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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 Jul - 28 Jul 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted July 21, 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
published June 8, 2000
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): AF-654
- Physical state: white powder
- Lot/batch No.: #CE-201
- Expiration date of the lot/batch: 01 Jan 2005
- Storage condition of test material: at room temperature in the dark

Method

Target gene:
his operon (S. typhimurium strains), trp operon (E. coli strains)
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254 (experiment I: S9 mix contained 5% (v/v) S9-fraction; experiment II: S9 mix contained 10% (v/v) S9-fraction)
Test concentrations with justification for top dose:
pre-experiment (with and without S9 mix): 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate for TA 100 and E. coli WP2 uvr A
experiment I (with and without S9 mix): 3, 10, 33, 100 and 333 µg/plate for TA 98, TA 1535 and TA 1537
experiment II (with and without S9 mix): 3, 10, 33, 100 and 333 µg/plate for all strains
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
sodium azide
methylmethanesulfonate
other: daunomycin; 2-aminoanthracene
Remarks:
-S9: sodium azide (SA, 5µg/plate, TA1535); 9-aminoacridine (9AC, 60µg/plate, TA1537); daunomycin (DM, 4µg/plate, TA98), methylmethanesulfonate (MMS, 650µg/plate, TA100); 4-nitroquinoline-N-oxide (4-NQO, 10µg/plate, E. coli); +S9: 2-aminoanthracene (2AA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: triplicates each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduction of bacterial background lawn, increase in size of microcolonies, reduction of revertant colonies

OTHER:
The dose range finding test was reported as part of the first experiment of the mutation assay.

Evaluation criteria:
Acceptability criteria:
1) The negative control data (number of spontaneous revertants per plate) should be within the laboratory background historical range for each tester strain.
2) The positive control chemicals should produce responses in all tester strains, which are within the laboratory historical range documented for each positive control substance. Furthermore the mean plate count should be at least two times the concurrent vehicle control group mean.
3) The selected dose range should induce a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.

Evaluation criteria:
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent value, with or without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) It induces a number of revertant colonies, dose related, greater than two-times the number of revertants induced by the solvent control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 is considered to be not significant.
b) The positive response should be reproducible in at least one independently repeated experiment.
Statistics:
Mean values and standard deviation were calculated.

Results and discussion

Test resultsopen allclose all
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:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation was observed at concentrations of 100 µg/plate and above in the top agar and on the plates at 333 µg/plate at the start and the end of the incubation period.

RANGE-FINDING/SCREENING STUDIES:
Selection of an adequate range of doses was based on a dose rane finding test with strain TA 100 and E. coli WP2uvrA in the presence and absence of S9 mix using eight concentrations (3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate). The highest concentration chosen for the main assay was the level at which the test substance showed limited solubility (333 µg/plate). This dose range finding test was reported as a part of the first experiment of the mutation assay.

COMPARISON WITH HISTORICAL CONTROL DATA:
All values are within the range of historical control data, except the response for TA98 in the absence of S9 mix (first experiment, negative control). Since this value was just outside the limit of the range the validity of the test was considered to be not affected.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. Test results of experiment 1 (plate incorporation).

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates ± Standard deviation)

Base-pair substitution type

Frameshift type

TA 100

TA1535

E. coli WP2

TA98

TA1537

DMSO

138 ± 6

 8 ± 2

9 ± 2

 11 ± 2

 4 ± 1

3

139 ± 1

 7 ± 2

11 ± 2

 10 ± 2

 6 ± 3

10

137 ± 15

 8 ± 2

8 ± 1

 11 ± 3

 4 ± 2

33

156 ± 6

 7 ± 2

8 ± 2

 11 ± 1

 5 ± 3

100

145 ± 20

 9 ± 1

12 ± 1

 14 ± 1

 3 ± 1

333SP

149 ± 5

 10 ± 3

12 ± 3

 12 ± 3

 3 ± 2

1000MP

129 ± 4

-

9 ± 2

-

-

3330MP

136 ± 21

-

11 ± 3

-

-

5000MP

128 ± 9

-

13 ± 2

-

-

Positive controls, –S9

Name

MMS

SA

4-NQO

DM

9AC

Concentrations

(μg/plate)

650

5

10

4

60

Mean No. of colonies/plate

(average of 3 ± SD)

962 ± 22

 244 ± 16

812 ± 73

 516 ± 71

 280 ± 46

+

DMSO

140 ± 21

 10 ± 1

10 ± 1

 17 ± 1

 4 ± 4

+

3

142 ± 4

 6 ± 2

10 ± 2

 14 ± 4

 6 ± 2

+

10

140 ± 16

 7 ± 4

10 ± 2

 13 ± 2

 6 ± 2

+

33

140 ± 8

 6 ± 1

12 ± 2

 17 ± 2

 4 ± 2

+

100

155 ± 6

 7 ± 2

12 ± 2

 20 ± 5

 4 ± 2

+

333SP

149 ± 9

 6 ± 1

9 ± 2

 15 ± 7

 5 ± 2

+

1000MP

134 ± 20

-

8 ± 2

-

-

+

3330MP

143 ± 11

-

10 ± 3

-

-

+

5000MP

151 ± 10

-

11 ± 2

-

-

Positive controls, +S91

Name

 2AA

 2AA

 2AA

 2AA

 2AA

Concentrations

(μg/plate)

1

 1

 5

 1

 2.5

Mean No. of colonies/plate

(average of 3 ± SD)

717 ± 50

 100 ± 4

 73 ± 11

 520 ± 7

 75 ± 16

Table 2. Test results of experiment 2 (plate incorporation).

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates ± Standard deviation)

Base-pair substitution type

Frameshift type

TA 100

TA1535

E. coli WP2

TA98

TA1537

DMSO

 138 ± 13

 10 ± 4

 13 ± 3

 15 ± 6

 5 ± 2

3

 142 ± 9

 11 ± 3

 17 ± 4

 13 ± 1

 4 ± 2

10

 137 ± 6

 9 ± 1

 12 ± 3

 17 ± 4

 6 ± 3

33

 132 ± 12

 11 ± 6

 14 ± 2

 12 ± 1

 5 ± 3

100

 151 ± 9

 9 ± 6

 16 ± 4

 13 ± 3

 5 ± 2

333SP

 139 ± 15

 9 ± 2

 13 ± 4

 15 ± 3

 5 ± 1

Positive controls, –S9

Name

MMS

SA

4-NQO

DM

9AC

Concentrations

(μg/plate)

650

5

5

4

60

Mean No. of colonies/plate

(average of 3 ± SD)

 1025 ± 27

 351 ± 20

 712 ± 6

 499 ± 79

 220 ± 34

+

DMSO

 141 ± 15

 10 ± 3

 19 ± 4

 23 ± 2

 6 ± 2

+

3

 137 ± 18

 6 ± 1

 16 ± 2

 21 ± 3

 7 ± 3

+

10

 127 ± 7

 9 ± 5

 14 ± 2

 18 ± 6

 6 ± 3

+

33

 139 ± 13

 6 ± 4

 13 ± 2

 20 ± 3

 5 ± 1

+

100

 130 ± 8

 8 ± 3

 12 ± 1

 19 ± 4

 4 ± 2

+

333SP

 121 ± 6

 8 ± 2

 16 ± 4

 21 ± 1

 6 ± 2

Positive controls, +S92

Name

 2AA

 2AA

 2AA

 2AA

 2AA

Concentrations

(μg/plate)

 1

 1

 5

 1

 2.5

Mean No. of colonies/plate

(average of 3 ± SD)

 766 ± 45

 128 ± 11

 122 ± 4

 436 ± 46

 201 ± 19

1: The S9 mix contained 5% (v/v) S9 fraction

2: The S9 mix contained 10% (v/v) S9 fraction

SP: slight precipitate

MP: moderate precipitate

2AA: 2-aminoanthracene

MMS: methylmethanesulfonate

SA: sodium azide

4-NQO: 4-nitroquinoline-N-oxide

DM: daunomycin

9AC: 9-aminoacridine

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative