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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames Test (OECD 471, GLP, K, rel.1): not mutagenic up to limit concentration in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. Coli WP2 uvrA

- Ames Test (OECD 471, GLP, S, rel.1): not mutagenic up to limit concentration in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. Coli WP2 uvrA

- CHO/HPRT assay on an analogue (OECD 476, GLP, K, rel.1): not mutagenic up to cytogenic concentrations in CHO cells

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From August 11 to 25, 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD test guideline No. 471 without any deviation.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Programme (inspected on December 02, 2002 /signed on February 13, 2003)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Storage condition of test material: Stored at approximately 4 °C in the dark under nitrogen.
Target gene:
Histidine and tryptophan for S. typhimurium and E. coli, respectively.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
10 % S9 mix: S9 from liver of male Sprague-Dawley rats orally received three consecutive daily doses of phenobarbitone/β-naphthoflavone (80/100 mg/kg bw/day) prior to S9 preparation on Day 4
Test concentrations with justification for top dose:
Preliminary toxicity study: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in TA100 and WP2uvrA-, with and without S9-mix using the direct plate incorporation method.

Mutation test (direct plate incorporation method):

- Experiment-1(range-finding test)
Salmonella strains (with and without S9-mix): 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
E.coli strain WP2uvrA- (with and without S9-mix): 50, 150, 500, 1500 and 5000 µg/plate

- Experiment-2 (main test)
Salmonella strains (with and without S9-mix): 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
E.coli strain WP2uvrA- (with and without S9-mix): 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: Test material was immiscible at 50 mg/mL in water, but miscible at 50 mg/mL in DMSO. Therefore, DMSO was selected as vehicle.
- Preparation of test materials: The test material was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer on the day of each experiment.
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
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 2-aminoanthracene
Remarks:
With S9-mix.
Details on test system and experimental conditions:
SOURCE OF TEST SYSTEM: Salmonella typhimurium strains were obtained from the University of California at Berkeley whilst Escherichia coli strain WP2uvrA- was obtained from the British Industrial Biological Research Association.

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: Approximately 48 h at 37 °C.

NUMBER OF REPLICATIONS:
- Preliminary toxicity study: One plate/dose
- Mutation study: 3 plates/dose

DETERMINATION OF CYTOTOXICITY
- Method: Evaluation of the toxicity was performed on the basis of growth of the bacterial background lawn.
Evaluation criteria:
- There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS (Kirkland, 1989) can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
- 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 a definitive judgement about the test material activity. Results of this type will be reported as equivocal.
Statistics:
Statistical methods, as recommended by the UKEMS (Kirkland, 1989) can be used as an aid to evaluation. However, statistical significance will not be the only determining factor for a positive response.
Key result
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not applicable
- Effects of osmolality: Not applicable
- Evaporation from medium: No data
- Water solubility: immiscible in water.
- Precipitation: A slight, oily precipitate was observed at 5000 µg/plate, but this did not prevent the scoring of revertant colonies.
- Other confounding effects: None

PRELIMINARY TOXICITY STUDY: Test material exhibited toxicity from 500 µg/plate to TA100 (without S9-mix) and was non-toxic to WP2uvrA- strain.

COMPARISON WITH HISTORICAL CONTROL DATA: The comparison was made with the historical control ranges for 2003 and 2004 of the corresponding testing laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The test material caused a visible reduction in the growth of the bacterial background lawn to all of the Salmonella strains, both with and without S9-mix, initially from 1500 µg/plate. No toxicity was observed to E.coli strain, WP2uvrA-. These results were not indicative of toxicity sufficiently severe enough to prevent the test material being tested up to the maximum recommended dose level of 5000 µg/plate.

Table 7.6.1/2: Preliminary toxicity test results

Metabolic activation

Strain

Dose (µg/plate)

0

0.15

0.5

1.5

5

15

50

150

500

1500

5000

-

TA100

79

76

86

93

104

87

78

88

73*

52*

0*P

+

TA100

95

81

93

93

80

81

98

78

85

40*

19*P

-

WP2uvrA-

19

25

26

30

20

30

23

24

26

22

20P

+

WP2uvrA-

34

24

22

27

30

24

43

31

15

22

18P

Key: *- Partial absence of bacterial background lawn

         P- Precipitate

See the attached document for information on tables of results – mutagenicity test

Conclusions:
Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium (strains TA 1535, TA 1537, TA 98 and TA 100) and E. coli WP2 uvr A- according to the criteria of the Annex VI of the of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.
Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, S. typhimurium strains TA1535, TA1537, TA98 and TA100 and E.coli strain WP2 uvrA- were exposed to test material diluted in DMSO, both in the presence and absence of metabolic activation system (10% liver S9 in standard co-factors), using the plate incorporation method. The dose range for the range-finding test was determined in a preliminary toxicity assay and was 50 to 5000 and 5 to 5000 μg/plate for WP2uvrA-and the Salmonella strains respectively (with and without S9-mix). The experiment was repeated on a separate day using the same extended dose range as the range-finding test. Negative, vehicle (dimethyl sulphoxide) and positive control groups were also included in mutagenicity tests.

The vehicle 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 test material caused a visible reduction in the growth of the bacterial background lawn to all of the Salmonella strains, with and without S9-mix, initially from 1500 µg/plate, although less toxicity was observed to TA 98. However, no toxicity was observed to E.coli WP2uvrA- strain. These results were not indicative of toxicity sufficiently severe enough to prevent the test material being tested up to the maximum recommended dose level of 5000 µg/plate. A slight, oily precipitate was observed at 5000 µg/plate but this did not prevent the scoring of revertant colonies. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation.

Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium strains TA1535, TA1537, TA98 and TA100, and E.coli WP2uvrA- according to the criteria of the Annex VI of the of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

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

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From April 12 to May 4, 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD test guideline No. 471 without any deviation.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese guideline: Notification No. 700 of the Environmental Agency, No. 1039 of the Ministry of Health and Welfare and No. 1014 of Ministry of International Trade and Industry, 9th December 1986.
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
OECD GLP (inspected on September 22, 1999/signed on January 18, 2000)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine and tryptophan for S. typhimurium and E. coli, respectively.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
10 % S9-mix: S9 fraction obtained from the liver of rats treated with Aroclor 1254 (500 mg/kg bw) by intraperitoneal route.
Test concentrations with justification for top dose:
- Preliminary toxicity test (direct plate incorporation method, with and without S9-mix): 10, 100, 500, 1000, 2500 and 5000 µg/plate in TA98, TA100 and WP2uvrA strains.

- Experiment 1 (Direct plate incorporation method):
Salmonella typhimurium strains TA 98 and TA 1537 (with and without S9 mix): 7.825, 15.625, 31.25, 62.5 and 125 µg/plate
Salmonella typhimurium strains TA 100 and TA 1535 (with and without S9 mix): 31.25, 62.5, 125, 250 and 500 µg/plate
E.coli strain WP2uvrA (with and without S9 mix): 312.5,625,1250, 2500 and 5000 µg/plate

- Experiment 2:
All Salmonella typhimurium strains (without S9 mix): 15.625, 31.25, 62.5, 125 and 250 µg/plate by Direct plate incorporation method
Salmonella typhimurium strains TA 98 and TA 1537 (with S9 mix): 15.625, 31.25, 62.5, 125 and 250 µg/plate by preincubation method
Salmonella typhimurium strains TA 100 and TA 1535 (with S9 mix): 31.25, 62.5, 125, 250 and 500 µg/plate by preincubation method
E.coli strain WP2uvrA : 312.5,625,1250, 2500 and 5000 µg/plate, by preincubation method with S9-mix and by Direct plate incorporation method without S9 mix

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: The test material was freely soluble in the DMSO at a concentration of 50 mg/mL. Therefore, DMSO was selected as the vehicle.
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
Remarks:
Without S9-mix.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-anthramine
Remarks:
With S9-mix.
Details on test system and experimental conditions:
SOURCE OF TEST SYSTEM: Salmonella typhimurium strains were obtained from the University of California (Berkeley, USA) whilst Escherichia coli strain WP2uvrA was obtained from S. Venitt's Laboratory (ICR, Sutton, England).

METHOD OF APPLICATION: in agar (plate incorporation); preincubation.

DURATION
- Preincubation period: 60 minutes at 37 °C
- Exposure duration: 48 to 72 h at 37 °C

NUMBER OF REPLICATIONS:
- Preliminary toxicity study: One plate/dose
- Mutation study (Experiment-1 and 2): 3 plates/dose

DETERMINATION OF CYTOTOXICITY
- Method: Evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.
Evaluation criteria:
- A reproducible two-fold increase in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result.
- Reference to historical data or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.
Statistics:
None
Key result
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not applicable
- Effects of osmolality: Not applicable
- Evaporation from medium: No data
- Water solubility: None
- Precipitation: A slight to moderate emulsion was noted in the Petri plates when scoring the revertants at dose-levels ≥ 2500 µg/plate in the preliminary toxicity test and Experiment-1.
- Other confounding effects: None

PRELIMINARY TOXICITY STUDY:
- In the TA98 strain a slight to marked toxicity was induced at dose-levels ≥ 100 µg/plate or ≥ 500 µg/plate, without and with S9-mix, respectively.
- In the TA100 strain, a moderate to marked toxicity was induced at dose-levels ≥ 500 µg/plate, both with and without S9-mix.
- In the WP2uvrA strain, slight toxicity was induced at dose-levels ≥ 2500 µg/plate, without S9-mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
The comparison was made with the historical control ranges for March, 2000 to August, 2000 of the corresponding Testing Laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A slight to marked toxicity was induced, depending on the tester strain, the dose-levels and the experimental conditions:
Experiment 1 (without S9 mix): Toxicity was observed at ≥250 µg/plate in TA 1535 and TA 100 strains
Experiment 2 (without S9 mix): Toxicity was observed at 250 µg/plate in TA 1535 and TA 1537 strains; at ≥62.5 µg/plate in TA 98 and TA 100 strains; at 5000 µg/plate in E. coli WP2uvrA strain
Experiment 1 (with S9 mix): Toxicity was observed at 500 µg/plate in TA 1535 and TA 100 strains
Experiment 2 (with S9 mix): Toxicity was observed at 250 µg/plate in TA 1537 and TA 98 strains; at ≥250 µg/plate in TA 1535 and TA 100 strains

OTHER:
- The sterility of the S9-mix was checked before the beginning and at the end of each experiment and was found to be satisfactory.

See the attached document for information on tables of results

Conclusions:
Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium strains TA1535, TA1537, TA98 and TA100 and E.coli WP2uvrA strain according to the criteria of the Annex VI of the of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.
Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, S. typhimurium strains TA1535, TA1537, TA98 and TA100 and E.coli strain WP2 uvrA were exposed to test material diluted in DMSO, both in the presence and absence of metabolic activation system (10 % liver S9-mix). The dose range was determined in a preliminary toxicity assay and ranged between 7.8125-125 µg/plate for TA98 and TA1537 strains, 31.25-500 µg/plate for TA100 and TA1535 strains, and 312.5-5000 µg/plate for WP2uvrA strain, with and without S9-mix. In Experiment-2, dose ranged between 15.625-250 µg/plate for all Salmonella strains without S9-mix as well as for the TA98 and TA1537 strains, with S9-mix; 31.25-500 µg/plate for TA100 and TA1535 strains, with S9-mix; and 312.5-5000 µg/plate for WP2uvrA strain, without and with S9-mix. All experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method. Vehicle (Dimethyl sulfoxide) and positive control groups were also included in mutagenicity tests.

The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. In the TA98 strain a slight to marked toxicity was induced at dose-levels ≥ 100 µg/plate or ≥ 500 µg/plate, without and with S9-mix, respectively. Test material also induced a moderate to marked toxicity at dose-levels ≥ 500 µg/plate, with and without S9-mix, to TA100 strain. In the WP2uvrA strain, slight toxicity was induced at dose-levels ≥ 2500 µg/plate, without S9-mix. A slight to moderate emulsion was observed in the Petri plates when scoring the revertants at dose-levels ≥ 2500 µg/plate in the preliminary toxicity test and Experiment-1. Test substance did not induce any noteworthy increase in the number of revertants, in any of the five tester strains, in both experiments.

Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium strains TA1535, TA1537, TA98 and TA100 and E. coli WP2uvrA strain according to the criteria of the Annex VI of the of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

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

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From July 30 to November 07, 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD test Guideline No. 476 without any deviation.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Program (inspected on March 12 to 14, 2014 / Signed on May 12, 2014)
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT locus
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Cells: CHO-K1 cells were obtained from ECACC, Salisbury, Wiltshire.
- Type and identity of media: The stocks of cells were stored in liquid nitrogen at approximately -196 °C. Cells were routinely cultured in Ham's F12 medium, supplemented with 5% foetal bovine serum (FBS) and antibiotics (Penicillin/Streptomycin at 100 units/100 μg per mL) at 37 °C with 5% CO2 in air.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes, the cells were supplied mycoplasma free.
- Periodically checked for karyotype stability: no, assumed to be stable
- Periodically "cleansed" against high spontaneous background: yes. Cell stocks spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen down they were cleansed of HPRT- mutants by culturing in HAT medium for 4 days. This is Ham's F12 growth medium supplemented with Hypoxanthine (13.6 μg/mL, 100 μM), Aminopterin (0.0178 μg/mL, 0.4 μM) and Thymidine (3.85 μg/mL, 16 μM). After 4 days in medium containing HAT, the cells were passaged into HAT-free medium and grown for 4 to 7 days. Bulk frozen stocks of HAT cleansed cells were frozen down, with fresh cultures being recovered from frozen before each experiment.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (2% final S9 concentration)
Test concentrations with justification for top dose:
Preliminary toxicity test: 7.43, 14.87, 29.73, 59.46, 118.93, 237.85, 475.7, 951.4 and 1902.8 μg/mL (up to 10 mM). The 24-hr was repeated : 0.25, 0.5, 1, 2, 4, 6, 8, 12 and 16 μg/mL.
Main test 1 (3hr -S9 mix): 1.75, 3.5, 7, 10.5, 14, 21, 28 µg/mL.
Main test 1 (3hr+S9 mix): 1.88 3.75, 7.5, 15, 20, 25, 30 µg/mL.
Main test 2 (24hr -S9 mix): 0.63, 1.25, 2.5, 5, 7.5, 10, 12 µg/mL.
Main test 2 (3hr +S9 mix): 1.25, 2.5, 5, 10, 20, 25, 30, µg/mL.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the substance was found to be soluble in dimethyl sulphoxide (DMSO).
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
500 and 750 µg/mL (4 hr), 200 and 300 µg/mL (24hr)
Positive control substance:
ethylmethanesulphonate
Remarks:
in the absence of S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
0.5 and 1 µg/mL
Positive control substance:
9,10-dimethylbenzanthracene
Remarks:
in the presence of S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hours 24 hours
- Expression time (cells in growth medium): 7 days, at 37°C, in a humidified atmosphere of 5% CO2 in air.

Fixation and staining of all flasks was achieved by aspirating off the media, washing with phosphate buffered saline, fixing for 5 minutes with methanol and finally staining with a 10% Giemsa solution for 5 minutes.


NUMBER OF REPLICATIONS: duplicate cultures for each concentration of the test compound, positive and negative controls.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency (200 cells/plate)

OTHER: ACCEPTANCE CRITERIA
An assay will normally be considered acceptable for the evaluation of the test results only if all the following criteria are satisfied. The with and without metabolic activation portions of mutation assays are usually performed concurrently, but each portion is, in fact, an independent assay with its own positive and negative controls. Activation or non-activation assays will be repeated independently, as needed, to satisfy the acceptance criteria.
i) The average absolute cloning efficiency of negative controls should be between 70 and 115% with allowances being made for errors in cell counts and dilutions during cloning and assay variables. Assays in the 50 to 70% range may be accepted but this will be dependent on the scientific judgement of the Study Director. All assays below 50% cloning efficiency will be unacceptable.
ii) The background (spontaneous) mutant frequency of the vehicle controls are generally in the range of 0 to 25 x 10-6. The background values for the with and without-activation segments of a test may vary even though the same stock populations of cells may be used for concurrent assays. Assays with backgrounds greater than 35 x 10-6 will not be used for the evaluation of a test item.
iii) Assays will only be acceptable without positive control data (loss due to contamination or technical error) if the test item clearly shows mutagenic activity. Negative or equivocal mutagenic responses by the test item must have a positive control mutant frequency that is markedly elevated over the concurrent negative control.
iv) Test items with little or no mutagenic activity, should include an acceptable assay where concentrations of the test item have reduced the clonal survival to approximately 10 to 15% of the average of the negative controls, reached the maximum recommended dose (10 mM or 5 mg/mL) or twice the solubility limit of the test item in culture medium. Where a test item is excessively toxic, with a steep response curve, a concentration that is
at least 75% of the toxic dose level should be used. There is no maximum toxicity requirement for test items that are clearly mutagenic.
v) Mutant frequencies are normally derived from sets of five dishes/flasks for mutant colony count and three flasks for viable colony counts. To allow for contamination losses it is acceptable to score a minimum of four mutant selection dishes and two viability dishes.
vi) Five dose levels of test item, in duplicate, in each assay will normally be assessed for mutant frequency. A minimum of four analysed duplicate dose levels is considered necessary in order to accept a single assay for evaluation of the test item.
Evaluation criteria:
See "any information on materials and methods incl. tables"
Statistics:
SPSS program or a suitable alternative.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Remarks:
HPRT gene locus
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no significant change in pH when the test item was dosed into media.
- Effects of osmolality: The osmolality did not increase by more than 50 mOsm at the dose levels investigated
- Evaporation from medium: not expected
- Water solubility: not soluble in water
- Precipitation: No precipitate of the test item was seen at the end of the exposure period in either exposure group in the 2 main experiments.
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: A dose range of 7.43, 14.87, 29.73, 59.46, 118.93, 237.85, 475.7, 951.4 and 1902.8 μg/mL was used in the preliminary cytotoxicity test for all three exposure groups. The maximum dose level tested was the maximum recommended dose level, the 10 mM concentration. Due to excessive toxicity being demonstrated in the 24-hour exposure group, with only one surviving dose level, this exposure group was repeated with a dose range of 0.25, 0.5, 1, 2, 4, 6, 8, 12 and 16 μg/mL.
A greasy/oily precipitate was seen at the end of the exposure period at and above 237.85 μg/mL in the 4-hour exposure in the absence of S9 and at and above 118.93 μg/mL in the 4-hour exposure in the presence of S9 and in the 24-hour exposure. Cloudy precipitate was also seen at 951.4 μg/mL in the 4-hour exposure in the absence of S9 and at and above 951.4 μg/mL in the presence of S9. A precipitate of the test item was observed at the end of exposure in the initial 24-hour experiment at and above 951.4 μg/mL. In the repeat of the 24-hour exposure group, no precipitate was observed at the end of the exposure period.
The results of the individual flask counts and their analysis are presented in Table 1 (see "attached background material). It can be seen that the test item was very toxic and there was a dose-related reduction in the cloning efficiency (CE) in all three exposure groups. The dose levels at and above 29.73 μg/mL and 59.46 μg/mL in the 4-hour exposure groups in the absence and presence of S9, respectively, had no viable cells remaining at the end of the exposure period. The 24-hour exposure group had only one surviving dose level and was therefore repeated with a revised dose range. The results of the repeat of the 24-hour exposure are presented in Table 2 (see "attached background material).
The selection of the maximum dose level for the main experiments was based on toxicity and was 28 μg/mL and 30 μg/mL for the 4-hour exposure groups in the absence and presence of S9, respectively, in Experiment 1. In Experiment 2 the maximum dose selected was 12 μg/mL for the 24-hour exposure and 30 μg/ml for the 4-hour exposure in the presence of S9 (2%).

COMPARISON WITH HISTORICAL CONTROL DATA:
It can be seen that the vehicle control values were all considered to be within an acceptable range, and that the positive controls all gave marked increases in mutant frequency, indicating the test and the metabolic activation system were operating as expected.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Test 1: The Day 0 and Day 7 cloning efficiencies for the 4-hour exposure with and without metabolic activation are presented in Table 3 and Table 4. The Day 0 toxicity in the absence and presence of S9 was similar to that seen in the Preliminary Toxicity Test with a dose related reduction in cloning efficiency. In the absence of S9 a reduction in the Day 0 cloning efficiency of 44% and 90% was achieved at 10.5 μg/mL and 14 μg/mL, respectively. There were no colonies surviving from the Day 0 cultures at 21 μg/mL and 28 μg/mL in the absence of S9 and these dose levels were not plated for mutant frequency or Day 7 viability. In the presence of S9 a dose related response was demonstrated and a reduction in the Day 0 cloning efficiency of 87% was achieved at 30 μg/mL. The 4-hour exposure group in the absence of S9 demonstrated a modest reduction in the Day 7 cloning efficiency of 28% at 14 μg/mL but no reduction was seen in the 4-hour exposure in the presence of S9.
The Day 0 and Day 7 vehicle control cloning efficiencies in the absence and presence of S9 did not achieve 70%, in all replicates, however, since they achieved at least 50% this was considered to be acceptable.
The mutation frequency counts and mean mutation frequency per survivor values are presented in Table 3 and Table 4 (see "attached background document"). There were no increases in mutation frequency per survivor that exceeded the vehicle control value by 20 x 10-6 at any dose level in either exposure group.
- Test 2: The Day 0 and Day 7 cloning efficiencies for the without and with metabolic activation exposure groups are presented in Tables 5 and 6. It can be seen that, as in Experiment 1, there was a dose related reduction in cloning efficiency at Day 0 in both exposure groups. The 24-hour exposure group achieved ideal maximum toxicity at 7.5 μg/mL with a reduction in cloning efficiency of 87%. The dose levels of 10 μg/mL and 12 μg/mL in the absence of S9 were too toxic for plating for mutant frequency. In the 4-hour exposure in the presence of S9 a reduction in cloning efficiency at Day 0 of 88% was achieved at 25 μg/mL which was within the ideal upper range of 80 to 90% toxicity. The dose level of 30 μg/mL in the presence of S9 had no surviving cells at the end of the exposure period and was therefore not maintained to be plated for Day 7 viability.
The Day 0 vehicle control cloning efficiencies in the absence of S9, did not achieve 70% in both replicates, however, since they achieved at least 50% this was considered to be acceptable. The mutation frequency counts and mean mutation frequency per survivor values are presented in Table 5 and Table 6. There were no increases in mutation frequency per survivor that exceeded the vehicle control value by 20 x 10-6 at any dose level in the absence or presence of S9.

None

Conclusions:
The test material did not induce any toxicologically significant or dose-related increases in the mutant frequency at the HPRT locus in CHO cells at any dose level, either in the presence or absence of metabolic activation, in both experiments, therefore the test material is not classified according to the annex VI of the Regulation EC No. 1272/2008 (CLP) and to the GHS.
Executive summary:

In an in vitro mammalian cell mutation assay performed according to the OECD test guideline No. 476 and in compliance with GLP, Chinese hamster ovary (CHO) cells were treated with the test item at up to eight dose levels, in duplicate, together with vehicle (dimethyl sulphoxide) and positive controls. Four treatment conditions were used for the test. In Experiment 1, a 4-hour exposure in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration and a 4-hour exposure in the absence of metabolic activation (S9). In Experiment 2, the 4-hour exposure in the presence of S9 was repeated (using a 2% final S9 concentration); whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

The dose range of the test item was selected based on the results of a preliminary cytotoxicity test and were as follows:

- 4-hour without S9: 1.75, 3.5, 7, 10.5, 14, 21, 28 µg/mL

- 4-hour with S9 (2%): 1.88, 3.75, 7.5, 15, 20, 25, 30 µg/mL

- 24-hour without S9: 0.63, 1.25, 2.5, 5, 7.5, 10, 12 µg/mL

- 4-hour with S9 (2%): 1.25, 2.5, 5, 10, 20, 25, 30 µg/mL

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

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

The test item demonstrated no significant increases in mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment.

Under the test conditions, the test material is not classified according to the annex VI of the Regulation EC No. 1272/2008 (CLP) and to the GHS.

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

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

Genetic toxicity in vivo

Description of key information

In vivo Micronucleus test (OECD 474, GLP, K, rel.1): not clastogenic in mice up to toxic concentrations

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From March 28 to November 20, 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD Guideline No. 474 (1997 version).
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
GLP principles of the German "Chemikaliengesetz" (inspected on May 15 and June 26, 2001/signed on September 24, 2001)
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland GmbH
- Age at study initiation: 5-8 weeks
- Weight at study initiation: 29 g (mean weight)
- Assigned to test groups randomly: yes, using an appropriate computer program.
- Housing: Animals were housed individually in Makrolon cages, type Ml.
- Diet: Standardized pelleted feed (Maus/Ratte Haltung "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland), ad libitum
- Water: Drinking water, ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Humidity: 30-70 %
- Photoperiod: 12 h dark/ 12 light

IN-LIFE DATES: From: March 28, 2003 To: November 20, 2003
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: olive oil
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, olive oil was selected as the vehicle, which had been demonstrated to be suitable in the in vivo micronucleus test and for which historical data are available.
- Concentration of test material in vehicle: 25, 50 and 75 mg/mL (main study).
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Test material was dissolved in olive oil. All test substance formulations were prepared immediately before administration. The stability of the test substance at 4°C in the vehicle over a period of 4 days was verified analytically.

ANALYSIS OF FORMULATION: For the determination of the test substance concentrations in the vehicle, 3 samples of each dose were taken from the test substance preparations, kept at room temperature until the treatment of the last animal (approximately 1 h) and then deep-frozen until they were determined analytically . The determination of the concentrations in the vehicle was carried out by HPLC.

DOSE VOLUME: 10 mL/kg bw
Duration of treatment / exposure:
Single intraperitoneal administration
Frequency of treatment:
Single intraperitoneal administration
Post exposure period:
250 and 500 mg/kg bw: 24 h
750 mg/kg bw: 24 and 48 h
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
actual administered by intraperitoneal injection
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
actual administered by intraperitoneal injection
Dose / conc.:
750 mg/kg bw/day (actual dose received)
Remarks:
actual administered by intraperitoneal injection
No. of animals per sex per dose:
Main study: 5 males/dose
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide and vincristine sulphate.
- Justification for choice of positive control(s): Cyclophosphamide and vincristine sulphate are well established reference clastogens and aneugens, respectively.
- Route of administration: Intraperitoneal
- Doses / concentrations: Cyclophosphamide: 20 mg/kg bw; vincristine sulphate: 0.15 mg/kg bw.
Tissues and cell types examined:
- 2000 polychromatic erythrocytes (PCEs) were evaluated per animal and investigated for micronuclei (MN).
- The normochromatic erythrocytes (NCEs) with and without micronuclei occurring per 2000 polychromatic erythrocytes were also recorded.
- Ratio of PCEs/NCEs.
- Number of small MN (diameter of MN < cell diameter/4) and of large MN (diameter of MN ≥ cell diameter/4).
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
- Dose selection was based on the range-finding test conducted in male and female animals at 500, 750, 1000 and 2000 mg/kg bw. Mortality was observed at 2000 mg/kg bw; evident signs of toxicity observed and some animals were sacrificed moribund at 750 and 1000 mg/kg bw; all animals survived but evident signs of toxicity were observed at 500 mg/kg bw. Therefore, a dose of 750 mg/kg bw was selected as the highest dose and 500 and 250 mg/kg bw were administered as further doses in the main study.

TREATMENT AND SAMPLING TIMES:
- The animals were sacrificed and the bone marrow of the femurs was prepared at 24 and 48 h after administration in the highest dose group of 750 mg/kg bw and in the vehicle controls.
- In the test groups of 500 and 250 mg/kg bw and in the positive control groups, only 24 h sacrifice interval was investigated.

DETAILS OF SLIDE PREPARATION:
- The bone marrow was prepared according to the method described by Schmid (1976, 1977) and Salamone et al (1980). Animals were sacrificed by cervical dislocation and femurs were prepared by dissection and removing all soft tissues. Femurs of the mice were removed and the bone marrow cells were extracted with fetal calf serum. After centrifugation, the supernatant was removed and a drop of the cell suspension was placed and spread on a slide.
- Slides were air-dried and stained in eosin and methylene blue (modified May-Grunwald solution or Wrights solution) for about 5 minutes, then followed by rinsing in purified water, stained in Giemsa solution for about 15 minutes and rinsed twice in purified water. Slides were clarified in xylene and then mounted in Corbit-Balsam.

METHOD OF ANALYSIS:
- Slides were examined under microscope to determine the frequency of micronuclei in 2000 polychromatic erythrocytes (PCEs) per animal. In addition, normochromatic erythrocytes (NCEs) with and without micronuclei occurring per 2000 polychromatic erythrocytes were also recorded. Ratio of PCEs to NCEs calculated. Number of small MN (diameter of MN < cell diameter/4) and of large MN (diameter of MN ≥ cell diameter/4) recorded.
Evaluation criteria:
- A test substance is considered positive if the following criteria are met:
Significant and dose-related increase in the number of PCEs containing micronuclei observed; the number of PCEs containing micronuclei has to exceed both the concurrent negative control and the highest value of the historical control range.
- A test substance is considered negative if the number of cells containing micronuclei in the dose groups is not significantly above the negative control and is within the historical control data.
Statistics:
- Statistical evaluation of the data was carried out using the program system MUKERN (BASF Aktiengesellschaft).
- The asymptotic U test according to MANN-WHITNEY (modified rank test according to WILCOXON) was carried out to clarify the question whether there were significant differences between the control group and dose groups with regard to the micronucleus rate in polychromatic erythrocytes.
- The relative frequencies of cells containing micronuclei of each animal were used as a criterion for the rank determination for the U test.
- Significances were identified as follows:
* p ≤ 0.05
** p ≤ 0.01
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
squatting posture, irregular respiration and poor general state
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 500, 750, 1000 and 2000 mg/kg bw
- Rationale for exposure: Determination of the acute intraperitoneal toxicity of test material.
- Clinical signs of toxicity in test animals: Mortality was observed at 2000 mg/kg bw; evident signs of toxicity observed and some animals were sacrificed moribund at 750 and 1000 mg/kg bw; all animals survived but evident signs of toxicity were observed at 500 mg/kg bw.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): Test material did not lead to any increase in the rate of micronuclei. The number of normochromatic or polychromatic erythrocytes containing small micronuclei (d < D/4) or large micronuclei (d ≥ D/4) did not deviate from the vehicle control value at any of the sacrifice intervals and was within the historical control range.

- Ratio of PCE/NCE (for Micronucleus assay): No dose-dependent inhibition of erythropoiesis induced by the treatment of mice with test material was detected; the ratio of polychromatic to normochromatic erythrocytes was always in the same range as that of the control values in all dose groups.

Table 7.6.2/1: Micronucleus data

 

 

Vehicle Control (0 mg/kg bw)

250 mg/kg bw

500 mg/kg bw

750 mg/kg bw

CCP

20 mg/kg bw

VCR

0.15 mg/kg bw

Interval

24 h

48 h

24 h

24 h

24 h

48 h

24 h

24 h

Total No. of PCEs

10000

10000

10000

10000

10000

10000

10000

10000

Total No. of NCEs

4594

3439

3755

4646

2476

2804

3920

5374

MN in PCEs (%)

1.4

0.7

1.6

1.8

1.2

1

11.1**

47.2**

MN in NCEs (%)

0.7

1.5

0.5

1.1

0.8

0.4

2

0.9

 

PCEs: Polychromatic erythrocytes

NCEs: Normochromatic erythrocytes

MN: Micronuclei

CCP: Cyclophosphamide

VCR: Vincristine

* : Statistically significant (Wilcoxon test, one-sided) vs. Vehicle control group, p<= 0.05

**: Statistically significant (Wilcoxon test, one-sided) vs. Vehicle control group, p<= 0.01

- The administration of the test substance at 250, 500 and 750 mg/kg bw led to squatting posture, poor general state and irregular respiration.

Conclusions:
Under the test conditions, test material is not classified as clastogenic or aneugenic according to the criteria of the Annex VI of the of the Regulation (EC) No.1272/2008 (CLP) and to the GHS.
Executive summary:

In an in vivo bone marrow micronucleus test, performed according to OECD guideline 474 (1997 version) and in compliance with GLP, Crl:NMRI mice (5 males/dose) were administered once intraperitoneally with test material, dissolved in olive oil, at the dose levels of 250, 500 and 750 mg/kg bw in a volume of 10 mL/kg bw. Vehicle control group was administered with olive oil and positive control groups were given cyclophosphamide (20 mg/kg bw) and vincristine (0.15 mg/kg bw) by the same route. Animals were sacrificed and the bone marrow of the femurs was prepared 24 and 48 h after administration in the 750 mg/kg bw dose group and in the vehicle controls. In the test groups of 250 and 500 mg/kg bw and in the positive control groups, only 24 h sacrifice interval was investigated. After staining of the preparations, 2000 polychromatic erythrocytes (PCEs) were evaluated per animal and investigated for micronuclei. The normocytes (NCEs) with and without micronuclei occurring per 2000 polychromatic erythrocytes were also recorded. Range-finding test was conducted to determine the dose levels for main study.

Test material did not lead to any increase in the number of PCEs containing either small or large micronuclei. The rate of micronuclei was always close to the range as that of the concurrent vehicle controls in all dose groups and at all sacrifice intervals and within the range of the historical control data. No inhibition of erythropoiesis determined from the ratio of PCE/NCE was detected. Both of the positive control chemicals, i.e. cyclophosphamide for clastogenicity and vincristine for spindle poison effects, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei indicating the validity of the study.

Under the test conditions, test material is not classified as clastogenic or aneugenic according to the criteria of the Annex VI of the of the Regulation (EC) No.1272/2008 (CLP).

This study is considered as acceptable and satisfies the requirement for mammalian erythrocyte micronucleus test.

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

Additional information

Table 7.6/1: Summary of genotoxicity tests

Test n°

Test substance

Test / Guideline

Reliability

Focus

Strains tested

Metabolic activation

Test concentration

Statement

1

 

Safepharm, 2005

Registered substance

Ames Test

(OECD 471)

K, rel. 1

Gene mutation

TA 1535,

TA 1537,

TA 98,

TA 100

E.coli WP2 uvrA

-S9

+S9

Up to limit concentration

-S9 : non mutagenic

+S9 : non mutagenic

2

 

CIT,

2001

Registered substance

Ames Test

(OECD 471)

S, rel. 1

Gene mutation

TA 1535,

TA 1537,

TA 98,

TA 100

E.coli WP2 uvrA

-S9

+S9

Up to limit concentration

-S9 : non mutagenic

+S9 : non mutagenic

3

 

Harlan, 2015

Read-across :

(2E)-1-(2,6,6-trimethyl-

1,3-cyclohexadien-1-yl)-

2-buten-1-one

CHO/HPRT test (OECD 476)

K, rel. 1

Gene mutation

Chinese hamster ovary (CHO)cells

-S9

+S9

Up to cytotoxic concentrations

-S9 : non mutagenic

+S9 : non mutagenic

4

 

BASF,

2003

Read-across :

(E)-4-(2,6,6-trimethyl-

1-cyclohexen-1-yl)-3-buten-2-one

In vivo MN

(OECD 474)

K, rel. 1

Chromosomal aberration

Mice

-

Up to toxic concentrations

-S9 : non clastogenic

+S9 : non clastogenic

Gene mutation Assays (Tests n° 1-3):

- Two Bacterial Reverse mutation Assays (Ames tests) were performed according to OECD guideline No. 471 with the substance (Test n°1 & 2 , see Table 7.6/1). The most recent study was selected as the key study. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains under the test condition, with any dose of the substance, either in the presence or absence of metabolic activation, in both assays. The substance does not induce gene mutations in bacteria whereas all positive control chemicals (with and without metabolic activation) induced significant increase of colonies. The substance is therefore considered as non-mutagenic according to the Ames test.

- Inability to produce gene mutation was confirmed in mammal cells using an in vitro gene mutation assay in Chinese hamster CHO cells (CHO/HPRT test) (Test n°3). None of the dose levels up to the cytotoxicity limit with the supporting substance which is considered adequate for read-across purpose (see Iuclid section 13 for additional justification), either in the presence or absence of metabolic activation, induced significant mutant frequency increases in the initial or repeat experiments. The supporting substance does not induce forward mutations at the HPRT locus in CHO chinese hamster cells under activation and non-activation conditions whereas both positive control chemicals (with and without metabolic activation) induced significant mutant frequency increases. Therefore both the supporting substance and the registered substance are considered as negative for inducing gene mutations at the HPRT locus in CHO chinese hamster cells under activation and non-activation conditions used in this assay. This result confirms the results of the Ames test and extends the non-mutagenic effect of both substances to mammalian cells.

Chromosomal aberration (Test n°4)

The clastogenic and aneugenic potential of the supporting substance, which is considered adequate for read-across purpose (see Iuclid section 13 for additional justification), was determined using an in vivo mammalian erythrocytes micronucleus assay (Test n°4), which identifies substances that cause micronuclei in erythroblasts. These micronuclei may originate from acentric fragments or whole chromosomes, and the test thus has the potential to identify both clastogenic and aneugenic chemicals. In this study erythroblasts were sampled from bone marrow cells of mice. None of the i.p. dose levels of up to 750 mg/kg bw, in males, induced increase in the frequency of micronucleated polychromatic erythrocytes (fMPCE), whereas the positive control chemical induced significant increases in the fMPCE. The supporting substance was therefore considered as negative for inducing chromosomal aberrations in mice bone marrow erythrocytes under the conditions used in this study. The supporting substance, and by analogy the registered substance, are therefore considered as non-clastogenic and non-aneugenic.

Justification for classification or non-classification

Harmonized classification:

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

Self-classification:

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