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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, unpublished report available, no restrictions, fully adequate for assessment

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Test substance/item: Sulphur 98.5% DP
Common name (active ingredient): Sulfur
Chemical name (IUPAC): Sulfur
Code by test facility: 115/1-SLR98.5DP
CAS no.: 7704-34-9
Batch no./ Lot no.: I-GLA
Batch produced by: SAPEC Agro S.A.; Rua Victor Cordon, 19; 1200-482 LISBOA Portugal
Supplier: ChemService srl Via F.lli Beltrami 15; 20026 Novate Milanese (MI) - Italy
Date of manufacture: 09/07/2004
Date of expiry: 09/07/2006
Receipt at test facility: The test item was received in good condition
Purity to be stated in report: 98.5% w/w
Bulk density: 0.84 g/ml
Storage conditions: ambient (+18 to +36°C)

Method

Target gene:
histidine and tryptopahn
Species / strain
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
S-9 homogenate of the liver of rats treated with Aroclor 1254
Test concentrations with justification for top dose:
first trial: 50, 158, 500, 1581 and 5000 µg/plate
second trial: 100, 266, 707, 1880 and 5000 µg/plate
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene; 9-aminoacridine; 2-nitrofluorene; 4-nitroquinoline-1-oxide; sodium azide
Details on test system and experimental conditions:
Preliminary Toxicity Test
One hundred microlitres each of the respective dilutions and the stock equivalent to a concentration of 16, 32, 64, 128, 256, 512, 1024, 2048 and 5000 µg was mixed with 2 ml of soft agar containing Histidine and Biotin, 500 µl of S-9 mix (for the test in the presence of metabolic activation) or 500 µl of PBS (for the test in the absence of metabolic activation), 0.1 ml of overnight TA 100 culture and overlaid onto pre-labeled VB agar plates in duplicate. A DMSO control, similarly treated, was maintained. After the agar had set, these plates were incubated at 37°C for 48 hours.
The number of revertant colonies on the VB agar plates was counted and the bacterial background lawn was observed. Toxicity was detectable as a decrease in the number of revertant colonies per plate and/or by a thinning or disappearance of the bacterial background lawn. If the test item is toxic, the highest concentration of the test item used in the subsequent mutagenicity assay will be that which gives a detectable reduction in the number of revertants on the selective agar plates and/or a thinning or disappearance of the bacterial background lawn.

Mutation Test
No. of Replicates: 3
The bacterial suspension of each tester strain was diluted up to 10-6 (six serial dilution) dilution in phosphate buffered saline. After dilution, 0.1 ml from the highest dilution of each tester strain was plated onto nutrient agar plates in triplicate. The plates were incubated at 37°C for 48 hours for both the trials mutation test. After incubation, the number of colonies in each plate were counted and colony forming units per ml of the suspension.

OBSERVATIONS
Effect on Bacterial Background Lawn: The condition of the bacterial background lawn was evaluated for evidence of the test item toxicity using the code system.
Number of Revertants: Revertant colonies for all given strains, for all groups were counted manually.
Viable Counts: Viable counts for the different bacterial strains on nutrient agar plates were counted manually.
Evaluation criteria:
Conditions necessary for determining a positive result are: there should be a concentration related increase over the range tested and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain, either in the presence or absence of the metabolic activation system.

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeds the threshold level of twice [strains TA 98, TA100 and WP2 uvrA (pKM 101)], or thrice (strains TA 1535 and TA 1537) the colony count when compared to the corresponding vehicle control and this should be evident at a minimum of three dose levels.
Statistics:
Data were analyzed for differences among vehicle control, treatment and positive control groups using ANOVA. Differences between individual treatment and vehicle control was tested by Dunnet's 't' test at a 5% level (p < 0.05) of significance.

Results and discussion

Test results
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
but tested up to the limit concentration
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
PRELIMINARY TOXICITY TEST
The number of revertant colonies was comparable to that of the respective vehicle control plates up to the highest tested concentration of 5000 µg/plate, both in the presence and absence of metabolic activation. Similarly, the intensity of the bacterial background lawn was comparable to that of the vehicle control plates up to 5000 µg/plate, both in the presence and absence of metabolic activation.

MUTATION TEST
Trial 1 was conducted using 5000 µg/plate as the maximum concentration, both in the presence and in the absence of metabolic activation. Since, the results of the Trial 1 were negative, a confirmatory trial (Trial 2) was conducted by modifying the method of exposure to treatment and also the concentration spacing of the test item up to a maximum of 5000 µg/plate, both in the presence and in the absence of metabolic activation.
Viable Counts:
The viable counts determined for all the tester strains were within the required range of 1 - 2 x 10E9 CFU/ml, in the first as well as the confirmatory trial.
Number of Revertants:
TRIAL 1: The mean number of revertant colonies/plate in the DMSO control was within the range of in-house spontaneous revertant counts for all the tester strains. For all the tester strains, the mean number of revertant colonies was statistically comparable to or lesser than that of the vehicle control at all the tested concentrations, both in the presence and absence of metabolic activation. The intensity of the bacterial background lawn was comparable to that of the vehicle control plates up to 5000 µg/plate, both in the presence and absence of metabolic activation. The specific positive control chemicals tested simultaneously produced a significantly high increase in the mean revertant colonies compared to the vehicle control plates.
TRIAL 2: The mean number of revertant colonies/plate in the DMSO control was within the range of in-house spontaneous revertant counts for all the tester strains. For all the tester strains, the mean number of revertant colonies was statistically comparable to or lesser than that of the vehicle control at all the tested concentrations, both in the presence and absence of metabolic activation. The intensity of the bacterial background lawn was comparable to that of the vehicle control plates up to 5000 µg/plate both in the presence and absence of metabolic activation. The specific positive control chemicals tested simultaneously produced a significantly high increase in the mean revertant colonies compared to the vehicle control plates.

Mean number of Revertant Colonies: The means and standard deviations of the two trials indicated no doubling of mean numbers of revertant colonies in strains TA98, TA100 and WP2 uvrA (pKM 101) or tripling of mean numbers of revertant colonies in strains TA1535 and TA1537 in any of the five concentrations tested as compared to the respective vehicle controls, presence or absence of metabolic activation.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Summary of Bacterial Reverse Mutation Test

 

Group

Test Item concentration (µg/plate)

No. of revertants/plate*

Presence of Metabolic activation

TA98

TA100

TA1535

TA1537

WP2 uvrA (pKM 101)

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

G1

Vehicle control – DMSO

19

1

113

5

16

1

16

0

102

6

G2

50

18

2

111

4

16

1

16

1

100

3

G3

158

18

1

105

5

15

0

15

1

98

5

G4

500

18

2

136

2

14-

1

14

1

95

2

G5

1581

17

2

93-

3

15

0

13-

2

87-

7

G6

5000

15-

1

90-

3

12-

1

11-

1

83-

7

G7

Positive control

615+

24

805+

37

118+

6

127+

13

563+

34

*: Values are means of three replicates                                           +/-: Significantly higher/lower than the vehicle control

 

Group

Test Item concentration (µg/plate)

No. of revertants/plate*

Absence of Metabolic activation

TA98

TA100

TA1535

TA1537

WP2 uvrA (pKM 101)

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

G1

Vehicle control – DMSO

18

1

109

3

15

1

15

1

98

7

G2

50

18

1

108

3

15

1

15

1

96

3

G3

158

17

1

103

3

14

2

14

0

97

5

G4

500

15

2

102

2

15

1

14

1

87

4

G5

1581

14-

2

98-

5

13

1

11-

2

85-

5

G6

5000

11-

2

83-

7

12-

1

8-

1

82-

4

G7

Positive control

172+

16

578+

38

139+

11

132+

9

589+

37

*: Values are means of three replicates                                           +/-: Significantly higher/lower than the vehicle control

 

 

Group

Test Item concentration (µg/plate)

No. of revertants/plate*

Presence of Metabolic activation

TA98

TA100

TA1535

TA1537

WP2 uvrA (pKM 101)

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

G1

Vehicle control – DMSO

17

1

109

3

15

2

13

2

98

6

G2

100

17

2

107

4

15

1

14

1

96

4

G3

266

17

1

104

9

15

1

14

2

94

2

G4

707

16

2

97

5

14

2

13

2

94

5

G5

1880

16

1

95

7

13

2

12

1

88

3

G6

5000

13-

2

85-

4

11

1

11

1

80-

7

G7

Positive control

+597

47

+776

24

+114

9

+138

18

+571

25

*: Values are means of three replicates                                           +/-: Significantly higher/lower than the vehicle control

 

Group

Test Item concentration (µg/plate)

No. of revertants/plate*

Absence of Metabolic activation

TA98

TA100

TA1535

TA1537

WP2 uvrA (pKM 101)

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

G1

Vehicle control – DMSO

17

1

105

6

14

1

14

2

99

5

G2

100

17

1

103

1

15

1

15

1

93

5

G3

266

14

2

102

4

15

1

13

2

90

4

G4

707

15

2

94-

3

13

1

12

2

84-

3

G5

1880

13-

2

89-

3

12-

1

11

1

81-

5

G6

5000

10-

1

80-

6

10-

1

8-

1

77-

2

G7

Positive control

160 +

14

565 +

48

135 +

8

129 +

10

590+

38

*: Values are means of three replicates                                           +/-: Significantly higher/lower than the vehicle control

Applicant's summary and conclusion

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

Sulfur dust was not mutagenic in the Ames test up to the highest tested concentration of 5000 µg/plate.
Executive summary:

The genotoxic effect of sulfur dust was studied using the Ames test. The study, according to OECD guideline 471 and under GLP, was conducted using TA98, TA100, TA1535 and TA1537 strains of Salmonella typhimurium and WP2uvrA (pKM 101) strain of Escherichia coll. Two trials were carried out (with two experiments in each trial, in the presence and in the absence of metabolic activation). The test item was tested in triplicate at the concentrations of 50, 158, 500, 1581 and 5000 µg/plate in the first trial and 100, 266, 707, 1880 and 5000 µg/plate in the second trial using DMSO as vehicle. The vehicle control and the appropriate positive controls were tested simultaneously. The mean numbers of revertant colonies for the different concentrations of the test item in the different tester strains was statistically comparable with or lower than those of the respective vehicle control plates, for both the first and the confirmatory trials, either in the presence or in the absence of the metabolic activation, while there was a statistically significant increase in the mean number of revertant colonies in the positive controls under identical conditions.

The study indicated that the test item sulfur dust is not mutagenic in this Ames test up to the highest tested concentration of 5000 µg/plate.