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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:
3 May to 26 May, 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test method according to OECD 471, OECD 472; EU B.14, EU B.13. GLP study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): OS-2200 (489-95A).
- Physical state: Clear to light yellow liquid.
- Analytical purity: >92%.
- Lot/batch No.:38659-14.
- Expiration date of the lot/batch: 1 January 1996.
- Storage condition of test material: room temperature in the dark under nitrogen.

Method

Target gene:
Histidine or tryptophan gene.
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
other: E. coli WP2 uvrA trp
Metabolic activation:
with and without
Metabolic activation system:
liver preparations (S9 mix) from rats treated with Aroclor 1254
Test concentrations with justification for top dose:
0 (control), 321.5, 625, 1250, 2500 and 5000 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone.
- Justification for choice of solvent/vehicle: Test item was fully miscible in acetone.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
without S9 mix
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
TA 1535, TA 100, WP2 uvrA
Positive controls:
yes
Remarks:
without S9 mix
Positive control substance:
9-aminoacridine
Remarks:
TA 1537
Positive controls:
yes
Remarks:
without S9 mix
Positive control substance:
2-nitrofluorene
Remarks:
TA 98
Positive controls:
yes
Remarks:
with S9 mix
Positive control substance:
other: 2-aminoanthracene
Remarks:
All strains
Details on test system and experimental conditions:
METHOD OF APPLICATION: In agar (plate incorporation) (Main test and confirmation test)
An aliquot of 0.1 mL of a 10 hour bacterial culture and 0.5 mL S9 mix or 0.5 mL 0.1 M phosphate buffer (at pH 7.4) were placed in glass bottles. An aliquot of 0.1 mL of the test solution was added, followed immediately by 2 mL of histidine/tryptophan deficient agar. The mixture was thoroughly shaken and overlaid onto previously prepared petri dishes containing 25 mL minimal agar. Three petri dishes were used for each dose level. A set of plates were also prepared containing only bacterial culture and S-9 mix or phosphate buffer (0 µg/plate). Plates were also prepared without the addition of bacteria in order to assess the sterility of the test substance, S-9 mix and phosphate buffer. All plates were incubated at 37°C for 3 days. After this period revertant colonies were counted using a Seescan Automatic Colony Counter.

DURATION
-Exposure duration: 3 days at 37ºC.

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY: Reductions in revertant colony counts or by the absence of a complete background bacterial lawn.
Evaluation criteria:
The mean number of revertant colonies for all treatment groups is compared with those obtained for solvent control groups. The mutagenic activity of a test substance is assessed by applying the following criteria:
a)If treatment with a test substance produces an increase in revertant colony numbers of at least twice the concurrent solvent controls, with some evidence of a positive dose-relationship, in two separate experiments, with any bacterial strain either in the presence or absence of S-9 mix, it is considered to show evidence of mutagenic activity in this test system. No statistical analysis is performed.
b)If treatment with a test substance does not produce reproducible increases of at least 1.5 times the concurrent solvent controls, at any dose level with any bacterial strain, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.
-If the results obtained fail to satisfy the criteria for a clear "positive" or "negative" response given in paragraphs a and b, the following approach is taken in order to resolve the issue of the substance's mutagenic activity in this test system.
-Repeat tests may be performed using modifications of the experimental method. These modifications include (but are not restricted to), the use of a narrower dose range than that already tested; the use of different levels of liver homogenate S-9 fraction in the S-9 mix. Should an increase in revertant colony numbers be observed which satisfies paragraph a), the substance is considered to show evidence of mutagenic activity in this test system. No statistical analysis is performed.
-If no clear "positive" response can be obtained the test data may be subjected to analysis to determine the statistical significance of any observed increases in revertant colony numbers. The statistical procedures used will be those described by Mahon et al. (1989)
Statistics:
No statistical analysis.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: No toxicity was observed in the preliminary test study up to 5000 µg/plate test item. Therefore 5000µg/plate was chosen as the top dose level in the mutation tests.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Mean reverntan colonies*:

Test 1:

Material

Concentrat.

(µg/plate)

Without metabolic activation

With metabolic activation

Base pair exchange type

Frameshift type

Base pair exchange type

Frameshift type

TA100

TA 1535

WP2 uvrA

TA 98

TA 1537

TA100

TA 1535

WP2 uvrA

TA 98

TA 1537

Solvent control

 

121

17

70

24

13

121

16

69

23

14

OS-2200

5000.0

100

15

39

21

14

111

19

50

24

8

2500.0

117

14

58

22

13

131

13

64

26

11

1250.0

118

14

62

19

9

101

14

62

25

11

625.0

132

11

75

23

11

112

15

66

27

11

312.5

120

14

61

20

10

119

13

63

26

12

Negative control

0.0

120

13

69

23

10

139

14

72

21

10

Positive control

EENG

355

210

745

-

-

-

-

-

-

-

NF

-

-

-

191

-

-

-

-

-

-

9 AC

-

-

-

-

X

-

-

-

-

-

AA

-

-

-

-

-

354

147

246

106

70

Test 2:

Material

Concentrat.

(µg/plate)

Without metabolic activation

With metabolic activation

Base pair exchange type

Frameshift type

Base pair exchange type

Frameshift type

TA 100#

TA1535#

WP2uvrA#

TA 98#

TA1537#

TA100#

TA1535#

WP2uvrA#

TA98#

TA1537#

Solvent control

 

108

12

66

26

10

120

14

75

24

8

OS-2200

5000.0

109

12

53

25

7

120

13

47

14

10

2500.0

115

11

67

20

8

107

14

81

23

6

1250.0

85

12

58

20

6

133

18

77

17

7

625.0

96

13

63

29

6

128

12

76

15

6

312.5

115

9

66

24

10

120

11

81

20

9

Negative control

0.0

112

9

68

27

8

129

21

90

15

5

Positive control

EENG

312 401$

200

567

-

-

-

-

-

-

-

NF

-

-

-

181

-

-

-

-

-

-

9 AC

-

-

-

-

X

-

-

-

-

-

AA

-

-

-

-

-

205

109

311

77

50

X Too many colonies to count accurately
ENNG N-Ethyl-N’-nitro-N-nitrosoguanidine
9 AC 9-Aminoacridine
NF 2-Nitrofluorene
AA 2-Aminoanthracene
* Values are the mean of 3 plates
# S-9 data obtained from a separate experiment due to a contamination
$ Additional control data obtained from the separate S-9 mix experiment

No substantial increases in revertant colony numbers of any of the tester strains were observed following treatment with OS-2200 (489-95A) at any dose level, in the presence or absence of S-9 mix, in either mutation test.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

OS-2200 when tested in acetone shows no evidence of mutagenic activity in the described bacterial system.
Executive summary:

An in-vitro bacterial reverse assay (Ames test) was performed on OS-2200 in accordance with OECD Guideline 471, 472 and EU methods B.13/14 with histidine dependent auxotrophic mutants of Salmonella typhimurium (strains TA 1535, TA 1537, TA 98 and TA 100) and a tryptophan dependent mutant of Escherichia coli (WP2 uvrA). In the preliminary toxicity test with dose levels of up to 5000 µg/plate no toxicity was observed. Therefore, the dose levels tested in the main test were 5000, 2500, 1250, 625, 312.5 µg/plate. Two independent mutation tests were performed, in the presence md absence of liver preparations from Aroclor 1254-induced rats. Negative controls, solvent controls and positive controls were included in each test. No signs of toxicity were observed towards the tester strains in either mutation tests following exposure to OS-2200 . No evidence of mutagenic activity was seen at any concentration of OS-2200 in either mutation test. The concurrent positive control compounds demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations. It was concluded that OS-2200 showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.