Registration Dossier

Administrative data

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

Referenceopen allclose all

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.

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.

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.

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

Additional information

Justification for classification or non-classification

Not classified.