Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 Apr - 13 May 2005
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Remarks:
/ Guideline study conducted according to the Japanese test guideline reported as "Standard of the Minister of Labor, based on the Industrial Safety and Health Law Article 57-2 Paragraph 1 (Notification No. 77 of JMOL on September 1, 1988) and Notification on partial revision of the standard (Notification No. 67 JMOL on June 2, 1997)", which in principle is similar to OECD TG 471.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Qualifier:
according to
Guideline:
other: Standard of the Minister of Labor, based on the Industrial Safety and Health Law Article 57-2 Paragraph 1 (Notification No. 77 of JMOL on September 1, 1988) and Notification on partial revision of the standard (Notification No. 67 JMOL on June 2, 1997)
Deviations:
not applicable
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, protected from light
- Solubility and stability of the test substance in the solvent/vehicle:stable in dimethylsulfoxid, acetone and tetrahydrofuran

FORM AS APPLIED IN THE TEST: liquid

Method

Target gene:
his operon, tryp operon
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 5,6-benzoflavone
Test concentrations with justification for top dose:
Based on the results of the range-finding study (all strains; doses applied 5, 20, 78, 313, 1250, 5000 μg/mL), the following concentrations were used in the main experiment:
- 39, 78, 156, 313, 625, 1250 µg/plate (TA strains, without metabolic activation)
- 313, 625, 1250, 2500, 5000 µg/plate (WP2uvrA, with and without metabolic activation, TA strains with metabolic activation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: tetrahydrofuran (THF)
- Justification for choice of solvent/vehicle: The solvent was chosen based on its solubility and stability properties.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
benzo(a)pyrene
other: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2), 2-Aminoanthracene (2AA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation
- Cell density at seeding:
in main test
TA100 and TA1535: 2.2 x 10^9
TA98: 3 x 10^9
TA1537: 1.7 x 10^9
WP2uvrA: 6.7 x 10^9

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

NUMBER OF REPLICATIONS: duplicate

Evaluation criteria:
The positive control should show a clear dose-dependend increase of at least two times compared to the negative control (spontaneous reversion rate). The negative and positive control as well as the test item should satisfy the acceptance criteria.

Acceptance criteria
The study was considered valid if:
- the number of revertant colonies of the negative (solvent) and positive controls were in the historical control range in all strains (value ± 2SD)
- no contamination in test material, S9 mix, Sodium phosphate buffer (analysis performed)
- the positive control led to an increase of at least two times compared to the negative control
Statistics:
No statistical analysis was performed. Mean values were calculated.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
number of revertant colonies decreased compared to solvent control starting at 1250 μg/plate without S9 mix (24%)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
number of revertant colonies decreased compared to solvent control starting at 1250 μg/plate without S9 mix (46%)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
number of revertant colonies decreased compared to solvent control starting at 625 μg/plate without S9 mix (22 - 78%)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
number of revertant colonies decreased compared to solvent control starting at 625 μg/plate without S9 mix (14 - 71%)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitate was detected in all examined strains with metabolic activation at all concentration tested and without metabolic activation starting at a concentration of 156 μg/plate (all TA strains) or 313 μg/plate (WP2uvrA).

RANGE-FINDING/SCREENING STUDIES: All bacterial strains showed negative responses over the entire dose range, i.e. no clear dose-dependent increase in the number of revertants of at least two times compared to the negative control. Precipitate was detected in all examined strains with and without metabolic activation starting at 313 μg/plate (for further details see "any other information on results incl tables").

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:

TA98, -S9: 444 - 679, mean ± SD: 561 ± 58.8
TA98, +S9: 249 - 448, mean ± SD: 349 ± 49.8

TA100, -S9: 398 - 689, mean ± SD: 544 ± 72.8
TA100, +S9: 843 - 1457, mean ± SD: 1150 ± 153.5

TA1535, -S9: 352 - 720, mean ± SD: 536 ± 92.0
TA1535, +S9: 116 - 320, mean ± SD: 218 ± 51.0

TA1537, -S9: 254 - 895, mean ± SD: 574 ± 160.4
TA1537, +S9: 52 - 104, mean ± SD: 78 ± 13.1

WP2uvrA, -S9: 87 - 137, mean ± SD: 107 ± 14.8
WP2uvrA, +S9: 523 - 1101, mean ± SD: 812 ± 144.4

- Negative (solvent/vehicle) historical control data:

TA98, -S9: 15 - 31, mean ± SD: 23 ± 4.0
TA98, +S9: 18 - 40, mean ± SD: 29 ± 5.5

TA100, -S9: 90 - 133, mean ± SD: 111 ± 11
TA100, +S9: 96 - 147, mean ± SD: 122 ± 13

TA1535, -S9: 3 - 14, mean ± SD: 9 ± 2.7
TA1535, +S9: 4 - 14, mean ± SD: 9 ± 2.6

TA1537, -S9: 3 - 12, mean ± SD: 8 ± 2.3
TA1537, +S9: 2 - 15, mean ± SD: 9 ± 3.1

WP2uvrA, -S9: 14 - 33, mean ± SD: 23 ± 4.8
WP2uvrA, +S9: 15 - 35, mean ± SD: 25 ± 5.0

The ranges are calculated as mean ± 2xSD.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: toxic effects were examined with a stereoscopic microscope

Any other information on results incl. tables

Table 1: Summary of test results (Range-finding study, experiment 1, pre-incubation method)

With or without S9-Mix

Test substance concentration (μg/plate)

Mean number of revertant colonies per plate (average of 2 plates)

Frameshift type

Base-pair substitution type

TA1537

TA98

TA100

TA1535

WP2 uvrA

Solvent control (THF)

9

25

129

10

22

5

8

22

132

8

23

20

7

22

134

10

27

78

10

30

130

9

24

313

9 P

26 P

121 P

8 P

20 P

1250

3 P T

7 P T

56 P T

6 P T

21 P

5000

3 P T

11 P T

62 P T

5 P T

21 P

Positive controls (µg/plate)

9AA
(80)

AF-2
(0.1)

AF-2
(0.01)

NaN3
(0.5)

AF-2
(0.01)

Mean No. of colonies/plate (average of 2 plates)

385

507

602

482

123

+

Solvent control (THF)

10

31

142

11

25

5

9

34

141

9

26

20

9

35

153

9

25

78

7

32

145

9

20

313

8 P

30 P

145 P

10 P

25 P

1250

10 P

30 P

140 P

8 P

21 P

5000

9 P

30 P

126 P

10 P

20 P

Positive controls 

(µg/plate)

B[α]P
(5)

B[α]P
(5)

B[α]P
(5)

2AA
(2)

2AA
(10)

Mean No. of colonies/plate (average of 2 plates)

85

384

1068

240

941

THF = tetrahydrofuran

AF-2 = 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide

NaN3= sodium azide

9AA = 9-aminoacridine

B[α]P = benzo[α]pyrene

2AA = 2-aminonathracene

P = precipitate

T = toxic effect observed

Table 2: Summary of test results (main study, experiment 2, pre-incubation method)

With or without S9-Mix

Test substance concentration (μg/plate)

Mean number of revertant colonies per plate (average of 2 plates)

Frameshift type

Base-pair substitution type

TA1537

TA98

TA100

TA1535

WP2 uvrA

Solvent control (THF)

7

29

132

9

26

39

7

23

124

7

-

78

8

29

117

8

-

156

7 P

28 P

137 P

8 P

-

313

7 P

29 P

116 P

8 P

22 P

625

6 P T

27 P

102 P

7 P T

26 P

1250

2 P T

22 P T

71 P T

2 P T

25 P

2500

-

-

-

-

23 P

5000

-

-

-

-

23 P

Positive controls (µg/plate)

9AA
(80)

AF-2
(0.1)

AF-2
(0.01)

NaN3
(0.5)

AF-2
(0.01)

Mean No. of colonies/plate (average of 2 plates)

430

554

465

484

127

+

Solvent control (THF)

7

28

139

9

25

313

8 P

32 P

144 P

10 P

25 P

625

8 P

36 P

142 P

9 P

26 P

1250

9 P

34 P

146 P

9 P

21 P

2500

7 P

32 P

137 P

8 P

25 P

5000

7 P

29 P

140 P

9 P

25 P

Positive controls 

(µg/plate)

B[α]P
(5)

B[α]P
(5)

B[α]P
(5)

2AA
(2)

2AA
(10)

Mean No. of

colonies/plate (average of 2 plates)

91

334

1044

220

898

THF = tetrahydrofuran

AF-2 = 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide

NaN3= sodium azide

9AA = 9-aminoacridine

B[α]P = benzo[α]pyrene

2AA = 2-aminonathracene

P = precipitate

T = toxic effect observed

Applicant's summary and conclusion