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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Refer to the Category Approach Justification document provided in IUCLID6 Section 13.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(1997)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
semi-solid (amorphous): gel
Remarks:
migrated information: paste

Method

Target gene:
his-operon
Species / strain
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain characteristics:
other: All strains are deep rough and have a reduced capability to repair DNA-damage (except for TA 102). Strains TA 98, TA 100 and TA 102 contain the R-factor plasmid pkM101 enhancing an error prone DNA repair system.
Metabolic activation:
with and without
Metabolic activation system:
S9 mix prepared from the livers of rats treated with phenobarbital/beta-naphthoflavone was achieved from Trinova Biochem GmbH and was cofactor supplemented.
Test concentrations with justification for top dose:
plate incorporation and preincubation test: 3.16, 10.0, 31.6, 100, 316, 1000, 2500, 5000 µg/plate
Vehicle:
- Vehicle used: distilled water
Controls
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9 mix: Sodium azide: 10 µg/plate (TA 100, TA 1535), 4-NOPD: 10 µg/plate (TA 98)/40 µg/plate (TA1537), MMS: 1 µL/plate (TA 102); +S9 mix: 2-AA: 2.5 µg/plate (TA 98, TA 100, TA 1535, TA 1537) / 10 µg/plate (TA 102)
Details on test system and conditions:
A preliminary plate incorporation experiment with the tester strains TA 98 and TA 100 was performed. The results were were in accordance with the acceptance criteria and are therfore reported as part of Experiment I. METHOD OF APPLICATION: Experiment I: in agar (plate incorporation); Experiment II: preincubationThe initial plate incorporation test was conducted as follows:Briefly, 0.1 mL test substance, 0.1 mL bacteria culture, 0.5 mL S9 mix (or buffer) and 2.0 mL soft agar were mixed and plated onto petri dishes with solid agar. After incubation at 37 °C for 48 h colonies were counted using an automatic counter. Each experiment was performed in triplicate. The independent repeat was performed as preincubation test. Briefly, 0.1 mL test substance, 0.1 mL bacteria culture and 0.5 mL S9 mix (or buffer) were preincubated at 37 °C for 60 min. At the end of the preincubation period 2 mL of molten soft agar was added. After mixing, the suspension was plated, incubated for 48 h at 37 °C and colonies were counted using an automatic counter. Each experiment was performed in triplicate.DETERMINATION OF CYTOTOXICITYThe reduction of background growth of bacteria on the plates as well as a reduction in the mutant count per plate to approximately 0.5 in comparison to control were used as marker for cytotoxicity.
Evaluation criteria:
The following criteria determined the acceptance of an assay: 1. The negative controls had to be within the expected range, as defined by the laboratories’ own historical data. 2. The positive controls had to show sufficient effects, as defined by the laboratories' experience 3. The bacteria strains TA 98, TA 100 and TA 102 demonstrate their typical response to ampicillin.4. Corresponding background growth on negative control, solvent control and test plates is obervedA clear and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 98, TA 100, and TA 102 this increase should be about twice that of negative controls. For TA 1535 and TA 1537 the increase should be about three times than that of solvent controls. Otherwise, the result is evaluated as negative. However, these criteria may be overruled by good scientific judgment.
Statistics:
Mean values and standard deviation were calculated.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
beginning at 1000 µg/plate in the plate incorporation experiment and 316 µg/plate in the preincubation experiment.
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Additional information on results:
No precipitation was observed in any tester strain at any concentration.

Any other information on results incl. tables

Table 1: Test results of experiment 1 (plate incorporation).

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates ± Standard deviation)

Base-pair substitution type

Frameshift type

TA 100

TA1535

TA102

TA98

TA1537

without S9-Mix

0

107 ± 7.5

6 ± 3.1

175 ± 10.6

25 ± 10.4

9 ± 2.5

3.16

128 ± 14.6

10 ± 6.7

196 ± 13.1

27 ± 6.4

14 ± 4.5

10.0

134 ± 4.2

7 ± 0.6

174 ± 13.4

21 ± 3.1

12 ± 3.1

31.6

134 ± 17.3

8 ± 1.7

185 ± 25.1

25 ± 2.5

9 ± 0.6

100

120 ± 15.0

8 ± 0.0

164 ± 15.1

28 ± 7.5

12 ± 2.5

316

95 ± 8.5

10 ± 3.2

182 ± 15.1

27 ± 9.7

8 ± 2.5

1000

74 ± 13.2 B

10 ± 4.2

215 ± 10.8

21± 3.1

9± 4.2

2500

38 ± 3.0 B

0± 0.0 B

179± 10.8

19± 6.5 B

6± 4.6 B

5000

0± 0.6 B

0± 0.0 B

148± 11.0

14± 2.5 B

0± 0.0 B

Positive controls:

Na-azide

MMS

4-NOPD

Concentration

(μg/plate)

10

1 µL/plate

10

40

Mean No. of colonies/plate

(average of 3 ± SD)

934 ± 22.0

758 ± 220.3

1782 ± 345.7

461 ± 23.8

119 ± 7.8

With S9-Mix

0

124 ± 13.8

7 ± 4.0

253 ± 5.3

26 ± 1.0

7 ± 3.5

3.16

120 ± 20.6

7 ± 7.1

272 ± 21.0

35 ± 5.2

11 ± 2.5

10.0

120 ± 8.1

10 ± 3.6

262 ± 2.5

35 ± 2.9

13 ± 4.5

31.6

113 ± 12.5

10 ± 3.2

262 ± 7.2

40 ± 7.2

10 ± 3.5

100

133 ± 16.7

7 ± 1.7

248 ± 19.7

35 ± 7.6

9 ± 1.0

316

121 ± 4.6

9 ± 1.0

277 ± 7.8

32 ± 3.5

12 ± 1.2

1000

117 ± 29.5

11 ± 1.7

274 ± 7.0

36 ± 5.3

13 ± 3.8

2500

100 ± 9.9

7 ± 1.5

233 ± 8.4

31 ± 3.1

6 ± 2.5

5000

61 ± 4.4 B

4 ± 1.0 B

238 ± 28.6

20 ± 5.5

9 ± 1.5

Positive controls:

2-AA

Concentrations

(μg/plate)

2.5

10

2.5

Mean No. of colonies/plate

(average of 3 ± SD)

1581 ±

229.7

102 ±
9.0

516 ±
44.6

1959 ±

504.5

261 ±
14.9

MMS = Methylmethanesulfonate

4-NOPD = 4-Nitro-o-phenylene diamine

2-AA = 2-Aminoanthracene

B = Reduction of background lawn

Table 2: Test results of experiment 2 (pre-incubationtion).

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates ± Standard deviation)

Base-pair substitution type

Frameshift type

TA 100

TA1535

TA102

TA98

TA1537

without S9-Mix

0

115± 23.1

10± 4.0

270± 12.2

22± 3.2

7± 1.5

3.16

129± 14.4

4± 2.1

241± 29.2

24± 1.2

10± 2.0

10.0

133± 9.0

9± 2.3

264± 29.4

31± 2.5

10± 6.0

31.6

119± 4.4

5± 1.0

278± 29.7

23± 2.6

12± 6.4

100

114± 15.9

8± 2.0

283± 23.1

26± 1.0

9± 3.2

316

100± 6.1

7± 1.2

262± 9.8

25± 2.1

8± 1.2 B

1000

58± 9.1 B

0± 0.0 B

280± 22.8

25 ± 0.6 B

0 ± 0.0 B

2500

0 ± 0.0 B

0 ± 0.0 B/N

155 ± 37.0

22 ± 6.1 B

0 ± 0.0 N

5000

0 ± 0.0 N

0 ± 0.0 N

125 ± 45.0 B

0 ± 0.0 B

0 ± 0.0 N

Positive controls:

Na-azide

MMS

4-NOPD

Concentration

(μg/plate)

10

1 µL/plate

10

40

Mean No. of colonies/plate

(average of 3 ± SD)

965± 120.2

1038± 227.1

2158± 67.5

389± 30.1

107± 7.0

With S9-Mix

0

108± 12.7

10± 2.5

292± 7.8

38± 4.5

9± 3.2

3.16

137± 30.7

5± 2.1

289± 15.9

39± 5.6

7± 2.1

10.0

139± 4.2

5± 2.5

323± 36.5

37± 2.0

4± 2.9

31.6

121± 14.0

7± 3.1

310± 21.2

32± 4.5

5± 4.9

100

134± 14.7

7± 1.5

297± 6.1

31± 6.0

7± 2.6

316

116± 7.6

8± 1.5

286± 24.0

31± 6.7

4 ± 1.2

1000

101± 15.5

7± 0.6

310± 7.4

30± 12.7

12± 3.5

2500

85± 11.8 B

4± 2.5 B

284± 15.0

22± 4.2 B

7± 3.5 B

5000

70± 4.6 B

0± 0.0 B

273± 10.7

21± 10.4 B

6± 3.6 B

Positive controls:

2-AA

Concentrations

(μg/plate)

2.5

10

2.5

Mean No. of colonies/plate

(average of 3 ± SD)

789±
158.7

60 ±
15.0

628 ±
14.3

2943 ±
399.9

128 ±
29.8

MMS = Methylmethanesulfonate

4-NOPD = 4-Nitro-o-phenylene diamine

2-AA = 2-Aminoanthracene

B = Reduction of background lawn

N = No background lawn

Applicant's summary and conclusion

Conclusions:
negative