Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a reliable GLP OECD Guideline 471 study, Harpin-ab Fermentation Extract was tested in a bacterial reverse mutation assay in Salmonella typhimurium (strains TA 98, TA 100, TA 102, TA 1535 and TA 1537) with and without metabolic activation (S9). The results show that Harpin-ab Fermentation Extract did not cause a positive increase in the mean number of revertants per plate with any of the tester strains either in the presence or absence of S9.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 February - 09 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
TEST MATERIAL
- Lot No. of test material: R134-2
- Expiration date of the lot: 17 July 2018
- Appearance: Tan, light brown liquid
- Active components: Harpin-ab proteins 0.4% in aqueous solution
- Storage: <-70°C, protected from light
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: MOLTOX, INC., NC 28607, USA (for TA 98, TA 1535 and TA 102) and Xenometrix AG, Switzerland (for TA 100 and TA 1537)
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 liver microsomal fraction
Test concentrations with justification for top dose:
The test item was dissolved in distilled water and diluted prior to treatment.

The following test concentrations were used: 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 µL/plate

The test item concentrations to be applied in the main experiment were chosen according to the results of the pre-experiment (which used test concentrations of 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 µL/plate).
Vehicle / solvent:
- Vehicle/ solvent used: water
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.
Untreated negative controls:
no
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: TA 98 and TA 1537 (without S9); 10 µg/plate for TA and 40 µg/plate for TA 1537; 2-aminoanthracene: TA 98, TA 100, TA 1535, TA 1537 and TA 102 (with S9); 2.5 µg/plate and 10 µg/plate for TA 102
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation test - EXPERIMENT I; pre-incubation test - EXPERIMENT II

DURATION
- Preincubation period: 60 minutes at 37°C
- Exposure duration: at least 48 hours at 37°C in the dark

NUMBER OF REPLICATIONS: For each strain and dose level, including the controls, three plates were used.

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity can be detected by a clearing or diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤0.5 in relation to the solvent control.
Evaluation criteria:
A test item is considered 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 trater strain with or without metabolic activation.

A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- 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.
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:
no cytotoxicity
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
Key result
Species / strain:
S. typhimurium TA 102
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:
RESULTS: See Tables 5 and 6

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item was observed in any tester strain used in experiments I and II (with and without metabolic activation).

HISTORICAL CONTROL DATA
- Positive historical control data: See Tables 2 and 4
- Negative historical control data: See Tables 1 and 3

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity: No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated with and without metabolic activation in experiment I and II.

Table 1: Historical laboratory control data of the negative control (in 2014 – 2016) without S9

 

TA 98

TA 100

TA 1535

TA 1537

TA 102

Mean

24.2

90.7

13.8

8.2

270.4

SD

6.7

15.6

6.7

2.9

55.0

Min

11

49

4

3

141

Max

58

155

41

35

472

RSD (%)

27.7

17.2

48.6

35.3

20.3

n

972

1191

929

931

682

 

Table 2: Historical laboratory control data of the positive control (in 2014 – 2016) without S9

 

TA 98

TA 100

TA 1535

TA 1537

TA 102

Substance

Conc/plate

4-NOPD

10 µg

NaN3

10 µg

NaN3

10 µg

4-NOPD

40 µg

MMS

1 µL

Mean

430.7

612.1

792.0

94.5

1729.2

SD

155.5

220.0

299.5

22.7

518.8

Min

141

132

38

35

272

Max

1830

1423

1854

273

3321

RSD (%)

36.1

35.9

37.8

24.0

30.0

n

971

1188

931

929

682

 

Table 3: Historical laboratory control data of the negative control (in 2014 – 2016) with S9

 

TA 98

TA 100

TA 1535

TA 1537

TA 102

Mean

29.0

96.4

10.5

8.3

339.7

SD

6.8

14.1

4.5

3.1

71.3

Min

15

62

3

3

157

Max

59

160

38

36

586

RSD (%)

23.4

14.6

42.7

37.4

21.0

n

967

1189

925

926

676

 

Table 4: Historical laboratory control data of the positive control (in 2014 – 2016) with S9

 

TA 98

TA 100

TA 1535

TA 1537

TA 102

Substance

Conc/plate

2-AA

2.5 µg

2-AA

2.5 µg

2-AA

2.5 µg

2-AA

2.5 µg

2-AA

10 µg

Mean

1880.5

1727.0

133.9

234.1

801.2

SD

708.5

522.0

134.9

101.4

223.7

Min

70

169

22

26

137

Max

3606

3132

1954

682

3588

RSD (%)

37.7

30.2

100.8

43.3

27.9

n

966

1184

927

925

678

 

Table 5: Results Experiment I (plate incorporation test):

Treatment

Dose/plate

Revertant colonies per plate

Mutation factor

Without S9

With S9

Mean

SD

Mean

SD

-S9

+S9

TA 98

Water

-

33

7.9

26

9.3

1.0

1.0

Test item

0.0316 µL

31

4.9

24

7.9

0.9

0.9

Test item

0.100 µL

34

4.7

32

2.5

1.0

1.2

Test item

0.316 µL

31

4.2

31

3.5

0.9

1.2

Test item

1.0 µL

29

7.4

30

8.2

0.9

1.2

Test item

2.5 µL

37

2.6

28

4.5

1.1

1.1

Test item

5.0 µL

33

1.5

32

3.6

1.0

1.2

4-NOPD

10 µg

480

16.0

-

-

14.6

-

2-AA

2.5 µg

-

-

1656

170.0

-

64.5

TA 100

Water

-

96

10.4

92

8.5

1.0

1.0

Test item

0.0316 µL

93

2.5

103

23.8

1.0

1.1

Test item

0.100 µL

98

1.2

91

1.0

1.0

1.0

Test item

0.316 µL

80

11.0

101

3.6

0.8

1.1

Test item

1.0 µL

99

17.0

109

13.2

1.0

1.2

Test item

2.5 µL

95

7.2

102

5.2

1.0

1.1

Test item

5.0 µL

111

17.0

104

14.0

1.2

1.1

NaN3

10 µg

891

6.7

-

-

9.3

-

2-AA

2.5 µg

-

-

1933

132.5

-

21.0

TA 1535

Water

-

20

2.5

22

1.5

1.0

1.0

Test item

0.0316 µL

20

2.5

20

2.5

1.0

0.9

Test item

0.100 µL

21

1.5

20

2.5

1.0

0.9

Test item

0.316 µL

22

2.3

21

2.6

1.1

1.0

Test item

1.0 µL

19

2.1

22

2.6

0.9

1.0

Test item

2.5 µL

21

2.1

19

1.0

1.0

0.9

Test item

5.0 µL

20

1.0

22

4.4

1.0

1.0

NaN3

10 µg

679

18.0

-

-

33.4

-

2-AA

2.5 µg

-

-

280

18.0

-

12.9

TA 1537

Water

-

19

4.0

20

1.2

1.0

1.0

Test item

0.0316 µL

21

1.5

18

1.7

1.1

0.9

Test item

0.100 µL

19

1.5

19

3.1

1.0

0.9

Test item

0.316 µL

21

1.7

20

2.5

1.1

1.0

Test item

1.0 µL

20

2.6

20

3.6

1.1

1.0

Test item

2.5 µL

19

3.5

17

1.7

1.0

0.9

Test item

5.0 µL

19

6.1

18

3.5

1.0

0.9

4-NOPD

40 µg

121

3.5

-

-

6.5

-

2-AA

2.5 µg

-

-

253

20.0

-

12.9

TA 102

Water

-

357

20.6

386

8.0

1.0

1.0

Test item

0.0316 µL

323

30.7

369

10.4

0.9

1.0

Test item

0.100 µL

330

10.4

389

13.1

0.9

1.0

Test item

0.316 µL

337

19.3

395

18.3

0.9

1.0

Test item

1.0 µL

319

20.5

363

18.6

0.9

0.9

Test item

2.5 µL

326

15.6

377

22.8

0.9

1.0

Test item

5.0 µL

360

23.7

376

2.3

1.0

1.0

MMS

1 µL

2195

438.2

-

-

6.2

-

2-AA

10 µg

-

-

908

161.7

-

2.4

Table 6: Results Experiment II (Pre-incubation test):

Treatment

Dose/plate

Revertant colonies per plate

Mutation factor

Without S9

With S9

Mean

SD

Mean

SD

-S9

+S9

TA 98

Water

-

20

3.6

27

2.5

1.0

1.0

Test item

0.0316 µL

25

4.0

46

34.5

1.2

1.7

Test item

0.100 µL

20

4.0

25

6.5

1.0

1.0

Test item

0.316 µL

24

2.1

21

5.5

1.2

0.8

Test item

1.0 µL

22

6.0

31

5.1

1.1

1.2

Test item

2.5 µL

25

4.2

30

5.0

1.2

1.1

Test item

5.0 µL

34

1.2

28

2.3

1.7

1.0

4-NOPD

10 µg

735

37.4

-

-

36.8

-

2-AA

2.5 µg

-

-

1626

107.2

-

61.0

TA 100

Water

-

124

11.5

98

10.5

1.0

1.0

Test item

0.0316 µL

115

13.9

86

1.5

0.9

0.9

Test item

0.100 µL

117

9.8

96

9.5

0.9

1.0

Test item

0.316 µL

96

1.2

76

3.5

0.8

0.8

Test item

1.0 µL

95

3.5

92

7.0

0.8

0.9

Test item

2.5 µL

109

2.9

113

7.5

0.9

1.1

Test item

5.0 µL

112

11.0

98

10.4

0.9

1.0

NaN3

10 µg

411

24.1

-

-

3.3

-

2-AA

2.5 µg

-

-

288

24.0

-

2.9

TA 1535

Water

-

22

2.0

19

1.7

1.0

1.0

Test item

0.0316 µL

20

2.5

19

2.5

0.9

1.0

Test item

0.100 µL

20

1.5

19

3.0

0.9

1.0

Test item

0.316 µL

20

7.6

19

2.1

0.9

1.0

Test item

1.0 µL

21

2.6

21

2.6

1.0

1.1

Test item

2.5 µL

20

1.0

19

2.0

0.9

1.0

Test item

5.0 µL

21

5.5

18

2.0

1.0

0.9

NaN3

10 µg

684

82.3

-

-

31.1

-

2-AA

2.5 µg

-

-

234

25.5

-

12.3

TA 1537

Water

-

22

1.5

19

1.0

1.0

1.0

Test item

0.0316 µL

23

1.5

21

3.2

1.1

1.1

Test item

0.100 µL

19

1.0

20

2.1

0.9

1.0

Test item

0.316 µL

22

1.5

20

3.1

1.0

1.0

Test item

1.0 µL

21

2.3

24

3.8

1.0

1.2

Test item

2.5 µL

22

4.0

18

1.5

1.0

0.9

Test item

5.0 µL

21

1.7

19

2.1

1.0

1.0

4-NOPD

40 µg

200

47.9

-

-

9.2

-

2-AA

2.5 µg

-

-

173

12.0

-

9.1

TA 102

Water

-

280

6.1

443

29.1

1.0

1.0

Test item

0.0316 µL

268

18.2

415

12.9

1.0

0.9

Test item

0.100 µL

252

4.5

476

27.0

0.9

1.1

Test item

0.316 µL

302

16.1

459

38.9

1.1

1.0

Test item

1.0 µL

276

33.7

451

21.6

1.0

1.0

Test item

2.5 µL

268

17.0

486

14.6

1.0

1.1

Test item

5.0 µL

309

46.6

474

10.0

1.1

1.1

MMS

1 µL

2167

88.3

-

-

7.7

-

2-AA

10 µg

-

-

945

23.0

-

2.1

Conclusions:
The test item, Harpin-ab Fermentation Extract is considered to be non-mutagenic.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

The result of an in vitro gene mutation study in bacteria was negative. Therefore, it is concluded that Harpin-ab Fermentation Extract is not genotoxic and does not warrant classification for mutagenicity in accordance with Regulation (EC) No. 1272/2008.