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Genetic toxicity: in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 September 2005 to 29 September 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: Yakusyoku No.1121002 of the Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: H15.11.13 seikyoku No.2 of the Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: kanpoki No.031121002 of the Environmental Policy Bureau, Ministry of the Environment
Version / remarks:
November 21, 2003
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Notification of Ministry of the Labour, Japan, No.77, September 1, 1988 and No.67 (revised), June 2, 1997.
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction product of salicylaldehyde and formaldehyde and phenol
EC Number:
632-556-0
Cas Number:
304859-44-7
Molecular formula:
C6H5O(C13H10O2)m(C7H6O)nH values on m and n are variable as this is a UVCB substance
IUPAC Name:
Reaction product of salicylaldehyde and formaldehyde and phenol
Test material form:
solid
Details on test material:
- Appearance at ordinary temperature: Red-brownish solid

Method

Target gene:
S. typhimurium: Histidine locus
E. coli: Tryptophan locus
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Dr. T. Matsushima, Japan Bioassay Research Center, Japan Industrial Safety and Association, Hadano-City, Kanagawa.
- Strains were stored at -80 °C
- Prior to the master strains being used, characterisation checks were carried out to confirm the amino-acid requirement, presence of rfa, R factors, uvrB or uvrA mutation and the spontaneous reversion rate.
- In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth and incubated at 37 C for 10 hours. Each culture was monitored spectrophotometrically for turbidity with titters determined by viable count analysis on nutrient agar plates.
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Dr. T. Matsushima, Japan Bioassay Research Center, Japan Industrial Safety and Association, Hadano-City, Kanagawa.
- Strains were stored at -80 °C
- Prior to the master strains being used, characterisation checks were carried out to confirm the amino-acid requirement, presence of rfa, R factors, uvrB or uvrA mutation and the spontaneous reversion rate.
- In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth and incubated at 37 C for 10 hours. Each culture was monitored spectrophotometrically for turbidity with titters determined by viable count analysis on nutrient agar plates.
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Dose-Determination test: 4.88, 19.5, 78.1, 313, 1250 and 5000 µg/plate
Mutagenicity tests: 9.77, 19.5, 39.1, 78.1, 156 and 313 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test material was not dissolved in water at 50 mg/mL, but was dissolved in DMSO at 100 mg/mL in solubility checks performed in-house. DMSO was therefore selected as the vehicle of choice.
- The test material was dissolved in DMSO to make a stock solution of 100 mg/mL and further diluted to obtain desired concentrations.
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: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (0.01 µg/ 50 µL/ plate for TA100 and WP2uvrA (-S9 mix), and 0.1 µg/ 50 µL/ plate for TA98 (-S9 mix))
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 minutes
- Measured aliquots (0.05 mL) of the test material formulation, vehicle or positive control were dispensed into sets of test tubes followed by either 0.5 mL of S9 mix or phosphate buffer, 0.1 mL of one of the bacterial cultures. The contents of each test tube were incubated at 37 °C for 20 min. and mixed with 2.0 mL of molten, trace histidine and biotin or tryptophan supplemented, top agar and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in duplicate, for each bacterial strain and for each concentration of test material both with and without S9 mix.
- Exposure duration: 48 hours at 37 °C

EVALUATION
- The frequency of revertant colonies was assessed using a colony analyser CA-11S (System Science Co., Ltd.).

OTHER
- Dose-Determination test: Six concentrations of the test material (4.88, 19.5, 78.1, 313, 1250 and 5000 µg/plate) were assayed in duplicate against each tester strain, using the pre-incubation method.
Evaluation criteria:
- The response was regarded positive if the maximum number of revertant colonies in the test material group increased ≥ 2 fold relative to the negative control group and dose-response and reproducibility were confirmed.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium, other: TA 1535, TA 1537 TA 98 & TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
- The test material did not induce ≥ 2 fold increase in the number of revertant colonies relative to the negative control value for any strain with or without metabolic activation, and the reproducibility of the results was confirmed. From these results, the test material was judged negative for the potential to induce gene mutation (mutagenicity).
- Cytotoxicity were observed at and above 313 µg/plate with or without metabolic activation on dose-determination test, at and above 156 µg/plate without metabolic activation, at and above 313 µg/plate with metabolic activation on mutagenicity test 1 and 2. No precipitate of the test material was observed at any dose level. In the sterility test, no growth of bacteria was observed under any test conditions.
- All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9 mix and the sensitivity of the bacterial strains.

Any other information on results incl. tables

Table 1: Summary of Mutagenicity Test 1

± S9 Mix

Concentration

(µg/plate)

Mean number of colonies/plate

Base-pair Substitution Type

Frameshift Type

TA100

TA1535

WP2uvrA

TA98

TA1537

-

Solvent

9.77

19.5

39.1

78.1

156

313

110

93

102

100

107

81*

65*

10

6

8

11

10

8*

0*

34

28

27

31

28

28*

0*

14

14

17

18

16

8*

0*

9

11

9

10

6

0*

0*

+

Solvent

9.77

19.5

39.1

78.1

156

313

114

128

115

115

118

108

102*

11

9

10

8

12

11

6*

31

32

26

33

29

37

39*

25

24

27

26

30

26

25*

10

11

12

11

14

16

9*

Positive Controls

-

Name

AF-2

SA

AF-2

AF-2

9AA

Concentration (µg/plate)

0.01

0.5

0.01

0.1

80

Mean no. colonies/plate

639

261

224

513

359

+

Name

2AA

2AA

2AA

2AA

2AA

Concentration (µg/plate)

1

2

10

0.5

2

Mean no. colonies/plate

1293

159

725

537

194

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

9AA = 9-aminoacridine

2AA = 2-aminoanthracene

SA = Sodium azide

When toxicity was observed, "*" was placed to the right of the number of the revertants.

Table 2: Summary of Mutagenicity Test 2

± S9 Mix

Concentration

(µg/plate)

Mean number of colonies/plate

Base-pair Substitution Type

Frameshift Type

TA100

TA1535

WP2uvrA

TA98

TA1537

-

Solvent

9.77

19.5

39.1

78.1

156

313

122

123

96

109

113

74*

0*

9

7

8

10

6

6*

0*

26

35

32

28

23

31*

0*

12

15

15

12

11

10*

0*

9

7

7

7

8

0*

0*

+

Solvent

9.77

19.5

39.1

78.1

156

313

115

127

136

124

113

129

77*

9

10

8

10

8

8

6*

29

29

26

35

34

38

25*

24

22

28

28

32

24

20*

13

13

13

9

11

13

9*

Positive Controls

-

Name

AF-2

SA

AF-2

AF-2

9AA

Concentration (µg/plate)

0.01

0.5

0.01

0.1

80

Mean no. colonies/plate

627

314

229

520

331

+

Name

2AA

2AA

2AA

2AA

2AA

Concentration (µg/plate)

1

2

10

0.5

2

Mean no. colonies/plate

1317

210

719

576

211

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

9AA = 9-aminoacridine

2AA = 2-aminoanthracene

SA = Sodium azide

When toxicity was observed, "*" was placed to the right of the number of the revertants.

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the test material was judged negative for the potential to induce gene mutation (mutagenicity).
Executive summary:

The genetic toxicity of the test material was investigated in accordance with Japanese guidelines under GLP conditions. The mutagenic potential was assessed with a bacterial reverse mutation assay.

Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and Escherichia coli WP2uvrA were treated with the test material using the pre-incubation method at six dose levels, in duplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the dose-determination test was 4.88 to 5000 µg/plate. The experiment was repeated on a separate day using the dose range, 9.77 to 313 µg/plate, fresh cultures of the bacterial strains and fresh test substance formulations.

Cytotoxicity was observed at and above 313 µg/plate with or without metabolic activation on dose-determination test, at and above 156 µg/plate without metabolic activation, at and above 313 µg/plate with metabolic activation on mutagenicity test 1 and mutagenicity test 2. No precipitate of the test material was observed at any dose level.

No significant increases in the frequency of revertant colonies were recorded for any strain with or without metabolic activation.

Under the conditions of this study, the test material was judged negative for the potential to induce gene mutation (mutagenicity).