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Genetic toxicity in vitro

Description of key information

Ames assay:

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.

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
Type of information:
experimental study
Adequacy of study:
key study
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
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
True negative controls validity:
not specified
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
True negative controls validity:
not specified
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
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 for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
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)
Remarks:
4
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
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Remarks:
(CHO-W-B1) / 5
Details on mammalian cell type (if applicable):
Details on mammalian cell line
- Type and identity of media: McCoy’s 5a medium with 10% fetal calf serum, L-glutamine, and antibiotics
- Properly maintained: No data available
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
Colcemid (10-6M)
Metabolic activation:
with and without
Metabolic activation system:
4. 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.
5. The S9 mix consisted of 15 µl/ml liver homogenate (from male Sprague-Dawley rats, induced with Aroclor 1254), 2.4 mg/ml NADP, and 4.5 mg/ml isocitric acid in serum-free medium.
Test concentrations with justification for top dose:
4. Without S9: 0, 49, 150, 499 µg/mL
With S9: 0, 500, 1500, 5000 µg/mL
5. 0, 125, 400 and 1250 µg/mL
Vehicle / solvent:
4. - Vehicle(s)/solvent(s) used: Water
- Justification for choice of solvent/vehicle: The test chemical was soluble in water
5. - Vehicle(s)/solvent(s) used: The chemical was dissolved immediately before use in water, dimethyl sulfoxide (DMSO), ethanol, or
acetone, in that order of preference. Details are not available
Untreated negative controls:
yes
Remarks:
medium-treated cultures
Negative solvent / vehicle controls:
yes
Remarks:
Water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Remarks:
4
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
triethylenemelamine
cyclophosphamide
mitomycin C
Remarks:
5
Details on test system and experimental conditions:
4. 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: 8.5 hrs; With S9: 10 hrs
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): 10.5 hrs (without S9), 12 hrs (with S9)
SELECTION AGENT (mutation assays): No data available
SPINDLE INHIBITOR (cytogenetic assays): colcemid (10-6 M)
STAIN (for cytogenetic assays): 5% Giemsa
NUMBER OF REPLICATIONS: Only one trial was performed
NUMBER OF CELLS EVALUATED: 100 cells were scored
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data
OTHER EXAMINATIONS:
- Determination of polyploidy: Yes, but it is not included in the total number of aberration and statistical analyses.
- Determination of endoreplication: Yes, but it is not included in the total number of aberration and statistical analyses.
5. 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
Rationale for test conditions:
No data
Evaluation criteria:
4. 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. 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.
5. 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).
Statistics:
4. A binomial sampling assumption as described by Margolin et al. 1983 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.
5. Linear regression analysis of the percentage of cells with aberrations vs the log-dose was used as the test for trend. To examine absolute increases over control levels at each dose, a binomial sampling assumption (as opposed to Poisson) was used, and the test was that described by Margolin et al. The P values were adjusted by Dunnett’s method to take into account the multiple dose comparisons. For data analysis, we used the “total” aberration category, and the criterion for a positive response was that the adjusted P value be < 0.05.
Species / strain:
Chinese hamster Ovary (CHO)
Remarks:
4
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 499 ug/ml without S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
Chinese hamster Ovary (CHO)
Remarks:
(CHO-W-B1) / 5
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
4. 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.
5. TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: Yes, precipitate or immiscibility was noted at 75-150 µg/mL
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: Dose selection was based on a preliminary growth inhibition test in which cells that excluded trypan blue were counted 24 hr after treatment. The top doses selected for the cytogenetics assays were those estimated to reduce growth by 50%. This approach was subsequently modified such that toxicity estimates were made from observations of cell monolayer confluence and mitotic activity in the same cultures used for analysis of SCEs or aberrations. The aim was to obtain results at the highest dose at which sufficient metaphase cells would be available for analysis. Observations on cell growth and cell cycle kinetics were used from the SCE test to select the doses and fixation times for the chromosome aberration tests. In the first SCE test with each chemical, cells were exposed to a range of doses spanning four to five orders of magnitude, in half-log increments, up to a maximum dose of 5-10 mg/ml or to the limits of solubility in culture medium. In some cases, test chemical precipitate was observed at the higher dose levels. Dose selection for repeat trials involved a range of doses based on observations from the first trial.
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 for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
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 cell gene mutation tests using the thymidine kinase gene
Target gene:
Thymidine kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Remarks:
6 and 7
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
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
6. S9 metabolic activation system was prepared from the livers of Aroclor 1254 induced male Fischer 344/N rats
7. Freshly prepared 89 metabolic activation factors were obtained from the liver of either Aroclor 1254-induced or noninduced male F344 rats
Test concentrations with justification for top dose:
6. 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
7. 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:
6 and 7. - 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
Remarks:
6 and 7
Details on test system and experimental conditions:
6. 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
7. 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:
6 and 7. 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:
6 and 7. All data were evaluated statistically for trend and peak responses.
Species / strain:
mouse lymphoma L5178Y cells
Remarks:
6
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Remarks:
7
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
No data
Remarks on result:
other: No mutagenic potential
Conclusions:
Based on the presented data, the test chemical is regarded to be non-mutagenic in mouse lymphoma cells both in the presence and absence of S9 metabolic activation system.
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, thetest 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.

Above study is supported with another In vitro mammalian cell gene mutation assay 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 in Mouse 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 presented data, the test chemical is regarded to be non-mutagenic in mouse lymphoma cells both in the presence and absence of S9 metabolic activation system.

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

Genetic toxicity in vivo

Description of key information

Genetic Toxicity (In-Vivo):

Based on all the available observations and results, it was concluded that the test chemical was non-mutagenic (negative) in sex-linked recessive lethal (SLRL) mutation induction test in Drosophila melanogaster.

Link to relevant study records
Reference
Endpoint:
in vivo insect germ cell study: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
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 a publication
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.20 (Sex-linked Recessive Lethal Test in Drosophila melanogaster)
Principles of method if other than guideline:
Equivalent or similar to EU Method B.20 (Sex-linked Recessive Lethal Test in Drosophila melanogaster)
GLP compliance:
not specified
Type of assay:
Drosophila SLRL assay
Species:
Drosophila melanogaster
Strain:
not specified
Sex:
male
Details on test animals or test system and environmental conditions:
The males to be treated were collected from a Canton-S wild-type stock within 8 hr of emergence and kept on regular culture medium until exposure.
Route of administration:
other: The chemical was first tested by feed. If non-mutagenic by this route, the chemical was injected.
Vehicle:
Dietary exposure: 5% aqueous sucrose
Injection: 0.7% aqueous NaCl
Details on exposure:
Males to be fed were shaken into vials containing glass fiber filter material that was soaked with the feeding solution or mixture. At 24 hr and again at 48 hr, the flies were transferred to vials with freshly prepared feeding mixture and were mated at 72 hr. Males to be injected were held on regular food for 1-3 days. They were then injected with 0.7% NaCl solution containing the test chemical. They were held for 24 hr to recover, then mated.
Duration of treatment / exposure:
1-2 days
Frequency of treatment:
Feeding: 1-2 days of exposure
Injection: one injection
Post exposure period:
Injection: 24 hr recovery period
Remarks:
0 and 50000 ppm (feed)
0 and 10000 ppm (injection)
No. of animals per sex per dose:
no data
Control animals:
yes, concurrent vehicle
yes, plain diet
Positive control(s):
AF-2
Tissues and cell types examined:
Germ cells
Evaluation criteria:
Generally, a test was considered positive if the frequency of lethals in the treated series exceeded 0.2% over the control frequency.
Statistics:
Data were then tested for significance using the normal test as described by Margolin et al (Environ Mutagen 1983; 5: 705-16). A SLRL result was called negative if P > 06, equivocal if P = 0.04-0.06, either questionable or positive if P = 0.01-0.04 (depending on the control frequency), and positive if P < 0.01.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
19% mortality bu injection, 17% mortality by feeding
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid
Remarks:
AF-2
Remarks on result:
other: Negative
Additional information on results:
The sex-linked recessive lethal frequency was similar between insects fed at 0 and 50000 ppm (0.09% vs 0.06%, respectively) and similar between insects injected at 0 and 10000 ppm (0.05% vs 0.08%, respectively). Mortality was in the range of 17-19% for insects treated with the test chemical. No cases of sterility were observed in the study.
Conclusions:
The test chemical was non-mutagenic (negative) in sex-linked recessive lethal (SLRL) mutation induction test in Drosophila melanogaster.
Executive summary:

The test chemical was tested for sex-linkedrecessive lethal (SLRL) mutation inductionin Drosophila melanogaster. Initially, the test chemical was tested by feeding, whenno significant mutagenicitywas observed,it was re-tested by injection.Doses used in the mutagenicity test was selected based on the results of a preliminary dose-finding study. The test substance was dissolved in water anddiluted with aqueous 5%sucrose for feedingand with aqueous 0.7% NaCl for injection. Toxicity tests were performed both forfeeding and injection treatments using a series of concentrations.Doses were chosen to induce mortalityof about 30% after 72 hoursof feeding or 24 hoursafter injection. The males to be treated werekept on regular culture medium until exposure. Males to befed were shaken into vials containing glass fiber filter material that was soaked withthe feeding solution ortest chemicalmixture. At 24 hoursand again at 48hours, the flies were transferredto vials with freshly prepared feeding mixture and were mated at 72 hours.Males to be injected were held on regular food for 1-3 days. They were theninjected with0.7% NaCl solution containing the test chemical. They were held for24 hoursto recover, then mated. The flies for the SLRL assay were exposed for 4hours. Treated and control males were matedindividuallyto threeharems ofBascvirgin females to produce three broods of 3,2, and 2 days.Thus,primarily post-meiotic germ cells were tested. To reduce the chances of recoveringseveral lethals from the same male, no more than 40 F1 females were mated individuallyfrom each brood of each male. Thus, no more than 120 chromosomes weretested from each P1male. F2 cultures were scored as presumptive lethals if thenumber of wild-type males recovered was 0, 1, or fewer than5% of the number ofBascmales (or Basc/+females). Presumptive lethals were confirmed by repeating the matings and scoring the F3.The test chemical was used at doses of 0 (solvent control) and 50000 ppm for feeding experiments and 10000 ppm for injection. No mutagenic response was observed in broods examined when the test chemical solution was fed or injected to male flies. The percents of lethals (total lethals) were statistically comparable in treated and control groups using both treatment methods. The mortality was 19 and 17% in experiments with injection and feeding treatment, respectively In conclusion, the test chemical was non-mutagenic (negative) insex-linkedrecessive lethal (SLRL) inductiontest in Drosophila melanogaster.

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

Additional 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:

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, thetest 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.

Above study is supported with another In vitro mammalian cell gene mutation assay 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 in Mouse 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 presented data, the test chemical is regarded to be non-mutagenic in mouse lymphoma cells both in the presence and absence of S9 metabolic activation system.

Genetic Toxicity (In-Vivo):

The test chemical was tested for sex-linkedrecessive lethal (SLRL) mutation inductionin Drosophila melanogaster. Initially, the test chemical was tested by feeding, whenno significant mutagenicitywas observed,it was re-tested by injection.Doses used in the mutagenicity test was selected based on the results of a preliminary dose-finding study. The test substance was dissolved in water anddiluted with aqueous 5%sucrose for feedingand with aqueous 0.7% NaCl for injection. Toxicity tests were performed both forfeeding and injection treatments using a series of concentrations.Doses were chosen to induce mortalityof about 30% after 72 hoursof feeding or 24 hoursafter injection. The males to be treated werekept on regular culture medium until exposure. Males to befed were shaken into vials containing glass fiber filter material that was soaked withthe feeding solution ortest chemicalmixture. At 24 hoursand again at 48hours, the flies were transferredto vials with freshly prepared feeding mixture and were mated at 72 hours.Males to be injected were held on regular food for 1-3 days. They were theninjected with0.7% NaCl solution containing the test chemical. They were held for24 hoursto recover, then mated. The flies for the SLRL assay were exposed for 4hours. Treated and control males were matedindividuallyto threeharems ofBascvirgin females to produce three broods of 3,2, and 2 days.Thus,primarily post-meiotic germ cells were tested. To reduce the chances of recoveringseveral lethals from the same male, no more than 40 F1 females were mated individuallyfrom each brood of each male. Thus, no more than 120 chromosomes weretested from each P1male. F2 cultures were scored as presumptive lethals if thenumber of wild-type males recovered was 0, 1, or fewer than5% of the number ofBascmales (orBasc/+females). Presumptive lethals were confirmed by repeating the matings and scoring the F3.The test chemical was used at doses of 0 (solvent control) and 50000 ppm for feeding experiments and 10000 ppm for injection. No mutagenic response was observed in broods examined when the test chemical solution was fed or injected to male flies. The percents of lethals (total lethals) were statistically comparable in treated and control groups using both treatment methods.The mortality was 19 and 17% in experiments with injection and feeding treatment, respectivelyIn conclusion, the test chemical was non-mutagenic (negative) insex-linkedrecessive lethal (SLRL) inductiontest in Drosophila melanogaster.

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