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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:
21 May - 12 Jun 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 21 July 1997
Deviations:
yes
Remarks:
Only 2-Aminoanthracene was used as positive control in the presence of S9 mix.
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
Only 2-Aminoanthracene was used as positive control in the presence of S9 mix.
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material: Cyrene™- Analytical purity: 99%- Expiration date of the lot/batch: 30 Sep 2015

Method

Target gene:
his operon
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
nitroreductase deficient
Remarks:
for uvrB- strains TA 1537, TA 98, TA 1535, TA 100
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from phenobarbital/β-naphtoflavone induced rat liver
Test concentrations with justification for top dose:
1st experiment: 3, 10, 33, 100, 333, 1000, 2500, and 5000 μg/plate with and without metabolic activation.
2nd experiment: 33, 100, 333, 1000, 2500, and 5000 μg/plate with and without metabolic activation.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: deionized water- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine (4-NOPD); 2-aminoanthracene (2-AA)
Remarks:
+S9: 2-aminoanthracene (2-AA; 2.5 or 10 µg/plate, in DMSO, all strains); -S9: 4-NOPD (10 or 50 µg/plate, in DMSO, TA1537/TA98), sodium azide (NaN3, 10 µg/plate, in water, TA1535/TA100); methylmethanesulfonate (MMS, 2 µg/plate, in water, TA102)
Details on test system and experimental conditions:
Experiment I: in agar (plate incorporation)
Experiment II: pre-incubation
DURATION Experiment I - Exposure duration: 48 h
DURATION Experiment II - Preincubation period: 60 min
NUMBER OF REPLICATIONS: each concentration was tested in triplicates in two independent experiments both with and without S9 mix

DETERMINATION OF CYTOTOXICITY:
Method: inspection of the bacterial background growth and reduction in the number of revertant colonies
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed. A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration. An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment. A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS:
Water solubility: The test item is dissolvable in water.
Precipitation: No precipitation of the test item occurred up to the highest investigated dose.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.
No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
In the pre-experiment the concentration range of the test item was 3 – 5000 μg/plate. The pre-experiment is reported as experiment I. Since no toxic effects were observed 5000 μg/plate were chosen as maximal concentration.

Any other information on results incl. tables

Pre-Experiment for Toxicity

To evaluate the toxicity of the test item a pre-experiment was performed with all strains used. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre-experiment were the same as described for the experiment I (plate incorporation test). Toxicity of the test item can be evident as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn. The pre-experiment is reported as main experiment I, since the following criteria are met: Evaluable plates (>0 colonies) at five concentrations or more in all strains used.

Table 1: Test Results of Experiment 1

 EXPERIMENT 1

S9-Mix

 

Without

 

Test item (µg/plate)

TA 1535

TA 1537

TA 98

TA 100

TA 102

 Verhicle control

17.7

10.7

24.7

175.0

443.3

Untreated control

2.0

1.2

8.7

166.7

468.7

Cyrene™

(3)

18.3

12.7

25.0

168.3

453.0

Cyrene™

(10)

18.0

11.7

27.3

183.7

447.0

Cyrene™

(33)

17.7

9.3

28.0

183.0

435.7

Cyrene™

(100)

19.7

9.0

23.0

174.7

440.7

Cyrene™

(333)

15.7

9.0

27.7

175.0

455.0

Cyrene™

(1000)

17.7

9.7

22.7

172.0

424.7

Cyrene™

(2500)

15.0

11.0

24.0

188.0

467.3

Cyrene™

(5000)

14.3

13.7

30.3

178.0

465.0

NaN3

(10)

2940.3

 

 

1906.7

 

4-NOPD

(50)

 

58.3

 

 

 

4-NOPD

(10)

 

 

273.7

 

 

MMS

(2.0 µL/plate)

 

 

 

 

5586.7

S9-Mix

 

With

 

Test item (µg/plate)

TA 1535

TA 1537

TA 98

TA 100

TA 102

Vehicle control

16.7

24.0

38.3

177.0

595.3

Untreated control

15.3

20.0

36.7

170.0

618.3

Cyrene™

(3)

14.7

21.3

42.7

181.0

573.3

Cyrene™

(10)

15.3

24.0

41.3

184.7

587.7

Cyrene™

(33)

13.3

20.3

38.7

187.7

651.0

Cyrene™

(100)

12.0

20.0

34.3

185.3

690.7

Cyrene™

(333)

14.7

19.7

41.3

179.7

603.0

Cyrene™

(1000)

13.7

20.3

46.3

177.0

595.7

Cyrene™

(2500)

15.3

20.7

35.7

174.3

589.3

Cyrene™

(5000)

17.0

15.0

35.7

167.3

645.7

2-AA

(2.5)

548.3

376.0

3912.0

3957.7

 

2-AA

(10.0)

 

 

 

 

1384.7

2 -AA: 2 -aminoanthracene; 4 -NOPD: 4 -nitro-o-phenylene-diamine; NaN3: sodium azide; MMS: methylmethanesulfonate

Table 2: Test Results of Experiment 2

EXPERIMENT 2

S9-Mix

 

Without

 

Test item (µg/plate)

TA 1535

TA 1537

TA 98

TA 100

TA 102

Vehicle control

12.0

11.3

26.7

152.0

362.3

Untreated control

9.7

7.7

31.0

161.3

407.7

Cyrene™

(33)

12.7

11.0

25.3

159.7

366.3

Cyrene™

(100)

12.3

12.7

29.3

164.0

394.0

Cyrene™

(333)

13.3

9.0

29.7

165.3

351.7

Cyrene™

(1000)

17.3

8.7

29.7

165.3

391.7

Cyrene™

(2500)

13.0

12.7

33.3

163.0

395.7

Cyrene™

(5000)

14.0

11.3

29.7

152.3

425.3

NaN3

(10)

2758.7

 

 

1706.3

 

4-NOPD

(50)

 

53.3

 

 

 

4-NOPD

(10)

 

 

242.0

 

 

MMS

(2.0 µL/plate)

 

 

 

 

1776.7

S9-Mix

 

With

 

Test item (µg/plate)

TA 1535

TA 1537

TA 98

TA 100

TA 102

Vehicle control

11.0

16.0

43.0

200.0

526.7

Untreated control

15.3

11.7

42.7

160.7

534.3

Cyrene™

(33)

14.7

14.0

44.3

158.7

550.3

Cyrene™

(100)

11.7

19.7

42.7

156.3

447.3

Cyrene™

(333)

10.7

20.0

41.3

178.7

577.0

Cyrene™

(1000)

10.0

16.0

35.0

180.3

530.7

Cyrene™

(2500)

13.0

15.0

41.3

176.3

517.3

Cyrene™

(5000)

14.7

12.7

43.7

188.0

593.3

2-AA

(2.5)

505.3

131.0

3724.3

2360.7

 

2-AA

(10.0)

 

 

 

 

5146.7

2 -AA: 2 -aminoanthracene; 4 -NOPD: 4 -nitro-o-phenylene-diamine; NaN3: sodium azide; MMS: methylmethanesulfonate

Applicant's summary and conclusion

Conclusions:
Negative with and without metabolic activation.
Executive summary:

Cyrene™ was tested in a valid bacterial reverse mutation assay, according to the OECD TG 471 (1997), and under GLP, using Salmonella typhimurium strains S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102. No increase in the number of revertants was observed in any test strain, with or without metabolic activation when tested up to limit concentration. Appropriate positive, negative and solvent controls were added and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.