 mix), employed a range of levels of the test material from 10 to 1 000 µg per plate, selected following a preliminary toxicity test in strain TA98. All tests included solvent (dimethyl sulphoxide) controls with and without S-9 mix.

No increases in reversion to prototrophy were obtained with any of the four bacterial strains at the test material levels tested, either in the presence or absence of S-9 mix. Inhibition of growth, observed as thinning of the background lawn of non-revertant cells, occurred in all strains following exposure to the test material at 1 000 µg per plate.

Marked increases in the number of revertant colonies were induced by the known mutagens benzo[a]pyrene, 2-nitrofluorene, 2-aminoanthracene, 9-aminoacridine and sodium azide when examined under similar conditions.

Under the conditions of the study, the test material was devoid of mutagenic activity.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 July 1992 to 31 July 1992

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

Qualifier:

according to guideline

Guideline:

other: US Environmental Protection Agency, Method: HG-Gene Muta (40CFR798.5265) - S. typhimurium: The Salmonella typhimurium reverse mutation assay.

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability of the test substance in the solvent: The test material was found to be stable in the solvent for at least 4 hours. The test substance stock solution was analysed for each test for achieved concentration and was found to be within 2 % of the stated concentration of the active ingredient in both tests.

Target gene:

Histidine

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9 : Male Sprague-Dawley derived rat aged 7 - 8 weeks, weight < 300 g.
- Method of preparation of S9 mix: Mixed function oxidase systems in the livers of a group of rats were stimulated by Aroclor 1254, administered as a single intra-peritoneal injection in Arachis oil at a dosage of 500 mg/kg bodyweight. On the fifth day after injection, following an overnight starvation, the rats were killed, and their livers aseptically removed.
The following steps were carried out at 0 - 4 °C under aseptic conditions. The livers were placed in 0.15 M KCI (3 mL KCI: 1 g liver) before being transferred to an Ultra-Turrax homogeniser. Following preparation, the homogenates were centrifuged at 9 000 g for 10 minutes. The supernatant fraction (S-9 fraction) was dispensed into aliquots and stored at -80 °C until required.
S-9 mix contained: S-9 fraction (10 % v/v), MgCl2 (8 mM), KCI (33 mM), sodium phosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), NADP (4 mM). All the cofactors were filter-sterilised before use.
- Quality controls of S9: The efficacy of each batch of S-9 fraction was tested with the carcinogens 7, 12-dimethylbenzanthracene and 2-aminoanthracene before use.

Test concentrations with justification for top dose:

5 000, 1 500, 500, 150 and 50 μg of active ingredient/plate.
5 000 μg/plate is the highest concentration recommended by the regulatory guidelines with which this study complies.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: Prior to commencing testing the solubility of the test material in ethanol was assessed. The test material was found to be soluble in ethanol at 50 mg/mL so ethanol was used as the solvent for the assay.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-aminoanthracene with S9 mix, 2 µg/plate for strains TA1535 and TA1537, 0.5 µg/plate for strain TA98 and 1 µg/plate for strain TA100.

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments : Two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 10 hour bacterial culture, cell density not specified.
- Test substance added in medium: In suspension.
- The test material was added to cultures of the four tester strains at five concentrations separated by ca. half-log^10 intervals. The highest concentration of the test material used was 5 000 μg/plate. The negative control was the chosen solvent, ethanol. The positive control compounds were also included. An aliquot of 0.1 mL of a 10 hour bacterial culture and 0.5 mL S-9 mix or 0.5 mL 0.1 M phosphate buffer (pH 7.4) were placed in glass bottles. An aliquot of 0.1 mL of the test solution was added, followed immediately by 2 mL of histidine deficient agar. The mixture was thoroughly shaken and overlaid onto previously prepared petri dishes containing 25 mL minimal agar. Three petri dishes were used for each dose level. A set of plates were also prepared containing only bacterial culture and S-9 mix or phosphate buffer (0 μg/plate). Plates were also prepared without the addition of bacteria in order to assess the sterility of the test material, S-9 mix and phosphate buffer. The strains were also tested for the presence of the following genotypic markers; rja, uvrB and pKM101.

FOR GENE MUTATION:
- Expression time: All plates were incubated at 37 °C for 3 days. After this period revertant colonies were counted using a Biotran Automatic Colony Counter. At a later date the main test was repeated using the procedures described above using the same concentrations of test material.
- Method used: Agar overlay
- If a selective agent is used: Histidine deficient agar

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Growth inhibition
- Preliminary toxicity test: Four concentrations of test material were assessed for toxicity using the four tester strains. The highest concentration was 50 mg/mL of test material in the chosen solvent, which provided a final concentration of 5 000 μg/plate. Three 10-fold serial dilutions of the highest concentration were also tested. For strain TA100 three interim dose levels at c. half-log^10 intervals were also tested. The chosen solvent, ethanol, was used as the negative control.
An aliquot of 0.1 mL of a 10 hour bacterial culture and 0.5 mL S-9 mix or 0.5 mL 0.1 M phosphate buffer (pH 7.4) were placed in glass bottles. An aliquot of 0.1 mL of the test solution was added, followed immediately by 2 mL of histidine deficient agar. The mixture was thoroughly shaken and overlaid onto previously prepared petri dishes containing 25 mL minimal agar. A single petri dish was used for each dose level. Plates were also prepared without the addition of bacteria in order to assess the sterility of the test substance, S-9 mix and phosphate buffer. All plates were incubated at 37 °C for 3 days. After this period the appearance of the background bacterial lawn was examined. Revertant colonies were counted using a Biotran Automatic Colony Counter.
Any toxic effects of the test material can be detected by a substantial reduction in revertant colony counts or by the absence of a complete background bacterial lawn. In the absence of any toxic effects the top concentration used in the main tests is the same as that used in the preliminary toxicity test. If toxic effects are observed a lower concentration may be chosen for the main assays. Ideally the concentrations chosen for the mutation tests should include a minimum of three non-toxic concentrations.

Evaluation criteria:

Criteria for a valid assay
For the test to be considered valid the following criteria must have been met:
The presence of the relevant genotypic markers should have been confirmed. These markers were his, rfa, uvrB and pKM101. The mean revertant colony counts for the solvent control for each strain should have been within the ranges given below (based on HRC's historical data base).
TA1535: 5 - 20
TA1537: 5 - 20
TA98: 15 - 40
TA100: 70 - 160

Statistics:

The mean number of revertant colonies for all treatment groups was compared with those obtained for the solvent control groups. A test material is considered to show evidence of mutagenic activity if a reproducible statistically significant increase (p < 0.05) in revertant colony numbers is observed. Some evidence of a positive dose relationship should also be apparent. The statistical procedures are those described by Mahon et al. (1989) and analysis of variance followed by Dunnett's test was used.
The data analysed consisted of revertant colony counts obtained using bacterial strains TA1535, TA1537, TA98 and TA100 with and without S-9 mix. There were seven treatment groups (5 000, 1 500, 500, 150, 50, 0 μg/plate and solvent) and the test was carried out twice. The revertant colony counts for the positive control groups were also analysed to ensure that they were significantly greater than the concurrent solvent control group. Square root transformation was applied to the data to stabilise variances. Colony counts classified as contaminated were treated as missing values.
One-way analyses of variance were carried out for each test day, strain, with and without S-9 mix separately. The treatment groups were compared with the solvent using Dunnett's test.
Significance level testing was carried out at the 5 % level using one-tailed tests.
Significant differences are marked on the tables using the conventional asterisk notation: * P < 0.05

Species / strain:

S. typhimurium TA 1535

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:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

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:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

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:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

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:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

STUDY RESULTS
- Concurrent vehicle negative and positive control data : The concurrent positive control compounds demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations.
- Signs of toxicity : In the preliminary toxicity test no visible effect on the background bacterial lawn or decrease in revertant colony numbers was observed at any dose level in either the presence or absence of S-9 mix. There was no observed precipitation of the test material. Therefore 5 000 μg/plate was chosen as the top dose level in the mutation tests.
- With the exception of one case, no significant increases in revertant colony numbers of any of the tester strains were observed following treatment with the test material at any dose level, either in the presence or absence of S-9 mix. The one exception was a small increase observed in the second mutation test with tester strain TA 98 in the presence of S-9 mix at 150 μg/plate. This was a single significant increase with no other significant increases observed at any of the higher non-toxic dose levels. No significant increases occurred in the first mutation test so it was assumed that the significant increase arose by chance and was therefore considered not to be important.
- The genotypic markers rfa, uvrB and pKM101 were shown to be present in the appropriate strains in both mutation tests. All the revertant colony counts for the solvent controls were within the stated ranges for a valid assay.

Conclusions:

Under the conditions of the study it was concluded that, when tested in ethanol, the test material was not mutagenic in this bacterial system.

Executive summary:

The genotoxicity of the test material was assessed according to OECD Test Guideline 471, EU Method B.14 and in compliance with GLP.

In this in vitro assessment of the mutagenic potential of the test material, histidine dependent auxotrophic mutants of Salmonella typhimurium (strains TA1535, TA1537, TA98 and TA100) were exposed to the test material, diluted in ethanol which was also used as a negative control.

Two independent mutation tests were performed, in the presence and absence of liver preparations from Aroclor 1254-induced rats.

In the preliminary dose range finding study with dose levels of up to 5 000 μg/plate no toxicity was observed. A top dose level of 5 000 μg/plate was chosen for the subsequent mutation study. Other dose levels used in the mutation assays were: 1 500, 500, 150, 50 μg/plate.

No evidence of mutagenic activity was seen at any dose level of the test material in either mutation test. The concurrent positive control compounds demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations.

Under the conditions of the study it was concluded that, when tested in ethanol, the test material was not mutagenic in this bacterial system.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 September 2009 to 28 September 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: The stability of the test material and the stability and homogeneity of the test material in the vehicle were not determined as part of this study. Analysis of achieved concentration was not performed as part of this study.

Target gene:

Histidine for the Salmonella strains and tryptophan for the E. coli strain.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9 : S9 fraction, prepared from male Sprague-Dawley derived rats, dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver, was purchased from a commercial source and stored at approximately -80 °C.
- Method of preparation of S9 mix: The S9 mix contained: S9 fraction (10 % v/v), MgCl2 (8 mM), KCl (33 mM), sodium phosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), NADPH (4 mM) and NADH (4 mM) in water.
- Quality controls of S9: All the cofactors were filter-sterilised before use.

Test concentrations with justification for top dose:

5, 15, 50, 150, 500, 1 500, 5 000 µg/plate (Test 1)
50, 150, 500, 1 500, 5 000 µg/plate (Test 2)

The highest concentration of the test material tested in this study was 50 mg/mL in the chosen vehicle, which provided a final concentration of 5 000 μg/plate. This is the standard limit concentration recommended in the regulatory guidelines that this assay follows. The highest concentration in each test was diluted with DMSO to produce a series of lower concentrations, separated by approximately half-log^10 intervals.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solubility of the test material was assessed at 50 mg/mL in aqueous buffer (pH-adjusted) and in dimethyl sulphoxide (DMSO). It was found to be insufficiently soluble in buffer, but was soluble in DMSO. DMSO (ACS spectrophotometric grade) was, therefore, used as the vehicle for this study.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-Aminoanthracene 5 μg/plate in DMSO for strains TA100 and TA1535, 10 μg/plate in DMSO for strain WP2 uvrA (pKM101) in the presence of S9 mix.

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments: Two

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium: First experiment was a plate incorporation assay; the second included a pre-incubation stage.
First Test: Aliquots of 0.1 mL of the test material solutions (seven concentrations up to 5 000 μg/plate), positive control or negative control were placed in glass vessels. S9 mix (0.5 mL) or 0.1 M pH 7.4 phosphate buffer (0.5 mL) was added, followed by 0.1 mL of a 10-hour bacterial culture and 2 mL of agar containing histidine (0.05 mM), biotin (0.05 mM) and tryptophan (0.05 mM). The mixture was thoroughly shaken and overlaid onto previously prepared Petri dishes containing 25 mL minimal agar. Each Petri dish was individually labelled with a unique code, identifying the contents of the dish. Three Petri dishes were used for each treatment. Plates were also prepared without the addition of bacteria in order to assess the sterility of the test substance, S9 mix and sodium phosphate buffer. All plates were incubated at approximately 37 °C for ca. 72 hours. After this period, the appearance of the background bacterial lawn was examined and revertant colonies counted using an automated colony counter (Perceptive Instruments Sorcerer).
Second Test: As a clear negative response was obtained in the first test, a variation to the test procedure was used for the second test. The variation used was the pre-incubation assay in which the tubes, which contained mixtures of bacteria, buffer or S9 mix and test dilution, were incubated at 37 °C for 30 minutes with shaking before the addition of the agar overlay. The maximum concentration chosen was again 5 000 μg/plate, but only five concentrations were used.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Growth inhibition
- Any supplementary information relevant to cytotoxicity: Any toxic effects of the test material would be detected by a substantial reduction in mean revertant colony counts or by a sparse or absent background bacterial lawn. In the absence of any toxic effects, the maximum concentration selected for use in the second test would be the same as that used in the first. If toxic effects were observed, a lower concentration might be chosen, ensuring that signs of bacterial inhibition were present at this maximum concentration. Ideally, a minimum of four non-toxic concentrations should be obtained. If precipitate were observed on the plates at the end of the incubation period, at least four non-precipitating concentrations should be obtained, unless otherwise justified by the Study Director.

Evaluation criteria:

Acceptance Criteria
For a test to be considered valid, the mean of the vehicle control revertant colony numbers for each strain should lie within or close to the 99 % confidence limits of the current historical control range of the laboratory unless otherwise justified by the Study Director. The historical range is maintained as a rolling record over a maximum of five years. Also, the positive control compounds must induce an increase in mean revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) the concurrent vehicle controls. Mean viable cell counts in the 10-hour bacterial cultures must be at least 10^9/mL.
Criteria for Assessing Mutagenic Potential
If exposure to a test substance produces a reproducible increase in revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) the concurrent vehicle controls, with some evidence of a positive dose-response relationship, it is considered to exhibit mutagenic activity in this test system. No statistical analysis is performed. If exposure to a test substance does not produce a reproducible increase in revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed. If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. In general, treatment-associated increases in revertant colony numbers below two or three times the vehicle controls are not considered biologically important. It should be noted that it is acceptable to conclude an equivocal response if no clear results can be obtained.

Statistics:

The mean number and standard deviation of revertant colonies were calculated for all groups. The “fold-increases” relative to the vehicle controls were calculated in order to compare the means for all treatment groups with those obtained for the vehicle control groups.
The statistical procedures used are those described by Mahon et al (1989) and are usually Dunnett’s test followed, if appropriate, by trend analysis. Biological importance should always be considered along with statistical significance.

Species / strain:

S. typhimurium TA 1535

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:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

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:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

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:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

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:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A pKM 101

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:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

STUDY RESULTS
- Concurrent vehicle negative and positive control data : The absence of colonies on sterility check plates confirmed the absence of microbial contamination of the S9 mix, buffer and test material formulation.
The viability counts confirmed that the viable cell density of the cultures of the individual organisms exceeded 10^9/mL in all cases, and therefore met the acceptance criteria.
The mean revertant colony counts for the vehicle controls were all within historical limits and within or close to the 99 % confidence limits of the current historical control range of the laboratory. Appropriate positive control chemicals (with S9 mix where required) induced substantial increases in revertant colony numbers with all strains in all reported tests, confirming sensitivity of the cultures and activity of the S9 mix.

First Test
- No clear evidence of toxicity was obtained following exposure to the test material. A maximum exposure concentration of 5 000 μg/plate was, therefore, selected for use in the second test. No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to the test material at any concentration up to 5 000 μg/plate in either the presence or absence of S9 mix.
Second Test
- No evidence of toxicity was obtained following exposure to the test material. No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to the test material at any concentration up to 5 000 μg/plate in either the presence or absence of S9 mix.

Conclusions:

Under the conditions of the study it is concluded that the test material showed no evidence of mutagenic activity in this bacterial system.

Executive summary:

The genotoxicity of the test material was assessed according to OECD Test Guideline 471 and EU Method B. 13/14 and in compliance with GLP.

In this in vitro assessment of the mutagenic potential of the test material, histidine-dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan-dependent mutant of Escherichia coli, strain WP2 uvrA (pKM101), were exposed to the test material diluted in dimethyl sulphoxide (DMSO). DMSO was also used as a negative (vehicle) control.

Two independent mutation tests were performed in the presence and absence of liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone. The first test was a standard plate incorporation assay; the second included a pre-incubation stage. Concentrations of the test material up to 5 000 μg/plate were tested. This is the standard limit concentration recommended in the regulatory guidelines that this assay follows. Other concentrations used were a series of ca. half-log^10 dilutions of the highest concentration. No signs of toxicity were observed towards the tester strains in either mutation test following exposure to the test material at 5 000 μg/plate.

No evidence of mutagenic activity was seen at any concentration of the test material in either mutation test.

The concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations. The mean revertant colony counts for the vehicle controls were within or close to the 99 % confidence limits of the current historical control range of the laboratory.

Under the conditions of the study it is therefore concluded that the test material showed no evidence of mutagenic activity in this bacterial system.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 October 2010 to 25 October 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: The stability of the test material and the stability and homogeneity of the test material in the vehicle were not determined as part of this study. Analysis of achieved concentration was not performed as part of this study.

Target gene:

Histidine for the Salmonella strains and tryptophan for the E. coli strain.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9 : S9 fraction, prepared from male Sprague-Dawley derived rats, dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver, was purchased from a commercial source and stored at approximately -80 °C.
- Method of preparation of S9 mix: The S9 mix contained: S9 fraction (10 % v/v), MgCl2 (8 mM), KCl (33 mM), sodium phosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), NADPH (4 mM) and NADH (4 mM) in water.
- Quality controls of S9: All the cofactors were filter-sterilised before use.

Test concentrations with justification for top dose:

5, 15, 50, 150, 500, 1 500, 5 000 µg/plate (Test 1)
50, 150, 500, 1 500, 5 000 µg/plate (Test 2)

The highest concentration of the test material tested in this study was 50 mg/mL in the chosen vehicle, which provided a final concentration of 5 000 μg/plate. This is the standard limit concentration recommended in the regulatory guidelines that this assay follows. The highest concentration in each test was diluted with DMSO to produce a series of lower concentrations, separated by approximately half-log^10 intervals.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-Aminoanthracene 5 μg/plate in DMSO for strains TA100 and TA1535 in the presence of S9 mix; 10 μg/plate in DMSO for strain WP2 uvrA (PKM101) in the presence of S9 mix.

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments : Two

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium: First experiment was a plate incorporation assay; the second included a pre-incubation stage.
First test
Aliquots of 0.1 mL of the test material solutions (seven concentrations up to 5 000 μg/plate), positive control or vehicle control were placed in glass vessels. The vehicle control was DMSO. S9 mix (0.5 mL) or 0.1 M pH 7.4 phosphate buffer (0.5 mL) was added, followed by 0.1 mL of a 10-hour bacterial culture and 2 mL of agar containing histidine (0.05 mM), biotin (0.05 mM) and tryptophan (0.05 mM). The mixture was thoroughly shaken and overlaid onto previously prepared Petri dishes containing 25 mL minimal agar. Each Petri dish was individually labelled with a unique code, identifying the contents of the dish. Three Petri dishes were used for each treatment. Plates were also prepared without the addition of bacteria in order to assess the sterility of the test material, S9 mix and sodium phosphate buffer. All plates were incubated at approximately 37 °C for ca. 72 hours. After this period, the appearance of the background bacterial lawn was examined and revertant colonies counted using an automated colony counter (Perceptive Instruments Sorcerer).
Second test
As a clear negative response was obtained in the first test, a variation to the test procedure was used for the second test. The variation used was the pre-incubation assay in which the tubes, which contained mixtures of bacteria, buffer or S9 mix and test dilution, were incubated at 37 °C for 30 minutes with shaking before the addition of the agar overlay. The maximum concentration chosen was again 5 000 μg/plate, but only five concentrations were used.


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Growth inhibition
- Any supplementary information relevant to cytotoxicity:
Any toxic effects of the test material would be detected by a substantial reduction in mean revertant colony counts or by a sparse or absent background bacterial lawn. In the absence of any toxic effects, the maximum concentration selected for use in the second test would be the same as that used in the first. If toxic effects were observed, a lower concentration might be chosen, ensuring that signs of bacterial inhibition were present at this maximum concentration. Ideally, a minimum of four non-toxic concentrations should be obtained. If precipitate were observed on the plates at the end of the incubation period, at least four non-precipitating concentrations should be obtained, unless otherwise justified by the Study Director.

Evaluation criteria:

Acceptance criteria
For a test to be considered valid, the mean of the vehicle control revertant colony numbers for each strain should lie within or close to the 99 % confidence limits of the current historical control range of the laboratory unless otherwise justified by the Study Director. The historical range is maintained as a rolling record over a maximum of five years. Also, the positive control compounds must induce an increase in mean revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) the concurrent vehicle controls. Mean viable cell counts in the 10-hour bacterial cultures must be at least 10^9/mL.

Criteria for Assessing Mutagenic Potential
If exposure to a test substance produces a reproducible increase in revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) the concurrent vehicle controls, with some evidence of a positive dose-response relationship, it is considered to exhibit mutagenic activity in this test system. No statistical analysis is performed. If exposure to a test substance does not produce a reproducible increase in revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed. In general, treatment-associated increases in revertant colony numbers below two or three times the vehicle controls are not considered biologically important. It should be noted that it is acceptable to conclude an equivocal response if no clear results can be obtained.

Statistics:

The mean number and standard deviation of revertant colonies were calculated for all groups. The “fold-increases” relative to the vehicle controls were calculated in order to compare the means for all treatment groups with those obtained for the vehicle control groups.
If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used are those described by Mahon et al. (1989) and are usually Dunnett’s test followed, if appropriate, by trend analysis. Biological importance should always be considered along with statistical significance.

Species / strain:

S. typhimurium TA 1535

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:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

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:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

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:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

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:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A pKM 101

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:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

STUDY RESULTS
- Concurrent vehicle negative and positive control data : The absence of colonies on sterility check plates confirmed the absence of microbial contamination of the S9 mix, buffer and test material formulation.
The viability counts confirmed that the viable cell density of the cultures of the individual organisms exceeded 10^9/mL in all cases, and therefore met the acceptance criteria.
The mean revertant colony counts for the vehicle controls were within or close to the 99 % confidence limits of the current historical control range of the laboratory. Appropriate positive control chemicals (with S9 mix where required) induced substantial increases in revertant colony numbers with all strains in all reported tests, confirming sensitivity of the cultures and activity of the S9 mix.

First test
- No evidence of toxicity was obtained following exposure to the test material. A maximum exposure concentration of 5 000 μg/plate was, therefore, selected for use in the second test. No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to the test material at any concentration up to and including 5 000 μg/plate in either the presence or absence of S9 mix.

Second test
- No evidence of toxicity was obtained following exposure to the test material. No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to the test material at any concentration up to and including 5 000 μg/plate in either the presence or absence of S9 mix.

Conclusions:

Under the conditions of the study it is concluded that the test material showed no evidence of mutagenic activity in this bacterial system.

Executive summary:

The genotoxicity of the test material was assessed according to OECD Test Guideline 471 and EU Method B.13/14 and in compliance with GLP.

In this in vitro assessment of the mutagenic potential of the test material, histidine-dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan-dependent mutant of Escherichia coli, strain WP2 uvrA (pKM101), were exposed to the test material diluted in dimethyl sulphoxide (DMSO). DMSO was also used as a negative (vehicle) control.

Two independent mutation tests were performed in the presence and absence of liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone. The first test was a standard plate incorporation assay; the second included a pre-incubation stage. Concentrations of the test material up to 5 000 μg/plate were tested. This is the standard limit concentration recommended in the regulatory guidelines that this assay follows. Other concentrations used were a series of ca. half-log^10 dilutions of the highest concentration. No signs of toxicity were observed towards the tester strains in either mutation test following exposure to the test material.

No evidence of mutagenic activity was seen at any concentration of the test material in either mutation test.

The concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations. The mean revertant colony counts for the vehicle controls were within or close to the 99 % confidence limits of the current historical control range of the laboratory.

Under the conditions of the study it is therefore concluded that the test material showed no evidence of mutagenic activity in this bacterial system.

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 July 2011 to 09 August 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: The stability of the test material and the stability and homogeneity of the test material in the vehicle were not determined as part of this study. Analysis of achieved concentration was not performed as part of this study.

Target gene:

Histidine for the Salmonella strains and tryptophan for the E. coli strain.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9 : S9 fraction, prepared from male Sprague-Dawley derived rats, dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver, was purchased from commercial source and stored at approximately -80 °C.
- Method of preparation of S9 mix: The S9 mix contained: S9 fraction (10 % v/v), MgCl2 (8 mM), KCl (33 mM), sodium phosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), NADPH (4 mM) and NADH (4 mM) in water.
- Quality controls of S9: All the cofactors were filter-sterilised before use.

Test concentrations with justification for top dose:

5, 15, 50, 150, 500, 1 500, 5 000 µg/plate (Test 1)
50, 150, 500, 1 500, 5 000 µg/plate (Test 2)

The highest concentration of the test material tested in this study was 5 000 μg/plate. This is the standard limit concentration recommended in the regulatory guidelines that this assay follows.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-Aminoanthracene 5 μg/plate in DMSO for strains TA100 and TA1535 in the presence of S9 mix, 10 μg/plate in DMSO for strain WP2 uvrA (pKM101) in the presence of S9 mix.

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments : Two

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added: The first test was a standard plate incorporation assay; the second included a pre-incubation stage.
First test
Aliquots of 0.1 mL of the test material solutions (seven concentrations up to 5 000 μg/plate), positive control or vehicle control were placed in glass vessels. The vehicle control was DMSO. S9 mix (0.5 mL) or 0.1 M pH 7.4 phosphate buffer (0.5 mL) was added, followed by 0.1 mL of a 10-hour bacterial culture and 2 mL of agar containing histidine (0.05 mM), biotin (0.05 mM) and tryptophan (0.05 mM). The mixture was thoroughly shaken and overlaid onto previously prepared Petri dishes containing 25 mL minimal agar. Each Petri dish was individually labelled with a unique code, identifying the contents of the dish. Three Petri dishes were used for each treatment. Plates were also prepared without the addition of bacteria in order to assess the sterility of the test material, S9 mix and sodium phosphate buffer. All plates were incubated at approximately 37 °C for ca. 72 hours. After this period, the appearance of the background bacterial lawn was examined and revertant colonies counted using an automated colony counter (Perceptive Instruments Sorcerer).
Any toxic effects of the test material would be detected by a substantial reduction in mean revertant colony counts or by a sparse or absent background bacterial lawn. In the absence of any toxic effects, the maximum concentration selected for use in the second test would be the same as that used in the first. If toxic effects were observed, a lower concentration might be chosen, ensuring that signs of bacterial inhibition were present at this maximum concentration. Ideally, a minimum of four non-toxic concentrations should be obtained. If precipitate were observed on the plates at the end of the incubation period, at least four non-precipitating concentrations should be obtained, unless otherwise justified by the Study Director.

Second test
As a clear negative response was obtained in the first test, a variation to the test procedure was used for the second test. The variation used was the pre-incubation assay in which the tubes, which contained mixtures of bacteria, buffer or S9 mix and test dilution, were incubated at 37 °C for 30 minutes with shaking before the addition of the agar overlay. The maximum concentration chosen was again 5 000 μg/plate, but only five concentrations were used.

Evaluation criteria:

Acceptance criteria
For a test to be considered valid, the mean of the vehicle control revertant colony numbers for each strain should lie within or close to the 99 % confidence limits of the current historical control range of the laboratory unless otherwise justified by the Study Director. The historical range is maintained as a rolling record over a maximum of five years. Also, the positive control compounds must induce an increase in mean revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) the concurrent vehicle controls. Mean viable cell counts in the 10-hour bacterial cultures must be at least 10^9/mL.

Criteria for assessing mutagenic potential
If exposure to a test substance produces a reproducible increase in revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) the concurrent vehicle controls, with some evidence of a positive dose-response relationship, it is considered to exhibit mutagenic activity in this test system. No statistical analysis is performed. If exposure to a test substance does not produce a reproducible increase in revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.
In general, treatment-associated increases in revertant colony numbers below two or three times the vehicle controls (as described above) are not considered biologically important. It should be noted that it is acceptable to conclude an equivocal response if no clear results can be obtained.

Statistics:

The mean number and standard deviation of revertant colonies were calculated for all groups. The “fold-increases” relative to the vehicle controls were calculated in order to compare the means for all treatment groups with those obtained for the vehicle control groups.

If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used are those described by Mahon et al. (1989) and are usually Dunnett’s test followed, if appropriate, by trend analysis. Biological importance should always be considered along with statistical significance

Species / strain:

S. typhimurium TA 1535

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:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

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:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

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:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

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:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A pKM 101

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:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

STUDY RESULTS
- Concurrent vehicle negative and positive control data : The absence of colonies on sterility check plates confirmed the absence of microbial contamination of the S9 mix, buffer and test material formulation.
The viability counts confirmed that the viable cell density of the cultures of the individual organisms exceeded 10^9/mL in all cases, and therefore met the acceptance criteria.
The mean revertant colony counts for the vehicle controls were within or close to the 99 % confidence limits of the current historical control range of the laboratory. Appropriate positive control chemicals (with S9 mix where required) induced substantial increases in revertant colony numbers with all strains in all reported tests, confirming sensitivity of the cultures and activity of the S9 mix.

First test
- No evidence of toxicity was obtained following exposure to the test material. A maximum exposure concentration of 5 000 μg/plate was, therefore, selected for use in the second test. No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to the test material at any concentration up to and including 5 000 μg/plate in either the presence or absence of S9 mix.

Second test
- No evidence of toxicity was obtained following exposure to the test material. No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to the test material at any concentration up to and including 5 000 μg/plate in either the presence or absence of S9 mix.

Conclusions:

Under the conditions of the study it is concluded that the test material showed no evidence of mutagenic activity in this bacterial system.

Executive summary:

The genotoxicity of the test material was assessed according to OECD Test Guideline 471 and EU Test Method B.13/14 and in compliance with GLP.

In this in vitro assessment of the mutagenic potential of the test material, histidine-dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan-dependent mutant of Escherichia coli, strain WP2 uvrA (pKM101), were exposed to the test material diluted in dimethyl sulphoxide (DMSO). DMSO was also used as a negative (vehicle) control.

Two independent mutation tests were performed in the presence and absence of liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone. The first test was a standard plate incorporation assay; the second included a pre-incubation stage. Concentrations of the test material up to 5 000 μg/plate were tested. This is the standard limit concentration recommended in the regulatory guidelines that this assay follows. Other concentrations used were a series of ca. half-log^10 dilutions of the highest concentration. No signs of toxicity were observed towards the tester strains in either mutation test following exposure to the test material.

No evidence of mutagenic activity was seen at any concentration of the test material in either mutation test.

The concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations. The mean revertant colony counts for the vehicle controls were within or close to the 99 % confidence limits of the current historical control range of the laboratory.

Under the conditions of the study it is therefore concluded that the test material showed no evidence of mutagenic activity in this bacterial system.

Reference 6

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 September 2014 to 03 October 2014

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

8147 Guideline 2-1-19-1

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test material was accurately weighed and approximate half-log dilutions prepared in acetone by mixing on an autovortex mixer on the day of each experiment. Formulated concentrations were adjusted to allow for the stated water/impurity content (8.37 %) of the test material. Acetone is toxic to the bacterial cells at 0.1 mL (100 μL) after employing the pre-incubation modification; therefore all of the formulations for Experiment 2 were prepared at concentrations two times greater than required on Vogel-Bonner agar plates. To compensate, each formulation was dosed using 0.05 mL (50 μL) aliquots. All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test material formulations is not a requirement of the test guidelines and was, therefore, not determined. This is an exception with regard to GLP and has been reflected in the GLP compliance statement. Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 x 10^4 microns.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9 : The S9 Microsomal fraction was prepared in-house from male rats induced with Phenobarbitone/β-Naphthoflavone at 80/100 mg/kg/day, orally, for 3 days prior to preparation on day 4. The S9 homogenate was produced by homogenising the liver in a 0.15M KCl solution (1 g liver to 3 mL KCl) followed by centrifugation at 9 000 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.
- Method of preparation of S9 mix: The S9-mix was prepared before use using sterilised co-factors and maintained on ice for the duration of the test.
5.0mL S9
1.0mL 1.65 M KCl/0.4 M MgCl2
2.5mL 0.1 M Glucose-6-phosphate
2.0mL 0.1 MNADP
25.0mL 0.2 M Sodium phosphate buffer (pH 7.4)
14.5 mL Sterile distilled water
- Quality controls of S9: Prior to use, each batch of S9 was tested for its capability to activate known mutagens in the Ames test.
0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment.

Test concentrations with justification for top dose:

Plate Incorporation Method (Experiment 1): 1.5, 5, 15, 50, 150, 500, 1 500 and 5 000 μg/plate
The maximum concentration was the maximum recommended dose level.

Pre-Incubation Method (Experiment 2): 5, 15, 50, 150, 500, 1 500, 5 000 μg/plate (Salmonella strains TAlOO, TA1535 and TA98 and E.coli strain WP2uvrA); 1.5, 5, 15, 50,150, 500, 1 500, 5 000 μg/plate (Salmonella strain TA1537).
Up to eight test material dose levels were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the toxic limit of the test material following the change in test methodology.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: The test material was insoluble in sterile distilled water at 50 mg/mL but fully soluble in dimethyl sulphoxide at the same concentration and acetone at 100 mg/mL in solubility checks performed in-house. Acetone was selected as the vehicle as it was immediately soluble with the test material.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene 1 µg/plate for strain TA100, 2 µg/plate for straina TA1535 and TA1537, 10 µg/plate for strain WP2 uvrA in the presence of S9 mix.

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments : Two

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added: First experiment - Plate incorporation method; second experiment - Pre-incubation method.

First experiment - Plate incorporation method: Concentrations of the test material (1.5, 5, 15, 150, 500, 1 500 and 5 000 µg/plate) were assayed in triplicate against each tester strain.
- Without metabolic activation: Aliquots of 0.1 mL of the test solutions, positive controls or vehicle control were added to 2 mL of molten amino-acid supplemented media containing 0.1mL of the bacterial strain culture and 0.5 mL phosphate buffer. There 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.
- With metabolic activation: The same procedure as without metabolic activation, except 0.5 mL of S-9 mix was added to the molten trace amino-acid supplemented media instead of phosphate buffer.
- All of the plates were incubated at 37 ± 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). Several manual counts were required, predominantly due to revertant colonies spreading slightly, thus distorting the actual plate count

Second experiment - Pre-incubation method:
In the first experiment a clear negative response was observed and therefore experiment 2 was performed using the pre-incubation method.
The dose range used was determined by experiment 1, therefore, Strains TA100, TA1535, TA98 and WP2uvrA was tested with: 5, 15, 50, 150, 500, 1 500 and 5 000 µg/plate dose. Strain TA1537 was tested with 1.5, 5, 15, 150, 500, 1 500 and 5 000 µg/plate dose.
- Without metabolic activation:
0.1mL of the bacterial strain culture, 0.5 mL of phosphate buffer and 0.05 mL of test material formulation, vehicle, or 0.1mL positive control were incubated at 37 ± 3 °C for 20 minutes (with shaking) prior to addition of 2m L molten amino-acid supplemented media and plated onto Vogel-Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method.
- With metabolic activation:
Same procedure as without metabolic activation except following the addition of the test material formulation and bacterial strain culture, 0.5 mL of S9-mix was added instead of phosphate buffer, prior to incubation at 37 ± 3 °C for 20 minutes and addition of molten amino-acid supplemented media.
- All of the plates were incubated at 37 ± 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:

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 material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test material activity. Results of this type will be reported as equivocal.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

STUDY 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.

First experiment:
- In the first mutation test (plate incorporation method), the test material caused a visible reduction in the growth of the bacterial background lawns and/or a substantial reduction in the revertant colony frequency of all of the tester strains, initially from 1 500 μg/plate (TA1537 dosed in both the absence and presence of S9-mix) and at 5 000 μg/plate to the remaining strains dosed in both the absence and presence of S9-mix. Consequently, the maximum recommended dose level of the test item was employed in the second mutation test.
There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

Second experiment:
- In the second mutation test (pre-incubation method), the test material induced a similar toxic response with weakened bacterial background lawns noted in both the presence and absence of S9-mix to TA1537 at and above 1 500 μg/plate and to the remaining strains at 5 000 μg/plate. A test material precipitate (greasy in appearance) was noted at 5 000 μg/plate, this observation did not prevent the scoring of revertant colonies.
There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

Conclusions:

Under the conditions of the study the test material was considered to be non-mutagenic.

Executive summary:

The genotoxicity of the test material was assessed according to OECD Test Guideline 471 and EU Method B.13/14 and in compliance with GLP.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvr A were treated with the test material using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10 % liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5 000 μg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test material formulations. The dose range was amended following the results of Experiment 1 and ranged between 1.5 and 5 000 μg/plate, depending on bacterial strain type and presence or absence of S9-mix. Up to eight test material dose levels were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the toxic limit of the test material following the change in test methodology.

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 material in the first experiment was selected as the maximum recommended dose level of 5 000 μg/plate. In the first mutation test (plate incorporation method), the test material caused a visible reduction in the growth of the bacterial background lawns and/or a substantial reduction in the revertant colony frequency of all of the tester strains, initially from 1 500 μg/plate (TA1537 dosed in both the absence and presence of S9-mix) and at 5 000 μg/plate to the remaining strains dosed in both the absence and presence of S9-mix. Consequently, the maximum recommended dose level of the test material was employed in the second mutation test. In the second mutation test (pre-incubation method), the test material induced a similar toxic response with weakened bacterial background lawns noted in both the presence and absence of S9-mix to TA1537 at and above 1500 μg/plate and to the remaining strains at 5 000 μg/plate. A test material precipitate (greasy in appearance) was noted at 5 000 μg/plate, this observation did not prevent the scoring of revertant colonies. There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation in Experiment 1 (plate incorporation method). Similarly, 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 with or without metabolic activation in Experiment 2 (pre-incubation method).

Under the conditions of the study the test material was considered to be non-mutagenic.

Reference 7

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 February 1990 to 17 March 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

1983

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Solubility and stability of the test material in the solvent/dispersant/vehicle/test medium: No determinations of homogeneity and concentration were performed on prepared solutions of test material.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The solution of maximum concentration was prepared initially; lower concentrations were prepared from this by serial dilution in DMSO. All concentrations and dosages cited in this report refer to the test material sample as received; no allowance has been made for purity or activity below 100 %.

Species / strain / cell type:

lymphocytes: Human peripheral blood lymphocytes

Details on mammalian cell type (if applicable):

For lymphocytes:
- Sex, age and number of blood donors: Human peripheral blood was obtained by venepuncture from a healthy, non-smoking, male, human volunteer not currently taking any medication, and collected in heparinised vessels. Small inocula of whole blood (0.5 mL) were added to tubes containing 9.0 mL of culture medium and 0.5 mL phytohaemagglutinin to stimulate lymphocytes to divide. The tubes were sealed and incubated at 37 °C with occasional shaking to prevent clumping. After 48 hours of incubation, cultures were treated.

MEDIA USED
Culture medium:
443 mL RPMI 1640 medium with HEPES, sodium bicarbonate, and L-glutamine
50 mL Foetal calf serum
5 mL Heparin (100 i.u./mL)
2 mL Penicillin/streptomycin (5000 i.u./mL; 5000 µg/mL)

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9 : Aroclor 1254 (500 mg/kg bodyweight in corn oil) was administered to young male CD rats (ca. 200 g bodyweight) as a single intraperitoneal injection to induce microsomal enzyme activity. Four days after treatment, the animals were fasted overnight and then killed by cervical dislocation. The livers were removed, washed in cold 0.15M KCl, then homogenised with one volume of the same medium in a Potter-Elvehjem homogeniser. Homogenates were centrifuged at 9 000 G for 10 minutes, and supernatants collected and stored at -196 °C until required for preparation of the S-9 mix. Supernatant is used within six months of preparation.

- Method of preparation of S9 mix:
7.4 mL 0.1M KH2PO4-Na2HPO4 buffer
0.2 mL 0.4M MgCl2.6H2O/1.65M KCl aqueous solution
0.4 mL 0.1M NADP, sodium salt, in aqueous solution
0.5 mL 0.1M glucose-6-phosphate, sodium salt, in aqueous solution
Supernatant from liver homogenate to final volume of 1.5 mL.

Test concentrations with justification for top dose:

Preliminary toxicity test: 8, 40, 200, 1 000 and 5 000 µg/mL with and without S9 mix.
Cytogenetic test: 100, 200, 400 and 800 µg/mL without S9 mix and 400, 800, 1 600 and 3 200 µg/mL with S9 mix.

The highest concentration of the test material employed was chosen following a preliminary toxicity test; the lower concentrations were chosen as fractions of the highest concentration.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test material was found to be freely soluble in DMSO. The test material was therefore freshly dissolved in DMSO before addition to test cultures.

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: Chlorambucil in ethanol in the absence of S9 mix.

Details on test system and experimental conditions:

- Number of cultures per concentration: For the preliminary toxicity test, each treatment was established in duplicate cultures; treatments were established in triplicate cultures for the main assay.
- Number of independent experiments : Two separate experiments were performed. Initially the toxicity of the test material was examined: Slide evaluation was restricted to determination of mitotic indices only. Subsequently, a cytogenetic test was performed.
- Harvest time after the end of treatment: Three hours before harvesting, cell division was arrested by the addition of the spindle poison Colcemid to each culture (to a final concentration of 0.4 µg/mL). The tubes were capped and left to incubate for a further three hours. The cells were then harvested by low speed centrifugation (1 000 r.p.m. for 5 minutes) and the pellets of cells thus collected were resuspended in hypotonic potassium chloride solution (0.56 %) for ten minutes, centrifuged again and later fixed in freshly prepared methanol : glacial acetic acid fixative (3: 1 v/v).

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Treatment of cultures
Each 48-hour culture was centrifuged, the supernatant removed and the cell pellet resuspended in culture medium (9.5 mL, or 8.5 mL for cultures subsequently to contain S-9 mix). Freshly prepared S-9 mix (1.0 mL) was then added to the appropriate cultures and an aliquot (50 µL) of test solution, solvent or positive control solution was added to the relevant cultures. For the preliminary toxicity test, each treatment was established in duplicate cultures; treatments were established in triplicate cultures for the main assay.
All cultures were incubated at 37 °C, in a shaking water bath, for three hours. After this initial exposure period, the cultures treated in the absence of S-9 mix were transferred to a 37 °C incubator for the remainder of the 24 hour exposure period. Cultures treated in the presence of S-9 mix were centrifuged and the cells washed twice with Hanks' Balanced Salt Solution (5 mL) to remove the test material and S-9 mix. The cells were then resuspended in culture medium (9.5 mL), and the cultures incubated at 37 °C, under static conditions, for a further 21 hours.
- Spindle inhibitor: Colcemid
- Methods of slide preparation and staining technique used including the stain used: After two further changes of fixative, the tubes were centrifuged, the supernatant removed and the cell pellet resuspended in a few drops of fresh fixative. Single drops of the cell suspension were transferred to clean, moist, grease-free glass slides, and the slides were left to air-dry. Two or four slides (for the preliminary toxicity test or main cytogenetic test respectively) were made from each culture, stained for ten minutes in Giemsa stain (1 in 10 in Sorensen's buffer, pH 6.8), washed in buffer and left to air-dry. Permanent mounts were made using DPX mountant after clearing in xylene.

Main Cytogenetic Test - chromosomal analysis and mitotic index.
- At least two slides from each culture were randomly assigned code numbers by a person not subsequently engaged in the study. Care was taken to ensure that all unique identifications remained concealed to eliminate bias. The slides were examined under a low power (x 10 objective) and those areas judged to be of sufficient technical quality to permit scoring were located and examined under high power (x 100, oil immersion objective).
From 100 metaphases (with 46 centromeres) the following characters were recorded:
- chromosome number
- all chromosomes normal or some aberrant
- specific types and numbers of aberrations
Scoring followed the recommendations of the Ad Hoc committee of the Environmental Mutagen Society and the Institute for Medical Research (1972). Morphological observations were scored.
To examine the toxicity of the test compound to dividing lymphocytes, approximately 1 000 cells were scored and the mitotic index calculated.
When all examinations had been completed, the coding was broken and the results collated. Frequencies of aberrant metaphases (cells showing one or more aberrations) were calculated for each culture scored, both including and excluding gap-type aberrations.

Statistics:

The Fisher Exact Probability test is a useful nonparametric technique for analysing data when comparing two small independent samples. It is used when the scores for the samples all fall into one or other of two mutually exclusive classes. The test determines whether the two groups differ in the proportions with which they fall into the two classifications.
Data from each treatment group was compared with the corresponding solvent control group values using a one-tailed test.

Species / strain:

lymphocytes: Human peripheral lymphocytes

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

Only at a concentration which induced marked toxicity.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

STUDY RESULTS
- Concurrent vehicle negative and positive control data :
Positive controls
Treatment with the known clastogen, chlorambucil, in the absence of S-9 mix, produced a range of aberrant metaphases of 9 to 12 % with a mean value of 10.7 % including gaps, and 8 % for each culture excluding gaps. Statistical analysis confirmed that this group had a significantly higher frequency of aberrant metaphases than corresponding solvent control cultures whether gaps were included or excluded (p < 0.001). This demonstrates the sensitivity of the test system to a direct-acting clastogen.
Treatment with the known clastogen, cyclophosphamide, in the presence of S-9 mix, produced a marked increase in aberrant cell frequency, giving a range of aberrant metaphases of 18 to 21 %, with a mean value of 19.3 % including gaps, and a range of 14 to 16 %, mean 14.7 % excluding gaps. Statistical analysis confirmed that this group had a significantly higher frequency of aberrant metaphases than corresponding solvent control cultures whether gap-type aberrations were excluded or not (p < 0.001). This demonstrates the effective functioning of the S-9 mix metabolic activation system employed.

Preliminary Toxicity Test
All cultures treated with the test material at 5 000 µg/mL were seen to contain a precipitate, and to be dark in colour. Slides were prepared and stained from all cultures.
In the absence of S-9 mix, no real reductions in mean mitotic index were observed for cultures treated with the test material at concentrations of 8 and 40 µg/mL. However, reductions of 20 and 65 % were produced at 200 and 1 000 µg/mL respectively, while at 5 000 µg/mL, the test material was highly toxic and few cells and no metaphases were observed.
In the presence of S-9 mix, no real reductions in mitotic activity were observed for cultures treated at 8, 40, 200 or 1 000 µg/mL, but at 5 000 µg/mL, the test material was highly toxic and few cells and no metaphases were observed.
On the basis of these data, the concentrations of the test material selected for testing in the main cytogenetic assay were as shown below:
- without S-9 mix: 100, 200, 400 and 800 µg/mL
- with S-9 mix: 400, 800, 1 600 and 3 200 µg/mL.
Four test material concentrations were tested in both the presence and absence of S-9 mix to ensure that an appropriate range of toxicity was covered; slides prepared from cultures exposed to three concentrations only were selected for chromosomal analysis.

Mitotic Index
Initially, mitotic indices were scored for all cultures. Treatment with the test material produced dose-related toxicity in the absence of S-9 mix, with reductions in mean mitotic index (compared to the solvent control value) of 19, 22, 47 and 94 % at 100, 200, 400 and 800 µg/mL respectively.
In the presence of S-9 mix, no reductions in mitotic activity were observed in cultures treated with the test material at 400 and 800 µg/mL, but at 1 600 µg/mL a reduction of 76 % (compared to solvent control values) was recorded. In cultures treated at 3 200 µg/mL test material was highly toxic and mitotic activity was almost completely inhibited. Slides prepared from cultures exposed to the test material at 800 µg/mL in the absence of S-9 mix and at 3 200 µg/mL in its presence were excluded from those selected for chromosome analysis due to the marked toxic response seen at these concentrations.

Incidence of chromosomal aberrations: Comparison of treated and control cultures
One hundred metaphases were scored from each culture. In the absence of S-9 mix, solvent control cultures showed incidences of aberrant metaphases ranging from 0 to 3 % with a mean of 2.0 %. Corresponding values for cultures treated with the test material were 1 to 3 %, mean 1.7 % (at 100 µg/mL), 0 to 3 %, mean 2.0 % (at 200 µg/mL) and 1 to 3 %, mean 2.0 % (at 400 µg/mL). A similar lack of response to treatment with the test material was apparent when gaps were excluded from consideration.
In the presence of S-9 mix, solvent control cultures showed incidences of aberrant metaphases ranging from 0 to 1 % with a mean of 0.7 %. Corresponding values for cultures treated with the test material were 0 to 1 %, mean 0.7 % (at 400 µg/mL), 1 to 2 %, mean 1.7 % (at 800 µg/mL) and 2 to 9 %, mean 5.3 % (at 1 600 µg/mL). When gaps were excluded from consideration, solvent control cultures showed incidences of aberrant metaphases ranging from 0 to 1 % with a mean of 0.3 %. Corresponding values for cultures treated with the test material were 0 to 1 %, mean 0.7 % (at 400 µg/mL), 0 to 1 %, mean 0.3 % (at 800 µg/mL) and 2 to 6 %, mean 4.0 % (at 1 600 µg/mL). Increases in the frequency of aberrant metaphases were seen in two of the three cultures treated with the test material at 1 600 µg/mL in the presence of S-9 mix. The aberrations observed in these two cultures included chromatid exchanges, a metaphase with multiple aberrations and a pulverised metaphase. These types of aberrations are extremely rare in solvent control cultures and are biologically significant. The mean increases were also statistically significant (p < 0.01) both including and excluding gaps. At lower concentrations of the test material in the presence of S-9 mix, and at all concentrations in its absence, no statistically significant increases in the frequency of aberrant metaphases were observed.

HISTORICAL CONTROL DATA
Although individual aberrant cell frequencies for most cultures treated with the test material were within the historical control range, aberrant cell freguencies in two cultures treated with the test material at 1 600 µg/mL were at the upper end of, or outside, the historical control range.

Conclusions:

Under the conditions of study, the test material showed some evidence of clastogenic activity in the presence of S-9 mix, but only at a concentration which induced marked toxicity.

Executive summary:

The mutagenicity of the test material was assessed according to OECD Test Guideline 473 and in compliance with GLP.

The effects on chromosomal structure of exposure to the test material were investigated in cultured human lymphocytes. Tests were conducted with and without the inclusion of a rat liver-derived metabolic activating system (S-9 mix): Without S-9 mix cells were exposed for 24 hours, with S-9 mix exposure was limited to three hours. Treatments were established by the addition of test solutions, in dimethyl sulphoxide, (DMSO) to 48 hour cultures established from whole, human blood. Cell division was arrested by the addition of the spindle poison, Colcemid (to a final concentration of 0.4 µg/mL), three hours before the cells were harvested; slides were then prepared for microscopic analysis.

Mitotic indices were calculated for each culture. These were based on the number of metaphases observed per 1 000 cells scored. Chromosome aberrations were scored by examination of 100 metaphases per culture, and the frequencies of cells with one or more aberrations were calculated both including and excluding gap-type aberrations.

A preliminary test was performed to investigate the toxicity of the test material to dividing lymphocytes. Subsequently the following concentrations of the test material were tested in the main cytogenetic test:

- Without S-9 mix: 100, 200, 400 and 800 µg/mL

- With S-9 mix: 400, 800, 1 600 and 3 200 µg/mL

Four test material concentrations were tested in both the presence and absence of S-9 mix to ensure that an appropriate range of toxicity was covered; slides prepared from cultures exposed to three concentrations only were selected for chromosome analysis. The main test also incorporated solvent and positive (cyclophosphamide and chlorambucil) control cultures. Cyclophosphamide is a known clastogen requiring biotransformation to achieve optimum activity; chlorambucil is a direct-acting clastogen. All treatments were established in triplicate.

The test material produced dose-related toxicity in the absence of S-9 mix, with reductions in mean mitotic index (compared to the solvent control value) of 19, 22, 47 and 94 % at 100, 200, 400 and 800 µg/mL respectively. In the presence of S-9 mix, no reductions in mitotic activity were observed in cultures treated with the test material at 400 and 800 µg/mL, but at 1 600 µg/mL a reduction of 76 % (compared to solvent control values) was recorded. In cultures treated at 3 200 µg/mL the test material was highly toxic and mitotic activity was almost completely inhibited. Subsequently slides prepared from cultures treated with the test material at 800 µg/mL in the absence of S-9 mix and 3 200 µg/mL in its presence, were not scored for chromosome aberrations. 

Treatment with the test material at 1 600 µg/mL in the presence of S-9 mix produced increases in the mean frequency of aberrant metaphases to 5.3 % (4 % excluding gaps), compared to 0.7 % (0.3 % excluding gaps) for solvent control cultures. The increases were apparent in two of the three treated cultures. The increases were statistically significant (p < 0.01) both including and excluding gaps. At lower concentrations of the test material in the presence of S-9 mix, and at all concentrations in the absence of S-9 mix, no statistically significant increases in the frequency of aberrant metaphases were observed.

The known clastogens, cyclophosphamide and chlorambucil, induced significant increases in the frequency of aberrant metaphases over the vehicle control values (p < 0.001 in both cases; as expected).

Under the conditions of study, the test material showed some evidence of clastogenic activity in the presence of S-9 mix, but only at a concentration which induced marked toxicity. The sensitivity of the test procedure, and the metabolic activity of the S-9 mix employed, were demonstrated by the clear responses to the positive control substances.

Reference 8

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 August 1993 to 06 October 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

1983

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: UKEMS: Basic mutageniciyt tests part 3: Metaphase chromorome aberration assay in vitro.

Version / remarks:

1990

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA Code of Federal Regulations, Title 4, Subpart F - Genetic Toxicity "in vitro mammalian cytogenetics".

Version / remarks:

1986

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: Human lymphocytes

Details on mammalian cell type (if applicable):

For lymphocytes:
- Sex, age and number of blood donors: Blood samples were obtained from healthy donors not receiving medication. For this study blood was collected from two female donors (exp. I: 44 years; exp. II: age 26 years). Blood samples were drawn by venous puncture and collected in heparinized tubes by medical technical assistants of the St. Johannis Hospital Landstuhl (D-66849 Landstuhl). The tubes were sent to the Test Facility to initiate cell cultures within 24 hours after blood collection. Before use the blood was stored under sterile conditions at 4 °C.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9 : The S9 liver microsomal fraction was obtained from the livers of 8 - 12 weeks old male rats, strain Wistar/WU (weight approx. 150 - 200 g) which received a single i.p. injection of 500 mg/kg b.w. Aroclor 1254 in olive oil 5 days previously.
After cervical dislocation, the livers of the animals were removed, washed in 150 mM KCl and homogenised. The homogenate, diluted 1+3 with KCl was centrifuged twice at 9 000 g for 10 minutes (4 °C). A stock of the supernatant containing the microsomes was frozen in ampoules of 2 or 3 mL and stored at -70 °C. Small numbers of the ampoules were kept at -20 °C for only several weeks before use. The protein content was determined using the analysis kit of Bio-Rad Laboratories. The protein concentration in the S9 preparation is usually between 20 and 45 mg/mL. The protein concentration of the S9 used in both experiments was 38.6 mg/mL.
- Method of preparation of S9 mix: An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.76 mg/mL in the cultures. Cofactors were added to the S9 mix to reach the following concentrations:
8 mM MgCl2
33 mM KCl
5 mM glucose-6-phosphate
4 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment, the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al.

Test concentrations with justification for top dose:

Pre-test
20h: 3; 10; 30; 100; 300; 600; 1 000; 2 000 μg/mL
44h: 30; 100; 300; 600; 1 000; 2 000 μg/mL

Main test
In both experiments cultures treated with 100; 300; 600 μg/mL (at interval 20 h); and 300.0 μg/mL (44 h) were chosen for cytogenetic evaluation in the absence of S9 mix; in the presence of S9 mix 300 and 1 000 μg/mL (20 h) and 2 000 μg/mL (44 h) were evaluated.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubility properties and its non-toxicity for the cells.
The concentration of ethanol in the medium did not exceed 1 % v/v.

Untreated negative controls:

yes

Remarks:

Culture medium

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Culture Initiation
Within 24 h after collection blood cultures were set up in plastic vessels. The culture medium was DMEM/F12 (Dulbecco's modified eagle medium/Ham's F12 medium; mixture 1:1) containing 10 % FCS (foetal calf serum). The antibiotic solution contained 10 000 U/mL penicillin and 10 000 μg/mL streptomycin. PHA (phytohaemagglutinin, final concentration 12.0 μg/mL) and the anticoagulant heparin (25 000 u.s.P.-E/mL). The following quantities were added to the culture vessels:
8.45 mL culture medium
0.90 mL whole blood
0.50 mL PHA
0.10 mL antibiotic solution
0.05 mL heparin
All incubations were done at 37 °C in a humidified atmosphere with 15 % CO2.

Treatment
Exposure time 4 hrs (with S9 mix):
In both independent experiments, after 48 h the culture medium was replaced with 10 mL serum-free medium containing the test material and 50 μL/mL S9 mix. After 4 h the cells were spun down by gentle centrifugation for 5 minutes. The supernatant with the dissolved test material was discarded and the cells were re-suspended in "saline G''. The washing procedure was repeated once as described.
The "saline G" solution was composed as follows (per Litre):
8 000 mg NaCl
400 mg KCl
1 100 mg Glucose
290 mg Na2HPO4.7H2O
150 mg KH2PO4
pH was adjusted to 7.2

- After the washing the cells were re-suspended in complete culture medium.
Exposure time 20 and 44 hrs (without S9 mix): After 48 h the culture medium was replaced with 10 mL complete medium containing the test article without S9 mix. This medium was not changed until preparation of the cells.

Preparation of the Cultures
- Three hours before harvesting, Colcemide (Fluka, D-89203 Neu-Ulm) was added to the cultures (final concentration 0.2 μg/mL). The cultures were harvested by centrifugation 20 h and 44 h after beginning of treatment. The supernatant was discarded and the cells were re-suspended in approximately 5 mL hypotonic solution (0.0375 M KCl). The cell suspension was then allowed to stand at 37 °C for 20 minutes. After removal of the hypotonic solution by centrifugation the cells were fixed with 3 + 1 absolute methanol + glacial acetic acid.

FOR CHROMOSOME ABERRATION:
- Spindle inhibitor: Colcemide, final concentration 0.2 μg/mL.
- Methods of slide preparation and staining technique used including the stain used: Per experiment slides were prepared by dropping the cell suspension on to a clean microscope slide. The cells were stained with Giemsa and according the Fluorescent plus Giemsa technique, respectively.
- Number of cells spread and analysed per concentration: The slides were evaluated (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON microscopes with 100 x oil immersion objectives. Breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates. A gap is defined as a an achromatic region (occurring in one or both chromatids) independent of its width. The remaining visible chromosome regions should not be dislocated either longitudinally or laterally. At least 100 well spread metaphases per culture were scored for cytogenetic damage on coded slides. Only metaphases with 46 centromer regions were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined. In addition, the number of polyploid cells (% polyploid metaphases) was scored.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
In a pre-test phase (cytotoxicity determination) the concentrations which should be chosen for cytogenetic scoring were determined. Cytotoxicity was characterised by the percentages of mitotic suppression in comparison with the controls by counting 1 000 cells per culture and duplicate. The experimental conditions in this pre-test phase were identical to those required and described for the mutagenicity assay.
In this pre-test phase cytotoxicity of the test material (and negative and positive controls) was determined. All cell cultures were set up at least in duplicate. Exposure times were 4 h (with S9 mix), 20 and 44 h (without S9 mix). The fixation intervals were 20 and 44 hours after start of the exposure. At the 20 h fixation interval, additional solvent control cultures (with and without S9 mix) were used in the presence of BrdU (5-bromodeoxyuridine; 6 μg/mL) to check the replication index of the stimulated lymphocytes.
Treatment in this pre-test phase was performed with the following concentrations:
With and without S9 mix (exposure period 20 and 44 h):
20 h: 3; 10; 30; 100; 300; 600; 1 000; 2 000 μg/mL
44 h: 30; 100; 300; 600; 1 000; 2 000 μg/mL
The highest concentration used in this pre-test phase was 2 000 μg/mL (limit of solubility in ethanol: 200 mg/mL). Precipitation of the test material in culture medium was observed starting with 1 000 μg/mL. The results of the pre-test phase (measuring mitotic index and a qualitative evaluation) indicate that only in the absence of S9 mix cytotoxic effects were observed starting with 300 μg/mL. Based on these results, in both experiments cultures treated with 100; 300; 600 μg/mL (at interval 20 h); and 300.0 μg/mL (44 h) were chosen for cytogenetic evaluation in the absence of S9 mix; in the presence of S9 mix 300 and 1 000 μg/mL (20 h) and 2 000 μg/mL (44 h) were evaluated.

Evaluation criteria:

ACCEPTABILITY OF THE ASSAY
The chromosomal aberration assay is considered acceptable if it meets the following criteria:
a) the numbers of chromosomal aberrations found in the negative and/or solvent control cultures should reasonably fall within the laboratory historical control data range.
b) the positive control substances should produce a significant increase in the frequencies of aberrations.

EVALUATION OF RESULTS
A test article is classified as mutagenic if it induces reproducibly either a concentration-related increase in the number of structural chromosomal aberrations or a significant and reproducible positive response for at least one of the test points. A test article producing reproducibly neither a concentration related increase in the number of structural chromosomal aberrations nor a significant and reproducibly positive response at any one of the test points is considered non-mutagenic in this system. This can be confirmed by means of the chi-square test. However, both biological and statistical significance should be considered together.

Statistics:

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the chi-square test. Evaluation was performed only for cells carrying aberrations exclusive gaps.

Species / strain:

lymphocytes: Human lymphocytes

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

lymphocytes: Human lymphocytes

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

Statistically significant increases (up to 8.5 %) in the aberration rates in three treatment groups as compared to the corresponding controls at cytotoxic concentrations. These increases could not be confirmed in the independent experiment II.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The test material was assessed for its potential to induce chromosomal aberrations in human lymphocytes in vitro in the absence and presence of an extrinsic metabolising system (S9 mix).
Two independent experiments were performed. In each experimental group two parallel cultures were used. 100 metaphases per culture were scored for structural chromosomal aberrations.
Preparation of chromosomes was done 20 h (low, intermediate and high concentration range) and 44 h (high concentration range) after start of the treatment with the test material which was dissolved in ethanol. The exposure period was 20 h and 44 h without S9 mix and 4 h with S9 mix.
The following concentrations were evaluated at fixation intervals 20 and 44 h:
Experiment I and II
without S9 mix (exposure period 20/44 h):
20 h: 100; 300; 600 μg/mL
44 h: 300 μg/mL
with S9 mix (exposure period 4 h):
20 h: 300; 1 000; 2 000 μg/mL
44 h: 2 000 μg/mL
In both experiments, the test material was tested up to concentration levels which were in the range were toxic effects (without S9 mix) or precipitation (with S9 mix) could be observed. Obvious reductions of the mitotic indices were found with 300 and 600 μg/mL in the absence of S9 mix (in both experiments at intervals 20 hand 44 h).
In the presence of S9 mix, in both experiments, with the test material no biologically relevant increase in the structural chromosomal aberration rate could be found when compared with the range of aberrations in the corresponding negative and solvent controls. These results were obtained at both fixation intervals. In experiment II at interval 20 h, the statistical evaluation revealed a significant difference between the aberration rates of cultures treated with 1 000 and 2 000 μg/mL (3.0 %) and its corresponding control. However, this statistical result was due to the extremely low aberration rate in the solvent control (0.0 %). As the aberration rates of the treated cultures were near to the actual control data of this study and the historical data of the last 12 studies the statistical evaluation is not regarded to be biologically relevant.
In this study the aberration rates after treatment with the test material in the presence of S9 mix (Exp. I: 1.5 %; Exp. II: 0.0 % - 3.0 %) were in or near to the range of the control data (Exp. I: 1.5 % - 2.5 %; Exp. II: 0.0 % - 2.5 %).
In the absence of S9 mix, in experiment I, statistically and biologically increased aberration rates (8.0 %; 8.5 % at 20 h; 7.0 % at 44 h) were found after treatment with 300 (20 hand 44 h) and 600 μg/mL (20 h) as compared to the corresponding solvent controls (2.5 % and 3.0 %). However, these increased aberration rates could not be confirmed in the independent experiment II in the same magnitude. Here, aberration rates in the comparable treatment groups were 2.5 % and 3.5 % at 20 h; 4.0 % at 44 h with control values of 2.0 % (20 h) and 1.5 % (44 h).
In both experiments no biologically relevant deviations from the control data (0.0 % - 1.0 %) were found after treatment with the test material (0.0 % - 1.0 %).

EMS (330 μg/mL) and CPA (Exp.I: 26.7 μg/mL; Exp. II: 30.0 μg/mL) were used as positive controls and showed distinct statistically significant increases (α = 0.05) in cells with structural chromosomal aberrations. The proliferation index in the solvent control cultures with (1.00 and 1.05) and without S9 mix (1.36 and 1.39) of the lymphocytes was checked by analysing the proportion of mitotic cells in their 1st, 2nd and 3rd metaphase (M1, M1+, M2 and M3) and showed that the lymphocytes divided 1 - 1.5 fold within the early fixation interval which is requested by international guidelines.

Conclusions:

Under the conditions of the study and in presence of S9 mix, the test material was not clastogenic. In absence of S9 mix a final assessment of the clastogenic potential of the test material is difficult due to divergent results at cytotoxic concentrations.

Executive summary:

The mutagenicity of the test material was assessed according to OECD Test Guideline 473 and EU Method B.10 and in compliance with GLP.

The test material was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro.

Two independent experiments were performed. Preparation of chromosomes was done 20 h (low, intermediate and high concentration), and 44 h (high concentration) after start of treatment with the test material which was dissolved in ethanol. Exposure periods were 20 h and 44 h (without S9 mix) and 4 h (with S9 mix). In each experimental group two parallel cultures were used. Per culture, 100 metaphases were scored for structural chromosomal aberrations. The following concentrations were evaluated at fixation intervals

Experiment I and II:

Without S9 mix (exposure period 20/33 h)

20 h: 100; 300; 600 µg/mL.

44 h: 300 µg/mL.

 

With S9 mix (exposure period 4 h)

20 h: 300, 1 000, 2 000 µg/mL.

44 h: 2 000 µg/mL.

 

In both experiments, the test material was tested up to toxic (without S9 mix, 300 and 600 μg/mL) and precipitating concentrations (with S9 mix).

There was no biologically relevant increase in cells with aberrations after treatment with the test material at any test point in the groups treated in the presence of S9 mix. However, in the absence of S9 mix (long term treatment), there were statistically significant increases (up to 8.5 %) in the aberration rates in three treatment groups as compared to the corresponding controls at cytotoxic concentrations. These increases could not be confirmed in the independent experiment II.

Appropriate reference mutagens were used as positive controls and showed distinct, statistically significant increases (α = 0.05) in cells with structural chromosomal aberrations.

Under the conditions of the study and in presence of S9 mix, the test material was not clastogenic. In absence of S9 mix a final assessment of the clastogenic potential of the test material is difficult due to divergent results at cytotoxic concentrations.

Reference 9

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 July 1990 to 11 September 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Freshly dissolved in DMSO before addition to cultures, for both the preliminary toxicity test and the main mutation assays.

Target gene:

Hypoxanthine-guanine-phosphoribosyl transferase (HGPRT) gene locus.

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: V79 Clone 6 cells were used; this clone is established from single wild-type colonies of V79 4-1 clone 9 3/12. This cell line was obtained from Shell Research Limited, Sittingbourne, Kent, England and is a sub-clone of a V79 cell line originally established from lung tissue of a male Chinese hamster (Ford and Yerganian, 1958).
The cells were held in the laboratory frozen down in medium containing 10 % DMSO and stored in a liquid nitrogen cryostat. When required, an ampoule of frozen cells was thawed, the cells washed in medium to remove the DMSO and maintained in Dulbecco's modification of Eagle's minimal essential medium containing 10 % foetal calf serum. Throughout the study test cultures were passaged, when necessary, in order to maintain the cells at sub-confluence (< 80 %).

- Absence of Mycoplasma contamination: Cells from this laboratory are regularly screened for mycoplasma infection. Samples of the laboratory stock culture of V79 cells were screened during January and August 1990 and were found to be mycoplasma free.


MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
DMEM - Treatment medium: 500 mL Dulbecco's MEM (1x), 5 mL Penicillin: streptomycin solution (5000 IU per mL 5000 µg/mL)
DMEM 10% FCS - complete medium: As for treatment medium plus 60 mL foetal calf serum (FCS).
6-Thioguanine (Stock solution): 10 mg 6-Thioguanine, 10 mL Sodium bicarbonate solution (0.5 % w:v) incubated at 50 °C for at least 12 hours and filter sterilised.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9 : Aroclor 1254 (500 mg/kg bodyweight in corn oil) was administered to young male CD rats (ca. 200 g bodyweight) as a single intraperitoneal injection to induce microsomal enzyme activity. Four days after treatment the animals were fasted overnight and killed by cervical dislocation. The livers were removed, washed in cold 0.15 M KCl, then homogenised with more of the same medium (approximately 1 mL per g wet liver) in a Potter-Elvehjem homogeniser. Homogenates were centrifuged at 9 000 g for 10 minutes and supernatants collected and stored at approximately -196 °C until required for preparation of the S-9 mix. Supernatant is used within 6 months of preparation.
- Method of preparation of S9 mix: S-9 mix
0.4 mL 0.1 M NADP, sodium salt, in aqueous solution
0.5 mL 0.1 M glucose-6-phosphate, sodium salt, in aqueous solution
0.2 mL 0.4 M MgCl2.6H2O/1.65 M KCl aqueous solution
3.0 mL Supernatant from liver homogenate
0.1 M KH2PO4-Na2HPO4 buffer (pH 7.4) to final volume of 10.0 mL.

Test concentrations with justification for top dose:

First mutation assay: 6.4, 32, 160, 800, 4 000 µg/mL (with and without metabolic activation).
Second mutation assay: 7.2, 36, 180, 900, 4 500 µg/mL (without metabolic activation); 6, 30, 150, 750 and 3 750 µg/mL (with metabolic activation).

The concentrations were chosen to produce a range of toxicities following a preliminary toxicity test.
Test material concentrations employed in the second mutation assay differed from those employed in the first assay because, in the first assay, very little toxicity (relative to solvent control values) was observed at any concentration tested in the absence of S-9 mix, but survival was reduced to below 10 % of the solvent control value in one of the two cultures exposed at 4 000 µg/mL in the presence of S-9 mix.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO.
- Justification for choice of solvent/vehicle: The compound was found to be soluble in DMSO up to a maximum concentration in the region of 959 mg/mL.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Duplicate
- Number of independent experiments: Two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: In each assay, sufficient 25 cm² flasks were seeded with 7.5 x 10^5 cells per flask approximately 24 h propr to treatment; on the day of treatment the cultures were approximately 70 % confluent.
- Test substance added: In medium

TREATMENT AND HARVEST SCHEDULE:
- The complete medium was removed from each culture and replaced with treatment medium, which was prepared as follows:
150 µL of test material, DMSO or positive control solutions
1.5 mL of S-9 mix (for activated treatments only)
Serum free medium to 15 mL and 5 mL of this mixture was added to each culture.
- Cultures were gassed with 5 % CO2 in air and incubated for three hours at 37 °C. Following treatment the cells were washed three times with HBSS. To assess the toxicity of the test exposures, cell survival was determined by plating 5 mL of a cell suspension containing 20 cells/mL, i.e. 100 cells, on each of three 60 mm plates to determine P.E. At the same time, the cultures were passaged, gassed with 5 % CO2 in air and re-incubated at 37 °C. Subsequently, cultures were studied each day and passaged when necessary to maintain sub-confluence.

FOR GENE MUTATION:
- Expression time: In order for induced mutations to be expressed, the cells were allowed to undergo several divisions. In each mutation assay, the cells were plated for survival and 6-TG resistance after a 7-day expression time. On each of these occasions, the cultures were trypsinised, re-suspended in DMEM containing 10 % foetal calf serum, and aliquots of cells removed from each culture. Using appropriate dilutions 10 mL of the cell suspension containing 10^4 cells/mL i.e. 10^5 cells, were seeded on to each of three 90 mm plates for the selection of 6-TGr mutants. At the same time, 10 mL of the cell suspension containing 20 cells/mL, i.e. 200 cells, were seeded on three 90 mm plates to determine P.E. 6-TG was added to the 6-TG^r. selection plates, 2-3 hours after seeding, at a final concentration of 10 µg/mL. P.E. and 6-TG^r plates were incubated at 37 °C in a 5 % CO2 atmosphere and 100 % relative humidity. After 6 days, the resultant colonies were fixed in methanol and stained with 10 % Giemsa. The plates were scored by eye and the mutation frequency calculated.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
A preliminary range-finding test was conducted in order to establish the levels of the test material to be used in the main mutation assays. The highest level tested was the maximum concentration normally tested in this laboratory (5 mg/mL). A solution of 500 mg/mL was therefore prepared in DMSO and serial dilutions were made from this solution to give four further test material solutions at concentrations of 100, 20, 4 and 0.8 mg/mL. All dilutions were made up immediately prior to treatment.
Sufficient 25 cm^2 flasks were seeded with 7.5 x 10^5 cells per flask, approximately 24 hours prior to treatment. Each treatment was established in duplicate and tested in the absence and presence of S-9 mix, a microsomal activating system derived from rat liver The complete medium was removed from each culture and replaced with treatment medium which was prepared as follows:
- 150 µL of test material solution or DMSO
- 1.5 mL of S-9 mix (for activated treatments only)
- serum free medium to 15 mL
and 5 mL of this mixture was added to each culture.
Cultures were gassed with 5 % CO2 in air, and incubated at 37 °C. After three hours of incubation, the treatment medium was removed and the cell sheet washed three times with Hanks' balanced salt solution (HBSS), trypsinised and re-suspended in DMEM containing 10 % foetal calf serum. The cell concentrations of each culture were measured using a haemocytometer and aliquots of cells removed from each culture. Using appropriate dilutions, cell suspensions containing 20 cells/mL were prepared for each culture and 5 mL of these, i.e. 100 cells, were seeded on each of three 60 mm plates to determine plating efficiency (P.E.). After incubation at 37 °C in a 5 % CO2 atmosphere for 6 days, the colonies were fixed in methanol and stained with 10 % Giemsa. The plates were scored by eye and cytotoxicity was determined by the calculation of percentage P.E. relative to the solvent control. Test material concentrations that were expected to produce a range of survival levels were selected for use in the main mutation assays.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

STUDY RESULTS
- Concurrent vehicle negative and positive control data : Cultures treated with EMS, at 1 000 µg/mL, had a mean mutation frequency of 78.7 (compared to a solvent control value of zero) in the first mutation assay, and 56.3 per 10 survivors (compared to a solvent control value of 0.1) in the second mutation assay. Large increases in mutant colony numbers were also observed.
DMBA (at 10 µg/mL) in the presence of S-9 mix induced increases in mutation frequencies on each occasion of testing; mean mutation frequencies in the two mutation assays were 36.9 and 19.8 per 10 survivors respectively, compared to the corresponding mean solvent control values of 1.5 and 2.1 per 10 survivors.

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements: In this assay, colour changes were noted at the two highest test material levels tested in the absence and presence of S-9 mix; at 1 000 µg/mL, the treatment medium changed from its normal red colouration to orange, while at 5 000 µg/mL the treatment medium was yellow (indicating a possible reduction in pH), and there was evidence of slight precipitation of the test material.
No evidence of toxicity (relative to solvent control values) was observed at test material concentrations up to and including 1 000 µg/mL in either the absence or presence of S-9 mix but at 5 000 µg/mL there was almost total cell death in both non-activated and activated cultures (survival was reduced to 0.2 % and zero, compared to the solvent controls).
The concentrations of the test material selected for use in the first mutation assay were 6.4, 32, 160, 800 and 4 000 µg/mL, with and without S-9 mix.
- In the first mutation assay, where the highest concentration tested was 4 000 µg/mL, very little evidence of toxicity (compared to solvent control values) was observed at any test material concentration tested in non-activated cultures. In activated cultures, no toxicity was observed at concentrations up to and including 800 µg/mL but, at 4 000 µg/mL, survival was reduced to 14.1 % of the solvent control value (mean value; individual values were 7.9 % and 20.2 %). The test material was, therefore, less toxic than anticipated in the absence of S-9 mix, but more toxic than anticipated in its presence. The concentrations selected for use in the second mutation assay were 7.2, 36, 180, 900 and 4 500 µg/mL without S-9 mix and 6, 30, 150, 750 and 3 750 µg/mL with S-9 mix.
- In the second mutation assay, no evidence of toxicity was observed in cultures exposed to the test material at concentrations up to and including 900 µg/mL in the absence of S-9 mix, but survival was markedly reduced (to 9.9 % of the solvent control value) at 4 500 µg/mL. In the presence of S-9 mix, no toxicity was observed at concentrations up to and including 750 µg/mL, but survival was reduced to 36.6 % of the solvent control value at 3750 µg/mL. Colour changes were again observed in treatment preparations of the test material in both assays. In the absence and presence of S-9 mix, the treatment medium was yellow, compared to its normal, red colouration, and there was also slight precipitation at the highest tested concentrations in each assay (4 000 and 4 500 µg/mL without S-9 mix, and 4 000 and 3 750 µg/mL with S-9 mix in the first and second assays respectively). Also, the treatment medium was orange at the second highest concentrations tested in each assay (800 and 900 µg/mL without S-9 mix, and 800 and 750 µg/mL with S-9 mix). These colour changes may be indicative of a reduction in pH.

Genotoxicity results:
- Mutagenic potential of the test material without S-9 mix.
The plating efficiencies observed in treated cultures after the expression period ranged from 40.0 % to 112.9 % (in the first assay) and from 81.5 % to 284.7 % (in the second assay) with no evidence of dose-related toxicity in either assay. There was an unusual range of plating efficiencies, some being higher or lower than normally expected, but this did not affect the validity of the Study, since very low mutant colony numbers were observed in all solvent and test material treated cultures.
No real increases in mutation frequency or mutant colony numbers (compared to the solvent control values) were observed in either of the two mutation assays.
In the first mutation assay, the highest recorded mutating frequency observed in any treated culture was 1.2 per 10 survivors, in one culture treated at 800 µg/mL, compared to the solvent control value of zero (from one culture only; the replicate culture died prior to plating out due to a cracked culture flask).
In the second mutation assay, the solvent control mutation frequencies were 0.2 and 0.0 per 10 survivors, while the highest mutation frequency recorded in any treated culture was 0.7 per 10 survivors (in one culture exposed at 4 500 µg/mL).
- Mutagenic potential of the test material with S-9 mix.
The plating efficiencies observed in treated cultures after the expression period ranged from 69.5 % to 93.2 % (in the first assay) and from 63.2 % to 221.9 % (in the second assay). There was again no evidence of a dose-related response, but an unusual range of plating efficiencies was observed in the second assay (where some values were higher than expected). However, this again did not affect the validity of the Study, since the mutant colony numbers in control and treated cultures were sufficiently low. There were again no increases in mutation frequencies or in mutant colony numbers, compared to the solvent control values, in any treated culture in either of the two mutation assays.
In the first mutation assay, the highest recorded mutation frequency in any treated culture was 3.2 per 10 survivors (in one culture exposed at 800 µg/mL) compared to the solvent control values of 3.0 and 0.0 per 10 survivors. In the second mutation assay, the solvent control mutation frequencies were 4.2 and 0.0 per 10 survivors, while the highest mutation frequency observed in any treated culture was 2.9 per 10 survivors, in one culture treated at 3 750 µg/mL.

Conclusions:

Under the conditions of the test, the test material induced no increases in mutation frequency at the HGPRT gene locus when cells were treated in the absence or presence of S-9 mix.

Executive summary:

The genotoxicity of the test material was assessed according to OECD Test Guideline 476 and in compliance with GLP.

The test material was examined for mutagenic potential by measuring its ability to induce mutation in Chinese hamster (V79) cells at the hypoxanthine-guanine-phosphoribosyl transferase (HGPRT) locus.

A preliminary toxicity test was first conducted: Cells were exposed to five test material concentrations ranging from 8 to 5 000 µg/mL in the presence and absence of a rat-liver derived metabolic activating system (S-9 mix). After three hours of exposure, the cells were washed and seeded in non-selective medium to assess toxicity: Plating efficiency was determined by colony counts.

Cultures of Chinese hamster cells were then exposed to five concentrations of the test material selected to permit a range of cell survival values (relative to solvent controls) in each of two mutation assays. The concentrations selected for the first mutation assay were 6.4, 32, 160, 800 and 4 000 µg /mL in the absence and presence of S-9 mix, but the test material was less toxic than expected in the absence of S-9 mix and more toxic than expected in its presence; consequently, the levels selected for the second mutation assay were 7.2, 36, 180, 900 and 4 500 µg /mL in the absence of S-9 mix and 6, 30, 150, 750 and 3 750 µg /mL in the presence of S-9 mix. Treatments were established for three hours. Solvent control cultures treated with dimethyl sulphoxide (DMSO) were included in all experiments.

Ethylmethanesulphonate (EMS), a known direct-acting mutagen, and 7,12-dimethylbenzanthracene (DMBA), a mutagen which requires metabolic activation to achieve optimal activity, were used as positive controls. All cultures were established in duplicate. After treatment, the cell sheet was washed and non-selective medium added. A sample of cells was taken from each culture and plated out to determine survival immediately post-treatment. The cultures were then incubated at 37 °C. Cultures were studied each day and passaged when necessary to maintain sub-confluence. After an expression time of 7 days, cells were plated out to assess survival and 6-thioguanine resistance (6-TG^r). After 6 days of incubation at 37 °C in a 5 % CO2 atmosphere, the resultant colonies were counted. Survival was estimated from the non-selective plates, and mutants were scored in selective plates; these latter contained 6-TG which was added to the plates 2-3 hours after seeding. Cultures exposed to the test material showed no increases in 6-TG^r colony numbers or mutant frequencies (per 10^5 survivors), compared to the solvent control cultures.

Under the same test conditions, EMS (1 000 µg/mL) produced increases in the incidence of mutant colonies. DMBA (10 µg/mL) increased the incidence of mutant colonies only in the presence of S-9 mix.

Under the conditions of the test, the test material induced no increases in mutation frequency at the HGPRT gene locus when cells were treated in the absence or presence of S-9 mix. The sensitivity of the assay system was proven by the observed responses to known mutagens.

Reference 10

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 April 1993 to 25 June 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: US Environmental Protection Agency, Method: HG-Gene Muta-Somatic Cells: Detection of gene mutations in somatic cells in culture.

Version / remarks:

1983

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test material was dissolved and diluted in ethanol on the morning of the test.

FORM AS APPLIED IN THE TEST
- The test material was formulated to account for the purity of the test substance to give concentrations in terms of active ingredient. All concentrations stated in this report are in terms of active ingredient.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability: The stability of the test material in the solvent was determined as part of this study. Analysis of achieved concentration was performed as part of this study.

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

CHO-Kl-BH4 cells

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Obtained from the British Industrial Biological Research Association. The cells were stored in polypropylene ampoules, at -196 °C, in heat-inactivated foetal calf serum containing 10 % dimethylsulphoxide (DMSO).
- Suitability of cells: These cells are functionally hemizygous at the HGPRT locus.

For cell lines:
- Absence of Mycoplasma contamination: The cells were screened for mycoplasma contamination prior to the commencement of the study.
- Periodically checked for karyotype stability: No. The karyotype of the cells was not examined and is assumed to be stable.

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: The media used were:
Ham's F12 medium, obtained from Imperial Laboratories. This medium was supplemented with 200 mM L-glutamine and 50 μg/mL gentamicin. The resulting medium is referred to as H0 medium.
H0 medium supplemented with 5 % HiFCS, referred to as H5 medium, was used for general cell culture purposes.
The selective medium, in which only HGPRT deficient cells will grow, consisted of H5 medium supplemented with 6-TG at a final concentration of 10 μg/mL.
During the week prior to treatment, spontaneous mutants were eliminated from the stock cultures by growing the cells in H5 medium containing 15 μg/mL hypoxanthine, 0.3 μg/mL amethopterin and 4 μg/mL thymidine. This medium is referred to as H5-HAT medium.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9 :
Species: Rat
Sex: Male
Strain: Sprague-Dawley derived
Age: 7 - 8 weeks
Weight: < 300 g
Diet: Biosure Rodent Diet LAD 1
S-9 fraction was prepared from a group of ca. 10 animals. Mixed function oxidase systems in the rat liver were stimulated by Aroclor 1254, administered as a single intraperitoneal injection in Arachis oil at a dosage of 500 mg/kg bodyweight. On the fifth day after injection, following an overnight starvation, the rats were killed and their livers aseptically removed. The following steps were carried out at 0 – 4 °C under aseptic conditions. The livers were placed in 250 mM sucrose solution (3 mL of sucrose solution: 1 g of liver) before being transferred to an UltraTurrax homogeniser. Following preparation the homogenate was centrifuged at 9 000 g for 10 minutes. The supernatant fraction (S-9 fraction) was dispensed into aliquots and stored at -80 °C until required.
- Method of preparation of S9 mix: S-9 mix contains: S-9 fraction (25 % v/v), isocitric acid (43.5 mM), NADP (8.0 mM) in ice cold H0 medium taken directly from a refrigerator. The co-factors were prepared on the morning of the test, neutralised with 1N NaOH and filter sterilised before adding to S-9 fraction.
- Quality controls of S9: All batches of S-9 fraction were tested using 20-methylcholanthrene before use.

Test concentrations with justification for top dose:

Preliminary toxicity test: 10, 25, 50, 100, 200, 400, 750, 1 125, 1 500 µg/mL

The final concentrations expressed as active ingredient were:
-S-9 mix
Test 1: 23.2, 46.3, 92.6, 185.2, 370.4, 463, 578.8, 694.5 μg/mL.
Test 2: 50, 100, 200, 400, 500, 625, 750, 800, 850 μg/mL.
+S-9 mix
Test 1: 46.3, 92.6, 185.2, 370.4, 694.5, 810.3, 926, 1041.8 μg/mL.
Test 2: 25, 50, 100, 200, 400, 750, 875, 925, 1 000 μg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: Prior to commencing testing, the solubility of the test material in ethanol was assessed. The maximum solubility of the test material in ethanol was 500 mg/mL. On dosing at 1 % precipitate was observed. A final concentration of 1 500 μg/mL in the culture medium was chosen as the top concentration to be used in the preliminary toxicity test based on solubility in the culture medium.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

other: 20-Methylcholanthrene: 5 µg/mL in DMSO with S9 mix.

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of independent experiments : Two

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium: In suspension

FOR GENE MUTATION:
Cell suspensions were prepared as described for the preliminary toxicity test except that duplicate cultures were used for each treatment and positive controls were included. At least five serial dilutions of the test material were used, when possible at concentrations expected to span the LC50 and LC0 range. Following treatment with the test material as described for the preliminary test, cells were again seeded at 200 cells/plate in 60 mm diameter tissue culture plates in 5 mL of H5 medium to determine cytotoxicity. In addition 10^6 viable cells from each culture, estimated on the basis of toxicity data, were seeded into a 175 cm^2 flask containing 50 mL of H5 medium and incubated for seven days to allow for expression of the mutant phenotype. The cultures were sub-cultured once during the expression period on Day 4 or 5 and, after a total of 7 days. were harvested by trypsinisation. For each treatment group, three 60 mm culture plates were each seeded with 200 cells in H5 medium to determine plating efficiency, and five 100 mm plates with 2 x 10^5 cells in selective medium, to determine the number of mutant colonies. The plates were returned to the incubator for a further 7 days at 37 °C in a humidified atmosphere of 5 % CO2 in air. At the end of this incubation period colonies growing in the plates were fixed, stained and counted. Two independent tests in the absence of S-9 mix and two independent tests in the presence of S-9 mix were carried out. In the preliminary toxicity test in the absence and the presence of S-9 mix, 750 μg/mL resulted in cell survival of 0 % and 125 % respectively. Based on these results the dose levels tested were 23.2, 46.3. 92.6, 185.2, 370.4, 463, 578.8 and 694.5 μg/mL in Test 1 and 50, 100, 200, 400, 500, 625, 750, 800 and 850 μg/mL in Test 2 in the absence of S-9 mix. In the presence of S-9 mix the dose levels tested were 46.3, 92.6, 185.2, 370.4, 694.5, 810.3, 926 and 1041.8 μg/mL in Test 1 and 25, 50, 100, 200, 400, 750. 875, 925 and 1 000 μg/mL in Test 2.
- Number of cells seeded: 200 cells/plate


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Cell survival
- A cell suspension was prepared at 7.5 x 10^5 cells/mL in H5 medium. 10 mL aliquots of this suspension were dispensed into 80 cm^2 flasks, two flasks per concentration of test material and four for the solvent controls. The cells were incubated for at least 20 hours at 37 °C in a humidified atmosphere of 5 % CO2 in air prior to exposure to the test material. 2 mL of H0 medium or S-9 mix was then added to one flask per treatment group followed by 120 μL of test material (at 100 times the desired final concentration) or solvent. The flasks were then returned to the incubator for a further four hours. The dose levels tested were 10, 25, 50, 100, 200, 400, 750, 1 125 and 1 500 μg/mL (final).
At the end of the treatment period the cells were harvested, washed and three 60 mm dishes per culture seeded with 200 cells in H5 medium. The plates were incubated as above for 7 days after which time colonies growing in the plates were fixed, stained and counted. Counting was done using an Optomax V Image analyser (Analytical Measuring Systems, Cambridge). Cell survival for each culture was expressed as the plating efficiency relative to that of the solvent controls. Concentrations of test material used in the main test were chosen on the basis of these results.

CALCULATIONS
The cytotoxic effect of each concentration of test substance will be calculated as follows:

(Total no. colonies on plates from treated cultures / Total no. colonies on plates from untreated cultures) x 100

The plating efficiency (PE) for each set of plates will be calculated as follows:

PE = (Total number of viable colonies for each treatment group / (Number of plates scored for colony formation x 200)) x 100

The number of plates scored will be three except in cases where contaminated plates are present. 200 is the number of cells originally added to each plate.

The mutant frequency (MF) per 10^6 viable cells for each set of plates will be calculated as follows:

MF = ((Total number of mutant colonies x5*) / (PE x number of uncontaminated plates)) x 100

* Represents a correction factor for the number of uncontaminated plates which normally equals 5, but may be less.

Normally five plates will be scored for the presence of mutant colonies for each replicate. Two replicates are used for each treatment and positive control, four for the solvent control.

Evaluation criteria:

Criteria for validity
For an assay to be accepted as valid the following criteria should be met:
The plating efficiency of the solvent control group should be 50 % or greater.
For the solvent control group, the mean mutant frequency per 10^6 viable cells should not exceed 20.
For the positive control group, the mean mutant frequency per 10^6 viable cells should exceed 100.
At least one of the dose levels should show a cytotoxic effect such that the relative cell survival is 40 % or less. This does not apply if the maximum advisable concentration (5 mg/ml) or the limit of solubility of the test material has been reached without toxic effect.

Statistics:

The statistical significance of the data was analysed by weighted analysis of variance following the methods described by Arlett et al. (1989). The criteria for a positive response were:
The demonstration of a statistically significant increase in mutant frequency in cultures treated with the test substance.
Evidence of a dose relationship over at least two consecutive doses, in the increase in mutant frequency.
Demonstration of reproducibility of any increase in mutant frequency in treated cultures.
The response must lie outside the historical control range of 20 mutant colonies per 10^6 surviving cells.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: In the preliminary toxicity test cells were treated with the test material at concentrations ranging from 10 – 1 500 μg/mL in the absence and presence of S-9 mix. This resulted in cell survivals relative to the controls of 122 - 0 % and 125 - 0 % respectively. Concentrations used in the main test were based upon this data.

STUDY RESULTS
Concurrent vehicle negative and positive control data :
- EMS, the positive control, induced highly significant increases in mutant frequency in both tests.
- 20-Methylcholanthrene, the positive control, induced highly significant increases in mutant frequency in both tests.

Gene mutation tests in mammalian cells:
- In the absence of S-9 mix, treatment of cells with 23.2 - 694.5 μg/mL in Test 1 and 50 - 850 μg/mL in Test 2 resulted in mean cell survivals of 116 - 51 % and 129 - 8 % respectively. Cultures treated with 23.2, 92.6, 370.4, 463 and 694.5 μg/mL in Test 1 and 100, 200, 500, 625, 750 and 800 μg/mL in Test 2 were assessed for viability and induced mutation.
No significant increases in mutant frequency were observed in either test after treatment with the test material. In Test 2 the observed solvent control mutant frequency of 19 was outside the previously established historical control range. However this was considered acceptable for the purpose of this test as the positive control was still clearly positive and all other mutant frequencies, except at the highest dose of the test material, were within the historical range. As the control mutant frequency may have been influenced by the loss of some plates due to contamination, these data have not been incorporated into the historical range which therefore remains unchanged at 15 mutants per 10^6 survivors.

- In the presence of S-9 mix, treatment of cells with 46.3 - 1 041.8 μg/mL in Test 1 and 25 - 1 000 μg/mL in Test 2 resulted in mean cell survivals of 122 - 1 % and 165 - 33 % respectively. Cultures treated with 92.6, 185.2, 370.4, 694.5 and 810.3 μg/mL in Test 1 and 100, 200, 400, 750 and 925 μg/mL in Test 2 were assessed for viability and induced mutation.
Although a significant increase was observed after treatment with 92.6 μg/mL in test 1, this was not reproduced at higher concentrations in either test and was not considered to be biologically significant. Therefore, it was concluded that the test material did not demonstrate mutagenic potential in the presence of S-9 mix.

Conclusions:

Under the conditions of the study the test material did not demonstrate mutagenic potential in this in vitro test system.

Executive summary:

The test material was assessed for genotoxicity according to OECD Test Guideline 476 and in compliance with GLP.

The test material was tested for its ability to induce forward mutation at the functionally hemizygous hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in Chinese hamster ovary (CHO) cells in vitro both in the presence and the absence of exogenous metabolic activation in the form of Aroclor 1254-induced rat liver S-9.

Toxicity was observed in all the tests after treatment with the test material, both in the absence and the presence of S-9 mix.

In the absence of S-9 mix, treatment of the cells with 23.2 - 694.5 μg/mL in Test 1 and 50 - 850 μg/mL in Test 2 resulted in mean cell survivals of 116 - 51 % and 129 - 8 % respectively. Cultures treated with 23.2, 92.6, 370.4, 463 and 694.5 μg/mL in Test 1 and 100, 200, 500, 625, 750 and 800 μg/mL in Test 2 were assessed for viability and induced mutation. No significant increases in mutant frequency were observed in either of the tests in the absence of S-9 mix.

In the presence of S-9 mix, treatment of the cells with 46.3 - 1 041.8 μg/mL in Test 1 and 25 – 1 000 μg/mL in Test 2 resulted in mean cell survivals of 122 - 1 % and 165 – 33 % respectively. Cultures treated with 92.6, 185.2, 370.4, 694.5 and 810.3 μg/mL in Test 1 and 100, 200, 400, 750 and 925 μg/mL in Test 2 were assessed for viability and induced mutation. No biologically significant increases in mutant frequency were observed in either of the tests in the presence of S-9 mix.

Under the conditions of the study the test material did not demonstrate mutagenic potential in this in vitro test system.

Reference 11

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

(Q)SAR

Adequacy of study:

supporting study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

results derived from a (Q)SAR model, with limited documentation / justification

Justification for type of information:

1. SOFTWARE : Lazar Toxicity Prediction

2. MODEL: Not reported.

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
- Test material: O=C(O)C(Oc(c(cc(c1)Cl)C)c1)C
- Impurities:
2-Propenoic acid, 2-chloro- (also known as 2-chloro acrylic acid): O=C(O)C(=C)Cl
(5-methylbenzene-1,3-dicarboxylic acid): Cc1cc(cc(c1)C(=O)O)C(=O)O
(2-methylpropyl 2-(4-chloro-2-methylphenoxy)propanoate): Cc1c(ccc(c1)Cl)OC(C)C(=O)OCC(C)C
(2-methylpropyl 2-(4-chloro-2-methylphenoxy)propanoate): O=C(O)C(Oc(c(cc(c2)Cl)C)c2)COC(=O)C(Oc(c(cc(c1)Cl)C)c1)C

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
The easiest and most consistent way of applying QSAR models is to use ready-made software that implements the models via a user interface (European Commission, 2010). The Lazar is an open-source software programme that makes predictions of toxicological endpoints in a training set (http://lazar.in-silico.de). This model has been used to predict the potential mutagenicity and carcinogenicity of the test material and selected impurities.
Lazar searches the training dataset for similar compounds (neighbors) and calculates the prediction from their measured activities. Lazar calculates predictions using:
· A majority vote (weighted by compound similarity) for classification.
· A local QSAR model based on neighbors for regression.
Significant fragments are highlighted in the structure display as follows:
- Features that occur predominately in compounds with activity: "Non-carcinogen".
- Features that occur predominately in compounds with activity: "Carcinogen".
- Regions, where fragments from different classes overlap inert parts.
Note that predictions are based on the measured activities of neighbours. Significant fragments are solely used to determine activity specific similarities of neighbours.
Key to interpretation of results from simulations of Lazar software tool:
- Confidence: Indicates the applicability domain of a model. Predictions with a high confidence can be expected to be more reliable than predictions with low confidence. Confidence values may take any value between 0 and 1. For most models confidence > 0.025 is a (hard) threshold to distinguish between reliable and unreliable predictions.
- Measured activity: Experimental result(s) from the training dataset.

5. APPLICABILITY DOMAIN
Not reported.

6. ADEQUACY OF THE RESULT
For all predictions the model confidence is > 0.025 i.e. above the cut off to distinguish between reliable and unreliable predictions. This means that, for all substances, the predictions are deemed as reliable.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline required

Principles of method if other than guideline:

QSAR prediction.

GLP compliance:

no

Remarks:

Not applicable

Type of assay:

other: TKazius-Bursi Salmonella mutagenicity

Results of Lazar Prediction

Chemical	SMILES	TKazius-Bursi Salmonella Mutagenicity
Test material	O=C(O)C(Oc(c(cc(c1)Cl)C)c1)C	Non-mutagenic (Measured activity)
2-Propenoic acid, 2-chloro- (also known as 2-chloro acrylic acid)	O=C(O)C(=C)Cl	Non-mutagenic (Confidence: 0.0372)
(5-methylbenzene-1,3-dicarboxylic acid)	Cc1cc(cc(c1)C(=O)O)C(=O)O	Non-mutagenic (Confidence: 0.136)
(2-methylpropyl 2- (4-chloro-2-methylphenoxy) propanoate)	Cc1c(ccc(c1)Cl)OC(C)C(=O)OCC(C)C	Non-mutagenic (Confidence: 0.0743)
(2-methylpropyl 2- (4-chloro-2-methylphenoxy) propanoate)	O=C(O)C(Oc(c(cc(c2)Cl)C)c2)COC(=O) C(Oc(c(cc(c1)Cl)C)c1)C	Non-mutagenic (Confidence: 0.0719)

 

Conclusions:

All substances examined were predicted to be non-mutagenic with the restraints of the models.

Executive summary:

Using the QSAR models within the Lazar software tool to predict the mammalian toxicity and mutagenicity for the test material and selected impurities, the results show that;

- For all predictions the model confidence is > 0.025 i.e. above the cut off to distinguish between reliable and unreliable predictions. This means that, for all substances, the predictions are deemed as reliable.

- All substances examined (i.e. the test material and 4 impurities) are predicted to be non-mutagenic with the restraints of the models.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Several genetic toxicity studies are available on the substance. All of the available studies were conducted to standardised guidelines which were relevant at the time of conduct. In one case, the method followed a draft guideline.

All of the studies were conducted in reputable laboratories and under GLP conditions. As such, every available study is regarded as a key study and contributes to the overall conclusion that the substance does not require classification with respect to genetic toxicity.

In Vivo Micronucleus Assay: Edwards (1991)

Under the conditions of study, there was no evidence of induced chromosomal or other damage leading to micronucleus formation in polychromatic erythrocytes of treated mice 24, 48 or 72 hours after oral administration of the test material.

In Vivo UDS: Fautz (1994)

Under the conditions of the study the test material did not induce DNA-damage leading to increased repair synthesis in the hepatocytes of the treated rats. Therefore, the test material is considered to be negative in this in vivo/in vitro UDS test system.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 October 1990 to 01 November 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability under test conditions: No determinations of homogeneity or concentration were performed on prepared suspensions of the test material.

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Approximately 4 - 5 weeks old
- Weight at study initiation: Animals weighed 19.2 - 23.6 g on arrival
- Housing: Housed in single-sex groups of two or five in high density polypropylene cages with stainless steel tops
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: All animals were acclimatised for at least four days prior to treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 21 °C
- Humidity (%): 40 - 64 % R.H
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour light: 12-hour dark cycle

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 0.5 % methyl cellulose
- Justification for choice of solvent/vehicle: The test material was found to form a doseable and apparently homogeneous suspension in 0.5 % methyl cellulose.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Dosing suspensions were freshly prepared in 0.5 % methyl cellulose on the day of dosing, each concentration being individually formulated, and mixed prior to use.
- The dosage-volume was 10 mL/kg.

Duration of treatment / exposure:

Single administration.

Frequency of treatment:

The animals received the test material once.

Post exposure period:

Five male and five female mice per group were scheduled to be killed 24 hours after treatment; further lots of five males and five females from groups 1 and 4 were scheduled to be killed 48 and 72 hours after treatment.

Dose / conc.:

20 mg/kg bw/day (actual dose received)

Remarks:

Main micronucleus test

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Main micronucleus test

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Remarks:

Main micronucleus test

No. of animals per sex per dose:

Five / sex / group

Control animals:

yes, concurrent vehicle

Positive control(s):

Chlorambucil (5 animals per sex) in aqueous 10 % ethanol.
- Route of administration: Oral gavage
- Doses / concentrations: 30 mg/kg

Tissues and cell types examined:

Animals were killed by cervical dislocation following carbon dioxide inhalation. Femurs from each animal were rapidly dissected out and cleaned of adherent tissue. The epiphyses were cut off to obtain access to the marrow canal. Marrow cells were flushed out with 2.5 mL foetal calf serum using a syringe and needle.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
- Preliminary toxicity test
Initially, the toxicity of the test material to dividing bone marrow erythrocytes was examined: Slide evaluation was restricted to determination of numbers of polychromatic and mature erythrocytes and calculation of the ratio of polychromatic to mature cells for each animal, and for each group. The selection of levels tested were based on an oral LD50 of approximately 800 mg/kg in the rat. Dosing was by the oral route, on one occasion and at a dosage-volume of 10 mL/kg. All animals were killed 72 hours after treatment. The experimental design was as follows: 62.5 125, 250 and 500 mg/kg bw (two males and two females per group).


DETAILS OF SLIDE PREPARATION: The recovered cells were centrifuged at 1 000 rpm for five minutes. The bulk of the supernatant fluid was discarded and the cell pellet re-suspended in the remaining fluid. Single drops of the cell suspension were transferred to clean, dry slides, two or three smears (for the preliminary toxicity test or main micronucleus test respectively) prepared, and the slides left to air-dry. Following fixation in methanol for ten minutes, they were stained manually, using the Schmid (May-Grunwald and Giemsa) staining technique.
When air-dried, permanent mounts were made using DPX mountant, after clearing for five minutes in xylene.


METHOD OF ANALYSIS:
- Preliminary toxicity test
The slides were examined under the light microscope, and regions judged to be of adequate technical quality to permit scoring were selected under low magnification. At high magnification (x 1 000, oil immersion) a total of at least 2 000 erythrocytes per animal were examined. Each erythrocyte scored was classed as polychromatic or mature: Polychromatic cells stain blue/pink and the older cells stain red/pink. At least 1 000 cells of each type were scored from each animal where possible, but where there was an appreciable deviation from unity in the ratio of polychromatic to mature erythrocytes, scoring continued until a minimum of 2 000 of the predominant cell type were counted.
- Main micronucleus test
At least one slide from each animal was randomly coded by a person not subsequently involved in the scoring of the study. Care was taken to ensure that no unique slide identifications remained visible in order to eliminate bias. Slides were examined as detailed for the preliminary toxicity test, but in addition each erythrocyte scored was examined for the presence or absence of micronuclei. When examination had been completed, the data were decoded. The number of micronucleated cells per 1 000 erythrocytes was then calculated. The ratio of polychromatic to mature cells was also determined; a decrease in this may indicate inhibition of cell division following treatment, and the incidence of micronuclei in the mature cell population 24 hours after treatment reflects the pre-treatment situation, since most of these cells were produced before treatment. The frequency of micronuclei in polychromatic cells provides an index of induced genetic damage.


OTHER: Animals were inspected daily throughout the acclimatisation period and the dosing period for signs of ill-health or reaction to treatment. Any deviation from normal was recorded. All animals were weighed on the day of treatment and again immediately before termination, and bodyweights were recorded. In addition, the animals in the preliminary toxicity test were weighed immediately prior to dosing and daily thereafter until termination.

Statistics:

The frequencies of micronucleated cells per 1 000 polychromatic erythrocytes scored were subjected to statistical analysis by the Mann-Whitney procedure (Mann and Whitney, 1942). A computer-based version of this test was employed and significance was determined by reference to tabulated values of R1.
Data from males and females within each group were compared using a two-tailed test. Where there was no significant difference within the group, the sexes were pooled for further analysis. For each sampling time (24, 48 or 72 hours), each treated group was compared with concurrent vehicle controls using a one-tailed test.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Clinical signs of toxicity in test animals: All animals survived to scheduled termination. Three of the mice treated at 500 mg/kg showed an unstable gait after dosing and two of these also showed a hunched posture. One male treated at 62.5 mg/kg showed a hunched posture and rales two hours after dosing and one female treated at this dosage showed piloerection. All the mice had recovered within six hours of dosing and showed no further adverse reactions to treatment.
- Evidence of cytotoxicity in tissue analysed: No evidence of toxicity to the bone marrow (as shown by a reduction in the ratio of polychromatic to mature erythrocytes) was observed for any group treated with the test material. There was no evidence of toxicity of the test material (as evidenced by depression in bone marrow proliferation) to the bone marrow of the treated mice.
- After consideration of these data, the highest test material dosage selected for the main micronucleus test was 500 mg/kg.

RESULTS OF DEFINITIVE STUDY
- Clinical signs of toxicity in test animals: Occasional incidences of slight weight loss were noted in groups treated with the test material and in the vehicle control group. In view of the low incidence and absence of a strict relationship to dosage, no major significance can be attached to these results. All ten mice given chlorambucil, the positive control agent, lost weight during the 24 hour period before termination. No real indication of bone marrow toxicity, as evidenced by depression of bone marrow proliferation, was noted in any group treated with the test material. A few hours after dosing, eight of the mice treated with the test material at 500 mg/kg showed adverse reactions to treatment including underactivity (5 mice), hunched posture (2) and rales (1). Two of these animals were also prone and were sacrificed in extremis approximately two hours after dosing. All of the surviving mice had recovered by the day after dosing and no further adverse reactions to treatment were observed.
- Induction of micronuclei (positive control): Chlorambucil treatment produced a range of micronucleated cells per 1 000 polychromatic erythrocytes of 44.0 - 87.7 with a mean of 71.0. Statistical analysis showed that animals treated with chlorambucil had a significantly higher frequency of micronucleated polychromatic cells than vehicle control animals (p < 0.01). This increase in chromosomal damage after exposure to a known mutagen demonstrates the sensitivity of the test system.
The recorded incidence of micronuclei per 1 000 mature erythrocytes varied between 0.0 and 3.9 throughout all groups. These findings demonstrate the normal status of the animals used in the study: In particular, the low incidence in animals killed 24 hours after treatment shows the absence of any pre-treatment abnormality in the bone marrow.
- Incidence of micronucleated cells (Test material): Among mice killed 24 hours after treatment, the mean incidence of micronucleated polychromatic erythrocytes (per 1 000 polychromatic cells scored) was 1.7 for the vehicle control group, with a range of 0.0 - 3.0. Corresponding values for animals given the test material at 20, 100 or 500 mg/kg were closely similar: 2.4, 2.0 or 1.6 with ranges of 0.0-5.9, 0.0 - 5.0 and 0.0 - 2.8 respectively. Among mice killed after 48 or 72 hours, the mean incidences of micronucleated polychromatic erythrocytes in vehicle control groups were 1.3 (range 0.0 - 2.9) and 2.3 (range 1.0 - 3.5) respectively. Corresponding values for animals given test material at 500 mg/kg were 1.0 (range 0.0 - 2.7) and 1.7 (range 0.0 - 4.0) respectively. Thus, mean values for groups treated with the test material were closely similar to mean control group values at all termination times.
Statistical analysis confirmed that there was no significant difference in the incidence of micronucleated polychromatic erythrocytes between the vehicle control group and any group treated with test material, at any termination time (p > 0.05).
- Ratio of PCE/NCE: The ratio of polychromatic to mature erythrocytes was 0.7 in the vehicle control group at 24 hours. Ratios for groups given the test material at 20, 100 or 500 mg/kg and terminated 24 hours later were 0.7, 0.7 or 0.8 respectively. Forty eight hours after treatment, the ratio for the vehicle control group was 0.8, and in animals given the test material at 500 mg/kg it was 0.7. Seventy two hours after treatment, the ratio of polychromatic to mature erythrocytes for the vehicle control group was 0.6 and in animals treated with the test material it was 0.7. Ratios for all treatment groups were therefore closely similar to those of their respective vehicle control groups. In animals treated with chlorambucil, the ratio between polychromatic and mature erythrocytes was reduced (0.5).

Conclusions:

Under the conditions of study, there was no evidence of induced chromosomal or other damage leading to micronucleus formation in polychromatic erythrocytes of treated mice 24, 48 or 72 hours after oral administration of the test material.

Executive summary:

The genotoxicity of the test material was assessed according to OECD Test Guideline 474 and in compliance with GLP.

The effect of the test material on chromosome structure in bone marrow cells was investigated following acute oral administration to mice. Chromosome damage was measured indirectly by counting micronuclei.

A preliminary toxicity test was conducted in which groups of two male and two female mice were treated with the test material at dosages of 62.5, 125, 250 and 500 mg/kg. In all cases the test material was dosed orally, suspended in aqueous 0.5 % (w/v) methyl cellulose (0.5 % MC). Three of the four mice treated at 500 mg/kg showed transient signs of reaction to treatment. No evidence of toxicity to the bone marrow (as shown by a reduction in the ratio of polychromatic to mature erythrocytes) was observed for any group treated with the test material.

In the main micronucleus test, male and female mice were given a single oral dose of the test material at 20, 100 or 500 mg/kg. Concurrent vehicle and positive control groups of mice were similarly dosed with 0.5 % MC or chlorambucil (30 mg/kg) respectively. Five males and five females from each group were scheduled to be killed 24 hours after treatment; further lots of five males and five females, given the test material at 500 mg/kg or the vehicle control, were scheduled to be killed 48 and 72 hours after treatment. Bone marrow smears on glass slides were made from each animal. These slides were then stained and prepared for examination.

A total of at least 2 000 erythrocytes per animal was then examined for the presence of micronuclei, using the light microscope. Calculated values of micronuclei per 1 000 polychromatic erythrocytes were analysed statistically using the Mann-Whitney U test. The ratio of polychromatic: mature cells was also calculated for each animal, as an indicator of gross toxicity.

No real indication of bone marrow toxicity, as evidenced by depression of bone marrow proliferation, was noted in any group treated with the test material. A few hours after dosing, eight of the mice treated with the test material at 500 mg/kg showed adverse reactions to treatment including underactivity (5 mice), hunched posture (2) and rales (1). Two of these animals were also prone and were sacrificed in extremis approximately two hours after dosing. All of the surviving mice had recovered by the day after dosing and no further adverse reactions to treatment were observed.

Frequencies of micronucleated polychromatic erythrocytes in animals killed 24, 48 or 72 hours after administration of the test material were similar to those in concurrent controls. This lack of treatment-related effect was apparent in both sexes, and was confirmed by statistical analysis. Statistically significant increases over controls were, however, seen in positive control group animals given chlorambucil at 30 mg/kg (p < 0.01). 

Under the conditions of study, there was no evidence of induced chromosomal or other damage leading to micronucleus formation in polychromatic erythrocytes of treated mice 24, 48 or 72 hours after oral administration of the test material. The test procedure was highly sensitive to the chromosome-damaging action of chlorambucil.

Reference 2

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 March 1994 to 31 May 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

Version / remarks:

Draft OECD Guideline 1991

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

unscheduled DNA synthesis

Species:

rat

Strain:

Wistar

Remarks:

Wistar WU (first pre-experiment) and Wistar HanIbm: WIST (SPF) (other experiments).

Details on species / strain selection:

The rat is an animal which has been used for many years as suitable experimental animal in genotoxicity investigations. There are many data available from such investigations which may be helpful in the interpretation of results from the UDS test. In addition, the rat is an experimental animal in many physiological, pharmacological, and toxicological studies. Data from such experiments may also be useful for the design and the performance of the UDS test.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 6 - 10 weeks
- Weight at study initiation: Mean value 180.77 g (SD ± 8.55 g)
- Assigned to test groups randomly: Yes
- Fasting period before study: The animals were starved overnight (2 hours treatment) or approximately 6 hours (16 hours treatment) before receiving the test material; water was available continuously.
- Housing: Singly housed in Makrolon Type I, with wire mesh top
- Water: Ad libitum
- Acclimation period: Minimum 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3 °C
- Humidity (%): 30 - 70 %
- Photoperiod (hrs dark / hrs light): Artificial light 6.00 a.m. - 6.00 p.m.

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: CMC (carboxymethyl cellulose); 0.5 % aqueous solution.
- Justification for choice of solvent/vehicle: The vehicle was chosen according to its relative non-toxicity for the animals.
- Amount of vehicle: The volume administered was 10 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- On the day of the experiment (immediately before treatment), the test material was formulated in CMC 0.5 %.

Duration of treatment / exposure:

Single administration.

Frequency of treatment:

The animals received the test material once.

Post exposure period:

2 h (500 mg/kg b.w) or 16 hours (50, 200 and 500 mg/kg b.w.)

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Dose / conc.:

500 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Five male rats were assigned to each test group.

Control animals:

yes, concurrent vehicle

Positive control(s):

2-AAF; 2-Acetylaminofluorene dissolved in dimethyl sulfoxide/polyethylene glycol 400 (1 + 9). Solution prepared on day of administration.
- Justification for choice of positive control(s): The stability of the positive control substance in vehicle was unknown, but a DNA repair response in the expected range demonstrates biological stability.
- Route of administration: Single oral administration
- Doses / concentrations: 100 mg/kg b.w., 10 mL/kg b.w.

Tissues and cell types examined:

The animals were sacrificed by liver perfusion. After anaesthetising the rats with Na-pentobarbital the liver was perfused through the vena portae with Hanks' balanced salt solution supplemented with collagenase (0.05 % w/v) adjusted to pH 7.4 and maintained at 37 °C.
The hepatocytes were isolated from the liver and washed twice with HBSS. The crude cell suspension was filtered through a stainless steel mesh to yield a single cell suspension. The quality of the actual performed perfusion was determined by the trypan blue dye exclusion method. In addition, the number of the isolated cells was determined.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
- A preliminary study on acute toxicity was performed with a small group of 4 male animals using the same conditions as in the UDS study concerning: Animal strain; starvation period; route, frequency, and volume of administration. The animals were challenged orally (gavage) and examined for acute toxic symptoms at intervals of 1 h and 24 h after administration of the test material.
- For genotoxicity investigations it is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly. The volume to be administered should be compatible with physiological space available.
The maximum tolerated dose is determined to be the dose that causes toxic reactions (e.g. reduced spontaneous activity, eyelid closure, apathy, etc.) without having major effects on survival within 24 hours. If no toxic reactions are observed the highest dose to be used should be 2 000 mg/kg bw. For the low dose one tenth and for the medium dose one third of the high dose level will be tested.


DETAILS OF SLIDE PREPARATION:
The washed hepatocytes were centrifuged and transferred into Williams medium E supplemented with:
2.38 mg/mL hepes
100 units/mL penicillin
0.10 mg/mL streptomycin
0.29 mg/mL glutamin
0.50 μg/mL insulin
100 μL/mL foetal Calf Serum (FCS)
The medium without the cells was adjusted to pH 7.6.
- At least three cultures were established for each animal. Aliquots of 2.5 mL with freshly isolated hepatocytes in complete culture medium (1.0 x 10^5 living cells/mL) were added to 35 mm six-well cluster dishes containing one gelatinised 25 mm round plastic coverslip per well.
- After an attachment period of approximately 1.5 h in a 95 % air/ 5 % CO2 humidified incubator at 37 °C the culture medium was discarded. Then the cell layer was rinsed once with PBS to remove non-adherent cells. Subsequently 3HTdR (5 μ.Ci/mL, specific activity 20 Ci/mmol) in 2.0 mL culture medium (WME, 1 % FCS) was added to the cultures. After a labelling time of 4 h the cells were washed twice with WME supplemented with 1 % FCS and 0.25 mM unlabelled thymidine. Cultures were incubated overnight using the same medium. To prepare for autoradiography the medium was replaced by a hypotonic solution of 1 % sodium citrate for 10 minutes to swell the nuclei for better grain quantification. The cells on the coverslips were then fixed by three changes of methanol: acetic acid (3+1 v/v) for 20 minutes each, rinsed with 96 % ethanol, and air dried.
- The cover slips were mounted the side carrying the cells up on glass slides and coated with KODAK NTB2 photographic emulsion in the dark. The coated slides were stored in light-proof boxes in the presence of a drying agent for 12 - 14 days at 4 °C. The photographic emulsion is then developed with KODAK Dektol Developer at room temperature, fixed in TETENAL and stained with haematoxylin/eosin.

METHOD OF ANALYSIS:
- Evaluation was performed microscopically on coded slides using NIKON microscopes with oil immersion objectives. The cells for scoring were randomly selected according to a fixed scheme. The number of silver grains above the nucleus was counted automatically using the ARTEK 880 or 982 counter. In addition, the number of grains of one nuclear-sized cytoplasm adjacent to the nucleus was counted. At least two slides per animal and 50 cells per slide were evaluated. Heavily labelled S-phase cells were excluded from counting.
- Four animals per group were evaluated as described above. The remaining animal per test group would be evaluated if an animal died spontaneously or in case of technical problems concerning the isolation of the hepatocytes.
- The nuclear and cytoplasm grain counts, as well as the net grain counts (nuclear - cytoplasmic grains) were reported separately. The mean counts with standard deviation were used to describe the distribution of 3HTdR incorporation in the nucleus, the cytoplasm, and for the net grains, respectively.

Evaluation criteria:

Nuclear and net grain counts were estimated together. Increased net grains should be based on enhanced nuclear grain counts rather than on decreased cytoplasmic grain counts.
A test material is classified as positive if the mean number of net grains is higher than five per nucleus at one of the test points.
A group average between 0 and 5 net grains is considered as a marginal response. A dose-related increase in nuclear and net grains and/or a substantial shift of the percentage distribution of the nuclear grain counts to higher values provide additional information to confirm a positive response with less than 5 net grains.
Statistical significance may give further evidence for a positive evaluation.
A test material producing net grains not greater than 0 at any one of the test points is considered non-effective in this system.

Statistics:

Statistical significance can be evaluated by means of the nonparametric Mann-Whitney test.

A statistical evaluation of the results was not necessary to perform, since the number of nuclear grain counts of the groups treated with the test article were in the range of the controls and net grain values obtained were consistently negative.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: In the first pre-experiment two male rats were treated orally with a single dose of 1 000 mg/kg bw of the test material formulated in PEG 400. Both treated animals died within 24 h after challenge.
As requested by the sponsor 0.5 % CMC should be used as vehicle instead of PEG 400. Therefore, in the second pre-experiment 2 male rats received orally a single dose of 500 mg/kg bw of the test material formulated in CMC 0.5 %.
- Clinical signs of toxicity in test animals: The treated animals expressed toxic reactions: One hour post-treatment two rats showed a reduction of spontaneous activity and two with pilo erection.
Twenty four hours post-treatment two rats showed a reduction of spontaneous activity, two rats showed eyelid closure, one rat showed apathy, one rat showed prostration, two showed pilo erection and one tremors.
After administration of higher doses animals died.
- On the basis of these data 500 mg/kg bw was estimated to be close to the maximum tolerated dose.

RESULTS OF DEFINITIVE STUDY
- The viability of the hepatocytes was not substantially affected due to the in vivo pre-treatment with the test material at any of the treatment periods or dose groups. The interindividual variations obtained for the numbers and the viabilities of the isolated hepatocytes were in the range of the historical laboratory controls.
- No dose level of the test material revealed UDS induction in the hepatocytes of the treated animals as compared to the current vehicle controls. Neither the nuclear grains nor the resulting net grains were distinctly enhanced due to the in vivo treatment of the animals with the test material for 2 hours or 16 hours, respectively. Therefore, the net grain values obtained after treatment with the test material were consistently negative.
- In addition, no substantial shift to higher values was obtained in the percentage distribution of the nuclear grain counts.
- An appropriate reference mutagen (2-AAF, 100 mg/kg bw) was used as positive control. In vivo treatment with 2-AAF revealed distinct increases in the number of nuclear and net grain counts.

Conclusions:

Under the conditions of the study the test material did not induce DNA-damage leading to increased repair synthesis in the hepatocytes of the treated rats.
Therefore, the test material is considered to be negative in this in vivo/in vitro UDS test system.

Executive summary:

The genotoxicity of the test material was assessed according to the draft OECD Test Guideline 486 and in compliance with GLP.

The test material was assessed in the in vivo/in vitro UDS assay for its potential to induce DNA repair (UDS) in the hepatocytes of rats.

The test material was formulated in CMC 0.5%.This suspending agent was used as vehicle control. The volume administered orally was 10 mL/kg bw. After a treatment period of 2 and 16 hours, respectively, the animals were narcotised and sacrificed by liver perfusion. Primary hepatocyte cultures were established and exposed for 4 hours to HTdR (methyl- Hthymidine) which is incorporated if UDS occurs.

The test material was tested at the following dose levels:

2 hour preparation interval: 500 mg/kg bw

16 hour preparation interval: 50, 200 and 500 mg/kg bw

The highest dose was estimated by a pre-experiment to be suitable. The animals expressed toxic reactions. After administration of higher dose levels animals died. For each dose level, including the controls, hepatocytes from four treated animals were assessed for the occurrence of UDS. The viability of the hepatocytes was not substantially affected by the in vivo treatment with the test material.

No dose level of the test material revealed UDS induction in the hepatocytes of the treated animals as compared to the current vehicle controls.

An appropriate reference mutagen (2-AAF, 100 mg/kg bw) was used as positive control. Treatment with 2-AAF revealed distinct increases in the number of nuclear and net grain counts.

Under the conditions of the study the test material did not induce DNA-damage leading to increased repair synthesis in the hepatocytes of the treated rats.

Therefore, the test material is considered to be negative in this in vivo/in vitro UDS test system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Several genetic toxicity studies are available on the substance. All of the available studies were conducted to standardised guidelines which were relevant at the time of conduct. In one case, the method followed a draft guideline.

All of the studies were conducted in reputable laboratories and under GLP conditions. As such, every available study is regarded as a key study and contributes to the overall conclusion that the substance does not require classification with respect to genetic toxicity.

 

A summary, on each available study is presented below:

 

Ames: Thompson (2014)

The genotoxicity of the test material was assessed according to OECD Test Guideline 471 and EU Method B.13/14 and in compliance with GLP.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvr A were treated with the test material using both the Ames plate incorporation and pre-incubation methods 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 pre-determined and was 1.5 to 5 000 μg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test material formulations. The dose range was amended following the results of Experiment 1 and ranged between 1.5 and 5 000 μg/plate, depending on bacterial strain type and presence or absence of S9-mix. Up to eight test material dose levels were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the toxic limit of the test material following the change in test methodology.

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 material in the first experiment was selected as the maximum recommended dose level of 5 000 μg/plate. In the first mutation test (plate incorporation method), the test material caused a visible reduction in the growth of the bacterial background lawns and/or a substantial reduction in the revertant colony frequency of all of the tester strains, initially from 1 500 μg/plate (TA1537 dosed in both the absence and presence of S9-mix) and at 5 000 μg/plate to the remaining strains dosed in both the absence and presence of S9-mix. Consequently, the maximum recommended dose level of the test material was employed in the second mutation test. In the second mutation test (pre-incubation method), the test material induced a similar toxic response with weakened bacterial background lawns noted in both the presence and absence of S9-mix to TA1537 at and above 1 500 μg/plate and to the remaining strains at 5 000 μg/plate. A test material precipitate (greasy in appearance) was noted at 5 000 μg/plate, this observation did not prevent the scoring of revertant colonies. There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation in Experiment 1 (plate incorporation method). Similarly, 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 with or without metabolic activation in Experiment 2 (pre-incubation method).

Under the conditions of the study the test material was considered to be non-mutagenic.

 

Ames: May (2011)

The genotoxicity of the test material was assessed according to OECD Test Guideline 471 and EU Test Method B.13/14 and in compliance with GLP.

In this in vitro assessment of the mutagenic potential of the test material, histidine-dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan-dependent mutant of Escherichia coli, strain WP2 uvrA (pKM101), were exposed to the test material diluted in dimethyl sulphoxide (DMSO). DMSO was also used as a vehicle control.

Two independent mutation tests were performed in the presence and absence of liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone. The first test was a standard plate incorporation assay; the second included a pre-incubation stage. Concentrations of the test material up to 5 000 μg/plate were tested. Other concentrations used were a series of ca. half-log^10 dilutions of the highest concentration. No signs of toxicity were observed towards the tester strains in either mutation test following exposure to the test material.

No evidence of mutagenic activity was seen at any concentration of the test material in either mutation test.

The concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations. The mean revertant colony counts for the vehicle controls were within or close to the 99 % confidence limits of the current historical control range of the laboratory.

Under the conditions of the study it is concluded that the test material showed no evidence of mutagenic activity in this bacterial system.

 

Ames: May (2010)

The genotoxicity of the test material was assessed according to OECD Test Guideline 471 and EU Method B.13/14 and in compliance with GLP.

In this in vitro assessment of the mutagenic potential of the test material, histidine-dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan-dependent mutant of Escherichia coli, strain WP2 uvrA (pKM101), were exposed to the test material diluted in dimethyl sulphoxide (DMSO). DMSO was also used as a vehicle control.

Two independent mutation tests were performed in the presence and absence of liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone. The first test was a standard plate incorporation assay; the second included a pre-incubation stage. Concentrations of the test material up to 5 000 μg/plate were tested. Other concentrations used were a series of ca half-log^10 dilutions of the highest concentration. No signs of toxicity were observed towards the tester strains in either mutation test following exposure to the test material.

No evidence of mutagenic activity was seen at any concentration of the test material in either mutation test.

The concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations. The mean revertant colony counts for the vehicle controls were within or close to the 99 % confidence limits of the current historical control range of the laboratory.

Under the conditions of the study it is concluded that the test material showed no evidence of mutagenic activity in this bacterial system.

 

Ames: May (2009)

The genotoxicity of the test material was assessed according to OECD Test Guideline 471 and EU Method B. 13/14 and in compliance with GLP.

In this in vitro assessment of the mutagenic potential of the test material, histidine-dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan-dependent mutant of Escherichia coli, strain WP2 uvrA (pKM101), were exposed to the test material diluted in dimethyl sulphoxide (DMSO). DMSO was also used as a negative control.

Two independent mutation tests were performed in the presence and absence of liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone. The first test was a standard plate incorporation assay; the second included a pre-incubation stage. Concentrations of the test material up to 5 000 μg/plate were tested. Other concentrations used were a series of ca. half-log^10 dilutions of the highest concentration. No signs of toxicity were observed towards the tester strains in either mutation test following exposure to the test material at 5 000 μg/plate.

No evidence of mutagenic activity was seen at any concentration of the test material in either mutation test.

The concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations. The mean revertant colony counts for the vehicle controls were within or close to the 99 % confidence limits of the current historical control range of the laboratory.

Under the conditions of the study it is concluded that the test material showed no evidence of mutagenic activity in this bacterial system.

 

Ames: May (1990)

The test material was examined for mutagenic activity in four histidine-dependent auxotrophs of Salmonella typhimurium, strains TA 98, TA 100, TA 1535 and TA 1537, using pour-plate assays according to OECD Test Guideline 471 and in compliance with GLP. Each test, in each strain, was conducted on two separate occasions.

The studies, which were conducted in the absence and presence of an activating system derived from rat liver (S-9 mix), employed a range of levels of the test material from 10 to 1 000 µg per plate, selected following a preliminary toxicity test in strain TA 98. All tests included solvent (dimethyl sulphoxide) controls with and without S-9 mix.

No increases in reversion to prototrophy were obtained with any of the four bacterial strains at the test material levels tested, either in the presence or absence of S-9 mix. Inhibition of growth, observed as thinning of the background lawn of non-revertant cells, occurred in all strains following exposure to the test material at 1 000 µg per plate.

Marked increases in the number of revertant colonies were induced by the known mutagens benzo[a]pyrene, 2-nitrofluorene, 2-aminoanthracene, 9-aminoacridine and sodium azide when examined under similar conditions.

Under the conditions of the study, the test material was devoid of mutagenic activity.

 

Ames: Jones et al. (1993)

The genotoxicity of the test material was assessed according to OECD Test Guideline 471, EU Method B.14 and in compliance with GLP.

In this in vitro assessment of the mutagenic potential of the test material, histidine dependent auxotrophic mutants of Salmonella typhimurium (strains TA 1535, TA 1537, TA 98 and TA 100) were exposed to the test material, diluted in ethanol which was also used as a negative control.

Two independent mutation tests were performed, in the presence and absence of liver preparations from Aroclor 1254-induced rats.

In the preliminary dose range finding study with dose levels of up to 5 000 μg/plate no toxicity was observed. A top dose level of 5 000 μg/plate was chosen for the subsequent mutation study. Other dose levels used in the mutation assays were: 1 500, 500, 150, 50 μg/plate.

No evidence of mutagenic activity was seen at any dose level of the test material in either mutation test. The concurrent positive control compounds demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations.

Under the conditions of the study it was concluded that, when tested in ethanol, the test material was not mutagenic in this bacterial system.

 

Chromosome Aberration: Heidemann (1994)

The mutagenicity of the test material was assessed according to OECD Test Guideline 473 and EU Method B.10 and in compliance with GLP.

The test material was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro.

Two independent experiments were performed. Preparation of chromosomes was done 20 h (low, intermediate and high concentration), and 44 h (high concentration) after start of treatment with the test article which was dissolved in ethanol. Exposure periods were 20 h and 44 h (without S9 mix) and 4 h (with S9 mix). In each experimental group two parallel cultures were used. Per culture, 100 metaphases were scored for structural chromosomal aberrations. The following concentrations were evaluated at fixation intervals

Experiment I and II:

Without S9 mix (exposure period 20/33 h)

20 h: 100; 300; 600 µg/mL.

44 h: 300 µg/mL.

With S9 mix (exposure period 4 h)

20 h: 300, 1 000, 2 000 µg/mL.

44 h: 2 000 µg/mL.

In both experiments, the test material was tested up to toxic (without S9 mix, 300 and 600 μg/mL) and precipitating concentrations (with S9 mix).

There was no biologically relevant increase in cells with aberrations after treatment with the test material at any test point in the groups treated in the presence of S9 mix. However, in the absence of S9 mix (long term treatment), there were statistically significant increases (up to 8.5 %) in the aberration rates in three treatment groups as compared to the corresponding controls at cytotoxic concentrations. These increases could not be confirmed in the independent experiment II.

Appropriate reference mutagens were used as positive controls and showed distinct, statistically significant increases (α = 0.05) in cells with structural chromosomal aberrations.

Under the conditions of the study and in presence of S9 mix, the test material was not clastogenic. In absence of S9 mix a final assessment of the clastogenic potential of the test material is difficult due to divergent results at cytotoxic concentrations.

 

Chromosome Aberration: Edwards (1990)

The mutagenicity of the test material was assessed according to OECD Test Guideline 473 and in compliance with GLP.

The effects on chromosomal structure of exposure to the test material were investigated in cultured human lymphocytes. Tests were conducted with and without the inclusion of a rat liver-derived metabolic activating system (S-9 mix): Without S-9 mix cells were exposed for 24 hours, with S-9 mix exposure was limited to three hours. Treatments were established by the addition of test solutions, in dimethyl sulphoxide, (DMSO) to 48-hour cultures established from whole, human blood. Cell division was arrested by the addition of the spindle poison, Colcemid (to a final concentration of 0.4 µg/mL), three hours before the cells were harvested; slides were then prepared for microscopic analysis.

Mitotic indices were calculated for each culture. These were based on the number of metaphases observed per 1 000 cells scored. Chromosome aberrations were scored by examination of 100 metaphases per culture, and the frequencies of cells with one or more aberrations were calculated both including and excluding gap-type aberrations.

A preliminary test was performed to investigate the toxicity of the test material to dividing lymphocytes. Subsequently the following concentrations of the test material were tested in the main cytogenetic test:

- Without S-9 mix: 100, 200, 400 and 800 µg/mL

- With S-9 mix: 400, 800, 1 600 and 3 200 µg/mL

Four the test material concentrations were tested in both the presence and absence of S-9 mix to ensure that an appropriate range of toxicity was covered; slides prepared from cultures exposed to three concentrations only were selected for chromosome analysis. The main test also incorporated solvent and positive (cyclophosphamide and chlorambucil) control cultures. Cyclophosphamide is a known clastogen requiring biotransformation to achieve optimum activity; chlorambucil is a direct-acting clastogen. All treatments were established in triplicate.

The test material produced dose-related toxicity in the absence of S-9 mix, with reductions in mean mitotic index (compared to the solvent control value) of 19, 22, 47 and 94 % at 100, 200, 400 and 800 µg/mL respectively. In the presence of S-9 mix, no reductions in mitotic activity were observed in cultures treated with the test material at 400 and 800 µg/mL, but at 1 600 µg/mL a reduction of 76 % (compared to solvent control values) was recorded. In cultures treated at 3 200 µg/mL the test material was highly toxic and mitotic activity was almost completely inhibited. Subsequently slides prepared from cultures treated with the test material at 800 µg/mL in the absence of S-9 mix and 3 200 µg/mL in its presence, were not scored for chromosome aberrations. 

Treatment with the test material at 1 600 µg/mL in the presence of S-9 mix produced increases in the mean frequency of aberrant metaphases to 5.3 % (4 % excluding gaps), compared to 0.7 % (0.3 % excluding gaps) for solvent control cultures. The increases were apparent in two of the three treated cultures. The increases were statistically significant (p < 0.01) both including and excluding gaps. At lower concentrations of the test material in the presence of S-9 mix, and at all concentrations in the absence of S-9 mix, no statistically significant increases in the frequency of aberrant metaphases were observed.

The known clastogens, cyclophosphamide and chlorambucil, induced significant increases in the frequency of aberrant metaphases over the vehicle control values (p < 0.001 in both cases; as expected).

Under the conditions of study, the test material showed some evidence of clastogenic activity in the presence of S-9 mix, but only at a concentration which induced marked toxicity. The sensitivity of the test procedure, and the metabolic activity of the S-9 mix employed, were demonstrated by the clear responses to the positive control agents.

 

In Vitro Mammalian Cell Gene Mutation Test: Adams et al. (1993)

The test material was assessed for genotoxicity according to OECD Test Guideline 476 and in compliance with GLP.

The test material acid was tested for its ability to induce forward mutation at the functionally hemizygous hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in Chinese hamster ovary (CHO) cells in vitro both in the presence and the absence of exogenous metabolic activation in the form of Aroclor 1254-induced rat liver S-9.

Toxicity was observed in all the tests after treatment with the test material, both in the absence and the presence of S-9 mix. In the absence of S-9 mix, treatment of the cells with 23.2 - 694.5 μg/mL in Test 1 and 50 - 850 μg/mL in Test 2 resulted in mean cell survivals of 116 - 51 % and 129 – 8 % respectively. Cultures treated with 23.2, 92.6, 370.4, 463 and 694.5 μg/mL in Test 1 and 100, 200, 500, 625, 750 and 800 μg/mL in Test 2 were assessed for viability and induced mutation. No significant increases in mutant frequency were observed in either of the tests in the absence of S-9 mix. In the presence of S-9 mix, treatment of the cells with 46.3 - 1041.8 μg/mL in Test 1 and 25 – 1 000 μg/mL in Test 2 resulted in mean cell survivals of 122 - 1 % and 165 – 33 % respectively. Cultures treated with 92.6, 185.2, 370.4, 694.5 and 810.3 μg/mL in Test 1 and 100, 200, 400, 750 and 925 μg/mL in Test 2 were assessed for viability and induced mutation. No biologically significant increases in mutant frequency were observed in either of the tests in the presence of S-9 mix.

Under the conditions of the study the test material acid did not demonstrate mutagenic potential in this in vitro test system.

 

In Vitro Mammalian Cell Gene Mutation Test: Lloyd (1990)

The genotoxicity of the test material was assessed according to OECD Test Guideline 476 and in compliance with GLP.

The test material was examined for mutagenic potential by measuring its ability to induce mutation in Chinese hamster (V79) cells at the hypoxanthine-guanine-phosphoribosyl transferase (HGPRT) locus.

A preliminary toxicity test was first conducted: cells were exposed to five test material concentrations ranging from 8 to 5 000 µg/mL in the presence and absence of a rat-liver derived metabolic activating system (S-9 mix). After three hours of exposure, the cells were washed and seeded in non-selective medium to assess toxicity: plating efficiency was determined by colony counts.

Cultures of Chinese hamster cells were then exposed to five concentrations of the test material selected to permit a range of cell survival values (relative to solvent controls) in each of two mutation assays. The concentrations selected for the first mutation assay were 6.4, 32, 160, 800 and 4 000 µg /mL in the absence and presence of S-9 mix, but the test material was less toxic than expected in the absence of S-9 mix and more toxic than expected in its presence; consequently, the levels selected for the second mutation assay were 7.2, 36, 180, 900 and 4 500 µg /mL in the absence of S-9 mix and 6, 30, 150, 750 and 3 750 µg /mL in the presence of S-9 mix. Treatments were established for three hours. Solvent control cultures treated with dimethyl sulphoxide (DMSO) were included in all experiments.

Ethylmethanesulphonate (EMS), a known direct-acting mutagen, and 7,12-dimethylbenzanthracene (DMBA), a mutagen which requires metabolic activation to achieve optimal activity, were used as positive controls. All cultures were established in duplicate. After treatment, the cell sheet was washed and non-selective medium added. A sample of cells was taken from each culture and plated out to determine survival immediately post-treatment. The cultures were then incubated at 37 °C. Cultures were studied each day and passaged when necessary to maintain sub-confluence. After an expression time of 7 days, cells were plated out to assess survival and 6-thioguanine resistance (6-TG^r). After 6 days of incubation at 37 °C in a 5 % CO2 atmosphere, the resultant colonies were counted. Survival was estimated from the non-selective plates, and mutants were scored in selective plates; these latter contained 6-TG which was added to the plates 2-3 hours after seeding. Cultures exposed to the test material showed no increases in 6-TG^r colony numbers or mutant frequencies (per 10^5 survivors), compared to the solvent control cultures.

Under the same test conditions, EMS (1 000 µg/mL) produced increases in the incidence of mutant colonies. DMBA (10 µg/mL) increased the incidence of mutant colonies only in the presence of S-9 mix.

Under the conditions of the test, the test material induced no increases in mutation frequency at the HGPRT gene locus when cells were treated in the absence or presence of S-9 mix. The sensitivity of the assay system was proven by the observed responses to known mutagens.

 

In Vivo Micronucleus Assay: Edwards (1991)

The genotoxicity of the test material was assessed according to OECD Test Guideline 474 and in compliance with GLP.

The effect of the test material on chromosome structure in bone marrow cells was investigated following acute oral administration to mice. Chromosome damage was measured indirectly by counting micronuclei.

A preliminary toxicity test was conducted in which groups of two male and two female mice were treated with the test material at dosages of 62.5, 125, 250 and 500 mg/kg. In all cases the test material was dosed orally, suspended in aqueous 0.5 % (w/v) methyl cellulose (0.5 % MC). Three of the four mice treated at 500 mg/kg showed transient signs of reaction to treatment. No evidence of toxicity to the bone marrow (as shown by a reduction in the ratio of polychromatic to mature erythrocytes) was observed for any group treated with the test material.

In the main micronucleus test, male and female mice were given a single oral dose of the test material at 20, 100 or 500 mg/kg. Concurrent vehicle and positive control groups of mice were similarly dosed with 0.5 % MC or chlorambucil (30 mg/kg) respectively. Five males and five females from each group were scheduled to be killed 24 hours after treatment; further lots of five males and five females, given the test material at 500 mg/kg or the vehicle control, were scheduled to be killed 48 and 72 hours after treatment. Bone marrow smears on glass slides were made from each animal. These slides were then stained and prepared for examination.

A total of at least 2 000 erythrocytes per animal was then examined for the presence of micronuclei, using the light microscope. Calculated values of micronuclei per 1 000 polychromatic erythrocytes were analysed statistically using the Mann-Whitney U test. The ratio of polychromatic: mature cells was also calculated for each animal, as an indicator of gross toxicity.

No real indication of bone marrow toxicity, as evidenced by depression of bone marrow proliferation, was noted in any group treated with the test material. A few hours after dosing, eight of the mice treated with the test material at 500 mg/kg showed adverse reactions to treatment including underactivity (5 mice), hunched posture (2) and rales (1). Two of these animals were also prone and were sacrificed in extremis approximately two hours after dosing. All of the surviving mice had recovered by the day after dosing and no further adverse reactions to treatment were observed.

Frequencies of micronucleated polychromatic erythrocytes in animals killed 24, 48 or 72 hours after administration of the test material were similar to those in concurrent controls. This lack of treatment-related effect was apparent in both sexes, and was confirmed by statistical analysis. Statistically significant increases over controls were, however, seen in positive control group animals given chlorambucil at 30 mg/kg (p < 0.01). 

Under the conditions of study, there was no evidence of induced chromosomal or other damage leading to micronucleus formation in polychromatic erythrocytes of treated mice 24, 48 or 72 hours after oral administration of the test material. The test procedure was highly sensitive to the chromosome-damaging action of chlorambucil.

 

In Vivo UDS: Fautz (1994)

The genotoxicity of the test material was assessed according to the draft OECD Test Guideline 486 and in compliance with GLP.

The test material was assessed in the in vivo/in vitro UDS assay for its potential to induce DNA repair (UDS) in the hepatocytes of rats.

The test material was formulated in CMC 0.5%. This suspending agent was used as vehicle control. The volume administered orally was 10 mL/kg bw. After a treatment period of 2 and 16 hours, respectively, the animals were narcotised and sacrificed by liver perfusion. Primary hepatocyte cultures were established and exposed for 4 hours to HTdR (methyl- Hthymidine) whitxh is incorporated if UDS occurs.

The test material was tested at the following dose levels:

2-hour preparation interval: 500 mg/kg bw

16-hour preparation interval: 50, 200 and 500 mg/kg bw

The highest dose was estimated by a pre-experiment to be suitable. The animals expressed toxic reactions. After administration of higher dose levels animals died. For each dose level, including the controls, hepatocytes from four treated animals were assessed for the occurrence of UDS. The viability of the hepatocytes was not substantially affected by the in vivo treatment with the test material.

No dose level of the test material revealed UDS induction in the hepatocytes of the treated animals as compared to the current vehicle controls.

An appropriate reference mutagen (2-AAF, 100 mg/kg bw) was used as positive control. Treatment with 2-AAF revealed distinct increases in the number of nuclear and net grain counts.

Under the conditions of the study the test material did not induce DNA-damage leading to increased repair synthesis in the hepatocytes of the treated rats.

Therefore, the test material is considered to be negative in this in vivo/in vitro UDS test system.

 

QSAR: Kye (2011)

Using the QSAR models within the Lazar software tool to predict the mammalian toxicity and mutagenicity for the test material and selected impurities, the results show that;

- For all predictions the model confidence is > 0.025 i.e. above the cut off to distinguish between reliable and unreliable predictions. This means that, for all substances, the predictions are deemed as reliable.

- All substances examined (i.e. the test material and 4 impurities) are predicted to be non-mutagenic with the restraints of the models.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to genetic toxicity.