Proteinase, Aspergillus acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The present test substance, aspergillopepsin I, has been investigated in two in vitro test systems, the Ames test and the in vitro chromosome aberration test. No evidence for genetic toxicitywas observed.  The results are supported by read-across from in vitro gene mutation studies in L5178Y mouse lymphoma cells performed with mucorpepsin.

Link to relevant study records

Reference

Reference 1

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

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

Target gene:

histidine or tryptophan operons

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

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.

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.

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

 

 

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

other: chromosome aberration in human lymphocytes

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

Primary human lymphocytes were obtained from 2 healthy, non-smoking, male volunteers. One volunteer provided the lymphocytes for the first test, and the other volunteer provided the lymphocytes for the second test. The lymphocytes were cultured in flasks containing whole blood (98%) and phytohaemagglutinin solution (1%) in culture medium. The culture medium was RPMI 1640 medium, supplemented with foetal calf serum (10%) and Gentamycin (45 µg/mL). The cells were cultured in sterile plastic culture flasks in an incubator at 37°C with an atmosphere containing 5% carbon dioxide in air.

The cell cycle times for the lymphocytes from each of the donors under the culture conditions employed in this study were determined previously. The cell cycle times for the 2 donors were found to be 15.7 and 13.9 hours; thus the sampling time of 20 hours corresponded to approximately 1.5 cell cycles.

Metabolic activation:

with and without

Metabolic activation system:

ARoclor-1254-induced rat liver

Test concentrations with justification for top dose:

First test (with and without S-9 mix): 39.1, 78.1, 156, 313, 625, 1250, 2500, 5000 μg/mL
Second test (without S-9 mix): 78.1, 156, 313, 625, 1250, 2500, 5000 μg/mL
Second test (with S-9 mix): 313, 625, 1250, 2500, 5000 μg/mL

5000 μg/mL is the maximum required by the OECD 473 guideline for material of low toxicity.

Vehicle / solvent:

test substance medium

Untreated negative controls:

yes

Remarks:

cell culture medium

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: daunomycin at 0.015 μg/mL (without S-9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION: the cultures were incubated at 37°C with gentle mixing for the appropriate treatment period. After 3 hours of treatment the appropriate cultures were centrifuged. The supernatant treatment medium was removed and the cells were resuspended in fresh medium (5 mL. The cultures were then incubated at 37°C for a further 17 hours until harvest. In the second test, cultures without S-9 mix were treated for 20 hours until harvest. Demecolcine (final concentration 0.1 µg/mL) was added to each culture for the last 2 hours before harvest.

CELL HARVEST: At the 20-hour harvest time, each culture was centrifuged and the supernatant was removed. The cell pellet was resuspended in warm 0.075 M KCl (37°C, 5 mL) and incubated at 37°C for 10 minutes. The cell suspensions were then centrifuged again and the supernatants were removed.

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The cells were fixed by addition of fixative (methanol/acetic acid, 3:1), washed 2 or 3 times with fresh fixative, and dropped onto clean glass slides. After drying in air, the chromosome preparations were stained in 3% Giemsa for 10 minutes. Cover glasses were mounted with Dammarxylen®.

NUMBER OF CELLS EVALUATED: The number of cells at metaphase was counted in 1000 cells from each culture.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: 100 metaphases with 46±2 chromosomes from most cultures were examined for the presence or absence of chromosomal aberrations. The scoring of the positive control slides was stopped before 100 metaphases had been scored when a large number of metaphases with aberrations had been observed (15/culture, excluding gaps). The aberrations were recorded as gaps, breaks, or exchanges, and classified as chromatid- or chromosome-type in each case. When the origin of a fragment was clear, it was recorded under that category. When the origin of the fragment was not clear, it was recorded as a chromatid break. Metaphases with 5 or more aberrations (excluding gaps) were recorded as showing multiple damage. Pulverised metaphases with gross fragmentation of the DNA were recorded separately and not included in the total of 100 scored for chromosomal aberrations because it was not possible to count the chromosomes. The Vernier co-ordinates of at least 5 metaphases were recorded for each culture.

DETERMINATION OF CYTOTOXICITY
- Method: The mitotic index was calculated as the percentage of cells at metaphase. mitotic index; cloning efficiency; relative total growth; other:
- Any supplementary information relevant to cytotoxicity:

OTHER EXAMINATIONS:
- Determination of polyploidy: The number of polyploid metaphases in 200 metaphases was counted for each culture. Polyploid metaphases were defined as metaphases with multiples of the haploid chromosome number (n), other than the diploid number (i.e., 3n, 4n, etc.)
- Determination of endoreplication: The numbers of endoreduplicated metaphases in 200 metaphases was counted for each culture. Endoreduplicated metaphases had chromosomes with 4, 8 chromatids. Marked reductions in the numbers of cells on the slides were recorded, if seen.

Evaluation criteria:

The test was considered valid if the negative control cultures showed a low frequency of metaphases with aberrant chromosomes and the positive controls showed clear increases in the frequency of metaphases with aberrant chromosomes.

It was concluded that the test substance had shown clastogenic activity if all of the following criteria had been met: (1) increases in the frequency of metaphases with aberrant chromosomes were observed at one or more test concentrations, (2) the increases were reproducible between replicate cultures and between tests (when treatment conditions were the same), (3) the increases were statistically significant, (4) the increases were not associated with large changes in pH or osmolarity of the treated cultures. The historical negative control data for the test facility was also considered in the evaluation. Evidence of a dose-response relationship would have been considered to support the conclusion. The test item was considered negative if no reproducible, statistically significant increases were observed. Results which failed to meet the stated criteria for a negative or positive response were considered equivocal.

Statistics:

The number of metaphases with aberrant chromosomes at each test concentration was compared to the concurrent negative control value. When appropriate, statistical analysis was performed using Fischer’s Exact Test. Gap-type aberrations were recorded during metaphase analysis and reported in the tables, but they were omitted form the statistical analysis because gaps are of questionable biological significance.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

dose-related toxicity in the absence and presence of S-9 mix in both tests

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test item caused dose-related toxicity in the absence and presence of S-9 mix in both tests: dose-related reductions in mean mitotic index were observed. Slides from cultures treated with the test item at the following concentrations were selected for the scoring of chromosomal aberrations: 625, 1250, and 2500 µg/mL (first test, without S-9 mix); 1250, 2500, and 5000 µg/mL (first test, with S-9 mix); 313, 625, and 1250 µg/mL (second test, without S-9 mix); 1250, 2500, 5000 µg/mL (second test, without S-9 mix).

In the absence of S-9 mix, the test item caused reductions in mean mitotic index of 52 and 53% at the highest concentrations scored for chromosomal aberrations in the first and second tests, respectively. This level of toxicity met the requirements of the OECD 473 guideline for the highest concentration to be scored for aberrations (greater than 50% reduction in mean mitotic index). The test item caused less toxicity in the presence of S-9 mix: reductions in mean mitotic index of 28 and 33% were observed at 5000 µg/mL in the first and second tests, respectively. The test concentration (5000 µg/mL) is the maximum required by the OECD 473 guideline for materials of low toxicity.

No biologically or statistically significant increases in the frequency of metaphases with chromosomal aberrations were observed in cultures treated with the test item. The frequencies of metaphases with chromosome aberrations in the negative and positive control cultures were within the normal ranges and compatible with the historical control data for the testing laboratory. The values for the positive control treatments without S-9 mix exceeded the historical control range for the previous 10 studies, but they were considered to be acceptable. The positive control treatments produced large, statistically significant increases in the frequency of aberrant metaphases in both tests, demonstrating the sensitivity of the tests and the efficacy of the S-9 mix. Two polyploid metaphases were observed in the study, but their incidence was not dose-related and it was concluded that they were not caused by the test item. No endoreduplicated metaphases were observed.

Test 1 Results for Treatments Without S-9 Mix
Treatment (µg/mL)	Mitotic Index	Redn. in mean MI (%)	No. aberrant metaphases		Number and types of aberrations observed								Pol	E
					Gaps		Breaks		Exchanges		M	Pul
					Ct	Cs	Ct	Cs	Ct	Cs
Vehicle	6.4	0	1	-			1
	6.7		0
TA (5000)	1.1	71	These slides were not selected for metaphase analysis.
	2.7
TA (2500)	3.2	52	1	NS	1		1
	3.1		2		1		1	1
TA (1250)	5.3	28	1	NS			1
	4.1		2				1	1
TA (625)	6.8	14	2	NS				2
	4.5		1				1
TA (313)	6.1	2 Inc	These slides were not selected for metaphase analysis.
	7.3
TA (156)	6.8	2	These slides were not selected for metaphase analysis.
	6.1
TA (78.1)	6.3	8	These slides were not selected for metaphase analysis.
	5.7
TA (39.1)	6.1	4	These slides were not selected for metaphase analysis.
	6.5												1
PC (0.015)	4.3	27	15a	**			4	4	17		4
	5.3		15b				7	2	21	2	3
Mitotic index: Percentage of cells at metaphase Redn. in mean MI (%): Percentage reduction in mean mitotic index for duplicate cultures compared to the negative control (Inc = Increase) No. aberrant metaphases; Number of metaphases with aberrant chromosomes (excluding gaps) 9Statistical analysis was performed on the total from both cultures) NS: not statistically significant, p>0.05, compared to negative control ** Statistically significant, p<0.01, compared to negative control Ct: Chromatid-type aberration Cs: Chromosome-type aberration M: Number of metaphases with multiple aberrations (>5, excluding gaps) Pul: Number of pulverized metaphases Pol: Number of polyploid metaphases, 200 metaphases scored where possible E: No. endoreduplicated metaphases, 200 metaphases scored where possible Vehicle: Vehicle control – Cell culture medium TA: Test substance PC: Positive control – Daunomycin a Only 30 metaphases were scored for this culture (15 aberrant = 50%) b Only 27 metaphases were scored for this culture (15 aberrant = 56%)

 

Test 1 Results for Treatments With S-9 Mix
Treatment (µg/mL)	Mitotic Index	Redn. in mean MI (%)	No. aberrant metaphases		Number and types of aberrations observed								Pol	E
					Gaps		Breaks		Exchanges		M	Pul
					Ct	Cs	Ct	Cs	Ct	Cs
Vehicle	5.4	0	1	-			1	1
	6.1		2				2		1
TA (5000)	4.5	28	2	NS			1		1
	3.8		1				1						1
TA (2500)	5.8	1Inc	3	NS			1	4
	5.8		1					1
TA (1250)	5.1	8	1	NS			1
	5.5		3				1	1		1
TA (625)	4.9	11	These slides were not selected for metaphase analysis.
	5.3
TA (313)	6.2	3 Inc	These slides were not selected for metaphase analysis.
	5.7
TA (156)	5.1	5	These slides were not selected for metaphase analysis.
	5.8
TA (78.1)	6.2	10 Inc	These slides were not selected for metaphase analysis.
	6.4
TA (39.1)	5.2	3	These slides were not selected for metaphase analysis.
	5.9
PC (0.06)	1.6	70	15a	**			11	1	5
	1.9		15b				11	1	2		2
Mitotic index: Percentage of cells at metaphase Redn. in mean MI (%): Percentage reduction in mean mitotic index for duplicate cultures compared to the negative control (Inc = Increase) No. aberrant metaphases; Number of metaphases with aberrant chromosomes (excluding gaps) 9Statistical analysis was performed on the total from both cultures) NS: not statistically significant, p>0.05, compared to negative control ** Statistically significant, p<0.01, compared to negative control Ct: Chromatid-type aberration Cs: Chromosome-type aberration M: Number of metaphases with multiple aberrations (>5, excluding gaps) Pul: Number of pulverized metaphases Pol: Number of polyploid metaphases, 200 metaphases scored where possible E: No. endoreduplicated metaphases, 200 metaphases scored where possible Vehicle: Vehicle control – Cell culture medium TA: Test substance PC: Positive control – Daunomycin a Only 30 metaphases were scored for this culture (15 aberrant = 50%) b Only 27 metaphases were scored for this culture (15 aberrant = 56%)

 

Test 2 Results for Treatments Without S-9 Mix
Treatment (µg/mL)	Mitotic Index	Redn. in mean MI (%)	No. aberrant metaphases		Number and types of aberrations observed								Pol	E
					Gaps		Breaks		Exchanges		M	Pul
					Ct	Cs	Ct	Cs	Ct	Cs
Vehicle	8.2	0	1	-	3		1
	8.9		0		2
TA (5000)	1.2	89	These slides were not selected for metaphase analysis.
	0.7
TA (2500)	2.1	72	These slides were not selected for metaphase analysis.
	2.7
TA (1250)	5.0	53	2	NS			1		1
	3.1		1		2				1
TA (625)	6.8	25	5	NS	4		3	1	2
	6.1		1		2			1
TA (313)	7.8	15	3	NS	1		3
	6.8		0		3
TA (156)	8.1	12	These slides were not selected for metaphase analysis.
	6.9
TA (78.1)	7.8	11	These slides were not selected for metaphase analysis.
	7.4
PC (0.015)	5.1	46	15a	**	3		6	3	3		5
	4.2		15b		10		13	3	10		2
Mitotic index: Percentage of cells at metaphase Redn. in mean MI (%): Percentage reduction in mean mitotic index for duplicate cultures compared to the negative control (Inc = Increase) No. aberrant metaphases; Number of metaphases with aberrant chromosomes (excluding gaps) 9Statistical analysis was performed on the total from both cultures) NS: not statistically significant, p>0.05, compared to negative control ** Statistically significant, p<0.01, compared to negative control Ct: Chromatid-type aberration Cs: Chromosome-type aberration M: Number of metaphases with multiple aberrations (>5, excluding gaps) Pul: Number of pulverized metaphases Pol: Number of polyploid metaphases, 200 metaphases scored where possible E: No. endoreduplicated metaphases, 200 metaphases scored where possible Vehicle: Vehicle control – Cell culture medium TA: Test substance PC: Positive control – Daunomycin a Only 30 metaphases were scored for this culture (15 aberrant = 50%) b Only 27 metaphases were scored for this culture (15 aberrant = 56%)

 

Test 2 Results for Treatments With S-9 Mix
Treatment (µg/mL)	Mitotic Index	Redn. in mean MI (%)	No. aberrant metaphases		Number and types of aberrations observed								Pol	E
					Gaps		Breaks		Exchanges		M	Pul
					Ct	Cs	Ct	Cs	Ct	Cs
Vehicle	6.3	0	1	-			1
	6.2		1		1		1
TA (5000)	4.5	33	1	NS			1
	3.9		0		1
TA (2500)	5.9	4	1	NS	1		1
	6.1		1					1
TA (1250)	6.3	7 Inc	0	NS	1
	7.1		2		2		1	2
TA (625)	6.8	1 Inc	These slides were not selected for metaphase analysis.
	5.8
TA (313)	6.1	2	These slides were not selected for metaphase analysis.
	6.1
PC (0.06)	2.1	58	15a	**	7		17	5		1
	3.1		15b		6	3	21	3	1
Mitotic index: Percentage of cells at metaphase Redn. in mean MI (%): Percentage reduction in mean mitotic index for duplicate cultures compared to the negative control (Inc = Increase) No. aberrant metaphases; Number of metaphases with aberrant chromosomes (excluding gaps) 9Statistical analysis was performed on the total from both cultures) NS: not statistically significant, p>0.05, compared to negative control ** Statistically significant, p<0.01, compared to negative control Ct: Chromatid-type aberration Cs: Chromosome-type aberration M: Number of metaphases with multiple aberrations (>5, excluding gaps) Pul: Number of pulverized metaphases Pol: Number of polyploid metaphases, 200 metaphases scored where possible E: No. endoreduplicated metaphases, 200 metaphases scored where possible Vehicle: Vehicle control – Cell culture medium TA: Test substance PC: Positive control – Daunomycin a Only 30 metaphases were scored for this culture (15 aberrant = 50%) b Only 27 metaphases were scored for this culture (15 aberrant = 56%)

Conclusions:

The test substance did not cause chromosomal aberrations using cultured human lymphocytes either in the absence or presence of S-9 mix.

Executive summary:

The test item was tested in primary cultures of human lymphocytes in the absence and presence of S-9 mix in accordance with OECD Guideline 473. The cultures were treated with formulations of the test item mixed with cell culture medium. Two tests were performed. In the first test, all cultures were treated for 3 hours. In the second test, the cultures were treated for 20 hours without S-9 mix and 3 hours with S-9 mix. All cultures were harvested 20 hours (approximately 1.5 normal cell cycles) after the start of treatment. The final concentration of S-9 homogenate used in the second test was twice as high as in the first test.

The test item caused dose-related toxicity to the test cells: reductions in mean mitotic index were observed at the higher concentrations tested. Slides from cultures treated with the test item at the following concentrations were scored for chromosomal aberrations: 625, 1250, and 2500 µg/mL without S-9 mix in the first test; 313, 625, and 1250 µg/mL without S-9 mix in the second test; and 1250, 2500, and 5000 µg/mL with S-9 mix in both tests. All of the concentrations reported are expressed in terms of the total protein content of the test item (stated by the Sponsor to be 124.7 mg/mL in the solution supplied). The highest concentrations scored for chromosomal aberrations in the absence of S-9 mix showed the appropriate level of toxicity (greater than 50%reductions in mean mitotic index), while the highest concentration in the presence of S-9 mix (5000 µg/mL) was the maximum required by the OECD 473 guideline for materials of low toxicity. No biologically or statistically significant increase in the frequency of metaphases with chromosomal aberrations were observed in cultures treated with the test item in either test. The positive control treatments produced large, statistically significant increases in the frequency of aberrant metaphases, demonstrating the sensitivity of the test and the efficacy of the S-9 mix. Two polyploid metaphases were observed in the study, but their incidence was not dose-related and it is concluded that they were not caused by the test item. No endoreduplicated metaphases were observed. It is concluded that the test substance did not cause chromosomal aberrations in this in vitro cytogenetic test using cultured human lymphocytes either in the absence or presence of S-9 mix.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 16 1991 - December 20 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT (6-thioguanine resistance)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Cells were obtained from the American Type Culture Collection.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Highest concentration tested was 5000 µg/mL and dilutions hereof (2500, 1250, 625, 312.5, 156.25 µg/mL).

Vehicle / solvent:

Vehicle for enzyme: Purified water.
Justification for choice of solvent/vehicle: Substance is water-soluble and any human exposure will be in aqueous solutions.
Solvent for the positive control: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 2 hours
- Expression time (cells in growth medium): 2 days

DETERMINATION OF CYTOTOXICITY
- Method: The plates were incubated at 37°C in a humidified incubator gassed with 5% v/v CO2 in air for 1-2 weeks. Wells containing viable clones were identified by eye using background illumination and counted.

Evaluation criteria:

Evaluation criteria
The test substance was considered to be mutagenic if:
1) the assay was valid.
2) the mutant frequency at 1 or more doses was significantly greater than that of the negative control.
3) there was a significant dose-relationship as indicated by the linear trend analysis.
4) the effects described above are reproducible.

Acceptance criteria
The assay was considered valid if the following criteria were met:
1) the mutant frequencies in the negative (solvent) control cultures fell within the normal range.
2) at least 1 concentration of each of the positive control chemicals induced a clear increase in mutant frequency.

Statistics:

Statistical significance of mutant frequencies (total wells with clones) was carried out according to the UKEMS guidelines. Thus, the control log mutant frequency (LMF) was compared with the LMF from each treatment dose, and secondly the data was checked for a linear trend in mutant frequency with treatment dose. These tests required the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not specified
- Effects of osmolality: There was no significant increase in osmolality (>50 mOsm/kg) in cultures treated with rennilase at the top dose of 5000 μg/mL.
- Evaporation from medium: Not specified
- Water solubility: The enzyme is water soluble
- Precipitation: Could be a confounding factor, but not an issue in this study.

RANGE-FINDING/SCREENING STUDIES: 15.8 - 5000 μg/mL

HISTORICAL CONTROL DATA
Sufficient data using identical test conditions were not available to enable historical mean values to be quoted. However, the mutant frequencies obtained in this study for the negative control cultures were in line with those obtained for recent assays performed in this laboratory. Furthermore, the positive control chemicals induced a clear increase in mutant frequency at all doses tested.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: The percentage relative survival (%RS) in each test culture was therefore determined by comparing plating efficiencies in test and control cultures thus:
%RS = [PE (test)/PE (control)] x 100

In the presence of S-9 no statistically significant increases were obtained in Experiment 1 or 2 following rennilase treatment. Although there were no significant increases in mutant frequency at any dose level in Experiment 1, a significant linear trend was indicated in this experiment. However, in Experiment 2, rennilase was tested over the same dose range and no linear trend was obtained. Therefore the dose-relationship indicated in Experiment 1 was not reproducible and is attributed to a chance effect of no biological significance.

Conclusions:

It was concluded that rennilase, under the conditions employed in this study, had no mutagenic activity in the presence and absence of S-9.

Executive summary:

Rennilase was assayed for its ability to induce mutation at the tk locus (5-trifluorothymidine resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a preliminary experiment followed by 2 independent experiments, each conducted in the absence and presence of metabolic activation by an Aroclor 1254 induced rat liver post-mitochondrial fraction (S-9). The preliminary experiment established that rennilase did not inactivate the enzymes of the S-9 mix and it could be tested as supplied.

Following a wide range of treatments, separated by 2-fold intervals and reaching 5000 μg/mL, cells survived all doses of rennilase in Experiment 1 yielding 98.6% relative survival in the absence and 101 .4% relative survival in the presence of S-9. Accordingly, the top 5 doses were plated for viability and 5- trifluorothymidine resistance 2 days after treatment. In the second experiment, the same dose range was selected, excluding the bottom dose. The top dose plated in this experiment was again 5000 μg/mL in the absence and presence of S-9, which yielded 58.1% and 77.4% relative survival, respectively. All 5 doses were plated for the determination of mutant frequency.

Negative (solvent) and positive control treatments were included in each experiment in the absence and presence of S-9. Mutant frequencies in negative control cultures fell within normal ranges, and statistically significant increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (without S-9) and benzo(a)pyrene (with S-9). Therefore, the study was accepted as valid.

In the absence of S-9, no significant increases in mutant frequency were observed following treatment with rennilase at any dose level in either experiment. Similarly, in the presence of S-9 no statistically significant increases were obtained in Experiment 1 or 2 following rennilase treatment. Although there were no significant increases in mutant frequency at any dose level in Experiment 1, a significant linear trend was indicated in this experiment. However, in Experiment 2, rennilase was tested over the same dose range and no linear trend was obtained. Therefore the dose-relationship indicated in Experiment 1 was not reproducible and is attributed to a chance effect of no biological significance.

It was concluded that rennilase, under the conditions employed in this study, had no mutagenic activity in the presence and absence of S-9.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

other information

Justification for type of information:

According to the ECHA Guidance Chapter R 7a: Endpoint specific guidance (version 2, Nov 2012), the following studies on genetic toxicity are required: In vitro gene mutation study in bacteria and one of the following, in vitro cytogenicity study in mammalian cells or an in vitro micronucleus study. In case these studies are both negative, an in vitro gene mutation study in mammalian cells is requested in addition. The present test substance, aspergillopepsin I, has been investigated in two in vitro test systems, the Ames test and the in vitro chromosome aberration test. The tests have been performed according to current OECD guidelines, and in compliance with GLP. No evidence for genetic toxicitywas observed. The results are supported by read-across from in vitro gene mutation studies in L5178Y mouse lymphoma cells performed with mucorpepsin.
The safety of the production strain is fully documented to belong to a safe strain lineage (Pariza and Johnson, 2001; Enzymes REACH Consortium, 2009) and the enzyme test material was well characterized. All enzyme classes are hydrophilic and readily biodegradable and in general, non-protease enzymes exhibit the same toxicological properties and although they are potential respiratory sensitizers, they are considered to be of low toxicity, confirmed by toxicity studies performed and published by the industry (summarized in Basketter et al. 2012a and 2012b). The physico-chemical properties of enzymes including logPow are very similar. They are further proteins built up of amino acids and the type, order and number of the amino acids in the polymer differs between enzymes, determining the 3-dimensional structure, the activity and specificity of the individual enzyme type. Industrial production strains typically have a history of safe use for many years in the production of technical and also often food grade enzymes. Because all enzymes are built up of the same amino acids the physical and chemical characteristics will be very similar for different enzymes, and hence read-across from other enzymes should be fully applicable. The overall conclusion is that beta-glucanase is not mutagenic and does not induce genotoxicity in the present test systems.
References - Pariza, M. W., and Johnson, E. A. (2001). Evaluating the Safety of Microbial Enzyme Preparations Used in Food Processing: Update for a New Century. Regulatory Toxicology and Pharmacology, 33: 173-186. - Enzymes REACH Consortium: Safety evaluation of technical enzyme products with regards to the REACH legislation. Document from Manufacturers, Importers and/or Only Representatives of one or more enzymes, who are subject to the registration requirements pursuant to REACH, 2009. http://www.enzymes-reach.org/documents.html - D. Basketter; N. Berg; F. Kruszewski; K. Sarlo; B. Concoby. The Toxicology and Immunology of Detergent Enzymes. 2012a. J. Immunotox 9(3): 320-6. - Basketter D., Berg N., Broekhuizen C., Fieldsend M., Kirkwood S., Kluin C., Mathieu S. and Rodriguez C. Enzymes in Cleaning Products: An Overview of Toxicological Properties and Risk Assessment/Management. 2012b. Reg. Toxicol. Pharmacol, 64/1: 117-123

Reason / purpose for cross-reference:

read-across source

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Not classified.