Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames assay:

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay. The study was performed using Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvrA. The test chemical was dissolved in DMSO and used at dose level of 0, 20, 100, 500, 2,500 or 5,000 µg/plate. Concurrent solvent and positive control chemicals were also included in the study. According to the results of the present study, the test substance pigment red 169 is not mutagenic in the Salmonella typhimurium /Escherichia coli reverse mutation assay with and without metabolic activation.

In vitro mammalian chromosome aberration study:

The test chemical did not induce chromosomal aberrations in the cultured in the mammalian cell line with and without metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In vitro mammalian cell gene mutation:

The test chemical did not induce gene mutation in Mouse lymphoma L5178Y cells in the presence and absence of S9 metabolic activation system and hence it is not likely to be mutagenic in vitro.

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:
weight of evidence
Study period:
January 1999
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:
other: * OECD No. 471 (July 21, 1997) * EEC Directive 92/69, B14 and B13 (December 1992)
Principles of method if other than guideline:
Standard plate test and Preincubation test was performed to determine the mutagenic nature of the test chemical
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine for Salmonella strains and Tryptophan for E. coli strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
No data
Additional strain / cell type characteristics:
other: histidine auxotrophs (his-)
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
No data
Additional strain / cell type characteristics:
other: tryptophan auxotrophy (trp-)
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S-9 mix
Test concentrations with justification for top dose:
0, 20, 100, 500, 2,500 or 5,000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, DMSO was selected as the vehicle, which had been
demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.
Untreated negative controls:
yes
Remarks:
Additional plates are treated with soft agar, S-9 mix, buffer, vehiele or the test substance but without the addition of tester strains
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Remarks:
with S9
Positive control substance:
other: 2-aminoanthracene (2-AA)
Remarks:
strains: TA 1535, TA 100, TA 1537, TA 98: 2.5 µg/plate, dissolved in DMSO; Strain: Escherichia coli WP2 uvrA: 60 µg/plate, dissolved in DMSO
Untreated negative controls:
yes
Remarks:
Additional plates are treated with soft agar, S-9 mix, buffer, vehicle or the test substance but without the addition of tester strains
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Remarks:
without S9
Positive control substance:
other: N-methyl-N'-nitro-N-nitrosuguanidine (MNNG)
Remarks:
Strains: TA 1535, TA 100: 5 µg/plate, dissolved in DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylendiamine (NOPD)
Remarks:
Strain: TA 98: 10 µg/plate, dissolved in DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Strain: TA 1537: 100 µg/plate, dissolved in DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Strain: E. coli WP2 uvrA: 10 µg/plate, dissolved in DMSO
Details on test system and experimental conditions:
Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (Aroclor-induced rat liver S-9 mix)

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk
- Cell density at seeding (if applicable):

DURATION
- Preincubation period: For PIT: 20 mins
- Exposure duration: 48 - 72 hours
- Expression time (cells in growth medium): 48 - 72 hours
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays):

SPINDLE INHIBITOR (cytogenetic assays):

STAIN (for cytogenetic assays):

NUMBER OF REPLICATIONS: Triplicate

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:

NUMBER OF CELLS EVALUATED:

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells):

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:
- Any supplementary information relevant to cytotoxicity:

OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable):

- OTHER:
Rationale for test conditions:
No data
Evaluation criteria:
The test chemical is considered positive in this assay if the following criteria are met:

A dose-related and reproducible increase in the nuinber of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S-9 mix or after adcling a metabolizing system.

A test substance is generally considered nonmutagenic in this test if:

The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.
Statistics:
No data
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
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: Precipitation of the test substance was found from about 500 µg/plate onward.
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: No data

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: No data

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: No data
- Indication whether binucleate or mononucleate where appropriate: No data

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No data
- Negative (solvent/vehicle) historical control data: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No data
- Other observations when applicable: No data
Remarks on result:
other: No mutagenic potential

Standard plate assay

Table: Mutagenic potential

Strain TA1535 Without S9

 

Dose

Rev

M

SD

FAC

DMSO

16

20

3

1.0

21

22

20 µg

20

20

1

1.0

19

20

100 µg

21

20

1

1.0

19

19

500 µg

18P

19

1

1.0

20P

19P

2500 µg

5P

5

1

0.3

4P

6P

5000 µg

5P

3

2

0.2

3P

1P

MNNG

5.0µg

1004

1002

24

50.9

977

1024

2-AA

2.5 µg

 

 

 

 


Strain TA1535 With S9

Dose

Rev

M

SD

FAC

TITRE

DMSO

18

19

2

1.0

29

18

22

21

27

20 µg

19

18

1

1.0

 

18

18

100 µg

15

14

1

0.7

 

13

14

500 µg

17P

17

2

0.9

 

19P

19P

2500 µg

7P

6

2

0.3

15

8P

14

4P

20

 

5000 µg

0P

 

 

 

0

0P

0

0P

0

2-AA

176

171

14

9.0

 

155

 

181

 

 

 

Strain TA100 Without S9

 

Dose

Rev

M

SD

FAC

DMSO

124

115

8

1.0

108

113

20 µg

104

101

8

0.9

107

94

100 µg

98

102

7

0.9

110

99

500 µg

55P

60

5

0.5

61P

65P

2500 µg

11P

8

3

0.1

5P

8P

5000 µg

0P

 

 

 

0P

0P

MNNG

5.0µg

1312

1319

49

11.5

1273

1371

2-AA

2.5 µg

 

 

 

 

 

Strain TA100 With S9

Dose

Rev

M

SD

FAC

TITRE

DMSO

112

111

9

1.0

22

102

21

120

29

20 µg

110

110

2

1.0

 

108

111

100 µg

104

114

8

1.0

 

118

119

500 µg

57P

52

17

0.5

 

66P

33P

2500 µg

5P

5

3

0.0

1

8P

2

2P

5

5000 µg

0P

 

 

 

0

0P

0

0P

0

2-AA

2.5 µg

1135

1274

180

11.4

 

1477

1210

 

Strain TA1537 Without S9

 

Dose

Rev

M

SD

FAC

DMSO

9

10

1

1.0

10

10

20

9

9

1

1.0

9

10

100

9

10

1

1.0

10

11

500

5P

5

2

0.6

7P

4P

2500

1P

1

0

0.1

1P

1P

5000

0P

 

 

 

0P

0P

AAC

100 µg

621

649

27

67.2

674

653

2-AA

2.5 µg

 

 

 

 

 

Strain TA1537 With S9

Dose

Rev

M

SD

FAC

TITRE

DMSO

11

11

1

1.0

38

11

29

10

28

20 µg

9

9

2

0.9

 

8

11

100 µg

7

8

1

0.8

 

9

9

500 µg

8P

8

0

0.8

 

8P

8P

2500 µg

1P

1

1

0.1

2

1P

5

2P

1

5000 µg

0P

 

 

 

0

0P

0

0P

0

AAC

100 µg

 

 

 

 

 

 

2-AA

2.5 µg

173

185

13

17.5

 

198

185

 

Strain TA 98 Without S9

 

Dose

Rev

M

SD

FAC

DMSO

20

27

7

1.0

29

33

20 µg

24

21

3

0.8

19

21

100 µg

21

23

2

0.9

24

25

500 µg

21P

20

1

0.7

19P

19P

2500 µg

2P

3

1

0.1

3P

4P

5000 µg

0P

 

 

 

0P

0P

NOPD

10 µg

937

945

25

34.6

925

973

2-AA

2.5 µg

 

 

 

 

 

Strain TA 98 With S9

Dose

Rev

M

SD

FAC

TITRE

DMSO

42

38

3

1.0

17

36

24

36

24

20 µg

35

33

2

0.9

 

31

32

100 µg

34

33

2

0.9

 

31

34

500 µg

20P

22

2

0.6

 

24P

23P

2500 µg

11P

8

3

0.2

2

8P

2

6P

2

5000 µg

0P

 

 

 

0

0P

0

0P

0

NOPD

10 µg

 

 

 

 

 

2-AA

2.5 µg

936

943

13

24.8

 

925

958

 

 

StrainE.coli WP2uvrA Without S9

 

Dose

Rev

M

SD

FAC

DMSO

36

36

1

1.0

37

35

20 µg

31

32

4

0.9

37

29

100 µg

34

32

3

0.9

33

29

500 µg

29P

32

3

0.9

31P

35P

2500 µg

25P

28

3

0.8

30P

30P

5000 µg

15P

18

3

0.5

17P

21P

ENNG

10 µg

999

1009

18

28.0

998

1029

2-AA

60 µg

 

 

 

 

 

Strain E.coli WP2 uvrAWith S9

Dose

Rev

M

SD

FAC

TITRE

DMSO

39

40

2

1.0

40

42

45

39

39

20 µg

35

32

3

0.8

 

31

29

100 µg

37

35

3

0.9

 

37

31

500 µg

31P

31

1

0.8

 

31P

30P

2500 µg

20P

24

4

0.6

8

26P

6

27P

7

5000 µg

12P

12

1

0.3

0

12P

0

13P

0

ENNG

10 µg

 

 

 

 

 

2-AA

60 µg

326

314

40

7.9

 

347

269

Conclusions:
According to the results of the present study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay with and without metabolic activation.
Executive summary:

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay. The study was performed using Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvrA. The test chemical was dissolved in DMSO and used at dose level of 0, 20, 100, 500, 2,500 or 5,000 µg/plate. Concurrent solvent and positive control chemicals were also included in the study. According to the results of the present study, the test substance pigment red 169 is not mutagenic in the Salmonella typhimurium /Escherichia coli reverse mutation assay with and without metabolic activation.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
Experimental data from various test chemicals
Justification for type of information:
Data for the target chemical is summarized based on the data from various test chemicals
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
other: Refer below principle
Principles of method if other than guideline:
WoE derived based on the experimental data from various test chemicals
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
No data
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: McCoy's 5A (modified) medium buffered with 20 mM HEPES and supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 50 IU/ml penicillin, and 50 µg/ml streptomycin
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- 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 rat liver microsomal fraction was prepared from Aroclor 1254-induced male Sprague-Dawley rats and was combined with cofactors and culture medium to form the metabolic activation system.
Test concentrations with justification for top dose:
1,Without S9: 0.0, 49.8000, 150.0000, 499.0000 µg/mL

With S9: 0.0, 500.0000, 1500.0000, 5000.0000 µg/mL

2,125,400 and 1250 µg/mL
Vehicle / solvent:
1,- Vehicle(s)/solvent(s) used: Water
- Justification for choice of solvent/vehicle: The test chemical was soluble in water
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
Water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
1,METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: No data available
- Exposure duration:
Without S9: 8 hrs
With S9: 2 hrs
- Expression time (cells in growth medium):
Without S9: 10-10.5 hrs
With S9: 12 hrs
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): Standard harvest time: 10-14 hr after addition of BrdUrd

SELECTION AGENT (mutation assays): No data available
SPINDLE INHIBITOR (cytogenetic assays): No data available
STAIN (for cytogenetic assays): 5% Giemsa

NUMBER OF REPLICATIONS: Only one trial was performed

NUMBER OF CELLS EVALUATED: 100-200 cells were scored

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

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

OTHER: No data available

2,Details on test system and conditions
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: No data
- Exposure duration: The chemical treatment periods were appoximately 25 hr without S9 and 2 hr with S9.
- Expression time (cells in growth medium): 25-26 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): 14 hour

SELECTION AGENT (mutation assays): After staining for 10 min in “concentrated” Hoechst 33258 (5 pg/ml in pH 6.8 buffer) and exposure to “black light” at 55 to 60°C for about 5 min, slides were stained in Giemsa
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: 50 cells per dose were scored from the three highest doses

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

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

OTHER: Cells were collected by mitotic shake off for evaluation
All types of aberrations were recorded separately, but for data analysis they were grouped into categories of “simple” (breaks and terminal deletions), “complex” (exchanges and rearrangements), “other” (includes pulverized chromosomes), and “total. ” Gaps and endoreduplications were recorded but were not included in the totals. We did not score aberrations in polyploidy cells but used metaphases with 19-23 chromosomes (the modal number being 21).

Rationale for test conditions:
No data
Evaluation criteria:
Selection of cells for scoring was based on well-spread chromosomes with good morphology and a chromosome number of 21 ± 2. Cells were analyzed for the following categories of chromosomal aberrations: “simple,” defined as a chromatid gap, break, fragment, and deletion or chromosome gap,break, or double minutes; “complex,” defined as interstitial deletions, triradials, quadriradials, rings, and dicentric chromosomes; and “other,” defined as pulverized chromosomesor cells with greater than 10 aberrations. Chromatid and chromosome gaps were recorded but were not used in the analysis.

A positive response at a single dose was designated “ + W”, weak evidence for clastogenicity. If there was a strong trend as the result of a large increase in ABs at a single dose only, we designated the result ‘‘ + W*”. A test was designated “ + ” if at least two doses gave significantly increased responses.
Statistics:
A binomial sampling assumption as described by Margolin et al. was used to examine absolute increases in ABs over solvent control levels at each dose. The P values were adjusted by Dunnett’s method to take into account the multiple dose comparisons. Only the “total” percent cells with aberrations were analyzed, and a positive response was defined as one for which the adjusted P value was <0.05.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and 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:
Chinese hamster Ovary (CHO)
Remarks:
(CHO-W-B1)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
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: Test concentrations for the AB assays were empirically chosen based on toxicity and cell cycle delay as noted in the SCE experiments. At least five
concentrations of the test chemical were selected; the concentrations were spaced using two merged half-log scales (e.g., 1,000, 500, 300, 150, 100, etc.), and the highest concentrations analyzed were those yielding a sufficient number of suitable metaphase cells. The concentrations analyzed generally covered a one-log range.

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data
Remarks on result:
other: No mutagenic potential
Conclusions:
The test chemical did not induce chromosomal aberrations in the cultured in the mammalian cell line with and without metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.
Executive summary:

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

In vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of the test chemical. Approximately 24 hr before chemical treatment, cultures were initiated at a density of 1.75 X 106cells/75 cm2flask. In the AB trials without S9, the cultures were treated with the test chemical in medium for 8 hr, washed to remove the test chemical, and treated with colcemid M) for 2-2.5 hr before cell harvest. In the experiments with activation, cultures were exposed to the test chemical in serum free medium with S9 and cofactors for 2 hr, washed to remove the test chemical and S9, and incubated at 37°C with fresh medium for 8 hr. Colcemid was then added, andthe cells were harvested 2 hr later. Thus the total durations of the nonactivated and activated AB experiments were 10hr and 12 hr, respectively, to give 10 hr growth in medium with serum for each experiment. For ABs, slides were stained in 5% Giemsa for 5 min. In early studies, one hundred cells were scored for each ofthree concentrations: the highest test concentration in whichsufficient metaphase cells could be scored and the next two lower concentrations, covering a one-log range. For later studies, 200 cells per dose were scored; however, fewer cells were scored if a test chemical produced a strong positive response or the chemical was toxic. Cells were analyzed for the following categories of chromosomal aberrations: “simple,” defined as a chromatid gap, break, fragment, and deletion or chromosome gap,break, or double minutes; “complex,” defined as interstitial deletions, triradials, quadriradials, rings, and dicentric chromosomes; and “other,” defined as pulverized chromosomesor cells with greater than 10 aberrations. Chromatid and chromosome gaps were recorded but were not used in the analysis. The test chemical did not induce chromosome aberrations in the CHO-LB cell line in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In another study, in vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of the test chemical. The test chemical was studied at a dose level of 0, 125, 400 and 1250 µg/mL in the absence and presence of S9 using Chinese hamster ovary cells (CHO-W-B1).Cells were exposed to the test chemical for 2 hr in the presence of S9 or throughout the incubation period without S9. 100 cells were scored from each of the three highest dose groups having sufficient metaphases for analysis. All types of aberrations were recorded separately, but for data analysis they were grouped into categories of “simple” (breaks and terminal deletions), “complex” (exchanges and rearrangements), “other” (includes pulverized chromosomes), and “total”. Gaps and endo-reduplications were recorded but were not included in the totals. Polyploid cells were not scored but used metaphases with 19-23 chromosomes (the modal number being 21). Based on the results noted, the test chemical did not induce chromosome aberrations in the Chinese hamster ovary cells (CHO-W-B1) in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

Based on the data available, the test chemical did not induce chromosomal aberrations in the cultured in the mammalian cell line with and without metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
Experimental data from various test chemicals
Justification for type of information:
Data for the target chemical is summarized based on the data from various test chemicals
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
other: Refer below principle
Principles of method if other than guideline:
WoE derived based on the experimental data from various test chemicals
GLP compliance:
not specified
Type of assay:
other: In vitro mammalian cell gene mutation assay
Target gene:
Thymidine kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Remarks:
1
Details on mammalian cell type (if applicable):
- Type and identity of media: Fischer's medium
- 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
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: Fischer's medium supplemented with l-glutamine, sodium pyruvate, pluronic F68, antibiotics, and heat-inactivated horse serum
- 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
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
S9 metabolic activation system was prepared from the livers of Aroclor 1254 induced male Fischer 344/N rats
Test concentrations with justification for top dose:
1. Without S9:
Trial 1: 0, 5, 10, 20, 30 , 40 or 60 nL/mL
Trial 2: 0, 20, 30, 40, 50, 60 or 80 nL/mL
Trial 3: 0, 10, 20, 30, 40, 60 or 80 nL/mL
Trial 4: 0, 10, 20, 30 or 40 nL/mL

With S9:
Trial 1: 0, 30, 40, 50, 60, 80 or 100 nL/mL
Trial 2: 0, 30, 40, 50, 60, 80 or 100 nL/mL

2. Without S9:
Trial 1: 0, 1.25, 2.5, 3.75, 5.0 or 7.5 µg/mL
Trial 2: 0, 2, 3, 4, 5, 6 or 8 µg/mL
Trial 3: 0, 2, 3, 4, 5, 6, 8 or 10 µg/mL

With S9:
Trial 1: 0, 2.5, 5.0, 7.5, 10 or 15 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: The test chemical was soluble in ethanol
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Details on test system and experimental conditions:
1. METHOD OF APPLICATION: in medium
Cells at start of experiment: 6000000 cells

DURATION
- Preincubation period: No data
- Exposure duration: 4 hrs
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): 10-12 days

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

NUMBER OF REPLICATIONS: 4 trial were performed without S9 and 2 trials were performed with S9.

NUMBER OF CELLS EVALUATED: No data

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

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

OTHER: No data

2. METHOD OF APPLICATION: in medium
Cells at start of experiment: 6000000 cells

DURATION
- Preincubation period: No data
- Exposure duration: 4 hrs
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): 10-12 days

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

NUMBER OF REPLICATIONS: Triplicate

NUMBER OF CELLS EVALUATED: No data

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

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 responses had to be significant (P<0.05) for the test chemical to be considered positive, i.e., capable of inducing TFT resistance. A single significant response led to a "questionable" conclusion, and the absence of both a trendand peak response resulted in a "negative" call
Statistics:
All data were evaluated statistically for trend and peak responses.
Species / strain:
mouse lymphoma L5178Y cells
Remarks:
1
Metabolic activation:
with and 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:
mouse lymphoma L5178Y cells
Remarks:
2
Metabolic activation:
with
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:
mouse lymphoma L5178Y cells
Remarks:
2
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
No data
Remarks on result:
other: No mutagenic potential
Conclusions:
The test chemical did not induce gene mutation in Mouse lymphoma L5178Y cells in the presence and absence of S9 metabolic activation system and hence it is not likely to be mutagenic in vitro.
Executive summary:

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

In vitro mammalian cell gene mutation assay was performed to determine the mutagenic nature of the test chemical. The study was performed using Mouse lymphoma L5178Y cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in ethanol and used at dose level of 0, 5, 10, 20, 30 , 40 or 60 nL/mL (trial 1), 0, 20, 30, 40, 50, 60 or 80 nL/mL (trial 2), 0, 10, 20, 30, 40, 60 or 80 nL/mL (trial 3) or 0, 10, 20, 30 or 40 nL/mL (trial 4) without S9 and 0, 30, 40, 50, 60, 80 or 100 nL/mL (trial 1) and 0, 30, 40, 50, 60, 80 or 100 nL/mL (trial 2) with S9. Mouse lymphoma L5178Y cells were maintained at 37˚C as suspension cultures in supplemented Fischer's medium; normal cycling time was approximately 10 hours. To reduce the number of spontaneously occurring trifluorothymidine-resistant cells, subcultures were exposed to medium containing THMG (thymidine, hypoxanthine, methotrexate, and glycine) for 1 day, to medium containing THG (thymidine, hypoxanthine, and glycine) for 1 day, and to normal medium for 3 to 5 days. For cloning, the horse serum content was increased and Noble agar was added. All treatment levels within an experiment, including concurrent positive and solvent controls, were replicated. Treated cultures contained 6 x 106cells in 10 mL medium. This volume included the S9 fraction in those experiments performed with metabolic activation. Incubation with butyl benzyl phthalate continued for 4 hours, at which time the medium plus butyl benzyl phthalate was removed and the cells were resuspended in fresh medium and incubated for an additional 2 days to express the mutant phenotype. Cell density was monitored so that log phase growth was maintained. After the 48-hour expression period, 3 x 106cells were plated in medium and soft agar supplemented with trifluorothymidine (TFT) for selection of TFT-resistant (TK-1) cells; 600 cells were plated in nonselective medium and soft agar to determine cloning efficiency. Plates were incubated at 37" C in 5% CO2 for 10 to 12 days. The test was initially performed without 59.' If a clearly positive response was not obtained, the test was repeated using freshly prepared S9 from the livers of Aroclor 1254 induced maleFischer344/N rats. Based on the observations made, the test chemical did not induce gene mutation inMouse lymphoma L5178Y cells in the presence and absence of S9 metabolic activation system and hence it is not likely to be mutagenic in vitro.

In vitro mammalian cell gene mutation assay was performed to determine the mutagenic nature of the test chemical. The study was performed using Mouse lymphoma L5178Y cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in ethanol and used at dose level of 0, 1.25, 2.5, 3.75, 5.0 or 7.5µg/mL (trial 1), 0, 2, 3, 4, 5, 6 or 8µg/mL (trial 2), 0, 2, 3, 4, 5, 6, 8 or 10µg/mL (trial 3) without S9 and 0, 2.5, 5.0, 7.5, 10 or 15µg/mL (trial 1) with S9. Mouse lymphoma L5178Y cells were maintained at 37˚C as suspension cultures in supplemented Fischer's medium; normal cycling time was approximately 10 hours. To reduce the number of spontaneously occurring trifluorothymidine-resistant cells, subcultures were exposed to medium containing THMG (thymidine, hypoxanthine, methotrexate, and glycine) for 1 day, to medium containing THG (thymidine, hypoxanthine, and glycine) for 1 day, and to normal medium for 3 to 5 days. For cloning, the horse serum content was increased and Noble agar was added. All treatment levels within an experiment, including concurrent positive and solvent controls, were replicated. Treated cultures contained 6 x 106cells in 10 mL medium. This volume included the S9 fraction in those experiments performed with metabolic activation. Incubation with butyl benzyl phthalate continued for 4 hours, at which time the medium plus butyl benzyl phthalate was removed and the cells were resuspended in fresh medium and incubated for an additional 2 days to express the mutant phenotype. Cell density was monitored so that log phase growth was maintained. After the 48-hour expression period, 3 x 106cells were plated in medium and soft agar supplemented with trifluorothymidine (TFT) for selection of TFT-resistant (TK-1) cells; 600 cells were plated in nonselective medium and soft agar to determine cloning efficiency. Plates were incubated at 37˚C in 5% CO2 for 10 to 12 days. Based on the observations made, thetest chemical did not induce gene mutation inMouse lymphoma L5178Y cells in the presence S9 metabolic activation system. It however induced gene mutation I the cell line in the absence of S9 metabolic activation system.

Based on the observations made, the test chemical did not induce gene mutation in Mouse lymphoma L5178Y cells in the presence and absence of S9 metabolic activation system and hence it is not likely to be mutagenic in vitro.

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

Additional information

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

Ames assay:

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay. The study was performed using Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvrA. The test chemical was dissolved in DMSO and used at dose level of 0, 20, 100, 500, 2,500 or 5,000 µg/plate. Concurrent solvent and positive control chemicals were also included in the study. According to the results of the present study, the test substance pigment red 169 is not mutagenic in the Salmonella typhimurium /Escherichia coli reverse mutation assay with and without metabolic activation.

In vitro mammalian chromosome aberration study:

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

In vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of the test chemical. Approximately 24 hr before chemical treatment, cultures were initiated at a density of 1.75 X 106cells/75 cm2flask. In the AB trials without S9, the cultures were treated with the test chemical in medium for 8 hr, washed to remove the test chemical, and treated with colcemid M) for 2-2.5 hr before cell harvest. In the experiments with activation, cultures were exposed to the test chemical in serum free medium with S9 and cofactors for 2 hr, washed to remove the test chemical and S9, and incubated at 37°C with fresh medium for 8 hr. Colcemid was then added, andthe cells were harvested 2 hr later. Thus the total durations of the nonactivated and activated AB experiments were 10hr and 12 hr, respectively, to give 10 hr growth in medium with serum for each experiment. For ABs, slides were stained in 5% Giemsa for 5 min. In early studies, one hundred cells were scored for each ofthree concentrations: the highest test concentration in whichsufficient metaphase cells could be scored and the next two lower concentrations, covering a one-log range. For later studies, 200 cells per dose were scored; however, fewer cells were scored if a test chemical produced a strong positive response or the chemical was toxic. Cells were analyzed for the following categories of chromosomal aberrations: “simple,” defined as a chromatid gap, break, fragment, and deletion or chromosome gap,break, or double minutes; “complex,” defined as interstitial deletions, triradials, quadriradials, rings, and dicentric chromosomes; and “other,” defined as pulverized chromosomesor cells with greater than 10 aberrations. Chromatid and chromosome gaps were recorded but were not used in the analysis. The test chemical did not induce chromosome aberrations in the CHO-LB cell line in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In another study, in vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of the test chemical. The test chemical was studied at a dose level of 0, 125, 400 and 1250 µg/mL in the absence and presence of S9 using Chinese hamster ovary cells (CHO-W-B1).Cells were exposed to the test chemical for 2 hr in the presence of S9 or throughout the incubation period without S9. 100 cells were scored from each of the three highest dose groups having sufficient metaphases for analysis. All types of aberrations were recorded separately, but for data analysis they were grouped into categories of “simple” (breaks and terminal deletions), “complex” (exchanges and rearrangements), “other” (includes pulverized chromosomes), and “total”. Gaps and endo-reduplications were recorded but were not included in the totals. Polyploid cells were not scored but used metaphases with 19-23 chromosomes (the modal number being 21). Based on the results noted, the test chemical did not induce chromosome aberrations in the Chinese hamster ovary cells (CHO-W-B1) in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

Based on the data available, the test chemical did not induce chromosomal aberrations in the cultured in the mammalian cell line with and without metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In vitro mammalian cell gene mutation assay:

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

In vitro mammalian cell gene mutation assay was performed to determine the mutagenic nature of the test chemical. The study was performed using Mouse lymphoma L5178Y cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in ethanol and used at dose level of 0, 5, 10, 20, 30 , 40 or 60 nL/mL (trial 1), 0, 20, 30, 40, 50, 60 or 80 nL/mL (trial 2), 0, 10, 20, 30, 40, 60 or 80 nL/mL (trial 3) or 0, 10, 20, 30 or 40 nL/mL (trial 4) without S9 and 0, 30, 40, 50, 60, 80 or 100 nL/mL (trial 1) and 0, 30, 40, 50, 60, 80 or 100 nL/mL (trial 2) with S9. Mouse lymphoma L5178Y cells were maintained at 37˚C as suspension cultures in supplemented Fischer's medium; normal cycling time was approximately 10 hours. To reduce the number of spontaneously occurring trifluorothymidine-resistant cells, subcultures were exposed to medium containing THMG (thymidine, hypoxanthine, methotrexate, and glycine) for 1 day, to medium containing THG (thymidine, hypoxanthine, and glycine) for 1 day, and to normal medium for 3 to 5 days. For cloning, the horse serum content was increased and Noble agar was added. All treatment levels within an experiment, including concurrent positive and solvent controls, were replicated. Treated cultures contained 6 x 106cells in 10 mL medium. This volume included the S9 fraction in those experiments performed with metabolic activation. Incubation with butyl benzyl phthalate continued for 4 hours, at which time the medium plus butyl benzyl phthalate was removed and the cells were resuspended in fresh medium and incubated for an additional 2 days to express the mutant phenotype. Cell density was monitored so that log phase growth was maintained. After the 48-hour expression period, 3 x 106cells were plated in medium and soft agar supplemented with trifluorothymidine (TFT) for selection of TFT-resistant (TK-1) cells; 600 cells were plated in nonselective medium and soft agar to determine cloning efficiency. Plates were incubated at 37" C in 5% CO2 for 10 to 12 days. The test was initially performed without 59.' If a clearly positive response was not obtained, the test was repeated using freshly prepared S9 from the livers of Aroclor 1254 induced maleFischer344/N rats. Based on the observations made, the test chemical did not induce gene mutation inMouse lymphoma L5178Y cells in the presence and absence of S9 metabolic activation system and hence it is not likely to be mutagenic in vitro.

In vitro mammalian cell gene mutation assay was performed to determine the mutagenic nature of the test chemical. The study was performed using Mouse lymphoma L5178Y cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in ethanol and used at dose level of 0, 1.25, 2.5, 3.75, 5.0 or 7.5µg/mL (trial 1), 0, 2, 3, 4, 5, 6 or 8µg/mL (trial 2), 0, 2, 3, 4, 5, 6, 8 or 10µg/mL (trial 3) without S9 and 0, 2.5, 5.0, 7.5, 10 or 15µg/mL (trial 1) with S9. Mouse lymphoma L5178Y cells were maintained at 37˚C as suspension cultures in supplemented Fischer's medium; normal cycling time was approximately 10 hours. To reduce the number of spontaneously occurring trifluorothymidine-resistant cells, subcultures were exposed to medium containing THMG (thymidine, hypoxanthine, methotrexate, and glycine) for 1 day, to medium containing THG (thymidine, hypoxanthine, and glycine) for 1 day, and to normal medium for 3 to 5 days. For cloning, the horse serum content was increased and Noble agar was added. All treatment levels within an experiment, including concurrent positive and solvent controls, were replicated. Treated cultures contained 6 x 106cells in 10 mL medium. This volume included the S9 fraction in those experiments performed with metabolic activation. Incubation with butyl benzyl phthalate continued for 4 hours, at which time the medium plus butyl benzyl phthalate was removed and the cells were resuspended in fresh medium and incubated for an additional 2 days to express the mutant phenotype. Cell density was monitored so that log phase growth was maintained. After the 48-hour expression period, 3 x 106cells were plated in medium and soft agar supplemented with trifluorothymidine (TFT) for selection of TFT-resistant (TK-1) cells; 600 cells were plated in nonselective medium and soft agar to determine cloning efficiency. Plates were incubated at 37˚C in 5% CO2 for 10 to 12 days. Based on the observations made, thetest chemical did not induce gene mutation inMouse lymphoma L5178Y cells in the presence S9 metabolic activation system. It however induced gene mutation I the cell line in the absence of S9 metabolic activation system.

Based on the observations made, the test chemical did not induce gene mutation in Mouse lymphoma L5178Y cells in the presence and absence of S9 metabolic activation system and hence it is not likely to be mutagenic in vitro.

Based on the data available, the test chemical does not exhibit gene mutation in vitro. Hence the test chemical is not likely to be mutagenic in vitro as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

Based on the data available, the test chemical does not exhibit gene mutation in vitro. Hence the test chemical is not likely to be mutagenic in vitro as per the criteria mentioned in CLP regulation.