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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other: Study conducted in compliance with GLP and OECD guideline, available as unpublished report, no restrictions, fully adequate for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
gas under pressure: liquefied gas
Details on test material:
- Storage Conditions: Room temperature, upright in the dark without desiccant

Method

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:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
Preliminary toxicity assay: 1, 3, 6, 8, 11, 21 and 37 mmoles/L (24,500; 73,400; 147,000; 196,000, 269,000; 513,000 and 905,000 ppm, respectively)
Mutagenicity and confirmatory assay: 3, 6, 8, 11, 21 and 37 mmoles/L (73,400; 147,000; 196,000, 269,000; 513,000 and 905,000 ppm, respectively).
Confirmatory retest: 0.7, 1, 3, 6, 8, 11, 21 and 37 mmoles/L (17,200; 24,500; 73,400; 147,000; 196,000, 269,000; 513,000 and 905,000 ppm, respectively)
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene
Remarks:
With S9-mix: 2-aminoanthracene; Without S9-mix: 2-nitrofluorene (TA98), sodium azide (TA 100, TA 1535), 9-aminoacridine (TA 1537), methylmethanesulfonate (E.Coli)
Details on test system and experimental conditions:
METHOD OF APPLICATION: test system was exposed to the test article via the desiccator methodology, a modification of the plate incorporation methodology originally described by Ames et al. (1975) and updated by Maron and Ames (1983). This test system has been shown to detect a wide range of classes of chemical mutagens (McCann et al., 1975; McCann and Ames, 1976). The desiccator methodology has been shown to be an effective method for detecting the genotoxic activity of volatile and gaseous test articles (Wagner et al., 1992). Briefly, once agar had solidified, the plates were inverted in 9 liter dessicators and an appropriate quanitity of test material was introduced into each dessicator by withdrawing the appropriate amount of air and replacing with test material.

DURATION
- Original assay: following 24 hour exposure to test article, the plates were removed from the dessicators and allowed to inclubate at 37°C for an additional 24 hours.
- Confirmatory assay: exposure duration 48 hours in dessicator.

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth; number of revertants
Evaluation criteria:
- Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean vehicle control value. Data sets for tester strains TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0-times the mean vehicle control value.
- An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited.
- A response will be evaluated as negative, if it is neither positive nor equivocal.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
See table
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
- Preliminary Toxicity Assay: No background lawn toxicity was observed however a reduction in revertant counts was observed beginning at 11, 21 or at 37 mmoles/L with some test conditions.
- Mutagenicity Assay: No positive mutagenic responses were observed with any of the tester strains in the presence or absence of S9 activation. No precipitate was observed. Toxicity was observed beginning at 11, 21 or at 37 mmoles/L.
- Confirmatory mutagenicity assay: No positive mutagenic response was observed with tester strains TA100, TA1535 and WP2 uvrA in the absence of S9 activation and with tester strains TA100, TA1535 and TA1537 in the presence of S9 activation. No precipitate was observed. Toxicity was observed beginning at 21 or 37 mmoles/L. Tester strain TA98 in the presence and absence of S9 was not evaluated due to confluent growth but was retested. Due to excessive toxicity, as a result of a drop in the revertant counts beginning at 8 mmoles/L, tester strain TA 1537 in the absence of S9 was retested. Due to an unacceptable vehicle control value, tester strain WP2 uvrA in the absence of S9 activation was not evaluated but was retested. In the retest of the confirmatory mutagenicity assay, no positive responses were observed with tester strains TA98 and TA1537 in the absence of S9 activation and with tester strains TA98 and WP2 uvrA in the presence of S9 activation. No precipitate was observed. Toxicity was observed beginning at 21 or 37 mmoles/L.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

 

 

-S9

 

 

+S9

 

 

Strain

Test conc

(mmol/L)

Average reverants original

Average revertants – confirmatory

Average reverants – original

Average revertants – confirmatory

 

 

 

 

 

 

TA98

Vehicle

35

30

24

12

 

0.7

NA

31

NA

9

 

1

NA

34

NA

9

 

3

27

29

15

10

 

6

28

24

25

9

 

8

22

27

18

11

 

11

19

22

13

10

 

21

4

15

3

10

 

37

0

0

0

0

 

Positive control

129

351

442

408

TA100

Vehicle

179

96

209

106

 

3

214

108

216

88

 

6

179

113

203

83

 

8

184

130

236

72

 

11

216

88

214

63

 

21

205

40

267

40

 

37

0

0

0

0

 

Positive control

 

772

 

407

TA1535

Vehicle

10

11

7

9

 

3

10

8

7

10

 

6

9

9

6

5

 

8

11

11

10

12

 

11

8

7

8

7

 

21

6

11

5

7

 

37

0

0

0

0

 

Positive control

293

666

73

140

TA1537

Vehicle

7

10

7

7

 

0.7

NA

6

NA

NA

 

1

NA

6

NA

NA

 

3

3

10

4

6

 

6

3

11

3

6

 

8

5

7

4

4

 

11

0

6

7

5

 

21

1

4

0

5

 

37

0

0

0

0

 

Positive control

992

633

36

473

WP2 uvrA

Vehicle

14

45

19

42

 

0.7

NA

NA

NA

39

 

1.0

NA

NA

NA

42

 

3

13

56

23

43

 

6

12

55

16

28

 

8

11

39

13

35

 

11

4

33

6

32

 

21

2

15

5

34

 

37

0

8

0

4

 

Positive control

168

372

181

198

Applicant's summary and conclusion

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

The results of the bacterial reverse mutation test using gas-phase exposure indicate that, under the conditions of this study, the test article did not cause a positive mutagenic response in either the presence or absence of Aroclor induced rat liver S9.
Executive summary:

In a reverse mutation assay, performed according to OECD Guideline 471 and GLP, salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test material using the desiccator methodology, a modification of the Ames plate incorporation methodology, in the presence and absence of S9 -mix. The assay was performed in three phases: a preliminary toxicity assay, a mutagenicity assay and a confirmatory mutagenicity assay. Based on the findings of the preliminary toxicity assay, the maximum dose plated in the confirmatory mutagenicity assay was 37 mmoles/L (905,000 ppm). In the mutagenicity and confirmation assay, no precipitation was observed and toxicity was observed beginning at 11, 21 or at 37 mmoles/L. In the mutagenicity and confirmation assay, no positive mutagenic response was observed. In conclusion, under the conditions of this study, the test article did not cause a positive mutagenic response in either the presence or absence of Aroclor-induced rat liver S9.