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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance is negative for causing gene mutation in bacteria cells. A substance of similar structure caused no gene mutations in mammalian cells in vitro. The positive clastogenic finding in vitro could not be confirmed in vivo. The substance is therefore considered to be not genotoxic.

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:
April 19, 1989 - May 29, 1989
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:
yes
Remarks:
(no statistics)
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
- Storage condition of test material: At room temperature
Target gene:
Salmonella typhimurium: His (-/-)
E. Coli: Tryp (-/-)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Species / strain / cell type:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S9
Test concentrations with justification for top dose:
Concentrations in the main test (with metabolic activation): 5, 10, 50, 100, 500, 1000 and 5000 µg/0.1 mL
Concentrations in the main test (without metabolic activation): 5, 10, 50, 100, 500, 1000 and 5000 µg/0.1 mL
Concentration range in the preliminary toxicity test: 20, 39, 78, 156, 313, 625, 1250, 2500, 5000 µg/0.1 mL
Vehicle / solvent:
Acetone (test article); DMSO (positive controls)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-aminoanthracene
Remarks:
with and without S9
Details on test system and experimental conditions:
METHOD OF APPLICATION (in agar, plate incorporation):

DURATION
- Incubation period: about 48 h at 37 ± 1.5°C in darkness

NUMBER OF REPLICATIONS:
- 3 Petri dishes per strain and per group per experiment

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
The test substance is considered to be positive in this test system if one or both of the following conditions are met:
- at least a reproducible doubling of the mean number of revertants per plate above that of the negative control at any concentration
level for one or more of the following strains: TA 98, TA 1535, TA 1537, TA 1538 and E. coli WP2uvrA,
- a reproducible increase of the mean number of revertants per plate for any concentration above that of the negative control by at least a factor of 1.5 for strain TA 100. Generally a concentration-related effect should be demonstrable
Statistics:
not performed
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no 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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at the concentrations of 500 µg/0.1 mL and above the test substance precipitated in soft agar

RANGE-FINDING/SCREENING STUDIES:
Nine concentrations ranging from 20 to 5000 ug/0.1 mL were tested to determine the highest concentration to be used in the mutagenicity assay. From the results obtained, the highest concentration suitable for the mutagenicity test was found to be 5000 ug/0.1 mL.

Experiment I

TA 98 TA 100 TA 1535 TA 1537 TA 1538 WP2uvrA
Dose (µg/0.1 ml) -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
solvent control 13 28 117 153 8 13 4 17 7 19 22 29
5 17 32 118 149 16 13 7 14 10 16 24 28
10 14 29 114 146 9 3 3 13 9 17 18 26
50 24 36 128 136 14 16 6 18 5 21 19 17
100 16 23 114 141 16 13 6 14 5 16 17 19
500 19 29 124 153 14 11 9 16 5 16 18 24
1000 15 32 126 137 15 13 7 15 8 18 19 26
5000 15 27 120 136 13 12 4 13 9 18 23 27
positive controls:
solvent control 14 23 100 139 8 14 7 14 8 19 17 21
concentration A 139 385 481 428 199 163 21 127 200 140 362 609
concentration B 222 376 754 542 313 94 149 124 358 123 674 718

Experiment II

TA 98 TA 100 TA 1535 TA 1537 TA 1538 WP2uvrA
Dose (µg/0.1 ml) -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
solvent control 17 28 135 185 14 9 6 10 7 20 24 34
5 15 26 118 186 15 10 4 18 5 23 21 33
10 11 28 120 188 10 9 8 17 3 15 27 27
50 22 27 105 156 9 8 6 11 11 14 25 22
100 14 21 108 145 19 8 5 17 5 15 19 29
500 23 29 124 133 9 11 8 14 11 16 25 24
1000 14 29 113 153 14 9 3 14 5 19 23 28
5000 20 24 123 133 17 12 5 15 9 19 24 35
positive controls:
solvent control 13 25 104 135 11 12 3 16 6 22 18 24
concentration A 137 455 318 289 219 144 30 191 189 179 392 498
concentration B 268 429 388 461 410 160 171 140 265 157 562 441

The slight increase in the number of back-mutant colonies observed with strain TA 1535 in the first experiment without microsomal activation at the concentrations of 5 and 100 µg/0.1 ml could not be confirmed in the second experiment and is therefore attributed to spontaneously occurring back-mutants.

Conclusions:
Based on the results of this study it is concluded that the test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

The test substance was tested in the Salmonella typhimurium reverse mutation assay with strains of Salmonella typhimurium (TA1535, TA1537, TA1538, TA100 and TA98) and in Escherichia coli WP2UvrA in two independent experiments. These tests were following GLP guidelines and were based on the OECD testing guideline 471. In a dose range finding test, the test article was tested up to concentrations of 5000 µg/0.1 ml in the absence and presence of S9-mix. At this dose level no toxicity was observed. In the mutation assays, the test substance was tested up to concentrations of 5000 µg/0.1 ml in the absence and presence of S9-mix. The test article precipitated at concentrations of 500 µg/0.1 ml and above. No decrease in the number of revertants was observed. No dose-related increase in the number of revertant both in the absence and presence of S9-metabolic activation was observed. These results were confirmed in an independently repeated experiment. Based on the results of this study it is concluded that the test article is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
other: in vitro mammalian cell chromosome aberration assay
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
Target gene:
not applicable
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Doubling time: about 15 h at 37°C
- Source: Dainippon Pharmaceutical Co.,Ltd. on January 14,1992
- Medium: Eagle's MEM medium (Nissui pharmaceutical Co.,Ltd. Lot No. 3161304 for chromosome aberration test, 323306 for additional test) supplemented with 10% calf serum (heat inactivate at 56°C; for 30 min, Irvine Scientific Lot No. 311702)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data
Metabolic activation:
with and without
Metabolic activation system:
S9:(Lot No. RAA-298; Kikkoman Co.), prepared from the livers of male 7-week old Sprague-Dawley rats, treated with phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
Positive controls:
- MMC: 0.05 µg/mL in physiological saline
- BP: 20 µg/mL in DMSO

Test article:
- 156 µg/mL, 313 µg/mL, 625 µg/mL, 1250 µg/mL, 2500 µg/mL, 5000 µg/mL
Vehicle / solvent:
acetone
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
(acetone)
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: continuous treatment (without S9) for 24 h and 48 h, puls treatment for 6 h with/without metabolic activation
- Expression time (cells in growth medium): 18 h (pulse treatment), 24 h and 48 h (continous treatment)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h (for 6 h pulse treatment and 24 h continuous treatment, respectively) and 48 h (48 h continuous treatment)

SPINDLE INHIBITOR (cytogenetic assays): colcemid, added 2 h before harvest
STAIN (for cytogenetic assays): giemsa

NUMBER OF REPLICATIONS: 2 experiments à 2 plates per concentration

NUMBER OF CELLS EVALUATED: 200 per dose per experiment

SCORING OF SLIDE PREPARATION:
Metaphase cells were examined under a microscope for structural and numerical abnormalities. In the case of numerical abnormalities, only polyploidy cells (more than 37 chromosomes ) were scored for these abnormalities. Structural abnormalities of chromosome were classified as gap (included chromosome type and chromatid type), chromatid break (ctb), chromosome break (csb), chromatid exchange (ete), chromosome exchange (cse), and other (fragmentation and so on). Observations of slides were carried out by the plural number of observers. The cell having at least one aberration was recorded as an aberrant cell.
Evaluation criteria:
Numbers of cells with chromosome aberration and polyploidy cells in the groups treated by the test substance are compared with those of the control groups for each dose. In principle, the test substance is judged to be clastogenic or aneugenic, if significant increase in aberration frequency, dose response and reproducibility of the result could be confirmed. The final judgment was decided as follows;
Negative (-): less than 5%
Inconclusive (+/-): from 5% to less than 10%
Positive (+): from 10% to less than 20%
Positive (++): from 20% to less than 50%
Positive (+++): 50% or more
The final evaluation was made on the basis of the total incidence of aberrant cells in which also cells with only gap were included. Gap is a clear existing discontinuity, but the distal part of the chromatid or chromosome showed no dislocation.
Statistics:
no data
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: due to the insolubility of the test article in aqueous media (water), emulsification occurs upon addition of its acetone solution to the cell culture during test substance treatment. This is discussed as possible reason as to why a definite dose reponse was not observed in the study.

- Significant increase in chromosome aberration for 24 h and 48 h continuous treatment and for 6 h pulse treatment without S9 Mix

- The major types of structural aberrations were chromatid break and chromatid exchange.

Conclusions:
The test substance is considered to be clastogenic under the experimental condition of this analysis.
Executive summary:

This study was designed to assess the mutagenicity of the test item in the metaphase chromosome analysis ofthe Chinese hamster lung cell line (CHL/IU). The test substance increased the frequency of structural chromosome aberrations in tlie continuous and pulse treatment. The increase in frequency of numerical chromosome aberrations was not observed with both continuous and pulse treatments. All negative (solvent) controls gave frequencies of aberrations within the range of negative values expected for the CHL/IU. The positive controls gave highly significant increases in the frequency of aberrations indicating the satisfactory performance of the test. The test substance is considered to be clastogenic under the experimental condition of this analysis.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-2011
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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: mammalian cell gene mutation assay (HPRT assay)
Target gene:
hprt
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/b-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
19.5, 39.1, 78.1, 156.3, 1250.0, 2500.0, 5000.0 mg/L (experiment 1; 4h, without S9)
25, 50, 100, 625, 1250, 2500, 5000 mg/L (experiment 2; 24h, without S9)
19.5, 39.1, 78.1, 156.3, 312, 1250, 2500 mg/L (experiment 1; 4h, with S9)
25, 50, 100, 200, 400, 600, 800 mg/L (experiment 2; 4h, with S9)
Vehicle / solvent:
Dimethylsulfoxide
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Without S9:300 μg/mL ethyl methanesulfonate, with S9: 20 μg/mL methylcholanthrene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 20 - 24h
- Exposure duration: 4h or 24h
- Expression time (cells in growth medium): 5 - 7 days
- Selection time (if incubation with a selection agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 16 days

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: Population doubling time 12 - 16h

NUMBER OF CELLS EVALUATED: not applicable

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
The HPRT assay is considered valid if the following criteria are met:
• The absolute cloning efficiencies of the negative/vehicle controls should not be less than 50% (with and without S9 mix).
• The background mutant frequency in the negative/vehicle controls should fall within our historical negative control data range of 0 – 15.95 mutants per 10exp6 clonable cells
• The positive controls both with and without S9 mix must induce distinctly increased mutant frequencies (historical positive control data given in Appendix to study report)
• At least 4 dose levels ranging up to a toxic concentration or up to or beyond the limit of solubility under culture conditions should be tested. Freely soluble and apparently non-toxic substances are not tested at concentrations higher than 5 mg/mL or 10 mM.

A finding is assessed as positive if the following criteria are met:
• Increase of the corrected mutation frequencies (MFcorr.) both above the concurrent negative control values and our historical negative control data range.
• Evidence of reproducibility of any increase in mutant frequencies.
• A statistically significant increase in mutant frequencies and the evidence of a dos-response relationship.
Statistics:
Due to the negative findings, a statistical evaluation was not carried out.
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 applicable
Positive controls validity:
valid
Additional information on results:
Cytotoxic effects indicated by clearly reduced cloning efficiencies of below 20% of control were observed in the 1st and 2nd Experiment in the presence of S9 mix at least in the highest applied concentrations. In both experimental parts in the absence of metabolic activation no distinct cytotoxicity was obtained.

In detail, with S9 mix, there was a decrease in the number of colonies from 625 μg/mL (CE1 relative: 8.3%) onward in the 1st Experiment and from 400 μg/mL (CE1 relative: 17.6%) onward in the 2nd Experiment. The cell densities were distinctly reduced.

Osmolarity and pH values were not influenced by test substance treatment.
In this study, under all test conditons, test substance precipitation in culture medium at the end of treatment was observed at about 50 μg/mL and above.

Summary of Results

Cytotoxicity***
Exp Exposure
period [h]
Test groups
[μg/mL]
S9
mix
Prec.* Genotoxicity**
MFcorr. [per 106cells]
CE1 [%] CE2 [%]
1 4 Vehicle control1 - - 2.03 100.00 100.00
19.5 - - 3.62 101.20 101.80
39.1 - - 2.74 108.40 106.60
78.1 - + 0.34 107.10 116.50
156.3 - + 0.00 84.80 95.30
1250.0 - + 0.00 62.30 115.90
2500.0 - + 0.00 45.30 105.40
5000.0 - + 3.58 50.80 115.80
Positive control2 - - 97.16 83.90 127.00
2 24 Vehicle control1 - - 0.00 100.00 100.00
25.0 - - 1.40 101.60 101.90
50.0 - + 7.92 96.70 94.20
100.0 - + 1.84 88.40 102.90
625.0 - + 0.90 68.60 105.40
1250.0 - + 3.86 58.60 113.30
2500.0 - + 0.00 40.30 106.60
5000.0 - + 11.87 60.30 114.70
Positive control2 - - 740.49 95.10 50.80
1 4 Vehicle control1 + - 4.49 100.00 100.00
19.5 + - 5.03 95.30 90.70
39.1 + - 3.62 84.80 91.20
78.1 + + 0.82 101.70 90.00
156.3 + + 2.21 93.40 89.20
312.5 + + 3.55 35.20 93.10
625.0 + + 2.30 8.30 106.20
1250.0 + + 0.70 3.60 108.90
Positive control3 + - 61.64 80.10 87.20
2 4 Vehicle control1 + - 2.95 100.00 100.00
25.0 + - 0.00 100.90 94.30
50.0 + + 0.84 100.70 99.00
100.0 + + 4.18 96.90 93.80
200.0 + + 1.17 64.10 101.60
400.0 + + n.c.1 17.6 n.c.1
600.0 + + n.c.1 8.5 n.c.1
800.0 + + n.c.1 6.6 n.c.1
Positive control3 + - 51.66 86.70 96.60

* Precipitation in culture medium at the end of exposure period

** Mutant frequency MFcorr.: mutant colonies per 106 cells corrected with the CE2 value

*** Cloning efficiency related to the respective vehicle control

n.c.1Culture was not continued due to strong cytotoxicity

1DMSO 1% (v/v)

2EMS 300 μg/mL

3MCA 20 μg/mL

Conclusions:
Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.
Executive summary:

The test substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Two independent experiments were carried out, both with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation). According to an initial range-finding cytotoxicity test for the determination of the experimental doses and taking into account the cytotoxicity actually found in the main experiments, the following doses were tested and the doses in bold type were evaluated in this study:

1st Experiment

without S9 mix (4-hour exposure period): 0; 19.5; 39.1; 78.1; 156.3; 1250.0; 2500.0; 5000.0 μg/mL

with S9 mix (4-hour exposure period): 0; 19.5; 39.1; 78.1; 156.3; 312.5; 625.0; 1250.0 μg/mL

2nd Experiment

without S9 mix (24-hour exposure period): 0; 25; 50; 100; 625; 1250; 2500; 5000 μg/mL

with S9 mix (4-hour exposure period): 0; 25; 50; 100; 200; 400; 600; 800 μg/mL

After an attachment period of 20-24 hours and a treatment period of 4 hours both with and without metabolic activation and 24 hours without metabolic activation, an expression phase of about 6-8 days and a selection period of about 1 week followed. The colonies of each test group were fixed with methanol, stained with Giemsa and counted. The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, EMS and MCA, led to the expected increase in the frequencies of forward mutations. Cytotoxicity indicated by reduced survival (CE1) of below 20% of control was observed in both experiments in the presence of metabolic activation only. On the basis from the results of the present study, the test substance did not cause any relevant increase in the mutant frequencies either without S9 mix or after adding a metabolizing system in two experiments performed independently of each other. Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

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

Genetic toxicity in vivo

Description of key information

The test substance is considered not to be clastogenic based on the negative result in the in vivo MNT Assay in hamster.

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

Additional information

Gene Mutation

In bacteria cells

In a reverse gene mutation assay in bacteria (OECD 471), strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 of S. typhimurium and E. coli WP2uvrA were exposed to the test article (96.2% pure) in acetone at concentrations of 5, 10, 50, 100, 500, 1000 and 5000 (limit concentration) µg/0.1 mL in the presence and absence of mammalian metabolic activation with a plate incorporation method. No dose related increase of revertants was seen. The positive controls induced the appropriate response in the corresponding strains. There was no evidence of induced mutant colonies over background. No cytotoxicity was observed up to the limit concentration. Substance precipitation that occurred from the concentration of 500 µg/0.1 mL and above did not affect scoring integrity. The study is classified as acceptable as it satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity data (bacterial reverse gene mutation) and GLP Guideline.

In mammalian cells

The analogue substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro (OECD 476, GLP). Two independent experiments were carried out, both with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation).

According to an initial range-finding cytotoxicity test for the determination of the experimental doses and taking into account the cytotoxicity actually found in the main experiments, the following doses were tested and the doses in bold type were evaluated in this study:

1st Experiment:

Without S9 mix (4-hour exposure period): 0; 19.5; 39.1; 78.1; 156.3; 1 250.0; 2 500.0; 5 000.0 μg/mL

With S9 mix (4-hour exposure period): 0; 19.5; 39.1; 78.1; 156.3; 312.5; 625.0; 1 250.0 μg/mL

2nd Experiment:

Without S9 mix (24-hour exposure period): 0; 25; 50; 100; 625; 1 250; 2 500; 5 000 μg/mL

With S9 mix (4-hour exposure period): 0; 25; 50; 100; 200; 400; 600; 800 μg/mL

After an attachment period of 20 - 24 hours and a treatment period of 4 hours both with and without metabolic activation and 24 hours without metabolic activation, an expression phase of about 6 - 8 days and a selection period of about 1 week followed. The colonies of each test group were fixed with methanol, stained with Giemsa and counted. The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, EMS and MCA, led to the expected increase in the frequencies of forward mutations. Cytotoxicity indicated by reduced survival (CE1) of below 20% of control was observed in both experiments in the presence of metabolic activation only. On the basis from the results of the present study, the test substance did not cause any relevant increase in the mutant frequencies either without S9 mix or after adding a metabolizing system in two experiments performed independently of each other.

The analogue substance has a lower degree of alkylation and is slightly better soluble. Neither the analogue substance nor the target substance carry a known structural alert for genotoxicity. For detailed information on read-across, it is referred to the attachment in IUCLID chapter 13.

 

Chromosomal Mutation

In vitro

In a mammalian cell cytogenetic assay, CHO/IU cell cultures were exposed to the test article (96.2% pure) in acetone at concentrations of 156, 313, 625, 1250, 2500 and 5000 (limit concentration) µg/mL with and without metabolic activation. Positive controls induced the appropriate response. There was evidence of structural chromosome aberration induced over background, but without dose-dependency. The test item is a liquid that was diluted in acetone. Upon addition to the cell culture medium, the test item formed droplets in the culture medium. The major types of aberrations were chromatid breaks and chromatid exchanges. There was no evidence of polyploidy induced over background. This study is classified as acceptable as it satisfies the requirement for Test Guideline in vitro mammalian cytogenics (Chromosome Aberration Test), OECD 473 for in vitro cytogenetic mutagenicity data and GLP Guideline. The test article is assumed to be clastogenic in this study.

 

In vivo

In a Chinese hamster bone marrow micronucleus assay, 8 animals/sex/dose/sacrifice time were given a single application of the test substance (96.2% pure) in arachis oil per gavage at a single dose 5000 mg/kg bw. The test substance was tested at an adequate dose based on a preliminary test. Bone marrow cells were harvested at 16, 24 and 48 hours post-treatment. There was no mortality nor were there any signs of systemic toxicity during the study. There was no cytotoxicity as evidenced in the ratio of PCR/NCR in the treated animals compared to controls. The positive control induced the appropriate response. There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any sampling time. This study is classified as acceptable as it satisfies the requirement for Test Guideline OECD 474 for in vivo cytogenetic mutagenicity data and GLP Guideline.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008.