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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 Mar - 2 Apr 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
no data on historical controls provided
GLP compliance:
yes
Remarks:
according to the Japanese GLP Standard (as described on JECDB)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in closed bottle at room temperature
- Solubility and stability of the test substance in the solvent/vehicle: water solubility 2.71%, easily soluble in DMSO and acetone


Method

Target gene:
his operon, trp operon
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 mix, prepared from the livers of rats treated with phenobarbital and 5-6-benzoflavone
Test concentrations with justification for top dose:
TA100 and TA1535, with and without metabolic activation: 39.1, 78.1, 156, 313, 652, 1250 and 2500 μg/mL (tested up to cytotoxic concentrations)
WP2uvrA, TA98 and TA1537, with and without metabolic activation: 156, 313, 652, 1250, 2500 and 5000 μg/mL (tested up to the limit dose)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, DMSO was selected as the vehicle.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: aminofluorene for TA100 and TA98, N-ethyl-N'-nitrosogunanidine for WP2uvrA, 2-aminoanthracene for strains with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 h

NUMBER OF REPLICATIONS:3 replications each in 2 independent experiments


Evaluation criteria:
Evaluation:
A test substance was considered positive in the Ames test if:
a) a more than 2-fold increase in the number of revertants was observed
b) the increase was dose-related
c) the positive result was reproducible.

A test substance was considered negative in the Ames test if none of the above criteria were observed under all experimental conditions.

Statistics:
Mean values and standard deviations were calculated.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Remarks:
TA100/ TA1535/ TA1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
starting at 1250 µg/mL +/-S9 in TA 100 and TA 1535; starting at 2500 µg/mL +/- S9 in WP2 uvrA, TA 98 and TA 1537
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
A preliminary test was performed with test concentrations ranging from 1.22 - 5000 μg/mL with/without S9. No increase in the number of revertants was observed. Furthermore, cytotoxicity was observed starting at 1250 μg/mL in TA100 and TA1535, and at 5000 μg/mL in WP2urvA, TA98 and TA1537 with and without metabolic activation.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: number of revertant colonies


Any other information on results incl. tables

Table 1. Test results for 2 -chlorphenol (Trial 1)

 

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

± standard deviation

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2urvA

TA98

TA1537

0

112 ± 7

 10 ± 3

24 ± 2

22 ± 5

7 ± 2

39.1

123 ± 5

8 ± 1

-

-

-

78.1

104 ± 18

12 ± 1

-

-

-

156

116 ± 15

11 ± 5

22 ± 3

24 ± 2

6 ± 2

313

103 ± 12

8 ± 2

33 ± 3

21 ± 3

7 ± 2

625

101 ± 12

12 ± 4

22 ± 2

23 ± 4

7 ± 2

1250

84 ± 9*

7 ± 2*

22 ± 2

20 ± 2

5 ± 1

2500

0 ± 0*

0 ± 0*

0 ± 0*

0 ± 0*

0 ± 0*

5000

-

-

0 ± 0*

0 ± 0*

0 ± 0*

Positive controls

AF-2

NaN3

ENNG

AF-2

9-AA

Mean No. of colonies/plate

± SD

541 ± 38

377 ± 12

821 ± 29

375 ± 29

351 ± 39

+

0

113 ± 4

7 ± 2

27 ± 5

26 ± 4

13 ± 5

+

39.1

111 ± 9

8 ± 1

-

-

-

+

78.1

110 ± 15

11 ± 3

-

-

-

+

156

122 ± 6

9 ± 2

25 ± 4

28 ± 3

10 ± 2

+

313

117 ± 9

7 ± 2

29 ± 9

29 ± 10

12 ± 3

+

625

109 ± 11

9 ± 2

32 ± 7

28 ± 7

14 ± 3

+

1250

85 ± 5*

12 ± 3*

31 ± 4

27 ± 6

12 ± 4

+

2500

0 ± 0*

0 ± 0*

0 ± 0*

0 ± 0*

0 ± 0*

+

5000

-

-

0 ± 0*

0 ± 0*

0 ± 0*

+

Positive controls

2-AA

2-AA

2-AA

2-AA

2-AA

Mean No. of colonies/plate

± SD

1161 ± 32

168 ± 7

1313 ± 38

443 ± 9

140 ± 6

AF-2= 2 -aminofluorene

NaN3 = sodium azid

ENNG = N-ethyl-N'-nitro-N'-nitrosogunanidine

9-AA = 9-aminoacridine

2AA = 2-aminoanthracene

SD = standard deviation

* = growth inhibition/cytotoxicity

Table 2. Test results for 2 -chlorphenol (Trial 2)

 

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

± standard deviation

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2urvA

TA98

TA1537

0

105 ± 4

8 ± 2

20 ± 5

23 ± 3

13 ± 1

39.1

106 ± 3

8 ± 2

-

-

-

78.1

103 ± 9

7 ± 1

-

-

-

156

103 ± 5

8 ± 2

29 ± 2

23 ± 2

15 ± 4

313

108 ± 11

11 ± 1

26 ± 4

18 ± 2

12 ± 2

625

94 ± 2

8 ± 1

23 ± 4

18 ± 2

15 ± 1

1250

87 ± 15*

8 ± 2*

19 ± 3

15 ± 2

14 ± 4

2500

0 ± 0*

0 ± 0*

0 ± 0*

0 ± 0*

0 ± 0*

5000

-

-

0 ± 0*

0 ± 0*

0 ± 0*

Positive controls

AF-2

NaN3

ENNG

AF-2

9-AA

Mean No. of colonies/plate

± SD

524 ± 14

514 ± 46

843 ± 33

505 ± 21

430 ± 34

+

0

105 ± 3

10 ± 2

25 ± 5

25 ± 5

19 ± 2

+

39.1

101 ± 6

9 ± 1

-

-

-

+

78.1

108 ± 10

8 ± 2

-

-

-

+

156

95 ± 8

11 ± 3

28 ± 5

25 ± 4

18 ± 5

+

313

96 ± 6

10 ± 1

27 ± 4

25 ± 6

16 ± 3

+

625

108 ± 10

9 ± 2

28 ± 6

26 ± 2

21 ± 3

+

1250

96 ± 5*

8 ± 3*

29 ± 3

23 ± 0

19 ± 2

+

2500

0 ± 0*

0 ± 0*

0 ± 0*

0 ± 0*

0 ± 0*

+

5000

-

-

0 ± 0*

0 ± 0*

0 ± 0*

+

Positive controls

2-AA

2-AA

2-AA

2-AA

2-AA

Mean No. of colonies/plate

± SD

1010 ± 58

210 ± 9

1477 ± 47

424 ± 12

192 ± 7

AF-2= 2 -aminofluorene

NaN3 = sodium azido

ENNG = N-ethyl-N'-nitro-N'-nitrosogunanidine

9-AA = 9-aminoacridine

2AA = 2-aminoanthracene

SD = standard deviation

* = growth inhibition/cytotoxicity

Applicant's summary and conclusion