[No public or meaningful name is available]

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Harpin-αß protein

IUPAC Name:

Harpin-αß protein

Test material form:

liquid

Details on test material:

The test material is identical to the registered substance, 'Cell Free Harpin Extract of Harpinαβ produced by fermentation'.

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

Method

Species / strain

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.

Controls

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.

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

Conclusions:

The test item, Harpin-ab Fermentation Extract is considered to be non-mutagenic.