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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:
2016
Report date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
2-AA inadvertently not added to WP2uvrA in the presence of S9. However, tester strain responded as expected to positive control in non-activated condition. All other tester strains responded as expected to 2-AA in the presence of S9.
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
2-AA inadvertently not added to WP2uvrA in the presence of S9. However, tester strain responded as expected to positive control in non-activated condition. All other tester strains responded as expected to 2-AA in the presence of S9.
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
yes
Remarks:
2-AA inadvertently not added to WP2uvrA in the presence of S9. However, tester strain responded as expected to positive control in non-activated condition. All other tester strains responded as expected to 2-AA in the presence of S9.
Qualifier:
according to guideline
Guideline:
other: Toxicological Principles for the Safety Assessment of Food Ingredients (Redbook 2000)
Deviations:
yes
Remarks:
2-AA inadvertently not added to WP2uvrA in the presence of S9. However, tester strain responded as expected to positive control in non-activated condition. All other tester strains responded as expected to 2-AA in the presence of S9.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Active enzyme protein of Aspergillopepsin I (EC no. 232-796-2, CAS no. 9025-49-4, EC name: Proteinase, Aspergillus acid, Enzyme Class no. 3.4.23.18)
Molecular formula:
Not applicable, see remarks
IUPAC Name:
Active enzyme protein of Aspergillopepsin I (EC no. 232-796-2, CAS no. 9025-49-4, EC name: Proteinase, Aspergillus acid, Enzyme Class no. 3.4.23.18)
Constituent 2
Reference substance name:
Protein as a constituent of enzyme deriving from the fermentation or extraction process
Molecular formula:
Not available
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. See remarks.
IUPAC Name:
Lipids as a constituent of enzyme deriving from the fermentation or extraction process
Test material form:
liquid
Details on test material:
- Lot/batch No.:2016208-00335

Method

Target gene:
histidine or tryptophan operons
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:
S-9 mix prepared form Aroclor®-1254-induced rat livers
Test concentrations with justification for top dose:
Toxicity Mutation test: 33.3, 66.7, 100, 333, 667, 1000, 3333, and 5000 μg per plate
Mutagenicity test: 333, 667, 1000, 3333, and 5000 µg/plate
The highest dose level tested is the maximum required by the OECD Guideline 471 for materials of low toxicity.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: sterile water
Controls
Untreated negative controls:
yes
Remarks:
sterile water
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
other: see remarks
Details on test system and experimental conditions:
A single pre-incubation treatment tube was prepared for each replicate group that was plated. The appropriate volume of each component of the treatment mixture was determined by multiplying the volume required per plate by the number of replicates plus one. Each tube was capped and incubated at approximately 37°C and 150 rpm, for a 60-minute treatment period. At the end of this treatment period the treatment mixture was transferred to a centrifuge. If a wash step was deemed necessary it was added prior to centrifugation. Treatment tubes were centrifuged for 10 minutes at 1500 rcf. The supernatant was aspirated down to ~50 μL. The pelleted bacteria were re-suspended with enough nutrient broth to match the original volume of bacterial culture. Plates were prepared by transferring 100 μL of re-suspended bacteria to a pre-heated (45-48°C) glass culture tube containing 2.5 mL of selective top agar. The bacteria were mixed with the agar by vortexing and then overlaid onto the surface of minimal glucose agar plates.
The positive control for WP2uvrA in the presence of S9 activation was tested using the plate incorporation method. One hundred 100 μL of the positive control was added to pre-heated (45-48°C) glass culture tubes containing 2 mL of selective top agar, followed by 100 μL of tester strain and 0.5 mL of S9 mix. The mixture was vortexed and overlaid onto the surface of a minimum glucose agar plate.
After the overlay solidified, the plates were inverted and incubated for approximately 48-52 hours at 37 ± 2°C. Plates that were not counted immediately following the incubation period were stored at 5 ± 3°C. All toxicity-mutation test dose preparations of negative (vehicle) controls, test substance, and positive controls were plated in duplicate. All mutagenicity test dose preparations of negative (vehicle) controls, test substance, and positive controls were plated in triplicate.
Rationale for test conditions:
The test was conducted using the treat and plate modification of the pre-incubation method. This method was selected due to the potential of the test substance to interfere with the selective conditions of the assay, potentially leading to false positive results.
Evaluation criteria:
Criteria for a positive response:
1. Strains TA1535 and TA1537: Data were judged positive if the increase in mean revertants at the highest numerical dose response was ≥ 3.0-fold the mean concurrent negative control value (vehicle control). This increase in the mean number of revertants per plate must be accompanied by a dose response associated with increasing concentrations of the test substance unless observed at the top dose level only.
2. Strains TA98, TA100 and WP2uvrA: Data sets were judged positive if the increase in mean revertants at the highest numerical dose response was ≥ 2.0-fold the mean concurrent negative control value (vehicle control). This increase in the mean number of revertants per plate must be accompanied by a dose response associated with increasing concentrations of the test substance unless observed at the top dose level only.
A data set may be judged equivocal if there is a biologically relevant increased response that only partially meets criteria for a positive response. A response was evaluated as negative if it is neither positive nor equivocal.
Statistics:
For all replicate plates, the results were presented as the mean revertants per plate and standard deviation.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
No appreciable toxicity or test substance precipitation was observed
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
No appreciable toxicity or test substance precipitation was observed
Untreated negative controls validity:
valid
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
Remarks:
No appreciable toxicity or test substance precipitation was observed
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
No appreciable toxicity or test substance precipitation was observed
Untreated negative controls validity:
valid
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
Remarks:
No appreciable toxicity or test substance precipitation was observed
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In the toxicity-mutation test, no positive mutagenic responses were observed at any dose level in any tester strain in the absence or presence of S9 metabolic activation. No appreciable toxicity or test substance precipitation was observed. A >50% reduction in the mean number of revertant colonies was observed with tester strain TA1537 in the absence of S9 activation at 100 and 667 μg/plate; however, this reduction occurred at intermediate dose levels with no dose related correlation and is not considered to be biologically relevant.

In the mutagenicity test, no positive mutagenic responses were observed at any dose level or with any tester strain in either the absence or presence of S9 metabolic activation. No appreciable toxicity or test substance precipitation was observed.

Any other information on results incl. tables

Mutagenicity test without S9 activation

Strain

Test substance

Dose level

µg/plate

Mean revertants/ plate

Standard deviation

Ratio treated/ solvent

Individual revertant colony counts and plate codes

WP2urvA

Water

-

46

5

-

50, 47, 40

 

Test substance

333

47

9

1.0

54, 49, 37

 

 

667

42

6

0.9

36, 44, 47

 

 

1000

50

8

1.1

43, 59, 48

 

 

3333

41

3

0.9

40, 39, 44

 

 

5000

41

5

0.9

47, 37, 40

 

4NQO

0.5

410

67

9.0

361, 383, 487

TA98

Water

-

26

6

-

26, 20, 31

 

Test substance

333

26

7

1.0

20, 23, 34

 

 

667

21

5

0.8

21, 17, 26

 

 

1000

30

6

1.2

26, 36, 27

 

 

3333

46

5

1.8

42, 46, 51

 

 

5000

18

6

0.7

12, 21, 22

 

2NF

2.0

286

3

11.2

288, 283, 288

TA100

Water

-

101

12

-

105, 111, 88

 

Test substance

333

99

10

1.0

102, 107, 88

 

 

667

94

6

0.9

87, 96, 98

 

 

1000

102

10

1.0

107, 109, 91

 

 

3333

103

4

1.0

102, 99, 107

 

 

5000

96

7

0.9

100, 99, 88

 

4NQO

0.5

3525

59

34.8

3485, 3592, 3497

TA1535

Water

-

11

5

-

16, 11, 7

 

Test substance

333

12

6

1.1

13, 18, 6

 

 

667

11

2

1.0

9, 11, 13

 

 

1000

14

3

1.3

16, 16, 11

 

 

3333

12

4

1.0

7, 15, 13

 

 

5000

14

4

1.2

9, 17, 16

 

MNNG

0.5

217

32

19.1

205, 253, 192

TA1537

Water

-

5

5

-

10, 4, 1

 

Test substance

333

6

2

1.1

6, 4, 7

 

 

667

6

3

1.3

6, 9, 4

 

 

1000

5

3

1.1

2, 7, 7

 

 

3333

8

3

1.6

11, 6, 7

 

 

5000

6

2

1.3

9, 5, 5

 

ICR

0.2

4695

30

938.9

4717, 4706, 4661

MMNG: N-Methyl, N’-Nitro-Nitrosoguanidine

ICR: Acridine ICR 191

4NQO: 4-Nitroquinoline-oxide

2NF: 2-Nitrofluorene

 

Mutagenicity test with S9 activation

Strain

Test substance

Dose level

µg/plate

Mean revertants/ plate

Standard deviation

Ratio treated/ solvent

Individual revertant colony counts and plate codes

WP2urvA

Water

-

52

4

-

49, 51, 56

 

Test substance

333

50

12

1.0

54, 36, 59

 

 

667

38

3

0.7

34, 40, 40

 

 

1000

45

2

0.9

45, 47, 43

 

 

3333

44

7

0.9

36, 49, 48

 

 

5000

48

8

0.9

40, 48, 55

 

2AA

25

222

22

4.3

239, 198, 230

TA98

Water

-

28

4

-

24, 31, 29

 

Test substance

333

30

5

1.1

33, 24, 33

 

 

667

21

10

0.8

33, 13, 18

 

 

1000

24

12

0.8

17, 37, 17

 

 

3333

27

6

1.0

34, 24, 24

 

 

5000

31

2

1.1

29, 32, 32

 

2AA

10

1357

85

48.5

1285, 1450, 1335

TA100

Water

-

106

16

-

92, 103, 124

 

Test substance

333

120

11

1.1

119, 131, 109

 

 

667

122

17

1.2

132, 103, 132

 

 

1000

122

12

1.1

135, 118, 113

 

 

3333

118

19

1.1

111, 14, 103

 

 

5000

129

21

1.2

136, 146, 105

 

2AA

10

2760

815

26.0

2404, 2183, 3692

TA1535

Water

-

17

1

-

17, 18, 16

 

Test substance

333

13

3

0.8

15, 10, 15

 

 

667

14

3

0.8

16, 15, 11

 

 

1000

18

3

1.1

20, 20, 15

 

 

3333

12

3

0.7

9, 13, 15

 

 

5000

17

1

1.0

16, 17, 18

 

2AA

10

126

10

7.4

119, 138, 121

TA1537

Water

-

10

3

-

12, 12, 6

 

Test substance

333

6

5

0.6

10, 6, 1

 

 

667

10

1

1.0

11, 10, 9

 

 

1000

9

2

0.9

10, 11, 7

 

 

3333

11

2

1.1

10, 10, 13

 

 

5000

12

3

1.2

13, 15, 9

 

2AA

10

87

9

8.7

77, 91, 93

2AA: 2-Amino-anthracene

 

 

Applicant's summary and conclusion

Conclusions:
The test substance was negative with or without S-9 activation.
Executive summary:

The test substance was evaluated for mutagenicity in the Bacterial Reverse Mutation Test using the treat and plate modification of the pre-incubation method. Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 and Escherichia coli strain WP2uvrA were tested in the absence and presence of an exogenous metabolic activation system (Aroclor-induced rat liver S9) in accordance with OECD Guideline 471. The test was performed in 2 phases. The first phase was the toxicity-mutation test, which established the dose range for the mutagenicity test, and provided a preliminary mutagenicity evaluation. The second phase was the mutagenicity test, which evaluated and confirmed the mutagenic potential of the test substance. The sponsor reported the total protein of the test substance to be 222.71 mg/mL. All test substance concentrations were prepared based on the total protein concentration. The test substance was dispensed volumetrically and formulated in sterile water. The tests substance formed a clear amber solution in sterile water at 50 mg of total protein /mL, the highest stock concentration prepared for use on this study. All reported dose concentrations represent the amount of total protein per unit.

In the toxicity-mutation test, the maximum dose evaluated was 5000 μg/plate for tester strains TA98, TA100, TA1535, TA1537, and WP2uvrA in the absence and presence of S9 metabolic activation. This dose was achieved using a concentration of 50 mg/mL and a 100 μL plating aliquot. The dose levels used in this test were 33.3, 66.7, 100, 333, 667, 1000, 3333, and 5000 μg/plate. No positive mutagenic responses were observed at any dose level in any tester strain in the absence or presence of S9 metabolic activation. No appreciable toxicity or test substance precipitation was observed.

Based on the toxicity-mutation test, the maximum dose evaluated in the mutagenicity test was 5000 μg/plate for tester strains TA98, TA100, TA1535, TA1537, and WP2uvrA in the absence and presence of S9 metabolic activation. This dose was achieved using a concentration of 50 mg/mL and a 100 μL plating aliquot. The dose levels used in this test were 333, 667, 1000, 3333, and 5000 μg/plate for all tester strains. No positive mutagenic responses were observed at any dose level or with any tester strain in either the absence or presence of S9 metabolic activation. No appreciable toxicity or test substance precipitation was observed.

All criteria for a valid study were met. Under the conditions of this study, the test substance showed no evidence of mutagenicity in the Bacterial Reverse Mutation Test either in the absence or presence of Aroclor-induced rat liver S9. It was concluded that the test substance was negative in this in vitro test.