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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 Mar - 17 Apr 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Landesinstitut für Arbeitsschutz und Produktsicherheit, München, Germany
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
his operon
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and beta-naphthoflavone
Test concentrations with justification for top dose:
Pre-experiment: 3.16, 10, 31.6, 100, 316, 1000, 2500, 5000 µg/plate
Experiment 1 (standard plate test): 3.16, 10, 31.6, 100, 316, 1000, 2500, 5000 µg/plate
Experiment 2 (pre-incubation test): 3.16, 10, 31.6, 100, 316, 1000, 2500, 5000 µg/plate
Vehicle / solvent:
- Vehicle/solvent used: DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: without S9: sodium azide (TA 100, TA 1535); 4-nitro-o-phenylenediamine (TA 98; TA 1537); methylmethanesulfonate (TA 102); with S9: 2-aminoanthracene (all strains without TA102; TA 102)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: diminution of the background lawn or a reduction in the number of revertants

METABOLIC ACTIVATION SYSTEM:
The quality of the S9 homogenate was routinely tested in S. typhimurium using 2-aminoanthracene and benzo[a]pyrene
Evaluation criteria:
A test item is considered mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
Biologically relevant means:
- number of reversion is at least twice the control (TA 98, TA 100, TA 102)
- number of reversion is at least thrice the control (TA 1535, TA 1537)
Statistics:
Mean and standard deviation (no. of revertant colonies)

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no precipitation was observed in any tester strain

RANGE-FINDING/SCREENING STUDIES:
No toxicity was observed in a range-finding assay (plate incorporation), tested up to 5000 µg/plate in TA 98 and TA 100.

COMPARISON WITH HISTORICAL CONTROL DATA:
The data were in accordance the historical control data.

Any other information on results incl. tables

Table 1: Test results of experiment 1 (standard plate test).

EXPERIMENT 1 (Standard Plate Test, SPT)

S9-Mix

Without

 

Test item (µg/plate)

TA 100

TA 1535

TA 98

TA 1537

TA 102

Water

142±34.9

32±2.5

28±2.5

8±4.0

270±20.0

Control (DMSO)

97±15.1

16±1.5

25±5.2

11±4.0

227±9.5

31.6

108±12.9

24±6.0

31±2.1

10±3.6

196±16.3

100

107±9.1

22±4.0

25±3.8

8±2.6

212±11.1

316

113±16.5

20±1.2

29±3.1

8±3.5

217±9.8

1000

103±3.2

19±3.8

21±8.7

10±3.2

234±19.5

2500

111±19.7

21±2.1

18±5.1

7±2.1

219±9.1

5000

103±11.0

16±1.7

24±6.1

10±3.2

225±4.9

NOPD (10 µg)

-

-

424±34.8

86±10.5

-

NaN3 (10 µg)

1006±78.6

962±52.5

-

-

-

MMS (1 µL)

-

-

-

-

946±306.0

 

 

 

 

 

 

S9-Mix

With

 

 

 

 

 

 

Test item (µg/plate)

TA 100

TA 1535

TA 98

TA 1537

TA 102

Water

129±12.5

25±1.5

34±2.1

13±2.6

309±31.2

NC (DMSO)

123±3.6

26±2.3

22±9.6

8±1.7

259±11.0

31.6

123±5.5

24±6.4

30±4.6

8±1.7

263±39.7

100

120±7.9

26±1.7

27±2.9

10±5.5

277±13.2

316

117±18.9

20±4.6

31±4.7

11±1.7

249±20.5

1000

119±9.1

23±2.6

27±4.6

10±2.3

241±21.8

2500

118±19.6

28±2.6

28±3.6

11±2.1

279±10.1

5000

108±0.7

20±4.0

33±10.6

8±3.8

275±13.3

2-AA (2.5 µg)

1808±119.8

166±17.5

3160±201.7

375±11.0

547±68.7

 

Table 2: Test results of experiment 2 (preincubation assay).

EXPERIMENT 2 (preincubation assay)

S9-Mix

Without

 

Test item (µg/plate)

TA 100

TA 1535

TA 98

TA 1537

TA 102

Water

135±7.6

16±2.5

26±9.0

6±2.9

287±14.9

Control (DMSO)

126±9.5

12±3.2

18±1.0

5±2.3

221±2.5

31.6

92±6.7

17±3.1

21±3.5

8±3.5

238±14.2

100

116±25.4

13±3.2

19±6.1

9±3.5

254±4.9

316

98±12.1

12±4.7

21±1.5

5±1.5

229±12.2

1000

100±12.5

12±1.5

23±6.0

6±1.2

248±13.7

2500

101±5.1

14±5.1

22±4.0

5±5.1

258±33.2

5000

100±14.2

16±2.9

22±2.9

5±2.9

255±28.0

NOPD (10 µg)

-

-

470±35.9

61±30.3

-

NaN3 (10 µg)

1065±64.5

1152±105.1

-

-

-

MMS (1 µL)

-

-

-

-

1851±10.4

S9-Mix

With

 

 

 

 

 

 

Test item (µg/plate)

TA 100

TA 1535

TA 98

TA 1537

TA 102

Water

166±6.7

11±4.6

23±1.5

11±1.7

412±11.6

NC (DMSO)

130±0.0

10±1.2

32±2.0

5±3.1

323±6.1

31.6

146±13.7

14±4.6

33±4.5

5±2.0

296±15.0

100

146±7.8

11±3.6

28±7.8

5±1.0

303±44.8

316

121±20.6

17±4.6

25±5.7

4±0.6

310±12.1

1000

132±4.7

12±3.5

25±2.5

7±0.0

298±17.7

2500

149±9.5

10±2.3

34±2.5

6±2.1

309±26.6

5000

139±3.5

10±4.9

20±2.9

6±2.1

319±42.2

2-AA (2.5 µg)

2208±69.0

194±17.5

1949±348.5

186±30.4

671±23.7

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
negative