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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:
06/11/1997-01/13/1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study done according to standard method.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guideline
Qualifier:
equivalent or similar 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

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: histidine and biotin requiring
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
other: histidine requiring
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Range finding: 8, 40, 200, 1000, 5000 µg/plate
Mutation Experiment 1 without metabolic activation: 0.5333, 2.67, 13.3, 66.7, 333.0 µg/plate
Mutation Experiment 1 with metabolic activation: 2.67, 8, 40, 200, 1000 µg/plate
Mutation Experiment 2 without metabolic activation: 31.25, 62.5, 125, 250, 500 µg/plate
Mutation Experiment 2 with metabolic activation, strains TA98 and TA100 initial treatments: 12.5, 25, 50, 100, 200 µg/plate
Mutation Experiment 2 with metabolic activation, strains TA1535, TA1537 and TA102 initial treatments: 62.5, 125, 250, 500, 1000 µg/plate
Mutation Experiment 2 with metabolic activation, strains TA1535 and TA102 repeat treatments: 7.8125, 15.625, 31.25, 62.5, 125, 250 µg/plate
Mutation Experiment 2 with metabolic activation, strain TA1535: 3.90625, 7.8125, 15.625, 31.25, 62.5, 125 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
other: not applicable
Positive controls:
yes
Positive control substance:
other: 2-nitrofluorene, sodium azide, 9-aminoacridine, gluteraldehyde, 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) - Platings were made by adding 0.1 ml bacterial culture, 0.1 ml test article solution, and 0.5 ml 10% S-9 mix or buffer to 2.5 ml molten agar at 46°C. The plating mixture was rapidly mixed and poured onto Minimal Davis agar plates. After setting, the plates were inverted and incubated for 3 days at 37°C in the dark. Plates were then examined for toxicity. In experiment 2, the test solution was reduced to 0.05 ml due to the toxicity of the solvent, DMSO.


DURATION
- Preincubation period: Experiment 2 - 1 hour
- Exposure duration: 3 days
- Expression time (cells in growth medium):
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):

NUMBER OF REPLICATIONS: 3

OTHER: Colonies were counted using a Seescan Colony Counter or manually when automatic counts could not be obtained.
Evaluation criteria:
Mean control counts fell within normal ranges.
Positive controls induced clear increases in revertant numbers.
No more than 5% of the plates lost through contamination.
Dunnett's test gave a significant response (p <= 0.01), and the data set showed a significant dose-correlation.
The positive responses were reproducible.
Statistics:
Individual plate counts were averaged. Dunnett's test was used to calculate significant responses.

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
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
200, 1000, 5000 µg/plate in TA100
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Mean revertant colonies (-S-9) - Experiment 1

Substance

Dose level (µg/plate)

TA98

TA100

TA1535

TA1537

TA102

Mean ± SD

Mean ± SD

Mean ± SD

Mean ± SD

Mean ± SD

DMSO

100 µl

28 ± 6

109 ± 6

19 ± 5

12 ± 3

271 ± 18

SHELLSOL

A

0.533

26 ± 6

101 ± 10

14 ± 2

10 ± 8

251 ± 10

2.67

31 ± 1

108 ± 10

14 ± 2

9 ± 3

255 ± 15

13.3

26 ± 6

98 ± 6

17 ± 1

10 ± 1

277 ± 4

66.7

24 ± 5

91 ± 14

18 ± 6

9 ± 2

214 ± 2

333 (V+Ppn)

21 ± 4

(V+Ppn)

86 ± 0

(V+Ppn)

13 ± 2

(V+Ppn)

6 ± 2

(S+Ppn)

184 ± 3

(S+Ppn)

Positive Controls

Compound

2NF

NaN3

NaN3

AAC

GLU

Dose Level

5 µg

2 µg

2 µg

50 µg

25 µg

Mean ± SD

754 ± 130

509 ± 29

312 ± 27

376 ± 27

435 ± 28

Mean Revertant Colonies (+S-9) - Experiment 1

Substance

Dose level (µg/plate)

TA98

TA100

TA1535

TA1537

TA102

Mean ± SD

Mean ± SD

Mean ± SD

Mean ± SD

Mean ± SD

DMSO

100 µl

32 ± 8

134 ± 9

17 ± 6

14 ± 5

318 ± 21

SHELLSOL

A

2.67

37 ± 5

143 ± 11

17 ± 7

11 ± 1

335 ± 45

8

41 ± 9

131 ± 7

21 ± 2

14 ± 3

315 ± 15

40

33 ± 6

133 ± 20

17 ± 3

15 ± 4

323 ± 12

200

34 ± 10 S

125 ± 2 S

23 ± 5

12 ± 2

275 ± 19

1000

31 ± 1 S

112 ± 6 S

15 ± 4 S

9 ± 2 S

252 ± 17 S

Positive Controls

Compound

AAN

AAN

-

-

-

Dose Level

5 µg

5 µg

-

-

-

Mean ± SD

925 ± 121

1226 ± 104

-

-

-

Mean Revertant Colonies (-S-9) - Experiment 2

Substance

Dose level (µg/plate)

TA98

TA100

TA1535

TA1537

TA102

Mean ± SD

Mean ± SD

Mean ± SD

Mean ± SD

Mean ± SD

DMSO

100 µl

29 ± 7

117 ± 7

22 ± 5

5 ± 3

331 ± 19

SHELLSOL

A

31.25

26 ± 6

131 ± 11

20 ± 7

10 ± 2

304 ± 6

62.5

30 ± 6

113 ± 7

20 ± 4

8 ± 2

276 ± 9

125

27 ± 5 S

113 ± 21 S

18 ± 3

10 ± 2

278 ± 28

250

16 ± 1 S

111 ± 19 S

16 ± 1 S

7 ± 2 S

180 ± 6 S

500

15 ± 2

M+Ppn+V

100 ± 19 M+Ppn+S/V

17 ± 6 M+S

 5 ± 2 S

199 ± 11 S/V

Positive Controls

Compound

2NF

NaN3

NaN3

AAC

GLU

Dose Level

5 µg

2 µg

2 µg

50 µg

25 µg

Mean ± SD

1004 ± 95

722 ± 15

438 ± 30

410 ± 112

595 ± 11

Mean Revertant Colonies (+S-9) Experiment 2

Substance

Dose level (µg/plate)

TA98

TA100

TA1535

TA1537

TA102

Mean ± SD

Mean ± SD

Mean ± SD

Mean ± SD

Mean ± SD

DMSO

100 µl

38 ± 4

140 ± 7

18 ± 5

7 ± 3

360 ± 19

SHELLSOL

A

3.90625

-

-

22 ± 2 M

-

-

7.8125

-

-

19 ± 2

10 ± 2

344 ± 13

12.5

36 ± 7

143 ± 9

-

-

-

15.625

-

-

12 ± 4 M

6 ± 3

323 ± 35

25

32 ± 9

133 ± 4

-

-

-

31.25

-

-

17 ± 5

9 ± 1

287 ± 66

50

34 ± 9

134 ± 3

-

-

-

62.5

-

-

17 ± 0

10 ± 1

307 ± 23

100

24 ± 3 S

133 ± 4 S

-

-

-

125

-

-

17 ± 5 S

4 ± 3 S

242 ± 15 S

200

26 ± 0 S

131 ± 4 S

-

-

-

250

-

-

-

5 ± 3 S

209 ± 27 S

Positive Controls

Compound

AAN

AAN

AAN

AAN

-

Dose Level

5 µg

5 µg

5 µg

5 µg

-

Mean ± SD

1109 ± 28

1045 ± 16

68 ± 3

63 ± 4

-

S - Slight thinning of background lawn

M - Plate counted manually

Ppn - Precipitation observed

V - Very thin background lawn

AAN - 2 -aminoanthracene

2NF - 2 -nitrofluorene

NaN3 - sodium azide

AAC - 9 -aminoacridine

GLU - gluteraldehyde

Summary of other bacterial mutagenicity studies.

End Point Study Reference  
REACH requirement IUCLID Section Study Name Data Waiving Waiving Justification Species Study Result Type Test Guideline/Qualifier Test Guideline/Guideline Test Guideline/Deviations Reliability Rational For Reliability GLP Compliance Test Materials/Identity Study Result Reference Type Reference Author Reference Year Reference Title Bibliographic Source Testing Laboratory Reference Report No. Owner Company Company Study No. Report Date Data access
8.4.1 In vitro bacterial mutagenicity 7.6.1 The mutagenic potential of high flash aromatic naptha Salmonella typhimurium experimental result According to  Ames, B.N., McCann, J., and Yamasaki, E. (1975).  Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutat. Res.31: 347-364. No 1 Well-documented journal article.  No Test material was specifically prepared to meet the composition requirements for High Flash Aromatic Naptha, Type 1 not mutagenic study report Schreiner, CA, Edwards, DA, McKee, RH, Swanson, M, Wong, ZA, Schmitt, S, Beatty, P 1989 The mutagenic potential of high flash aromatic naptha Cell Biology and Toxicology, Vol. 5, No. 2, 169-188 yes

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

It was concluded that the substance did not induce mutation in five strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537, and TA102 ) when tested under the conditions employed in this study, which included treatment at concentrations up to the lower limit of toxicity, both in the absence and in the presence of rat liver metabolic activation system (S-9).
Executive summary:

A bacterial reverse mutation assay was performed on five strains of Salmonella typhimurium. The study was performed both with (maximum concentration of 1000 microgram/ml) and without metabolic activation (maximum concentration of 500 microgram/ml). After 3 days of exposure to the test substance, the number of mean revertants per plate was calculated. Toxicity was seen at the highest doses. Most notably in the strains TA1535, TA1537, and TA102 during Experiment 2. These strains were retested at lower doses. There was no significant increase in the number of revertants in any of the test strains. There was a significant increase in the number of revertants in the positive control treatment. The study is therefore valid, and the test substance is not mutagenic.