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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames assay:

Ames assay was performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium TA1537, TA1535, TA158, TA98 and TA100 both with and without rat liver S-9 microsomal activation system. In the study reported here, the test chemical was tested in the Ames bioassay at levels upto 13.9 mg/L (0.450 mg/plate) without S-9 activation 82.3 mg/L (2.7mg/plate) with S-9 activation. The maximum concentrations used in these studies resulted in reduced viability of the test organisms, indicating that the test chemical was being tested at biologically significant concentrations. The difference in maximum test concentrations as a function of the presence of S-9 indicates that the metabolite(s) of the test chemical formed by microsomal enzymes in the s-9 have less biological impact on the test organism than the test chemical itself. In the Ames bioassay , no test chemical- related increases in revertants were noted in any test strain either with or without S-9 liver enzymes. HQ was negative when tested in the standard Ames test in five strains of Salmonella typhimurium , both with and without rat liver S-9 microsomal activation.

In vitro mammalian chromosome aberration study:

In vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical. The study was performed using Chinese hamster lung fibroblast cell line (CHL)in the presence and absence of S9 metabolic activation system. The chemical was dissolved in DMSO as solvent and used at dose levels 0, 0.0125, 0.025 or 0.05 mg/mL Concurrent solvent and positive controls were included in the study. The cells were treated for 24 and 48 h without S9 mix with a test compound at 3 dose levels at least. After treatment for 6 h with S9, the reaction mixture was replaced with fresh medium, and then the cells were recultured for 18 h. Both with and without the metabolic activation system, the cells were treated with Colcemid for 2 h, and chromosome preparations were made using a standard air-drying method. The frequency of cells with chromosomal aberrations was scored in 100 well-spread metaphases for each dose. Structural chromosomal aberrations were classified into 6 groups: chromatid gaps (ctg) including chromosome gaps, chromatid breaks (ctb), chromatid exchanges (cte), fragmentation (frg), chromosome breaks (csb), and chromosome exchanges (cse) including dicentric and ring chromosomes.Polyploid cells were also recorded.Solvent-treated cells served as controls. The experimental groups were judged as negative (-) if the total frequency was less than 5.0%, suspicious (+) if 5.0-< 10.0% and positive (+) if 10% or more. The test chemicaldid not induce chromosome aberration in Chinese hamster lung fibroblast cell line (CHL) in the absence of S9 metabolic activation system. It however induced gene mutation in the presence of S9 metabolic activation system.

In vitro mammalian cell gene mutation assay:

In vitro mammalian cell gene mutation test was performed to evaluate the mutagenic nature of the test chemical. The study was performed using V79 Chinese hamster lung cells with and without rat and hamster hepatocytes. The test chemical was dissolved in DMSO and used at appropriate dose levels of 0, 0.17, 1.70 or 3.40 µg/mL with and without hepatocytes. The period of exposure to chemicals, with or without activation by rat or hamster hepatocytes, was 48 h, after which the chemicals were removed and the cultures reseeded into flasks (75 cm*) at a density of 1.9 x 10” viable cells per 15 ml of Williams’ E medium. Following a 6-day period of expression, the cells were reseeded into 20 culture dishes (Corning, 60 mm) at 20 x lo3 viable cells per dish. For the selection of mutants, 6-thioguanine was added immediately after seeding to give a final concentration per dish of 10 pg/ml. Mutant colonies were counted 8 days later. Without hepatocytes, the test chemical was non-mutagenic at the HGPRT (TGR) gene locus at all concentrations. The test chemical in the presence of hepatocytes for TGR produced sporadic positive results (p < 0.01) when compared with concurrent controls.The chemical showed slight but increasing cytotoxicity to V79 cells at concentrations ranging from 2.0 to 3.4 µg/ml. Colony formation was reduced to 50% of the control at about 4.2 pg/ml and the antioxidant was lethal to 100% of the cells at dose levels above 5 µg/ml. Based on the observations made, test chemical did not induce gene mutation in the HGPRT locus in V79 Chinese hamster lung cells in the absence of rat and hamster hepatocytes. It however induced gene mutation in V79 Chinese hamster lung cells in the presence of rat and hamster hepatocytes.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
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:
guideline study
Justification for type of information:
Data is from study report
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
Ames assay was performed to determine the mutagenic nature of the test chemical
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine
Species / strain / cell type:
S. typhimurium, other: TA1537, TA1535, TA1538, TA98, TA100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
RAT, LIVER, S-9, AROCLOR 1254
Test concentrations with justification for top dose:
The test chemical was tested in the Ames bioassay at levels upto 13.9 mg/L (0.450 mg/plate) without S-9 activation 82.3 mg/L (2.7 mg/plate) with S-9 activation.
Vehicle / solvent:
Dimethyl sulfoxide (DMSO)
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulfoxide (DMSO)
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
With S9 activation
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulfoxide (DMSO)
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Without S9 activation for S. typhimurium TA 1535
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulfoxide (DMSO)
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Without S9 activation for S. typhimurium TA 1537
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulfoxide (DMSO)
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: picrolonic acid
Remarks:
Without S9 activation for S. typhimurium TA 1538
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulfoxide (DMSO)
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: ICR-191
Remarks:
Without S9 activation for S. typhimurium TA 98
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulfoxide (DMSO)
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Without S9 activation for S. typhimurium TA 100
Details on test system and experimental conditions:
No data
Rationale for test conditions:
No data
Evaluation criteria:
The plates were observed for an increase in number of revertants/plate
Statistics:
No data
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
Without the metabolic mixture, the 50% population survival dose was observed to be between 31 and 9.8 mg/L. A significant decrease in 2-tert-butylhydroquinone (TBHQ) toxicity was observed in the presence of the S-9 metabolic mixture with 50% population survival occurring at between 30.5 and 96.3 mg/L.

Remarks on result:
other: No mutagenic potential
Conclusions:
The test chemical was negative when tested in the standard Ames test in five strains of Salmonella typhimurium TA1537, TA1535, TA158, TA98 and TA100 both with and without rat liver S-9 microsomal activation and hence it is not likely to classify as a gene mutant in vitro.
Executive summary:

Ames assay was performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium TA1537, TA1535, TA158, TA98 and TA100 both with and without rat liver S-9 microsomal activation system. In the study reported here, the test chemical was tested in the Ames bioassay at levels upto 13.9 mg/L (0.450 mg/plate) without S-9 activation 82.3 mg/L (2.7mg/plate) with S-9 activation. The maximum concentrations used in these studies resulted in reduced viability of the test organisms, indicating that the test chemical was being tested at biologically significant concentrations. The difference in maximum test concentrations as a function of the presence of S-9 indicates that the metabolite(s) of the test chemical formed by microsomal enzymes in the s-9 have less biological impact on the test organism than the test chemical itself. In the Ames bioassay , no test chemical- related increases in revertants were noted in any test strain either with or without S-9 liver enzymes. HQ was negative when tested in the standard Ames test in five strains of Salmonella typhimurium , both with and without rat liver S-9 microsomal activation.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from peer reviewed publication
Qualifier:
according to guideline
Guideline:
other: Refer below principle
Principles of method if other than guideline:
In vitro mammalian chromosome aberration test was performed to evaluate the mutagenic nature of the test chemical
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
No data
Species / strain / cell type:
mammalian cell line, other: A Chinese hamster lung fibroblast cell line (CHL)
Details on mammalian cell type (if applicable):
- Type and identity of media: The cells were cultured with Eagle's minimal essential medium supplemented with 10% heat-inactivated calf serum.
- Properly maintained: No data
- Periodically checked for Mycoplasma contamination: No data
- Periodically checked for karyotype stability: The doubling time of the cells was estimated to be about 15 h, and the modal chromosome number was 25.
- Periodically "cleansed" against high spontaneous background: No data
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
The post-mitochondrial supernatant (S9) was prepared from the livers of Fischer rats pretreated with polychlorinated biphenyls
Test concentrations with justification for top dose:
For 24 and 48 hrs: 0, 0.0125, 0.025 or 0.05 mg/mL
For 6 hrs: 0, 0.02, 0.03 or 0.04 mg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: No data
- Exposure duration: Without S9: 6, 24 and 48 hrs
With S9: 6 hrs
- Expression time (cells in growth medium): 18 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: Once

NUMBER OF CELLS EVALUATED: 100 well-spread metaphases for each dose.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: No data
- Other: No data

OTHER:
Rationale for test conditions:
No data
Evaluation criteria:
Structural chromosomal aberrations
were classified into 6 groups: chromatid gaps (ctg) including chromosome gaps, chromatid breaks (ctb), chromatid exchanges (cte), fragmentation (frg), chromosome breaks (csb), and chromosome exchanges (cse) including dicentric and ring chromosomes.

The experimental groups were judged as negative (-) if the total frequency was less than 5.0%, suspicious (+) if 5.0-< 10.0% and positive (+) if 10% or more.
Statistics:
No data
Species / strain:
mammalian cell line, other: A Chinese hamster lung fibroblast cell line (CHL)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
mammalian cell line, other: A Chinese hamster lung fibroblast cell line (CHL)
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 0.05 mg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: The maximum dose of each test compound was estimated from the dose at which distinct cytotoxic effects were observed in a preliminary test on inhibition of cell growth. When the test compound did not show any cytotoxic effects, the maximum dose was limited to around 10 mM

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data
Remarks on result:
other: No mutagenic potential

Table: Results for the test chemical

Compound

Dose (mg/mL)

S9 mix

Time (h)

Pol (%)

Cells with chromosome aberrations (%)

Judgement

Ctg

Ctb

Cte

Frg

Csb

Cse

Total

Test chemical

0

-

24-0

0

2

2

0

0

1

0

4

 

0.0125

-

24-0

1

2

0

0

0

0

0

2

-

0.025

-

24-0

3

3

0

0

0

0

0

3

-

0.05

-

24-0

Tox

Tox

 

 

 

 

 

Tox

 

0

-

48-0

1

1

1

0

0

0

0

1

 

0.0125

-

48-0

0

0

2

0

0

0

0

2

-

0.025

-

48-0

1

1

1

0

1

0

0

2

-

0.05

-

48-0

Tox

Tox

 

 

 

 

 

Tox

 

0

-

6-18

0

0

0

0

0

0

0

0

 

0.02

-

6-18

1

2

0

0

0

0

0

2

-

0.03

-

6-18

0

0

1

0

0

0

0

1

-

0.04

-

6-18

Tox

Tox

 

 

 

 

 

Tox

 

0

+

6-18

0

0

2

0

0

0

0

2

 

0.02

+

6-18

0

1

3

2

0

0

0

5

±

0.03

+

6-18

1

3

4

9

0

0

0

12

+

0.04

+

6-18

2

6

7

12

0

0

1

19

+

 

Conclusions:
The test chemical did not induce chromosome aberration in Chinese hamster lung fibroblast cell line (CHL) in the absence of S9 metabolic activation system. It however induced gene mutation in the presence of S9 metabolic activation system.
Executive summary:

In vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical. The study was performed using Chinese hamster lung fibroblast cell line (CHL)in the presence and absence of S9 metabolic activation system. The chemical was dissolved in DMSO as solvent and used at dose levels 0, 0.0125, 0.025 or 0.05 mg/mL Concurrent solvent and positive controls were included in the study. The cells were treated for 24 and 48 h without S9 mix with a test compound at 3 dose levels at least. After treatment for 6 h with S9, the reaction mixture was replaced with fresh medium, and then the cells were recultured for 18 h. Both with and without the metabolic activation system, the cells were treated with Colcemid for 2 h, and chromosome preparations were made using a standard air-drying method. The frequency of cells with chromosomal aberrations was scored in 100 well-spread metaphases for each dose. Structural chromosomal aberrations were classified into 6 groups: chromatid gaps (ctg) including chromosome gaps, chromatid breaks (ctb), chromatid exchanges (cte), fragmentation (frg), chromosome breaks (csb), and chromosome exchanges (cse) including dicentric and ring chromosomes.Polyploid cells were also recorded.Solvent-treated cells served as controls. The experimental groups were judged as negative (-) if the total frequency was less than 5.0%, suspicious (+) if 5.0-< 10.0% and positive (+) if 10% or more. The test chemicaldid not induce chromosome aberration in Chinese hamster lung fibroblast cell line (CHL) in the absence of S9 metabolic activation system. It however induced gene mutation in the presence of S9 metabolic activation system.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from peer reviewed publication
Qualifier:
according to guideline
Guideline:
other: Refer below principle
Principles of method if other than guideline:
In vitro mammalian cell gene mutation test was performed to evaluate the mutagenic nature of the test chemical
GLP compliance:
not specified
Type of assay:
other: In vitro mammalian cell gene mutation test
Target gene:
HGPRT
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: Williams’ medium E (WE) supplemented with 10% fetal bovine serum L-glutamine and gentamicin
- Properly maintained: No data
- Periodically checked for Mycoplasma contamination: Yes, the cells were free from mycoplasma as determined by the Hoechst fluorochrome stain technique
- Periodically checked for karyotype stability: No data
- Periodically "cleansed" against high spontaneous background: No data
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
Rat and hamster liver hepatocytes
Test concentrations with justification for top dose:
0, 0.17, 1.70 or 3.40 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
Details on test system and conditions
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: No data
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 6 days
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): 6-thioguanine
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: Once

NUMBER OF CELLS EVALUATED: For thioguanine resistance data, the average number of mutant colonies/dish, based on 20 replicated dishes per treatment group, was analyzed.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Yes, The cytotoxicity of the test chemical was determined in the absence of hepatocytes by measurement of the effect on plating efficiency as described by
Rogers and Heroux-Metcalf (1983). The test chemical was tested at dose levels ranging from 0 to 8.3 µg/ml of medium.

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: No data

OTHER: No data
Rationale for test conditions:
No data
Evaluation criteria:
The cell line was noted for gene mutation at HGPRT ocus
Statistics:
No data
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
No data
Remarks on result:
other: Not mutagenic (in the absence of metabolic activation system) and mutagenic (in the absence of S9 metabolic activation system)

Table: Results for the test chemical

 

Treatment

Cloning efficiency (%)

Rat

 

 

Hamster

 

 

Exp 1

Exp 2

Exp 3

Exp 4

Exp 5

Exp 6

Without hepatocytes

 

 

 

 

 

 

Control

92

112

107

111

102

67

EMS

79

112

90

114

74

68

DMBA

86

93

101

128

102

77

Test chemical

 

 

 

 

 

 

0.17

81

119

99

102

106

101

1.70

100

52

75

69

85

128

3.40

92

75

86

77

88

119

With hepatocytes

 

 

 

 

 

 

Control

81

101

98

107

123

80

DMBA

64

105

105

98

93

81

Test chemical

 

 

 

 

 

 

0.17

94

92

92

112

103

141

1.70

95

73

75

62

83

99

3.40

103

60

72

55

67

122
Conclusions:
The test chemical did not induce gene mutation in the HGPRT locus in V79 Chinese hamster lung cells in the absence of rat and hamster hepatocytes. It however induced gene mutation in V79 Chinese hamster lung cells in the presence of rat and hamster hepatocytes.
Executive summary:

In vitro mammalian cell gene mutation test was performed to evaluate the mutagenic nature of the test chemical. The study was performed using V79 Chinese hamster lung cells with and without rat and hamster hepatocytes. The test chemical was dissolved in DMSO and used at appropriate dose levels of 0, 0.17, 1.70 or 3.40 µg/mL with and without hepatocytes. The period of exposure to chemicals, with or without activation by rat or hamster hepatocytes, was 48 h, after which the chemicals were removed and the cultures reseeded into flasks (75 cm*) at a density of 1.9 x 10” viable cells per 15 ml of Williams’ E medium. Following a 6-day period of expression, the cells were reseeded into 20 culture dishes (Corning, 60 mm) at 20 x lo3 viable cells per dish. For the selection of mutants, 6-thioguanine was added immediately after seeding to give a final concentration per dish of 10 pg/ml. Mutant colonies were counted 8 days later. Without hepatocytes, the test chemical was non-mutagenic at the HGPRT (TGR) gene locus at all concentrations. The test chemical in the presence of hepatocytes for TGR produced sporadic positive results (p < 0.01) when compared with concurrent controls.The chemical showed slight but increasing cytotoxicity to V79 cells at concentrations ranging from 2.0 to 3.4 µg/ml. Colony formation was reduced to 50% of the control at about 4.2 pg/ml and the antioxidant was lethal to 100% of the cells at dose levels above 5 µg/ml. Based on the observations made, test chemical did not induce gene mutation in the HGPRT locus in V79 Chinese hamster lung cells in the absence of rat and hamster hepatocytes. It however induced gene mutation inV79 Chinese hamster lung cells in the presence of rat and hamster hepatocytes.

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

Genetic toxicity in vivo

Description of key information

In vivo micronucleus assay was performed to determine the mutagenic nature of the test chemical. The study was performed using male B6C3F1 mice. Male B6C3F1 mice received three intraperitoneal injections of the test chemical dissolved in com oil at 24-hour intervals. Up to five mice were treated per exposure group and the highest dose administered was 400 mg/kg. Solvent control animals received com oil only, and the positive control mice received injections of 25 mg/kg cyclophosphamide. The mice were killed 24 hours after the final injection and slides were prepared from bone marrow smears obtained from the femurs. Slides were air-dried, fixed, and stained. Two thousand polychromatic erythrocytes (PCEs) were scored per animal for frequency of micronucleated cells. No animals survived in the 400 mg/kg group and only one mouse survived in the 300 mg/kg dose group. Based on the observations made, the test chemical did not induce micronuclei formation in bone marrow smears of B6C3F1 mice.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from peer reviewed publication
Qualifier:
according to guideline
Guideline:
other: Refer below principle
Principles of method if other than guideline:
In vivo sister chromatid exchange assay was performed to determine the mutagenic nature of the test chemical
GLP compliance:
not specified
Type of assay:
sister chromatid exchange assay
Species:
mouse
Strain:
Swiss
Remarks:
Albino
Sex:
male
Details on test animals or test system and environmental conditions:
Details on test animals and env conditions
TEST ANIMALS
- Source: Departmental Animal House Colony
- Age at study initiation: 6-8 weeks
- Weight at study initiation: 20-25 g
- Assigned to test groups randomly: No data
- Fasting period before study: No data
- Housing: Polycarbonated cages with bedding of rice husks were used
- Diet (e.g. ad libitum): Food (Lipton India, Bangalore) ad libitum
- Water (e.g. ad libitum): Water ad libitum
- Acclimation period: No data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 28 ± 2˚C
- Humidity (%): 60 ± 5%
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12 hrs photoperod

IN-LIFE DATES: From: To: No data
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: Corn oil
- Justification for choice of solvent/vehicle: The test chemical was soluble in corn oil
- Concentration of test material in vehicle: 0, 0.5, 2, 20, 50, 100, and 200 mg/kg
- Amount of vehicle (if gavage or dermal): 10 mL/Kg
- Type and concentration of dispersant aid (if powder): No data
- Lot/batch no. (if required): No data
- Purity: No data
Details on exposure:
No data
Duration of treatment / exposure:
24 hrs
Frequency of treatment:
Once
Post exposure period:
No data
Remarks:
0, 0.5, 2, 20, 50, 100, and 200 mg/kg
No. of animals per sex per dose:
No data
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Justification for choice of positive control(s): No data
- Route of administration: Intraperitoneally
- Doses / concentrations: 20 mg/Kg
Tissues and cell types examined:
Bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: No data

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Each animal was lightly anaesthetized with ether, and a small incision (- 6 mm) was made on the right lateral aspect of the abdomen. A paraffin-coated (- 70%) BrdUrd tablet was implanted subcutaneously, and the incision was closed with wound clips. Immediately after the tablet implantation, different concentrations of the test chemical, that is, 0.5, 2, 20, 50, 100, and 200 mg/kg of the test chemical dissolved in corn oil, were injected intraperitoneally (i.p.), in a volume equivalent to 10 ml/kg of body weight, to the different groups of mice accordingly, the volume injected varied from 0.2 to 0.4 ml. After 22 hr, each animal was injected i.p. with 2 mg/kg colchicine. Two hours later, they were killed by cervical dislocation and the bone marrow from both femurs was removed by flushing with phosphate buffered saline, pH 7.4. The aspirated bone marrow was pelleted by centrifugation, incubated in 0.075 M KCI for 20 min at 37˚C, repelleted, and fixed with ice-cold 3: 1 methanol: glacial acetic acid. The process of centrifugation and fixation was repeated twice, and finally flame-dried slides were prepared from each sample.

DETAILS OF SLIDE PREPARATION: Differential staining of SCE was carried by a modification of the fluorescence plus Giemsa (FPG) technique. Slides were stained for 10 min in Hoechst 33258 dissolved in NaCl solution, rinsed, and mounted with M/15 Sorenson's phosphate buffer, pH 6.8. The slides were then irradiated with a 254-nm UV mineralogic lamp for 30 min, rinsed in distilled water, and stained with 7% Giemsa in Sorenson's buffer for 10-15 min

METHOD OF ANALYSIS: Twenty second-generation metaphase cells (only those with 40 chromosomes) were scored for SCE in each mouse using coded slides. To evaluate the effects of the test chemical on cellular proliferation, the frequencies of the first-, second- and third-generation metaphases were determined
in 100 randomly selected consecutive metaphases on one slide from each animal

OTHER: No data
Evaluation criteria:
No data
Statistics:
The level of significance was established at an alpha of 0.05. A one-tailed trend test was used to determine if a treatment-related change occurred for SCE and PRI. In addition, pairwise comparisons between each treated group and the control group were conducted using the “t”-test with alpha level Bonferroni-corrected for multiple comparisons to determine the minimal effective dose.
Sex:
male
Genotoxicity:
positive
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
No data

Table: Mutagenic potential of the test chemical

Treatment (mg/Kg)

SCE/cell a

Range/cell

PRI b

0 (corn oil)

2.24±0.32(20)

0-4

2.00

 

2.40± 0.27(20)

0-4

2.06

 

2.10± 0.24(20)

1-3

2.00

 

2.34±0.33(20)

1-3

1.98

 

2.12± 0.29(20)

0-3

2.33

 

2.24c± 0.06

 

2.07c±0.07

0.5

2.40±0.24(20)

0-4

2.10

 

2.00± 0.32(20)

0-4

2.05

 

2.80± 0.28(20)

1-3

1.90

 

2.00±0.26(20)

1-4

2.00

 

2.40± 0.34(20)

1-3

1.95

 

2.32c±0.15

 

2.00±0.04

2

2.80± 0.32(20)

0-4

2.10

 

3.00± 0.26(20)

0-6

1.83

 

3.20± 0.35(20)

1-5

1.88

 

2.40± 0.22(20)

0-4

1.90

 

2.65± 0.34(20)

1-6

1.79

 

2.81±0.14

 

1.90±0.06

20

3.75±0.45(20)

1-7

1.91

 

4.30± 0.34(20)

1-7

1.98

 

4.70± 0.45(20)

0-7

1.89

 

4.35± 0.44(20)

2-8

2.00

 

4.45±0.40(20)

1-7

2.00

 

4.31c± 0.17

 

1.95c± 0.03

50

5.10± 0.62(20)

2-8

1.90

 

5.50± 0.53(20)

1-8

1.78

 

6.00± 0.63(20)

0-7

1.80

 

5.40± 0.57(20)

0-8

2.00

 

5.50± 0.50(20)

1-8

1.72

 

5.50c± 0.16

 

1.84c±0.05

100

6.90± 0.53(20)

3-10

1-76

 

6.70± 0.38(20)

3-9

1-68

 

6.60± 0.53(20)

3-12

1.91

 

7.60± 0.45(20)

4-12

1.65

 

7.60± 0.51(20)

3-11

2.00

 

7.08c± 0.22

 

1.80c±0.07

200

7.55± 0.51(20)

4-13

2.00

 

7.70± 0.57(20)

4-13

1.60

 

7.65± 0.58(20)

3-13

1.74

 

7.40± 0.63(20)

3-13

1.72

 

7.95± 0.56(20)

4-12

1.78

 

7.65c± 0.10

 

1.77c±0.07

20 CPA

15.10± 0.91(20)

8-26

1.90

 

14.45± 0.73(20)

10-20

1.78

 

13.75± 0.65(20)

11-20

1.80

 

16.00± 0.92(20)

9-24

2.00

 

15.20± 0.96(20)

9-26

1.72

 

14.89c± 0.38

 

1.84c±0.05

aIndividual animal mean SCE frequency -+ the standard error of the mean for (N) cells.

bProliferating rate index based on 100 metaphase cells analyzed per animal.

c Group mean -+ the standard error of the mean among animals.

Conclusions:
The test chemical induced sister chromatic exchanges in the bone marrow cells of male Swiss albino male mice and hence the test chemical is likely to be mutagenic.
Executive summary:

In vivo sister chromatid exchange assay was performed to determine the mutagenic nature of the test chemical. The study was performed using 6-8 weeks oldmale Swiss albino male mice. Each animal was lightly anaesthetized with ether, and a small incision (- 6 mm) was made on the right lateral aspect of the abdomen. A paraffin-coated (- 70%) BrdUrd tablet was implanted subcutaneously, and the incision was closed with wound clips. Immediately after the tablet implantation, different concentrations of the test chemical, that is, 0.5, 2, 20, 50, 100, and 200 mg/kg of the test chemical dissolved in corn oil, were injected intraperitoneally (i.p.), in a volume equivalent to 10 ml/kg of body weight, to the different groups of mice accordingly, the volume injected varied from 0.2 to 0.4 ml. After 22 hr, each animal was injected i.p. with 2 mg/kg colchicine. Two hours later, they were killed by cervical dislocation and the bone marrow from both femurs was removed by flushing with phosphate buffered saline, pH 7.4. The aspirated bone marrow was pelleted by centrifugation, incubated in 0.075 M KCI for 20 min at 37˚C, repelleted, and fixed with ice-cold 3: 1 methanol: glacial acetic acid. The process of centrifugation and fixation was repeated twice, and finally flame-dried slides were prepared from each sample. Differential staining of SCE was carried by a modification of the fluorescence plus Giemsa (FPG)  technique. Slides were stained for 10 min in Hoechst 33258 dissolved in NaCl solution, rinsed, and mounted with M/15 Sorenson's phosphate buffer, pH 6.8. The slides were then irradiated with a 254-nm UV mineralogic lamp for 30 min, rinsed in distilled water, and stained with 7% Giemsa in Sorenson's buffer for 10-15 min. Twenty second-generation metaphase cells (only those with 40 chromosomes) were scored for SCE in each mouse using coded slides. To evaluate the effects of the test chemical on cellular proliferation, the frequencies of the first-, second- and third-generation metaphases were determined in 100 randomly selected consecutive metaphases on one slide from each animal. The frequency of SCE/cell increased with the increase in concentration and was significant (one-tailed trend test P < .0001). Pairwise comparisons between each dose and the negative control gave significant differences except for the lowest dose. Two milligrams per kilogram was the minimum effective concentration for the induction of SCE. The proliferation rate indices did not show significant dose-response effects (one-tailed trend test). The three higher concentrations, 50, 100, and 200 mg/kg of the test chemical, caused significant delays in the cell cycle when compared to the negative control. Based on the observations made,the test chemical induced sister chromatic exchanges in the bone marrow cells of male Swiss albino male mice and hence the test chemical is likely to be mutagenic.

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

Additional information

Data available for the target chemical was reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

 

Gene mutation in vitro:

Ames assay was performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium TA1537, TA1535, TA158, TA98 and TA100 both with and without rat liver S-9 microsomal activation system. In the study reported here, the test chemical was tested in the Ames bioassay at levels upto 13.9 mg/L (0.450 mg/plate) without S-9 activation 82.3 mg/L (2.7mg/plate) with S-9 activation. The maximum concentrations used in these studies resulted in reduced viability of the test organisms, indicating that the test chemical was being tested at biologically significant concentrations. The difference in maximum test concentrations as a function of the presence of S-9 indicates that the metabolite(s) of the test chemical formed by microsomal enzymes in the s-9 have less biological impact on the test organism than the test chemical itself. In the Ames bioassay , no test chemical- related increases in revertants were noted in any test strain either with or without S-9 liver enzymes. HQ was negative when tested in the standard Ames test in five strains of Salmonella typhimurium , both with and without rat liver S-9 microsomal activation.

Salmonella/microsome assay was performed to determine the mutagenic nature of the test chemical using Salmonella typhimurium strain TA97, TA100, TA102 and TA104 with and without S9 metabolic activation system. Plate incorporation assay was performed at dose levels of 1, 10, 100, 200, 500 or 1000µg/plate in triplicate. Concurrent positive controls were also incorporated in the study.Toxicity of the phenolic antioxidant was also determined by plating each concentration in triplicate with 0.1 ml of a diluted culture (106times) and top agar enriched with 10µmole of L-histidine and 0.05µmole of D-biotin. The test chemical did not induce mutation in Salmonella typhimurium strain TA97, TA100, TA102 and TA104 both in the presence and absence of S9 metabolic activation system. Hence it is not likely to classify for gene mutation in vitro.

Gene mutation toxicity study was also performed to determine the mutagenic nature of the test chemical. The study was performed usingSalmonella typhimurium strains TA102, TA100, TA97 and TA98 in the presence and absence of S9 metabolic activation system. The chemical was dissolved in DMSO as solvent and used at dose levels 0, 3, 10, 33, 100, 166, 333, 666, 1000, 1666 or 3333 µg/plate by the preincubation method. The doses were selected on the basis of preliminary dose range finding study and concurrent solvent and positive controls were included in the study. The test chemical did not induce gene mutation in Salmonella typhimurium TA98, TA100, TA1538 in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The study was performed usingSalmonella typhimurium strainsTA100, TA98, TA102 and TA97 in the presence and absence of S9 metabolic activation system. The chemical was dissolved in acetone as solvent and used at dose levels 0, 0.5, 1, 2.5, 5, 10, 25, 50, 100, 250, 500 or 1000µg/plate by the preincubation method. Concurrent solvent and positive controls were included in the study. The test chemicaldid not induce gene mutation in Salmonella typhimurium TA98, TA100, TA1538 in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

In another study, WP2 Mutoxitest was performed to determine the mutagenic nature of the test chemical. The study was performed using E. coli strain IC203, deficient in OxyR, and its oxyR (with and without metabolic activation system) and its parent strain WP2 uvrA/pKM101 (IC188) (without metabolic activation system). The test chemical was dissolved in water as solvent and used at dose level of 0, 50, 100 or 150 ug/plate. The test chemical was considered as positive if a reproducible, dose-related increase in the number of revertants was noted. The increase should reach at least a doubling of the number of spontaneous revertants. Based on the observations made, the test chemical is non-mutagenic to E. coli strain IC188 in the absence of S9 metabolic activation system. It hoever induce gene mutation in strain IC203, deficient in OxyR, and its oxyR+ parent WP2 uvrA/pKM101 in the presence of rat liver S9 activation.

In vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical. The study was performed using Chinese hamster lung fibroblast cell line (CHL)in the presence and absence of S9 metabolic activation system. The chemical was dissolved in DMSO as solvent and used at dose levels 0, 0.0125, 0.025 or 0.05 mg/mL Concurrent solvent and positive controls were included in the study. The cells were treated for 24 and 48 h without S9 mix with a test compound at 3 dose levels at least. After treatment for 6 h with S9, the reaction mixture was replaced with fresh medium, and then the cells were recultured for 18 h. Both with and without the metabolic activation system, the cells were treated with Colcemid for 2 h, and chromosome preparations were made using a standard air-drying method. The frequency of cells with chromosomal aberrations was scored in 100 well-spread metaphases for each dose. Structural chromosomal aberrations were classified into 6 groups: chromatid gaps (ctg) including chromosome gaps, chromatid breaks (ctb), chromatid exchanges (cte), fragmentation (frg), chromosome breaks (csb), and chromosome exchanges (cse) including dicentric and ring chromosomes.Polyploid cells were also recorded.Solvent-treated cells served as controls. The experimental groups were judged as negative (-) if the total frequency was less than 5.0%, suspicious (+) if 5.0-< 10.0% and positive (+) if 10% or more. The test chemicaldid not induce chromosome aberration in Chinese hamster lung fibroblast cell line (CHL) in the absence of S9 metabolic activation system. It however induced gene mutation in the presence of S9 metabolic activation system.

Yet another in vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical. The study was performed using Chinese hamster ovary cells (CHO) in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose level of 0, 5.0, 7.6, 10.1 or 25.2 without S9 and 0, 100.5, 150.0 or 200.0 (Trial 1- with S9) and 0, 149.4, 199.2, 249.0, 300.0µg/mL (Trial 2- with S9). In the Abs test without S9, cells were incubated in McCoy's SA medium with the test chemical for 8 hours; Colcemid was added and incubation continued for 2 hours. The cells were then harvested by mitotic shake-off, fixed, and stained with Giemsa. For the Abs test with S9, cells were treated with the test chemical and S9 for 2 hours, after which the treatment medium was removed and the cells incubated for an additional 18 hours in fresh medium, with Colcemid present for the final 2 hours. Cells were harvested in the same manner as for the treatment without S9. The harvest time for the Abs test was based on the cell cycle information obtained in the SCE test; because cell cycle delay was anticipated in the presence of S9, the incubation period was extended beyond the normal 10 to 12 hour period. Cells were selected for scoring on the basis of good morphology and completeness of karyotype (21 ± 2 chromosomes). Two hundred first-division metaphase cells were scored at each dose level in the trial without S9. Because large numbers of aberrations were observed in the cells treated in the presence of S9, fewer cells were scored per dose level. Classes of aberrations scored included "simple" (breaks and terminal deletions), "complex" (rearrangements and translocations), and "other" (pulverized cells, despiralized chromosomes, and cells containing ten or more aberrations). Based on the observations made, the test chemical did not induce chromosome aberration in CHO cells in the absence of S9. It however induced chromosome aberration in the CHO cells in the presence of S9 metabolic activation system.

Another vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical. The study was performed using Chinese hamster ovary cells, clone CHO-K1-BH4 in the presence and absence of S9 metabolic activation system. The chemical was dissolved in DMSO as solvent and used at dose levels 0, 100, 150, 225, 335 or 500µM. cultures were seeded at confluent density (2 × 105/cm2) 4 h before treatment. The cells were suspended immediately after treatment and re-plated at 3.2 × 104//cm2to allow cell multiplication. Concurrent solvent and positive controls were included in the study. The cells were treated for 5 hrs with the test chemical. After removal of the test solutions, the cells were incubated in fresh medium until arrest of mitosis by addition of colcemid (0.1µg/ml) for 2 h. Chromosome aberrations were scored in 100 metaphases (with 18-21 chromosomes) from each culture. Chromatid and chromosome gaps were recorded in addition to breaks and exchanges but since no treatment-related changes in the frequency of gaps were observed they were not reported. the toxicity of the test chemical was greatly reduced, with a moderate reduction in mitotic index at 335µM. Chromosome damage was clearly demonstrated with increases in most types of aberration. Based on the observations made, the test chemicalinduced chromosome aberration in CHO-K1-BH4 in the presence and absence of S9 metabolic activation system.

Sister chromatid exchange assay was also performed to determine the mutagenic nature of the test chemical. The study was performed using Chinese hamster ovary cells (CHO) in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose level of 0, 0.5, 1.7, 5.0 or 16.7µg/mL – Without S9; 0, 5.0, 16.7, 50.0 or 166.7µg/mL – With S9 (Trial 1); 0, 49.8, 100.5 or 150.0µg/mL – With S9 (Trial 2). In the SCE test without S9, CHO cells were incubated for 25.5 hours with the test chemical in supplemented McCoy's 5A medium. Bromodeoxyuridine (BrdU) was added 2 hours after culture initiation.' After 25.5 hours, the medium containing the test chemical was removed and replaced with fresh medium plus BrdU and Colcemid, and incubation was continued for 2 hours. Cells were then harvested by mitotic shake-off, fixed, and stained with Hoechst 33258 and Giemsa. In the, SCE test with S9, cells were incubated with the test chemical, serum-free medium, and S9 for 2 hours. The medium was then removed and replaced with medium containing serum and BrdU and the test chemical, and incubation proceeded for an additional 25.5 hours, with Colcemid present for the final 2 hours. Harvesting and staining were the same as for cells treated without S9. All slides were scored blind and those from a single test were read by the same person. Fifty second-division metaphase cells were scored for frequency of SCEs/cell from each dose level. Because significant chemical-induced cell cycle delay was observed in the presence of S9,incubation time in the second trial with S9 was lengthened to ensure a sufficient number of scorable (second-division metaphase) cells. Based on the observations made, the test chemical did not induce sister chromatid exchange in CHO cells in the absence of S9. It however induced sister chromatid exchanges in CHO in the presence of S9 metabolic activation system.

In vitro mammalian cell gene mutation test was performed to evaluate the mutagenic nature of the test chemical. The study was performed using V79 Chinese hamster lung cells with and without rat and hamster hepatocytes. The test chemical was dissolved in DMSO and used at appropriate dose levels of 0, 0.17, 1.70 or 3.40 µg/mL with and without hepatocytes. The period of exposure to chemicals, with or without activation by rat or hamster hepatocytes, was 48 h, after which the chemicals were removed and the cultures reseeded into flasks (75 cm*) at a density of 1.9 x 10” viable cells per 15 ml of Williams’ E medium. Following a 6-day period of expression, the cells were reseeded into 20 culture dishes (Corning, 60 mm) at 20 x lo3 viable cells per dish. For the selection of mutants, 6-thioguanine was added immediately after seeding to give a final concentration per dish of 10 pg/ml. Mutant colonies were counted 8 days later. Without hepatocytes, the test chemical was non-mutagenic at the HGPRT (TGR) gene locus at all concentrations. The test chemical in the presence of hepatocytes for TGR produced sporadic positive results (p < 0.01) when compared with concurrent controls.The chemical showed slight but increasing cytotoxicity to V79 cells at concentrations ranging from 2.0 to 3.4 µg/ml. Colony formation was reduced to 50% of the control at about 4.2 pg/ml and the antioxidant was lethal to 100% of the cells at dose levels above 5 µg/ml. Based on the observations made, test chemical did not induce gene mutation in the HGPRT locus in V79 Chinese hamster lung cells in the absence of rat and hamster hepatocytes. It however induced gene mutation in V79 Chinese hamster lung cells in the presence of rat and hamster hepatocytes.

Gene mutation in vivo:

In vivo micronucleus assay was performed to determine the mutagenic nature of the test chemical. The study was performed using male B6C3F1 mice. Male B6C3F1 mice received three intraperitoneal injections of the test chemical dissolved in com oil at 24-hour intervals. Up to five mice were treated per exposure group and the highest dose administered was 400 mg/kg. Solvent control animals received com oil only, and the positive control mice received injections of 25 mg/kg cyclophosphamide. The mice were killed 24 hours after the final injection and slides were prepared from bone marrow smears obtained from the femurs. Slides were air-dried, fixed, and stained. Two thousand polychromatic erythrocytes (PCEs) were scored per animal for frequency of micronucleated cells. No animals survived in the 400 mg/kg group and only one mouse survived in the 300 mg/kg dose group. Based on the observations made, the test chemical did not induce micronuclei formation in bone marrow smears of B6C3F1 mice.

In another study, In vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical in vivo. For the oral treatment, the test chemical was dissolved in olive oil and administered by gavage in a daily dose of 2 mg/kg body weight, given at the same time each day for 30 successive days. The animals were killed 24 hr after receiving the final (30th) dose. Control mice were given the same volume (0.05 ml) of olive oil each day. Each experiment was carried out in duplicate. For preparations of bone-marrow chromosomes the animals were injected with 0.04% colchicine in a dose of 10 ml/kg body weight 90 min before being killed. Slides were prepared by the standard flame-drying Giemsa schedule. From each of the five animals in each experiment, 300 cells were scanned. Mitotic index was also noted. The metaphase abnormalities induced by the test chemical can be classified into three groups as- a disturbance in spindle formation, indicated by stickiness, C-mitosis and diplochromosomes in the late metaphase (Group 1), chromosomal abnormalities (breaks, gaps, centric fusion and others) but only one or two per cell (Group 2), gross abnormalities, in which a number of chromosomal abnormalities could be observed in the same metaphase together with spindle disturbances (Group 3). A slight increase in the mitotic index was observed alter the 30-day treatment. No significant differences were observed in the proportion of group l abnormalities with either treatment compared with the controls. Significant increases in both group II and group Ill abnormalities were observed. Based on the observations made, the test chemicalinduced chromosome aberrations in bone marrow cells of male mice upon repeated oral treatment and hence the test chemical is likely to be mutagenic.

In the same study as mentioned above, In vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical in vivo. The study was performed by giving a single intraperitoneal dose of the test chemical at a concentration of 0 or 200 mg/Kg being dissolved in DMSO to male mice. The mice were killed 24 hr later. Control mice were injected with an equal volume (0.05 ml) of DMSO. For preparations of bone-marrow chromosomes the animals were injected with 0.04% of colchicine in a dose of 10 ml/kg body weight 90 min before being killed.Slides were prepared by the standard flame-drying Giemsa schedule. From each of the five animals in each experiment, 300 cells were scanned. Mitotic index was also noted. The metaphase abnormalities induced by the test chemical can be classified into three groups as- a disturbance in spindle formation, indicated by stickiness, C-mitosis and diplochromosomes in the late metaphase (Group 1), chromosomal abnormalities (breaks, gaps, centric fusion and others) but only one or two per cell (Group 2), gross abnormalities, in which a number of chromosomal abnormalities could be observed in the same metaphase together with spindle disturbances (Group 3). The mitotic index was decreased in the preparations from treated animals and the decrease was statistically significant. No significant differences were observed in the proportion of group l abnormalities with either treatment compared with the controls. Significant increases in both group II and group Ill abnormalities were observed. Based on the observations made, the test chemicalinduced chromosome aberrations in bone marrow cells of male mice and hence the test chemical is likely to be mutagenic.

In another in vivo sister chromatid exchange assay, the study was performed to determine the mutagenic nature of the test chemical. The study was performed using 6-8 weeks oldmale Swiss albino male mice. Each animal was lightly anaesthetized with ether, and a small incision (- 6 mm) was made on the right lateral aspect of the abdomen. A paraffin-coated (- 70%) BrdUrd tablet was implanted subcutaneously, and the incision was closed with wound clips. Immediately after the tablet implantation, different concentrations of the test chemical, that is, 0.5, 2, 20, 50, 100, and 200 mg/kg of the test chemical dissolved in corn oil, were injected intraperitoneally (i.p.), in a volume equivalent to 10 ml/kg of body weight, to the different groups of mice accordingly, the volume injected varied from 0.2 to 0.4 ml. After 22 hr, each animal was injected i.p. with 2 mg/kg colchicine. Two hours later, they were killed by cervical dislocation and the bone marrow from both femurs was removed by flushing with phosphate buffered saline, pH 7.4. The aspirated bone marrow was pelleted by centrifugation, incubated in 0.075 M KCI for 20 min at 37˚C, repelleted, and fixed with ice-cold 3: 1 methanol: glacial acetic acid. The process of centrifugation and fixation was repeated twice, and finally flame-dried slides were prepared from each sample. Differential staining of SCE was carried by a modification of the fluorescence plus Giemsa (FPG)  technique. Slides were stained for 10 min in Hoechst 33258 dissolved in NaCl solution, rinsed, and mounted with M/15 Sorenson's phosphate buffer, pH 6.8. The slides were then irradiated with a 254-nm UV mineralogic lamp for 30 min, rinsed in distilled water, and stained with 7% Giemsa in Sorenson's buffer for 10-15 min. Twenty second-generation metaphase cells (only those with 40 chromosomes) were scored for SCE in each mouse using coded slides. To evaluate the effects of the test chemical on cellular proliferation, the frequencies of the first-, second- and third-generation metaphases were determined in 100 randomly selected consecutive metaphases on one slide from each animal. The frequency of SCE/cell increased with the increase in concentration and was significant (one-tailed trend test P < .0001). Pairwise comparisons between each dose and the negative control gave significant differences except for the lowest dose. Two milligrams per kilogram was the minimum effective concentration for the induction of SCE. The proliferation rate indices did not show significant dose-response effects (one-tailed trend test). The three higher concentrations, 50, 100, and 200 mg/kg of the test chemical, caused significant delays in the cell cycle when compared to the negative control. Based on the observations made,the test chemical induced sister chromatic exchanges in the bone marrow cells of male Swiss albino male mice and hence the test chemical is likely to be mutagenic.

 

The test chemical is non-mutagenic in multiple strains of Salmonella typhimurium based on the results from studies equivalent or similar to OECD Test Guideline 471. In chromosomal aberration studies, the test chemical has tested positive for clastogenic effects in Chinese hamster lung cells and Chinese hamster ovary cells. In vivo, the test chemical has been evaluated for clastogenic effects in a study by Giri et al (1984) and in a report by the National Toxicology Program (Report No. 459). The results in the study by Giri et al (1984) are inconclusive according to the European Food Safety Authority (2004; 84: 1-50) partly because the types of aberrations scored in the study were not detailed and because the frequencies of chromosomal aberrations in the control groups were unusually high. The results from the micronucleus test in mouse bone marrow outlined in the National Toxicology Program (Report No. 459) were conclusive according to EFSA (2004; 84: 1-50) and those results indicated no clastogenic effects of the test chemical. Data from a HPRT assay in Chinese hamster lungs cells have indicated that the test chemical is non-mutagenic although the highest dose tested did not achieve a 50% decrease in cloning efficiency. By weight of evidence, the test chemical is regarded to be classified as "Not Classified" for Germ cell mutagenicity.   

Justification for classification or non-classification

The test chemical is non-mutagenic in multiple strains of Salmonella typhimurium based on the results from studies equivalent or similar to OECD Test Guideline 471. In chromosomal aberration studies, the test chemical has tested positive for clastogenic effects in Chinese hamster lung cells and Chinese hamster ovary cells. In vivo, the test chemical has been evaluated for clastogenic effects in a study by Giri et al (1984) and in a report by the National Toxicology Program (Report No. 459). The results in the study by Giri et al (1984) are inconclusive according to the European Food Safety Authority (2004; 84: 1-50) partly because the types of aberrations scored in the study were not detailed and because the frequencies of chromosomal aberrations in the control groups were unusually high. The results from the micronucleus test in mouse bone marrow outlined in the National Toxicology Program (Report No. 459) were conclusive according to EFSA (2004; 84: 1-50) and those results indicated no clastogenic effects of the test chemical. Data from a HPRT assay in Chinese hamster lungs cells have indicated that the test chemical is non-mutagenic although the highest dose tested did not achieve a 50% decrease in cloning efficiency. By weight of evidence, the test chemical is regarded to be classified as "Not Classified" for Germ cell mutagenicity.