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Genetic toxicity in vitro

Description of key information

The test substance was found as non-mutagenic in three in vitro studies:

- Gene mutagenicity in bacteria Ames test (S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537, TA 1538 and E. coli WP2 uvrA), with or without metabolic activation: negative (OECD 471, BASF, 1995). 

- Gene mutagenicity in mammalian cells Mouse lymphoma assay, L5178Y cells, with or without metabolic activation: negative (OECD 476; CHV, 1996) 

- Cytogenicity in mammalian cells Chromosome aberration, CHL cells, with or without metabolic activation: negative (OECD 473; BASF 2002). 

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
- Batch No.: 4879
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
rat liver induced with phenobarbital and 5,6-benzoflavone.
Test concentrations with justification for top dose:
short-term treatment (-S9 mix): 4.88, 9.77, 19.5, 39.1, 78.1 µg/mL;
short-term treatment (+S9 mix): 19.5, 39.1, 78.1, 156, 313, 625, 1250 µg/mL;
continuous treatment (-S9 mix): 4.88, 9.77, 19.5, 39.1, 78.1 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9: Mitomycin C; +S9: Cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

NUMBER OF CELLS EVALUATED: 200
Evaluation criteria:
The results were judged to be: negative in the case where the appearance frequency of abnormal cells was less than 5 %; equivocal in the case where the frequency was 5 % or more and less than 10 % and also the reproducibility was observed; and positive in the case where the frequency was 10 % or more and also the reproducibility or the dependency on the test substance dose was observed.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxicity was observed at more than 39.1, 78.1, and 19.5 µg/mL in -S9 mix, +S9 mix and continuous treatments, respectively.
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
Clive et al., 1987, Mutation Research, 189, 143-156
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
thymidine kinase (TK)
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: culture medium: RPMI 1640 supplemented with Pluronic® F68, L-glutamine, sodium pyruvate, antibiotics and 10% horse serum; treatment medium: Fischer's medium with same supplements but 5% horse serum; cloning medium: same culture medium but with 20% horse serum and without Pluronic® F68 and addition of BBL purified agar (0.24%); selection medium: cloning medium containing 3 µg/ml of TFT (5-trifluorothymidine)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver
Test concentrations with justification for top dose:
3.13, 6.25, 12.5, 25, 50, 100 µg/mL
Vehicle / solvent:
- Vehicle used: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 2 h
- Selection time (if incubation with a selection agent): 10-14 h

SELECTION AGENT: 5-trifluorothymidine (TIFT)

NUMBER OF REPLICATIONS: 2 independent trials, 3 vehicle controls, 2 positive controls in each trial

NUMBER OF CELLS EVALUATED: approx. 3*10e5
Evaluation criteria:
- The minimum criterion considered necessary to demonstrate mutagenesis for any given treatment is a mutant frequency that is ≥ 2 times the concurrent background mutant frequency (as the average of the vehicle control cultures).
- A dose-related or toxicity-related increase in mutant frequency should be observed.
- If the mutant frequency obtained for a single dose at or near the highest testable toxicity is about two or more times the minimum criterion, the test substance will be considered mutagenic in a single trial.
- Treatments that induce less than ten percent relative growth are included in the assay, but are not used as primary evidence for mutagenicity.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: above 100 mg/L

RANGE-FINDING/SCREENING STUDIES: In preliminary range finding tests no cytotoxicity at 1000 µg/mL was observed.

Trial 1 was not considered acceptable because of cell culture problems (slower growth; positive control cultures were excessively cytotoxic; the vehicle control mutant frequencies, while still in the acceptable range, were higher than usual). However, no mutagenicity of the test substance was observed.

Trial 2

Treatment (µg/mL)

Metabolic activation

Suspension growth *a

Cloning efficiency *b

Relative growth *c

Mutant frequency (10e-6units)*d

3.13

no

109

85

93

92

 

yes

110

80

88

108

6.25

no

102

77

79

115

 

yes

105

76

80

81

12.5

no

104

75

78

111

 

yes

105

89

93

84

25

no

86

79

68

105

 

yes

86

92

79

91

50

no

92

87

79

97

 

yes

89

85

76

110

100

no

82

73

60

100

 

yes

67

90

61

123

*a = relative to vehicle control

*b = relative to vehicle control, total viable colony count/ number of cells seeded * 100

*c = (relative suspension growth + relative cloning efficiency)/ 100

*d = relative to vehicle control, (total mutant colonies/ total viable colonies) * 2 * 10e-4; decimal is moved to express the frequency in units for 10e-6

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
purity unknown, no second independent trial
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
No second independent trial was performed.
Principles of method if other than guideline:
Ames et al. (1975)/ Maron and Ames (1983)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his-
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 1254 induced rat liver
Test concentrations with justification for top dose:
100, 250, 500, 1000, 2500, 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Remarks:
sterility control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9 mix : 2-nitrofluorene (TA98, TA1538), sodium azide (TA1535, TA100), ICR-191 (TA1537 ); +S9 mix : 2-Aminoanthracene (five strains)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 ± 8 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
TA98 and TA100 considered positive if the TS produced at least a 2-fold increase in revertants per plate over vehicle control and a dose response to increasing concentrations; same criteria for the other strains but 3-fold increase.
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 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 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:
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:
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

Table 1: Plate Incorporation Test

Strain

Metabolic activation system

Replicates

Maximum revertant factor

Dose dependency

Assessment

TA 98

no

3

1.0

no

negative

yes

3

0.9

no

negative

TA 100

no

3

1.2

no

negative

yes

3

1.0

no

negative

TA 1535

no

3

1.2

no

negative

yes

3

1.1

no

negative

TA 1537

no

3

0.9

no

negative

yes

3

1.1

no

negative

TA 1538

no

3

1.4

no

negative

yes

3

1.0

no

negative

WP2ucrA

no

3

0.9

no

negative

yes

3

1.5

no

negative

Solvent: DMSO

Precipitation occurred in doses of ≥1000 µg/plate

In preliminary dose range-finding studies (TA100, 6.8-5000 µg/plate, 10 doses) no cytotoxicity with and without S9-mix; precipitates at 1000 µg/plate (background lawn could not be evaluated).

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Gene mutagenicity in bacteria 

For the gene mutagenicity in bacteria, a GLP compliant study is available, which was performed according to OECD guideline 471 (BASF, 1995). The study was performed as plate incorporation tests with S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537, TA 1538 and E. coli WP2 uvrA with or without metabolic activation up to 5000 µg/plate. All strains gave negative results. This result is supported by the negative result of another bacterial reverse mutation assay conducted with  S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and E. coli strain WP2 uvrA with and without metabolic activation at doses of 2.29 -5000 µg/plate (MHLW, 2001).  


Gene mutagenicity in mammalian cells 

In an in vitro mammalian cell gene mutation assay according to OECD guideline 476, L5178Y cells cultured in vitro were exposed to C.I. Pigment Yellow 53 at concentrations of 3.13, 6.25, 12.5, 25, 50 and 100 µg/mL (suspension with DMSO) in the presence and absence of mammalian metabolic activation (Aroclor-induce rat liver (S9)) (BASF, 1996). Higher concentrations were not tested because of the insoluble nature of the test substance. All dosing solutions were washed to remove the visible precipitate before application. Two trials of the non-activation and the S9 metabolic activation mutation assays were performed but the first trial was disregarded because of problems in the cell cultures. In Trial 2, six treatments from 3.13 µg/mL to 100 µg/mL were initiated and all doses were cloned for mutant analysis. No cytotoxicity was observed under either activation conditions. None of the six analysed treatments with or without metabolic activation induced a mutant frequency that exceeded the minimum criterion for a positive response. Nickel Antimony Titanate was therefore evaluated as negative with and without metabolic activation at the TK locus in L5178Y mouse lymphoma cells under the conditions used in this study.

Cytogenicity in mammalian cells 
In a mammalian chromosomal aberration test according to OECD guideline 473 (MHLW 2002), Chinese hamster lung cells (CHL/IU) were exposed to the test substance at concentrations of:
- 9.79, 19.5, 39.1 μg/mL without metabolic activation (short term treatment, -S9)
- 19.5, 39.1, 78.1 μg/mL with metabolic activation (short term treatment,+S9)
- 4.88, 9.75, 19.5 μg/mL without metabolic activation (continuous treatment, 24 hours)
The S9 mix was composed of phenobarbital- and 5,6-benzoflavone-induced rat liver. No increase in chromosomal aberrations was observed in the test with either the short term treatment (-S9 mix and +S9 mix) or the continuous treatment.

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. Based on the available in vitro data, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the ninth time in Regulation (EC) No 2016/1179.