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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
Study period:
1986
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Minor restrictions in design and/or reporting but otherwise adequate for assessment

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Unnamed
Year:
1986
Reference Type:
publication
Title:
Unnamed
Year:
1982

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
Salmonella Mutation Test (Ames) according to Haworth, S. et al.: Environ.Mutagen. 5, Suppl. 1, 3-142
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Purity 99%
Supplier: Strem Chemicals

Method

Target gene:
TA1537 hisC3076
TA1535 hisG46
TA100 hisG46
TA98 hisD3052
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 liver fraction from rats and hamsters treated with AROCLOR 1254
Test concentrations with justification for top dose:
100, 333, 1000, 3333, 10000 µg/plate
Vehicle / solvent:
water
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Sodium azide for TA1535 and TA100, 4-nitro-0-phenylenediamine for TA98, and 9-aminoacridine for TA1537 were used in assays without metabolic activation; 2-aminoanthracene was used in all assays as positive control with metabolic activation
Details on test system and experimental conditions:
All chemicals were assayed for mutagenicity in the preincubation assay. To each of 13 x 100-mm test tubes maintained at 37°C were added in the following order: 0.5 ml of S-9 mix or 0.1 M PO4 buffer (pH 7.4), 0.05 ml of the overnight culture, and 0.05 ml of solvent of chemical dilution. The mixture was mixed and allowed to incubate without shaking at 37°C for 20 minute, at which time 2.5 ml or 2.0 ml of molten (45°C) top agar supplemented with 0.5 mM L-histidine and 0.5 mM D-biotin were added. The contents of the tubes were mixed and poured onto 25 ml. of minimal glucose bottom agar in 15 x 100-mm plastic Petri dishes and Fisher Scientific plates. When the top agar had solidified, the plates were inverted an incubated at 37°C for 48 hr.

Concurrent solvent and positive controls were tested with and without the metabolic activation systems. At least five dose levels of the chemicals were tested, with three plates per dose level. All assays were repeated no less than 1 wk after the completion of the initial test.
Evaluation criteria:
1) mutagenic response: a dose-related, reproducible increase in the number of revertants over background even if the increase was less than
twofold, 2) nonmutagenic response: when no increase in the number of revertantts was elicited by the chemical and 3) questionable response: when there was an absence of a clear-cut dose-related increase in the number of revertants, when the dose-related increases in the number of revertants were not reproducible or when the response was of insufficient magnitude to support a determination of mutagenicity
Statistics:
Mean and Standard Error of the Mean was calculated.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
When tested at the highest concentration of 10000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
At the highest dose tested there was a slight to complete clearing of the lawn indicating cytotoxicity at 10,000 ug/plate
Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

At the highest dose tested there was a slight to complete clearing of the lawn indicating cytotoxicity at 10,000 ug/plate. DIPA was not mutagenic in any of the strains of S. Typhimurium tested up to 5,000 ug/plate with or without metabolic activation.

Strain TA1535:

Dose

No Activation
(Negative)

No Activation
(Negative)

No Activation
(Negative)

10% HLI
(Equivocal)

10% HLI
(Negative)

10% RLI
(Equivocal)

10% RLI
(Negative)

Dose units

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

0         

64

4.5

24

1.2

27

2.3

9

2.1

11

0.7

11

2.1

14

4.6

100         

51

8.1

22

2.4

27

5.5

9

2

11

1.5

9

1.3

10

1.5

333         

39

4.4

24

1

25

2.6

12

1.2

7

1.9

10

1

8

0.9

1000         

50

4.7

27

4.7

27

0.9

11

0.3

11

0.9

12

3.4

12

2.2

3333         

36

4.7

18

1.9

28

2.4

15

0.7

9

0.9

12

2.3

10

1.9

10000         

28S

4.3

12S

1.2

7S

2.9

24S

2.2

15S

0.6

22S

2.6

14S

1.5

Positive Control

1287

27.7

1228

29.6

736

47.3

160

15

161

1.9

143

10.3

151

7.1

Strain TA100:

Dose

No Activation
(Negative)

No Activation
(Negative)

10% HLI
(Negative)

10% HLI
(Negative)

10% RLI
(Negative)

10% RLI
(Negative)

Dose units

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

0         

162

9.8

128

2.6

113

6

123

5.6

115

8.7

109

8.9

100         

169

13

120

7.8

86

6.4

121

7.5

97

2.2

123

4.1

333         

180

7.5

125

5.1

100

5

119

9.3

122

11

124

4.1

1000         

175

6.6

129

5.5

115

8.5

100

0.9

108

5

123

2.5

3333         

164

6.7

115

7.2

102

0.6

105

8

122

14

123

8.4

10000         

160S

5.6

112S

5.9

144S

12.1

127S

1.9

133S

7.3

128S

3.8

Positive Control

1453

7.6

1280

75.4

1198

46.4

1299

68.2

1821

72.7

567

58.9

Strain TA98:

Dose

No Activation
(Negative)

No Activation
(Negative)

10% HLI
(Negative)

10% HLI
(Negative)

10% RLI
(Negative)

10% RLI
(Negative)

Dose units

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

0         

17

3.4

18

2.8

30

3.5

27

3

25

2.9

29

0.9

100         

19

0.9

17

1.8

26

3.5

23

3.5

28

1.2

31

1.7

333         

20

2.1

16

2.1

26

2.7

27

0.3

33

3.6

29

1.5

1000         

19

2

18

3.2

25

0.9

25

2.4

29

2.5

27

1.7

3333         

25

1.5

14

0.9

23

3.2

32

2

24

2.6

29

2.1

10000         

18S

1.2

14S

1.3

29S

4.7

28S

2.1

23S

3.7

23S

3.5

Positive Control

1590

5.4

928

22.7

957

14.7

932

31.7

1221

43.7

866

14.9

Strain TA1537:

Dose

No Activation
(Negative)

No Activation
(Negative)

10% HLI
(Negative)

10% HLI
(Negative)

10% RLI
(Negative)

10% RLI
(Negative)

Dose units

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

0         

9

1.5

7

0.3

5

1.8

8

1.2

7

2.6

5

1.3

100         

6

1.2

4

0.7

7

1.5

5

0.9

8

1.2

5

2

333         

8

2

5

1

6

0.9

9

0.3

5

1.2

4

1.3

1000         

8

0.7

4

1.2

7

0.7

7

1.5

7C

1.5

8

1.2

3333         

6

0.9

6

3.6

8

3

9

1.7

7

1.2

7

2

10000         

7S

1.7

T

10S

0.9

5S

0.6

5S

1.9

7S

1

Positive Control

375

89.7

536

36.7

117

8.7

130

3.8

161

8.9

98

6.8

Abbreviations:
RLI = induced male Sprague Dawley rat liver S9
HLI = induced male Syrian hamster liver S9
s = Slight Toxicity; p = Precipitate; x = Slight Toxicity and Precipitate; T = Toxic; c = Contamination

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

DIPA was not mutagenic in any of the strains of S. Typhimurium tested up to 5,000 ug/plate.
Executive summary:

This publication includes data of Salmonella mutagenicity results on 270 coded chemicals, encompassing 329 tests performed by three laboratories under contract to the National Toxicology Program (NTP). The preincubation modification of the Salmonella mammalian microsome assay was used to test chemicals in up to five Salmonella strains in the presence and absence of rat and hamster liver S-9. With a few exceptions, inter- and intralaboratory reproducibility was good.

DIPA was not mutagenic in any of the strains of S. Typhimurium tested up to 5,000 ug/plate with or without metabolic activation. At higher concentrations inhibition of bacterial lawn growth was observed.