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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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Active enzyme protein of Lipase, triacylglycerol (EC no. 232-619-9, CAS no. 9001-62-1, EC name: Lipase, triacylglycerol, Enzyme Class No.: 3.1.1.3)
Molecular formula:
Not applicable, see remarks
IUPAC Name:
Active enzyme protein of Lipase, triacylglycerol (EC no. 232-619-9, CAS no. 9001-62-1, EC name: Lipase, triacylglycerol, Enzyme Class No.: 3.1.1.3)
Constituent 2
Reference substance name:
Protein as a constituent of enzyme deriving from the fermentation or extraction process
Molecular formula:
Not available. See remarks.
IUPAC Name:
Protein as a constituent of enzyme deriving from the fermentation or extraction process
Constituent 3
Reference substance name:
Inorganic salts as a constituent of enzyme deriving from the fermentation or extraction process
Molecular formula:
Not available. See remarks.
IUPAC Name:
Inorganic salts as a constituent of enzyme deriving from the fermentation or extraction process
Constituent 4
Reference substance name:
Carbohydrates constituent of enzyme deriving from the fermentation or extraction process
Molecular formula:
Not available. See remarks.
IUPAC Name:
Carbohydrates constituent of enzyme deriving from the fermentation or extraction process
Constituent 5
Reference substance name:
Lipids as a constituent of enzyme deriving from the fermentation or extraction process
Molecular formula:
Not available.
IUPAC Name:
Lipids as a constituent of enzyme deriving from the fermentation or extraction process
Test material form:
liquid
Details on test material:
- Substance type: UVCB
- Physical state: translucent tan liquid

Method

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:
Aroclor 1254 induced rat liver S9
Test concentrations with justification for top dose:
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 relevant toxic effects were observed, 5000 µg/plate was chosen as maximal concentration. Due to observed minor contamination at higher concentrations eight concentrations were tested in Experiment II.

The concentration range included two logarithmic decades. The following concentrations (based on the total protein for this test item) were tested in Experiment II: 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate.
Vehicle / solvent:
Deionized water
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Deionized water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-Nitro-o-phenylenediamine: TA 1537 and TA 98 without S9 Aminoanthracene (2-AA): TA 98, TA 100, TA 1535, TA 1537, and E. coli
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable): 10E8-10E9 cells/mL

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3 plates per strain and dose, both with and without metabolic activation, and the same number of plates for the controls

DETERMINATION OF CYTOTOXICITY: Performed between 3 to 5000 µg/plate
Evaluation criteria:
The assay is considered to be valid if all of the following criteria are met:
• The negative and positive control data are consistent with the historical control data for the testing laboratory
• The positive control data show marked increases over the concurrent negative control values
• The evaluation of the test data is not restricted by the loss of plates (e.g., through contamination).

A test item is considered as a mutagen if all of the following criteria are met:
• A biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) 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.
• The increases are reproducible between replicate plates
• An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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:
In Experiment I, a minor reduction in the number of revertants (below the indication factor of 0.5), was observed in strain TA 98 at 5000 µg/plate with S9 mix.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
No toxic effects, evident as a reduction in the number of revertanst (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation. only in experiment I a minor reduction in the number of revertants (below the indication factor of 0.5), was observed in strain TA98 at 5000 µg/plate with S9 mix.
No substantial increase in revertant colony numbers of any of the five testerr strains was observed following treatment with the test substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.
In experiment I with S09 mix, the data in the negative control of strian WP2 uvrA were slightly above the test facility historical control range. Since the deviation is rather small, the effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies.

Any other information on results incl. tables

Table 1a - Summary of Results Pre-Experiment and Experiment I (Without activation)

 

                                                         Revertant Colony Counts (Mean ±SD)

 

Substance

µg/plate

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

Vehicle

 

29 ± 2

155 ± 3

22 ± 3

12 ± 3

69 ± 7

Untreated

 

35 ± 6

138 ± 10

19 ± 8

11 ± 5

68 ± 4

TS

3

32 ± 3

155 ± 8

18 ± 10

13 ± 4

77 ± 5

TS

10

35 ± 7

145 ± 2

22 ± 3

14 ± 1

80 ± 5

TS

33

36 ± 1

139 ± 7

23 ± 4

12 ± 2

73 ± 4

TS

100

37 ± 7

155 ± 19

23 ± 6

13 ± 3

72 ± 9

TS

333

29 ± 1

150 ± 2

20 ± 3

15 ± 3

71 ± 6

TS

1000

38 ± 7

154 ± 8

21 ± 4

13 ± 6

65 ± 8

TS

2500

21 ± 4*

111 ± 8*

15 ± 4*

7 ± 2*

53 ± 5*

TS

5000

14 ± 1*

106 ± 5*

16 ± 2*

7 ± 3*

55 ± 6*

NaN3

10

 

2161 ± 7

2141 ± 41

 

 

4-NOPD

10

306 ± 13

 

 

 

 

4-NOPD

50

 

 

 

94 ± 3

 

MMS

3.0 µL

 

 

 

 

1541 ± 141

 

TS = Test substance

* = Contaminated and Manual count

 

 

Table 1b - Summary of Results Pre-Experiment and Experiment I (With activation)

 

                                         Revertant Colony Counts (Mean ±SD)

 

Substance

µg/plate

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

Vehicle

0

39 ± 6

170 ± 11

26 ± 7

12 ± 3

79 ± 6

Untreated

 

31 ± 5

172 ± 2

22 ± 6

16 ± 6

87 ± 6

TS

3

39 ± 6

164 ± 8

23 ± 9

15 ± 2

84 ± 11

TS

10

38 ± 3

146 ± 17

17 ± 4

13 ± 1

76 ± 5

TS

33

40 ± 3

167 ± 4

20 ± 1

13 ± 3

87 ± 7

TS

100

40 ± 2

161 ± 2

22 ± 3

14 ± 2

72 ± 8

TS

333

34 ± 2

161 ± 17

25 ± 8

11 ± 4

77 ± 8

TS

1000

38 ± 5

149 ± 9

21 ± 7

16 ± 3

76 ± 4

TS

2500

23 ± 4*

140 ± 7*

16 ± 5*

11 ± 1*

57 ± 7*

TS

5000

14 ± 4*

117 ± 6*

18 ± 4*

13 ± 2*

71 ± 7*

2-AA

2.5

1363 ± 198

1368 ± 100

189 ± 22

155 ± 12

 

2-AA

10

 

 

 

 

219 ± 3

 

TS = Test substance

* = Contaminated and Manual count

 

Table 2a - Summary of Experiment II (Without activation)

 

                                                              Revertant Colony Counts (Mean ±SD)

 

Substance

µg/plate

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

Vehicle

0

34 ± 9

127 ± 13

21 ± 2

13 ± 4

46 ± 9

Untreated

 

33 ± 8

142 ± 10

28 ± 9

16 ± 2

52 ± 5

TS

3

30 ± 7

145 ± 15

29 ± 6

13 ± 3

52 ± 7

TS

10

34 ± 5

143 ± 15

20 ± 5

13 ± 8

48 ± 8

TS

33

36 ± 10

155 ± 32

23 ± 5

19 ± 7

55 ± 9

TS

100

44 ± 11

140 ± 8

24 ± 4

16 ± 6

53 ± 3

TS

333

29 ± 11

154 ± 33

29 ± 12

14 ± 1

48 ± 4

TS

1000

34 ± 5

145 ± 14

24 ± 3

11 ± 2

51 ± 2

TS

2500

31 ± 6

151 ± 17

31 ± 8

13 ± 4

67 ± 17

TS

5000

39 ± 9

158 ± 18

29 ± 1

17 ± 1

59 ± 7

NaN3

10

 

1766 ± 85

1615 ± 160

 

 

4-NOPD

10

354 ± 17

 

 

 

 

4-NOPD

50

 

 

 

114 ± 23

 

MMS

3.0µL

 

 

 

 

1453 ± 79

 

TS = Test substance

 

Table 2b - Summary of Experiment II (With activation)

 

                                       Revertant Colony Counts (Mean ±SD)

 

Substance

µg/plate

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

Vehicle

 

40 ± 4

166 ± 8

38 ± 6

17 ± 5

63 ± 14

Untreated

 

37 ± 4

156 ± 4

30 ± 11

18 ± 7

68 ± 11

TS

3

42 ± 9

185 ± 27

38 ± 6

22 ± 4

67 ± 9

TS

10

34 ± 14

161 ± 17

31 ± 4

22 ± 2

62 ± 6

TS

33

43 ± 11

171 ± 1

28 ± 8

20 ± 3

68 ± 1

TS

100

37 ± 8

160 ± 2

39 ± 15

23 ± 4

71 ± 6

TS

333

45 ± 9

170 ± 15

42 ± 5

22 ± 10

61 ± 7

TS

1000

43 ± 6

167 ± 21

30 ± 6

24 ± 3

72 ± 14

TS

2500

46 ± 4

194 ± 26

43 ± 8

24 ± 3

83 ± 31

TS

5000

50 ± 8

169 ± 17

43 ± 1

24 ± 1

70 ± 3

2-AA

2.5

482 ± 20

828 ± 35

748 ± 95

127 ± 6

 

2-AA

10

 

 

 

 

314 ± 21

 

TS = Test substance

Applicant's summary and conclusion

Conclusions:
The test substance is considered to be non-mutagenic in this Salmonella typhimurium and E.coli reverse mutation assay.
Executive summary:

This study was performed to investigate the potential of the test substance to induce gene mutations in the plate incorporation test (Experiment I) and the pre-incubation test (Experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA in accordance with OECD Test Guideline 471. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations (based on the total protein for this test item): Pre-Experiment/Experiment I and II: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate.

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments. 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. Only in experiment I a minor reduction in the number of revertants (below the indication factor of 0.5), was observed in strain TA 98 at 5000 µg/plate with S9 mix. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test substance did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.