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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:
19-JAN-1995 (preliminary test-treatment) to 14-FEB-1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD guideline 471 (Bacterial Reverse Mutation Assay: Salmonella typhimurium) and OECD guideline 472 (Bacterial Reverse mutation Assay: Escherichia coli)
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
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): RHODIASTAB X2
- Substance type: no data
- Physical state: opaque dark amber liquid and gold solid
- Analytical purity: the analytical purity was the responsibility of the Sponsor
- Purity test date: the purity test date was the responsibility of the Sponsor
- Lot/batch No.: 9330912
- Expiration date of the lot/batch: no data
- Stability under test conditions: the stability under the storage conditions below was the responsibility of the Sponsor
- Storage condition of test material: the test substance was stored at ambient temperature in the Sponsor original container
- Other: the test substance was received at the facility on 30 December 1994.

Rhodiastab X2 is the previous name of the same test substance currently commercialised as Rhodiastab 92.

Method

Target gene:
Histidine auxotrophy in S. typhimurium
Tryptophan auxotrophy in E.coli
Species / strain
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvrA
Metabolic activation:
with and without
Metabolic activation system:
liver microsomal preparation from 1254-aroclor induced rat (S9-mix).
Test concentrations with justification for top dose:
Preliminary toxicity test (S. typhimurium TA 98 and E. coli WP2 uvrA in the absence of S9-mix):
- 0 (DMSO) and 0 (top-agar and culture alone),
- 2.5, 25, 250, 2500 µg/plate (first test),
- 5, 50, 500 5000 µg/plate (second test)

Main test:
- 0, 50, 158, 500, 1580, 5000 µg/plate with and without metabolic activation in all bacteria strains (two independent tests)
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO ( batch 04047HZ, Aldrich) for test substance dilution and all positive control compounds except sodium azide which was dissolved in purified water
- Justification for choice of solvent/vehicle: no data
- Volume of vehicle/solvent in the medium: 0.1 mL of DMSO (as vehicle negative control) or 0.1 mL of test substance concentrations (in DMSO)
Controls
Negative controls:
yes
Solvent controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: see "any other information on materials and methods"
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: no
- Exposure duration: 48 hours at 37°C

SELECTION AGENT (mutation assays): histidine auxotrophy for S. typhymurium strains and tryptophan auxotrophy for E. coli

NUMBER OF REPLICATIONS: triplicates


DETERMINATION OF CYTOTOXICITY
- Method: observation of the visible thinning of the background lawn of non-revertant cells in following exposure to test substance


OTHER: no data
Evaluation criteria:
No data
Statistics:
No data

Results and discussion

Test results
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS: no data



RANGE-FINDING/SCREENING STUDIES: no visible thinning of the background lawn of non-revertant cells was obtained following exposure to RHODIASTAB X2 up to the highest tested concentrations in the preliminary range-finding test. Therefore,a top exposure level of 5000 µg/plate was selected for use in the main test.


COMPARISON WITH HISTORICAL CONTROL DATA: no


ADDITIONAL INFORMATION ON CYTOTOXICITY: no

Any other information on results incl. tables

Table 7.6.1/1: Number of revertants per plate (mean of triplicates) in the absence of metabolic activation (test 1)

Conc.
(µg/plate)

TA 98

TA 100

TA 1535

TA 1537

E. coli WP2 uvrA/pKM101

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

0*

23

1

114

5

16

1

17

4

45

8

50**

23

3

93

6

17

4

15

2

38

12

158**

21

5

99

2

16

1

16

2

48

1

500**

19

3

111

1

15

5

16

4

38

7

1580**

20

2

111

23

17

4

13

2

33

8

5000**

21

6

117

22

14

1

15

2

32

4

Positive controls***

143

10

851

47

561

45

588

66

846

47

34

6

110

10

24

4

15

2

42

3

Table 7.6.1/2: Number of revertants per plate (mean of triplicates) in the absence of metabolic activation (test 2)

Conc.
(µg/plate)

TA 98

TA 100

TA 1535

TA 1537

E. coli WP2 uvrA/pKM101

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

0*

28

2

98

10

16

4

12

3

50

7

50**

26

9

106

9

12

3

9

2

45

6

158**

31

0

90

6

11

2

9

1

42

0

500**

27

5

104

11

9

3

12

2

45

6

1580**

21

7

86

7

12

5

11

1

42

11

5000**

21

8

74

5

9

3

13

2

29

6

Positive controls***

123

6

834

72

502

14

540

11

698

19

22

4

104

5

9

1

14

2

53

3

* Solvent control = negative control: 100 µL DMSO/plate

** Test substance solution volume: 100 µL/plate

*** Mutagens positive controls:

-Sodium azide (2 µg/plate) in TA1535 and TA 100 strains

-2-Aminoanthracene in TA1535 (2 µg/plate) and WP2 uvrA (10 µg/plate)

-9-aminoacridine (80 µg/plate) in TA1537 strain

-2-nitrofluorene (1 µg/plate) in TA 98 strain

-Benzo[a]pyrene (5 µg/plate) in TA1537, TA 100, TA 98 strains

-N-Ethyl-N’-Nitro-N-nitrosoguanidine (2 µg/plate) in WP2 uvrA

Table 7.6.1/3: Number of revertants per plate (mean of triplicates) in the presence of metabolic activation (test 1)

Conc.
(µg/plate)

TA 98

TA 100

TA 1535

TA 1537

E. coli WP2 uvrA/pKM101

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

0*

27

1

125

11

21

4

18

1

43

3

50**

21

6

104

3

20

1

19

3

47

4

158**

23

2

109

4

18

2

18

3

43

8

500**

21

4

80

10

17

1

14

5

46

2

1580**

21

1

103

23

17

1

13

1

56

9

5000**

24

4

95

11

18

2

17

4

41

13

Positive controls***

478

34

598

43

486

78

114

1

553

18

Table 7.6.1/4: Number of revertants per plate (mean of triplicates) in the presence of metabolic activation (test 2)

Conc.
(µg/plate)

TA 98

TA 100

TA 1535

TA 1537

E. coli WP2 uvrA/pKM101

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

0*

29

3

103

10

16

3

12

2

47

6

50**

33

7

113

16

11

4

12

3

52

7

158**

40

2

110

16

10

4

11

2

45

13

500**

29

2

100

12

8

2

11

1

49

7

1580**

35

7

89

9

11

7

8

3

51

4

5000**

24

2

82

3

7

1

8

1

50

2

Positive controls***

482

13

338

4

237

11

202

8

500

33

* Solvent control = negative control: 100 µL DMSO/plate

** Test substance solution volume: 100 µL/plate

*** Mutagens positive controls:

-2-Aminoanthracene in TA1535 (2 µg/plate) and WP2 uvrA (10 µg/plate)

-Benzo[a]pyrene (5 µg/plate) in TA1537, TA 100, TA 98 strains

Applicant's summary and conclusion

Conclusions:
There was no evidence of mutagenic activity in the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and Escherichia coli WP2 uvrA at any dose level of Rhodiastab X2 in the presence and the absence od S9 -mix. Hence, Rhodiastab X2 was not mutagenic in these bacterial test systems.
Executive summary:

The mutagenic potential of Rhodiastab X2 (former commercial name of the registered substance) was assessed in the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and Escherichia coli WP2 uvrA, according to OECD guidelines 471-472, Japan MITI/MHW and US/EPA-TSCA 798.5265 and in compliance with Good Laboratory Practices. Tests were carried out in presence and in absence of liver preparations from Aroclor 1254-induced rats (S9-mix). Rhodiastab X2 was dissolved in dimethyl sulphoxide (DMSO) and tested at concentrations up to 5000 µg/plate without S9 -mix in a range-finding preliminary test to determine dose-level cytotoxicity. In the absence of cytotoxicity at the highest RHODIASTAB X2 concentrations, dose levels used in the main assays were: 50, 158, 500, 1580 and 5000 µg/plate with and without metabolic activation system. Two independent tests were performed .

Under conditions of both tests, no evidence of mutagenic activity was observed at any dose level of Rhodiastab X2 in the presence and the absence od S9 -mix. Hence, Rhodiastab X2 was not mutagenic in these bacterial test systems.