Proteinase, Aspergillus acid

Genetic toxicity: in vitro

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 guideline

Test guideline 1

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.

Test guideline 2

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.

Test guideline 3

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.

Test guideline 4

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

Test material information

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 / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

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 results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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.