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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:
20 Oct 2009 to 26 Jan 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Deviation: The highest concentration tested was based on growth inhibition. The study report does not specify which parameter was used to determine "growth inhibition".

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 21 July 1997
Deviations:
yes
Remarks:
The highest concentration tested was based on growth inhibition. The study report does not specify which parameter was used to determine "growth inhibition".
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed

Method

Target gene:
his operon (for S. typhimurium) and trp operon (for E. coli)
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male SD rats treated with phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
19.5, 78.1, 313, 1250 and 5000 µg/plate (Dose-finding test)

313, 625, 1250, 2500 and 5000 µg/plate (Main test: TA 100 and WP2 uvra +/-S9, TA 98 +S9)
2.44, 4.88, 9.77, 19.5, 39.1 and 78.1 (Main test: 1535 -S9)
9.77, 19.5, 39.1, 78.1, 156 and 313 (Main test: 1537 +/- S9, TA 98 -S9, TA 1535 +S9

Justification for top dose: In the dose-finding experiment growth inhibition was observed at 78.1 µg/plate and above for S. typhimurium TA1535 without metabolic activation, and at 313 µg/plate and above for S. typhimurium TA98 and TA1537 without metabolic activation and for S. typhimurium TA1535 and TA1537 with metabolic activation. The maximum dose level was set at 78.1 µg/plate for S. typhimurium TA1535 without metabolic activation and at 313 µg/plate for S. typhimurium TA98 and TA1537 without metabolic activation and for S. typhimurium TA1535 and TA1537 with metabolic activation and a total of 6 dose levels were selected by diluting five times at a common ratio of two. For S. typhimurium TA100 and E. coli WP2 uvrA without metabolic activation and S. typhimurium TA100, TA98 and E. coli WP2 uvrA with metabolic activation, the maximum dose level was set at 5000 µg/plate and a total of five dose levels obtained by diluting four times at a common ratio of two because no growth inhibition was observed. Precipitates were observed at 1250 µg/plate and above without metabolic activation.
Vehicle / solvent:
- Vehicle used: DMSO
- Justification for choice of vehicle: Since the test article is insoluble in water, a solubility test in DMSO was performed at the test facility. Based on these results, DMSO was used as the vehicle in this study because the test article was dissolved in DMSO at 50 mg/mL and there were no reactions such as exothermic reaction or generation of gasses.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) , 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)- aminopropylamino]acridine.2HCl (ICR-191) and 2-Aminoanthracene (2AA) solved in DMSO
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation method

DURATION
- Preincubation period: 20 min
- Exposure duration: 50 h

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: growth inhibition
Rationale for test conditions:
based on dose-range finding study.
Evaluation criteria:
If a twofold or more increase in the number of revertant colonies to that of natural revertant colonies (the negative control) was observed on paralle to a dose-response and reproducibility was noted, or even if no clear dose-response was observed but there was at least twofold increase in comparison with the number of natural revertant colonies and reproducibility was observed in the two main tests, the test article was judged to be positive.
Statistics:
Mean numbers and standard deviations of the revertant colonies of each concentration of each test system was calculated and compared to that of the solvent control.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
growth inhibition at 78.1 and 39. 1 (first and second experiment -S9), 313 and 156 µg/plate (first and second experiment +S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
growth inhibition at 313 and 156 µg/plate (first and second experiment +/-S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
growth inhibition at 313 and 156 µg/plate (first and second experiment -S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
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:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
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:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In both the first and second main tests, oily precipitation of the test article on the plate was observed at 1250 μg/plate and above without metabolic activation. Coloration by the test article in the test systems was not observed with or without metabolic activation.
- Cytotoxicity: In the observation of bacteria with a stereoscopic microscope, growth inhibition was observed at 39.1 µg/plate and above for S. typhimurium TA1535 without metabolic activation, at 156 µg/plate and above for S. typhimurium TA98 and TA1537 without metabolic activation and S. typhimurium TA1535 and TA1537 with metabolic activation.


Any other information on results incl. tables

 Table 1: Dose-range-finding test (mean)

Compound

S-9 Mix

concentration

TA 100

TA 1535

WP2 uvra

TA 98

TA 1537

DMSO

-

0

111

9

23

20

10

Test substance

-

5000#

110

8*

30

27*

5*

-

1250#

97

8*

31

31*

10*

-

313

101

7*

29

26*

8*

-

78.1

114

5*

24

23

6

-

19.5

106

9

31

29

8

DMSO

+

 

111

9

33

42

7

Test substance

+

5000

111

6*

31

33

10*

+

1250

106

7*

32

38

6*

+

313

104

9*

32

42

6*

+

78.1

126

12

32

35

7

+

19.5

127

8

25

32

12

Positive control

-

 

650 (a)

343 (b)

79 (a)

429 (a)

2081(c)

Positive control

+

 

1144 (d)

372 (e)

895 (e)

398 (d)

118 (d)

a)AF-2:2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

b) SAZ:Sodium azide

c) ICR-191:2-Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino]acridine.2HCl

d) B[a]P:Benzo[a]pyrene

e) 2AA:2-Aminoanthracene

*:Growth inhibition of tester strains was observed.

#:Precipitant was observed on the surface of agar plates.

Table 2: Results of the first experiment (without S-9)

Compound

S-9 Mix

concentration

TA 100

TA 1535

WP2 uvra

TA 98

TA 1537

DMSO

-

0

105 ± 4-6

8 ± 1.5

26 ± 4.7

27 ± 3.5

7 ± 3.1

Test substance

-

5000#

88 ± 10.8

NT

25 ± 2.6

NT

NT

-

2500#

90 ± 4.2

NT

26 ± 4.0

NT

NT

-

1250#

96 ± 3.1

NT

35 ± 11.6

NT

NT

-

625

80 ± 14.8

NT

26 ± 7.5

NT

NT

-

313

81 ± 4.4

NT

24 ± 1.5

24 ± 3.5*

4 ± 2.0*

 

156

NT

NT

NT

26 ± 2.1*

4 ± 2.5*

 

78.1

NT

7 ± 4.0*

NT

32 ± 1.2

5 ± 1.5

 

39.1

NT

8 ± 4.6*

NT

31 ± 3.8

7 ± 1.5

 

19.5

NT

11 ± 1.7

NT

30 ± 5.7

6 ± 1.0

 

9.77

NT

12 ± 2.1

NT

30 ± 10.7

5 ± 1.7

 

4.88

NT

12 ± 5.1

NT

NT

NT

 

2.44

NT

11 ± 0.6

NT

NT

NT

Positive control

-

 

565 ± 38.5 (a)

271 ± 38.9

(b)

83 ± 7.4 (a)

493 ± 11.5 (a)

1665 ± 291.5 (c)

NT: not tested

* growth inhibition

#: Precipitation was observed on the surface of agar plates.

a) 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2)

b) Sodium azide (SAZ)

c) 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino]acridine.2HCl (ICR-191)

  

 Table 3: Results of the first experiment (with S+9)

Compound

S-9 Mix

concentration

TA 100

TA 1535

WP2 uvra

TA 98

TA 1537

DMSO

+

0

110 ± 7.5

10 ± 1.2

26 ± 4.0

33 ± 6.1

6 ± 1.7

Test substance

+

5000

98 ± 8.5

NT

27 ± 6.1

35 ± 4.9

NT

+

2500

91 ± 6.0

NT

25 ± 3.1

37 ± 7.4

NT

+

1250

93 ± 4.7

NT

26 ± 6.1

37 ± 5.0

NT

+

625

102 ± 18

NT

27 ± 0.6

38 ± 3.6

NT

+

313

100 ± 8.6

NT

22 ± 5.1

34 ± 3.1

8 ± 2.0*

 

156

NT

NT

NT

NT

8 ± 0.0*

 

78.1

NT

9 ± 1.2*

NT

NT

7 ± 3.1

 

39.1

NT

13 ± 1.5*

NT

NT

6 ± 0.6

 

19.5

NT

8 ± 2.1

NT

NT

5 ± 2.5

 

9.77

NT

11 ± 2.5

NT

NT

7 ± 4.2

 

4.88

NT

10 ± 1.5

NT

NT

NT

 

2.44

NT

11 ± 4.4

NT

NT

NT

Positive control

+

 

1116 ± 55.4(d)

339 ± 15.5 (e)

898 ± 20.3(e)

354 ± 33.0(d)

98 ± 6.0 (d)

NT: not tested

* growth inhibition

d) B[a]P:Benzo[a]pyrene

e) 2AA:2-Aminoanthracene

 

Table 4: Results of the second experiment (without S-9)

Compound

S-9 Mix

concentration

TA 100

TA 1535

WP2 uvra

TA 98

TA 1537

DMSO

-

0

110 ± 8.6

12 ± 3.8

16 ± 4.0

31 ± 5.1

8 ± 1.2

Test substance

-

5000#

117 ± 4.6

NT

19 ± 7.6

NT

NT

-

2500#

102 ± 11.6

NT

15 ± 7.5

NT

NT

-

1250#

104 ± 12.1

NT

20 ± 6.0

NT

NT

-

625

104 ± 9.1

NT

15 ± 3.5

NT

NT

-

313

100 ± 7.2

NT

14 ± 3.5

31 ± 8.4*

8 ± 3.5*

 

156

NT

NT

NT

31 ± 7.6*

5 ± 4.2*

 

78.1

NT

12 ± 2.1*

NT

34 ± 5.3

9 ± 2.6

 

39.1

NT

9 ± 4.0*

NT

38 ± 3.5

6 ± 1.7

 

19.5

NT

11 ± 0.6

NT

37 ± 2.9

6 ± 1.0

 

9.77

NT

11 ± 1.5

NT

25 ± 3.5

5 ± 0.6

 

4.88

NT

12 ± 4.2

NT

NT

NT

 

2.44

NT

13 ± 4.0

NT

NT

NT

Positive control

-

 

624 ± 38.4 (a)

299 ± 12.9 (b)

65 ± 1.7 (a)

497 ± 12.3 (a)

1541 ± 190.3 (c)

NT: not tested

* growth inhibition

#: Precipitation was observed on the surface of agar plates.

a) 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2)

b) Sodium azide(SAZ)

c) 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino]acridine.2HCl(ICR-191)

   

Table 5: The result (mean) of the second experiment (with S-9)

Compound

S-9 Mix

concentration

TA 100

TA 1535

WP2 uvra

TA 98

TA 1537

DMSO

-

0

99 ± 3.5

8 ± 1.5

15 ± 4.0

42 ± 8.0

10 ± 4.0

Test substance

-

5000

108 ± 12.0

NT

19 ± 4.6

38 ± 5.3

NT

-

2500

107 ± 9.9

NT

23 ± 2.3

40 ± 1.2

NT

-

1250

99 ± 4.4

NT

16 ± 4.9

33 ± 7.8

NT

-

625

112 ± 14.0

NT

15 ± 5.5

41 ± 6.6

NT

-

313

108 ± 17.1

NT

19 ± 8.1

41 ± 11.3

7 ± 1.0*

 

156

NT

NT

NT

NT

7 ± 0.0*

 

78.1

NT

9 ± 3.1*

NT

NT

13 ± 2.6

 

39.1

NT

11 ± 0.6*

NT

NT

7 ± 1.7

 

19.5

NT

12 ± 3.6

NT

NT

11 ± 1.0

 

9.77

NT

9 ± 2.5

NT

NT

13 ± 2.5

 

4.88

NT

12 ± 1.0

NT

NT

NT

 

2.44

NT

12 ± 3.5

NT

NT

NT

Positive control

-

 

1018 ± 35.9 (d)

357 ± 41.0 (e)

1141 ± 42.0 (e)

361 ± 15.6 (d)

113 ± 3.5 (d)

NT: not tested

* growth inhibition

d) B[a]P:Benzo[a]pyrene

e) 2AA:2-Aminoanthracene

Applicant's summary and conclusion

Conclusions:
Under the conditions of the conducted test, the substance was not mutagenic in any of the five tester strains tested with and without metabolic activation up to 5000 µg/plate or tested up to cytotoxic concentrations.