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Toxicological information

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: GLP-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)
Version / remarks:
adopted in 1997
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
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 male Wistar rats treated with phenobarbital (80 mg/kg bw) and β-naphtoflavone (100 mg/kg bw) for three consecutive days
Test concentrations with justification for top dose:
31.6, 100, 316, 1000, 2500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
yes
Remarks:
Aqua dest.
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9: sodium azide (10 µg/pl.; TA 100, TA 1535); 4-nitro-o-phenylene-diamine (10 µg/pl. for TA 98 and 40 µg/pl. for TA 1537); methylmethanesulfonate (1 µL/pl.; TA 102); +S9: 2-aminoanthracene (10 µg/pl. for TA 102; 2.5 µg/pl. for all other strains)
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Experiment I: in agar (plate incorporation)
Experiment II: preincubation

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

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: clearing of the background lawn or reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control
Evaluation criteria:
A substance is considered to be 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. A biologically relevant positive response will be assumed if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high than the reversion rate of the solvent control and/or if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.

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 / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation was observed in Experiment I in all tester strains at 1000 µg/plate and higher without metabolic activation and at 5000 µg/plate with metabolic activation. In Experiment II, precipitation of the test substance was noticed in all tester strains at 2500 and 5000 µg/plate with and without metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA: The positive and negative control data of both experiments were in the range of the historical control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY: A reduction of the background lawn was only observed in tester strain TA1537 in Experiment II at concentrations of 2500 and 5000 µg/plate without metabolic activation.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. Experiment I: Plate-incorporation Test

Bacterial Reverse Mutation Assay, mean revertants colonies/plate (mutation factor)

EXPERIMENT I (Plate incorporation test)

S9-Mix

Without

 

Test item (µg/plate)

TA 98

TA 100

TA 1535

TA 1537

TA 102

NC

18 ± 2.0 (1.0)

87 ± 11.0 (1.1)

7 ± 2.1 (1.0)

8 ± 1.7 (1.6)

178 ± 5.6 (1.2)

VC

18 ± 4.4 (1.0)

76 ± 9.0 (1.0)

7 ± 4.0 (1.0)

5 ± 0.0 (1.0)

153 ± 9.0 (1.0)

31.6

16 ± 3.5 (0.9)

80 ± 14.7 (1.0)

5 ± 1.0 (0.8)

8 ± 2.0 (1.6)

172 ± 5.5 (1.1)

100

19 ± 3.8 (1.1)

87 ± 15.5 (1.1)

6 ± 3.5 (1.0)

9 ± 3.2 (1.9)

165 ± 15.0 (1.1)

316

23 ± 4.0 (1.3)

74 ± 6.6 (1.0)

6 ± 3.5 (0.9)

10 ± 3.6 (2.0)

167 ± 3.0 (1.1)

1000

15 ± 2.3 P (0.9)

86 ± 12.3 P (1.1)

5 ± 0.6 P (0.8)

13 ± 7.1 P (2.5)

166 ± 9.0 P (1.1)

2500

13 ± 2.5 P (0.7)

70 ± 6.0 P (0.9)

4 ± 3.1 P (0.7)

8 ± 1.0 P (1.6)

189 ± 12.3 P (1.2)

5000

18 ± 1.0 P (1.0)

82 ± 6.2 P (1.1)

6 ± 2.9 P (0.9)

8 ± 3.5 P (1.5)

176 ± 7.5 P (1.1)

4-NOPD

425 ± 56.3 (23.6)

---

---

86 ± 4.7 (17.1)

---

NaN3

---

318 ± 102.2 (3.7)

612 ± 193.7 (91.9)

---

---

MMS

---

---

---

---

1454 ± 246.3 (8.2)

S9-Mix

 

With

Test item (µg/plate)

TA 98

TA 100

TA 1535

TA 1537

TA 102

NC

23 ± 4.7 (0.8)

118 ± 15.0 (1.0)

6 ± 1.5 (1.4)

8 ± 3.2 (1.4)

216 ± 40.0 (1.3)

VC

28 ± 4.7 (1.0)

115 ± 15.6 (1.0)

5 ± 2.1 (1.0)

6 ± 3.2 (1.0)

169 ± 19.7 (1.0)

31.6

24 ± 5.5 (0.9)

123 ± 14.9 (1.1)

4 ± 2.5 (0.9)

9 ± 6.4 (1.6)

206 ± 18.1 (1.2)

100

32 ± 8.1 (1.1)

116 ± 10.7 (1.0)

5 ± 2.0 (1.1)

8 ± 5.5 (1.5)

209 ± 15.3 (1.2)

316

29 ± 4.0 (1.0)

116 ± 10.0 (1.0)

5 ± 2.1 (1.1)

9 ± 1.5 (1.5)

172 ± 13.1 (1.0)

1000

31 ± 10.0 (1.1)

107 ± 5.0 (0.9)

5 ± 1.7 (1.1)

5 ± 3.5 (0.9)

176 ± 15.5 (1.0)

2500

29 ± 2.1 (1.0)

123 ± 8.1 (1.1)

8 ± 3.2 (1.6)

8 ± 2.1 (1.4)

187 ± 12.1 (1.1)

5000

25 ± 4.6 P (0.9)

119 ± 18.0 P (1.0)

6 ± 2.1 P (1.2)

6 ± 1.0 P (1.1)

214 ± 1.7 P (1.3)

2-AA

2278 ± 372.4 (82.3)

1805 ± 347.6 (15.7)

74 ± 18.6 (15.9)

276 ± 38.6 (48.7)

389 ± 30.8 (2.3)

NC = Negative Control (Aqua dest.)

VC = Vehicle Control (DMSO)

4-NOPD = 4-nitro-o-phenylene-diamine

NaN3= sodium azide

MMS = methylmethanesulfonate

2-AA = 2-aminoanthracene

P = Precipitation

Table 2. Experiment II: Preincubation Test

Bacterial Reverse Mutation Assay, mean revertants colonies/plate (mutation factor)

EXPERIMENT II (Preincubation test)

S9-Mix

Without

 

Test item (µg/plate)

TA 98

TA 100

TA 1535

TA 1537

TA 102

NC

23 ± 3.8 (1.2)

87 ± 8.2 (1.4)

6 ± 1.0 (1.1)

8 ± 0.0 (1.7)

178 ± 17.8 (1.0)

VC

18 ± 6.5 (1.0)

61 ± 9.5 (1.0)

5 ± 0.6 (1.0)

5 ± 2.3 (1.0)

175 ± 13.8 (1.0)

31.6

21 ± 7.0 (1.1)

69 ± 6.7 (1.1)

6 ± 1.0 (1.1)

6 ± 1.5 (1.4)

173 ± 16.1 (1.0)

100

22 ± 8.3 (1.2)

81 ± 2.1 (1.3)

3 ± 3.5 (0.6)

7 ± 4.0 (1.6)

161 ± 13.4 (0.9)

316

25 ± 4.6 (1.4)

77 ± 7.6 (1.3)

4 ± 2.1 (0.7)

4 ± 1.2 (0.9)

162 ± 8.6 (0.9)

1000

20 ± 4.4 (1.1)

72 ± 4.6 (1.2)

3 ± 1.5 (0.5)

6 ± 1.0 (1.3)

155 ± 2.9 (0.9)

2500

12 ± 2.6 P (0.7)

77 ± 5.9 P (1.3)

5 ± 2.1 P (0.9)

8 ± 2.1 BP (1.6)

160 ± 14.9 P (0.9)

5000

27 ± 9.3 P (1.5)

79 ± 7.6 P (1.3)

4 ± 2.1 P (0.8)

6 ± 1.5 BP (1.2)

171 ± 25.0 P (1.0)

4-NOPD

266 ± 37.5 (14.5)

---

---

78 ± 7.0 (16.7)

---

NaN3

---

894 ± 219.3 (10.3)

417 ± 4.4 (69.5)

---

---

MMS

---

---

---

---

1430 ± 407.6 (8.0)

S9-Mix

 

With

Test item (µg/plate)

TA 98

TA 100

TA 1535

TA 1537

TA 102

NC

28 ± 5.8 (1.2)

97 ± 19.1 (1.1)

5 ± 1.0 (1.0)

5 ± 2.5 (0.8)

263 ± 36.5 (1.1)

VC

23 ± 4.0 (1.0)

88 ± 9.0 (1.0)

5 ± 2.0 (1.0)

6 ± 0.6 (1.0)

235 ± 9.5 (1.0)

31.6

27 ± 2.1 (1.2)

89 ± 5.1 (1.0)

6 ± 2.1 (1.1)

6 ± 1.2 (1.0)

257 ± 36.8 (1.1)

100

28 ± 1.2 (1.2)

100 ± 13.3 (1.1)

5 ± 1.0 (1.0)

6 ± 1.5 (1.0)

259 ± 2.5 (1.1)

316

21 ± 5.3 (0.9)

84 ± 27.3 (1.0)

4 ± 0.6 (0.7)

8 ± 0.0 (1.3)

255 ± 6.4 (1.1)

1000

26 ± 8.5 (1.1)

88 ± 22.1 (1.0)

6 ± 2.1 (1.3)

9 ± 4.4 (1.4)

259 ± 5.3 (1.1)

2500

26 ± 7.6 P (1.1)

97 ± 6.7 P (1.1)

8 ± 4.0 P (1.5)

10 ± 4.0 P (1.6)

223 ± 6.8 P (0.9)

5000

29 ± 4.5 P (1.3)

155 ± 27.4 P (1.8)

4 ± 2.1 P (0.7)

6 ± 3.2 P (1.0)

333 ± 31.5 P (1.4)

2-AA

2005 ± 694.8 (85.9)

1443 ± 57.5 (16.5)

53 ± 2.0 (10.6)

209 ± 26.9 (33.0)

696 ± 69.9 (3.0)

NC = Negative Control (Aqua dest.)

VC = Vehicle Control (DMSO)

4-NOPD = 4-nitro-o-phenylene-diamine

NaN3= sodium azide

MMS = methylmethanesulfonate

2-AA = 2-aminoanthracene

P = Precipitation

B = Background lawn reduced

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative