Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

bacterial reverse mutation assay (Ames test RA from the source substance aldolase, OECD 471, GLP): negative
in-vitro mammalian chromosome aberration test RA from the source substance aldolase
(OECD 473, GLP): positive

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 29-SEP-2004 to 25-OCT-2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The test was performed according to OECD guideline and GLP
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: Cultured peripheral human lymphocytes
Metabolic activation:
with and without
Metabolic activation system:
Rat liver microsomal enzymes were routinely prepared from adult male Wistar rats, which were obtained from Charles River (Sulzfeld, Germany)
Test concentrations with justification for top dose:
First cytogenetic assay:
- With and without S9-mix : 1000, 3330 and 5000 µg Aldolase/ml culture medium (3 h exposure time, 24 h fixation time)

Second cytogenetic assay:
- Without S9-mix : 333, 1000, 1500, 2000,2500 and 3330 µg Aldolase/ml culture medium (24 h exposure time, 24 h fixation time) ; 333, 1000, 2000, 3330, 4000 and 5000 µg Aldolase/ml culture medium (48 h exposure time, 48 h fixation time)
- With S9-mix : 1000, 3330 and 5000 µg Aldolase/ml culture medium (3 h exposure time, 48 h fixation time)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: culture medium
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
culture medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Without metabolic activation: Mitomycin C (0.5 µg/ml for a 3 h exposure period, 0.2 µg/ml for a 24 h exposure period and 0.1 µg/ml for a 48 h exposure period. With metabolic activation: Cyclophosphamid (15 µg/ml for a 3 h exposure period)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: 3, 24 or 48 hours
- Expression time (cells in growth medium): 24 or 48 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 24 or 48 hours


SPINDLE INHIBITOR : colchicine (0.5 µg/ml medium)
STAIN (for cytogenetic assays): 5% (v/v) Giemsa (Merck) solution in tap water


NUMBER OF REPLICATES: two


NUMBER OF CELLS EVALUATED:
- The mitotic index of each culture was determined by counting the number of metaphases per 1000 cells
- 100 metaphase chromosome spreads per culture were examined by light microscopy for chromosome aberrations


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
Evaluation criteria:
A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) it induced a dose-related statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations
b) a statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship

A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations.

The preceding criteria are not absolute and other modifying factors might enter into the final evaluation decision.
Statistics:
The incidence of aberrant cells (cells with one or more chromosome aberrations, inclusive or exclusive gaps) for each exposure group was compared to that of the solvent control using Chi-square statistics.
Species / strain:
lymphocytes:
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
after 48 hours of exposure
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
lymphocytes:
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: the pH of a concentration of 5000 µg/ml was 7.68 (compared to 7.87 in the solvent control)
- Effects of osmolality: the osmolarity of a concentration of 5000 µg/ml was 274 mOsm/kg (compared to 282 mOsm/kg in the solvent control)
- Precipitation: a concentration of 5000 µg Aldolase/ml showed no precipitation in the culture medium


RANGE-FINDING/SCREENING STUDIES:
In order to select the appropriate dose levels for the chromosome aberration test cytotoxicity data were obtained in a dose range finding test. Aldolase was tested in the absence and in the presence of 1.8% (v/v) S9-fraction. The highest tested concentration was 5000 µg/ml.

COMPARISON WITH HISTORICAL CONTROL DATA:
The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range.
Remarks on result:
other: strain/cell type: cultured human lymphocytes
Remarks:
Migrated from field 'Test system'.

Table 1: Chromosome aberrations with Aldolasein the absence of S9 mixin the first cytogenetic assay (3 h exposure time, 24 h fixation time)

Conc

µg/ml

Culture medium

1000

µg/ml

3330

µg/ml

5000

µg/ml

MMC-C

0.5 µg/ml

Culture

A

B

A+B

A

B

A+B

A

B

A+B

A

B

A+B

A

B

A+B

Mitotic

Index (%)

100

102

102

96

70

No. of cells scored

100

100

200

100

100

200

100

100

200

100

100

200

100

100

200

No. Of cells with

aberrations

(+ gaps)

0

2

2

0

1

1

2

4

6

0

2

2

24

35

59

(***)

No. of

cells with

aberrations

(- gaps)

0

2

2

0

0

0

1

2

3

0

2

2

24

35

59

(***)

 

Table 2: Chromosome aberrations with Aldolase in the presence of S9-mix in the first cytogenetic assay (3 h exposure time, 24 h fixation time)

Conc

µg/ml

Culture medium

1000

µg/ml

3330

µg/ml

5000

µg/ml

CP

15 µg/ml

Culture

A

B

A+B

A

B

A+B

A

B

A+B

A

B

A+B

A

B

A+B

Mitotic

Index (%)

100

102

102

101

47

No. of cells scored

100

100

200

100

100

200

100

100

200

100

100

200

100

100

200

No. Of cells with

aberrations

(+ gaps)

1

1

2

3

0

3

5

0

5

2

3

5

29

31

60

(***)

No. of

cells with

aberrations

(- gaps)

1

0

1

3

0

3

5

0

1

2

3

5

28

31

59

(***)

 

Table 3: Chromosome aberrations with Aldolase in the absence of S9-mix in the second cytogenetic assay (24 h exposure time, 24 h fixation time)

Conc

µg/ml

Culture medium

333

µg/ml

1500

µg/ml

3330

µg/ml

MMC-C

0.2 µg/ml

Culture

A

B

A+B

A

B

A+B

A

B

A+B

A

B

A+B

A

B

A+B

Mitotic

Index (%)

100

87

74

45

29

No. of cells scored

100

100

200

100

100

200

100

100

200

100

100

200

100

100

200

No. Of cells with

aberrations

(+ gaps)

0

1

1

1

0

1

1

1

2

1

0

1

35

36

71

(***)

No. of

cells with

aberrations

(- gaps)

0

1

1

1

0

1

1

1

2

1

0

1

35

36

71

(***)

 

Table 4:Chromosome aberrations with Aldolase in the absence of S9-mix in the second cytogenetic assay (48 h exposure time, 48 h fixation time)

Conc

µg/ml

Culture medium

1000

µg/ml

3330

µg/ml

5000

µg/ml

MMC-C

0.1 µg/ml

Culture

A

B

A+B

A

B

A+B

A

B

A+B

A

B

A+B

A

B

A+B

Mitotic

Index (%)

100

86

63

42

54

No. of cells scored

100

100

200

100

100

200

100

100

200

100

100

200

100

100

200

No. Of cells with

aberrations

(+ gaps)

1

0

1

2

2

4

4

6

10

(**)

9

10

19

(***)

40

31

71

(***)

No. of

cells with

aberrations

(- gaps)

0

0

0

2

1

3

3

4

7

(*)

7

8

15

(***)

38

30

68

(***)

 

Table 5: Chromosome aberrations with Aldolase in the presence of S9-mix in the second cytogenetic assay (3 h exposure time, 48 h fixation time)

Conc

µg/ml

Culture medium

1000

µg/ml

3330

µg/ml

5000

µg/ml

CP

15 µg/ml

Culture

A

B

A+B

A

B

A+B

A

B

A+B

A

B

A+B

A

B

A+B

Mitotic

Index (%)

100

140

137

184

Not calculated

No. of cells scored

100

100

200

100

100

200

100

100

200

100

100

200

100

100

200

No. Of cells with

aberrations

(+ gaps)

0

1

1

0

0

0

0

1

1

0

0

0

24

25

49

(***)

No. of

cells with

aberrations

(- gaps)

0

1

1

0

0

0

0

1

1

0

0

0

23

25

48

(***)

Conclusions:
Interpretation of results (migrated information):
positive without metabolic activation after 48 hours of exposure
negative with metabolic activation

Aldolase is clastogenic in human lymphocytes under the experimental conditions described in this report since at the 48 h continuous exposure time a dose related significant increase in the number of chromosome aberrations was observed.
Executive summary:

This report describes the effect of Aldolase on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (Aroclor-1254 induced rat liver S9-mix). The possible clastogenicity of Aldolase was tested in two independent experiments. The study procedures described in this report were based on the most recent OECD and EEC guidelines.

In the first cytogenetic assay, Aldolase was tested up to 5000 µg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of S9-mix. In the second cytogenetic assay, Aldolase was tested up to 3330 µg/ml for a 24 h continuous exposure time with a 24 h fixation time and up to 5000 µg/ml for a 48 h continuous exposure time with a 48 h fixation time in the absence of S9-mix. Appropriate toxicity was reached at these dose levels. In the presence of 1.8% (v/v) S9-fraction Aldolase was tested up to the recommended 5000 µg/ml for a 3 h exposure time with a 48 h fixation time.

Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

First cytogenetic assay:

Aldolase did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix.

Second cytogenetic assay:

In the absence of S9-mix, at the 24 h continuous exposure time, Aldolase did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.

In the absence of S9-mix, at the 48 h continuous exposure time, Aldolase induced a statistically significant, dose dependent increase in the number of cells with chromosome aberrations, both when gaps were included and excluded.

In the presence of S9 mix, Aldolase did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.

Finally, it is concluded that this test is valid and that Aldolase is clastogenic in human lymphocytes under the experimental conditions described in this report since at the 48 h continuous exposure time a dose related significant increase in the number of chromosome aberrations was observed.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 03-JUN-2002 to 17-JUN-2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The test was performed according to OECD guideline and GLP
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
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:
Rat liver microsomal enzymes were routinely prepared from adult male Wistar rats
Test concentrations with justification for top dose:
at least 5 differents doses (increasing with approximately half-log steps) up to 3330 µg protein/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: data not available
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
dimethyl sulfoxide (DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: WITHOUT S9-mix: sodium azide (5µg/pl, TA1535), 9-aminoacridine (60µg/pl, TA1537), daunornycine (4µg/pl, TA98), methylmethanesulfonate (650µg/pl, TA100), 4-nitroquinoline (10µg/pl, WP2uvrA). WITH S9-mix: 2-aminoanthracene (2.5µg/pl TA1537, 1µg/pl TA1535 &
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)


DURATION
- Exposure duration: 48h at 37 +/- 1°C

NUMBER OF REPLITES: three in each strain


OTHER: The revertant colonies were counted automatically with a Protos model 50000 colony counter or manually, if less than 40 colonies per plate were present. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually.
Evaluation criteria:
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) It induces a number of revertant colonies, dose related, greater than two-times the number of revertants induced by the solvent control in any of the tester strains, either with or without metabolic activation.
However, any mean plate count of less than 20 is considered to be not significant.
b) The positive response should be reproducible in at least one independently repeated experiment.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.
Statistics:
not applicable
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: ALDOLASE (IUB 4.1.2.4) precipitated in the top agar at concentrations of 1000 and 3330 µg protein/plate.
Precipitation of ALDOLASE (IUB 4.1.2.4) on the plates was observed at the start of the incubation period at the concentration of 3330 µg protein/plate in the first mutation experiment and at 1000 and 3330 µg protein/plate in the second mutation experiment. Precipitation of ALDOLASE (IUB 4.1.2.4) on the plates was observed at the end of the incubation period at 1000 and 3330 µg protein/plate in both experiments.
- Effects of pH, effects of osmolality, evaporation from medium, water solubility: data not available



RANGE-FINDING/SCREENING STUDIES:
ALDOLASE (IUB 4.1.2.4) was tested in the tester strains TA100 and WP2uvrA with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg protein/plate in the absence and presence of S9-mix.
Precipitate: the test substance precipitated in the top agar at concentrations of 1000 µg protein/plate and upwards. Precipitation of ALDOLASE (IUB 4.1.2.4) on the plates was observed at the start and at the end of the incubation period at concentrations of 3330 and 5000 µg protein/plate.
Toxicity: to determine the toxicity of ALDOLASE (IUB 4.1.2.4), the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined. No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.


COMPARISON WITH HISTORICAL CONTROL DATA:
The negative control values were within our laboratory background historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
The strain-specific positive control values were within our laboratory background historical control data ranges, except for TA1537 in the presence of S9-mix (second experiment). However, since this value was just outside the limit of the range and a more than three-fold increase was observed compared to the solvent control, the validity of the test was considered to be not affected.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1: Result (Experiment 1)

Dose (µg protein/plate)

Mean number of revertant colonies/3 replicate plates with different strains ofSalmonella typhimuriumand oneEscherichia colistrain

 

TA1535

TA1537

TA98

TA100

WP2uvrA

Without S9-mix

Positive control

898

478

478

859

809

Solvent control

9

6

13

142

15

3

 

 

 

156

16

10

 

 

 

136

17

33

5

5

12

147

17

100

7

7

12

154

15

333

11

4

11

137

10

1000

11

4

17

143

15

3330

7

7

15

147

13

5000

 

 

 

158

15

With S9-mix (5% v/v S9 fraction)

Positive control

214

278

627

987

80

Solvent control

11

6

18

130

13

3

 

 

 

142

17

10

 

 

 

124

10

33

7

6

18

145

14

100

7

4

11

127

17

333

9

5

14

139

15

1000

6

3

15

117

12

3330

6

6

16

149

14

5000

 

 

 

150

13

 

Table 2: Result (Experiment 2)

Dose (µg protein/plate)

Mean number of revertant colonies/3 replicate plates with different strains ofSalmonella typhimuriumand oneEscherichia colistrain

 

TA1535

TA1537

TA98

TA100

WP2uvrA

Without S9-mix

Positive control

841

399

380

906

685

Solvent control

12

4

18

158

12

10

10

5

14

148

15

33

7

7

15

186

15

100

11

8

18

170

9

333

13

6

16

138

15

1000

6

6

16

123

11

3330

10

8

19

128

11

With S9-mix (10% v/v S9 fraction)

Positive control

115

54

356

741

162

Solvent control

9

6

28

95

9

10

10

7

22

110

14

33

10

6

22

80

12

100

6

8

23

83

8

333

7

5

25

79

11

1000

6

4

20

78

11

3330

8

7

24

92

10

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

Based on the results of this study it is concluded that ALDOLASE (IUB 4.1.2.4) is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

ALDOLASE (IUB 4.1.2.4) was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli WP2uvrA. The test was performed in two independent experiments in the presence and absence of S9-mix (Aroclor-1254 induced rat liver S9-mix).

In the first and in the second mutation assay, ALDOLASE (IUB 4.1.2.4) was tested up to concentrations of 3330 µg protein /plate in the absence and presence of S9-mix. ALDOLASE (IUB 4.1.2.4) precipitated on the plates at the dose levels of 1000 and 3330 µg protein /plate. The bacterial background lawn was not reduced at any of the concentrations tested and no decrease in the number of revertants was observed.

The presence of 5 and 10% (v/v) liver rnicrosomal activation did not influence these findings.

ALDOLASE (IUB 4.1.2.4) did not induce a dose-related, two-fold increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. Based on the results of this study it is concluded that ALDOLASE (IUB 4.1.2.4) is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
from 29-SEP-2004 to 25-OCT-2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The test was performed according to OECD guideline and GLP
Justification for type of information:
Please refer to the analogue justification attached to IUCLID section 13.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain:
lymphocytes:
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
after 48 hours of exposure
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
lymphocytes:
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: strain/cell type: cultured human lymphocytes
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
positive without metabolic activation after 48 hours of exposure
negative with metabolic activation

Penicillin Amidohydrolase IUB 3.5.1.11 (CAS 9014-06-6) is clastogenic in human lymphocytes under the experimental conditions described in this report on the source substance Aldolase since at the 48 h continuous exposure time a dose related significant increase in the number of chromosome aberrations was observed.
Executive summary:

No data on the chromosome aberration of Penicillin Amidohydrolase IUB 3.5.1.11 (CAS 9014 -06 -6) are available. Therefore, the risk assessment was performed based on the available data from the source substance Aldolase enzyme concentrate; deoxyribose-phosphate aldolase IUB 4.1.2.4 (CAS 9026-97-5). In accordance with Regulation (EC) No. 1907/2006 Annex XI, 1.5 “Grouping of substances and read across” and following the Read across assessment framework (RAAF, ECHA 2017) read across from analogue substances has been applied to support the human health hazard assessment of Penicillin Amidohydrolase IUB 3.5.1.11 (CAS 9014 -06 -6).

This report describes the effect of Aldolase on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (Aroclor-1254 induced rat liver S9-mix). The possible clastogenicity of Aldolase was tested in two independent experiments. The study procedures described in this report were based on the most recent OECD and EEC guidelines.

In the first cytogenetic assay, Aldolase was tested up to 5000 µg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of S9-mix. In the second cytogenetic assay, Aldolase was tested up to 3330 µg/ml for a 24 h continuous exposure time with a 24 h fixation time and up to 5000 µg/ml for a 48 h continuous exposure time with a 48 h fixation time in the absence of S9-mix. Appropriate toxicity was reached at these dose levels. In the presence of 1.8% (v/v) S9-fraction Aldolase was tested up to the recommended 5000 µg/ml for a 3 h exposure time with a 48 h fixation time.

Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

First cytogenetic assay:

Aldolase did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix.

Second cytogenetic assay:

In the absence of S9-mix, at the 24 h continuous exposure time, Aldolase did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.

In the absence of S9-mix, at the 48 h continuous exposure time, Aldolase induced a statistically significant, dose dependent increase in the number of cells with chromosome aberrations, both when gaps were included and excluded.

In the presence of S9 mix, Aldolase did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.

Finally, it is concluded that this test is valid and that Aldolase is clastogenic in human lymphocytes under the experimental conditions described in this report since at the 48 h continuous exposure time a dose related significant increase in the number of chromosome aberrations was observed.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
from 03-JUN-2002 to 17-JUN-2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The test was performed according to OECD guideline and GLP
Justification for type of information:
Please refer to the analogue justification attached to IUCLID section 13.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

The target substance Penicillin Amidohydrolase IUB 3.5.1.11 (CAS 9014-06-6) is not mutagenic based on the results of the study on the source substance ALDOLASE (IUB 4.1.2.4)
Executive summary:

No data on the bacterial mutagenicity of Penicillin Amidohydrolase IUB 3.5.1.11 (CAS 9014-06 -6) are available. Therefore, the risk assessment was performed based on the available data from the source substance Aldolase enzyme concentrate; deoxyribose-phosphate aldolase IUB 4.1.2.4 (CAS 9026-97-5). In accordance with Regulation (EC) No. 1907/2006 Annex XI, 1.5 “Grouping of substances and read across” and following the Read across assessment framework (RAAF, ECHA 2017) read across from analogue substances has been applied to support the human health hazard assessment of Penicillin Amidohydrolase IUB 3.5.1.11 (CAS 9014-06-6).

ALDOLASE (IUB 4.1.2.4) was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli WP2uvrA. The test was performed in two independent experiments in the presence and absence of S9-mix (Aroclor-1254 induced rat liver S9-mix).

In the first and in the second mutation assay, ALDOLASE (IUB 4.1.2.4) was tested up to concentrations of 3330 µg protein /plate in the absence and presence of S9-mix. ALDOLASE (IUB 4.1.2.4) precipitated on the plates at the dose levels of 1000 and 3330 µg protein /plate. The bacterial background lawn was not reduced at any of the concentrations tested and no decrease in the number of revertants was observed.

The presence of 5 and 10% (v/v) liver rnicrosomal activation did not influence these findings.

ALDOLASE (IUB 4.1.2.4) did not induce a dose-related, two-fold increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. Based on the results of this study it is concluded that ALDOLASE (IUB 4.1.2.4) is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

No data on the genetic toxicity of penicillin amidohydrolase IUB 3.5.1.11 (CAS 9014 -06 -6) are available. Therefore, the risk assessment was performed based on the available data from the source substance Aldolase enzyme concentrate; deoxyribose-phosphate aldolase IUB 4.1.2.4 (CAS 9026-97-5). In accordance with Regulation (EC) No. 1907/2006 Annex XI, 1.5 “Grouping of substances and read across” and following the Read across assessment framework (RAAF, ECHA 2017) read across from the analogue substance has been applied to support the human health hazard assessment of penicillin Amidohydrolase IUB 3.5.1.11 (CAS 9014 -06 -6).

The source substance Aldolase was tested in the S. typhimurium reverse mutation assay with four histidine-requiring strains of S. typhimurium (TA1535, TA1537, TA100 and TA 98) and in the E. coli reverse mutation assay with a tryptophan-requiring strain of E. coli WP2 uvrA. The test was performed in two independent experiments in the presence and absence of S9-mix (Aroclor-1254 induced rat liver S9-mix). The maximum concentrations tested were 3330 µg/plate. At concentrations of 1000 and 3330 µg/plate the protein precipitated but toxicity for the test organisms was not observed. Aldolase did not induce a dose related two-fold increase in the number of revertant (His+) colonies in each of the 4 tester strains (TA1535, TA1537, TA100 and TA 98) and in the number of revertant (Trp+) colonies in tester strain WP2 uvrA both in the absence and presence of S9-metabolic activation at any of the tested concentrations. These results were confirmed in an independently repeated experiment. Based on the results of this study it is concluded that aldolase is not mutagenic in the S. typhimurium reverse mutation assay and in the E. coli reverse mutation assay.

The potential of the source substance aldolase to induce chromosome aberrations in cultured human peripheral lymphocytes has been investigated in a study conducted according to OECD 473 and in compliance with GLP (Buskens, 2004). Aldolase was tested up to 5000 µg/mL for a 3 h exposure time with a 24 h and 48 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction, respectively. Furthermore, aldolase was tested up to 3330 µg/mL for a 24 h continuous exposure time with a 24 h fixation time and up to 5000 µg/mL for a 48 h continuous exposure time with a 48 h fixation time in the absence of S9-mix, respectively. No evidence of a test substance related increase in the number of cells with aberrations was observed with or without metabolic activation in short- and long-term treatments in the main experiments. Except in the absence of S9-mix, at the 48 h continuous exposure time, aldolase induced a statistically significant, dose dependent increase in the number of cells with chromosome aberrations, both when gaps were included and excluded. The results of the solvent and positive controls were within the range of the historical control data. In conclusion, aldolase was found to be clastogenic to human lymphocytes under the experimental conditions described in this report.

Microbial enzyme preparations are used extensively in food processing and therefore their hazard to human health has been studied thoroughly. There is no data to suggest that enzymes are in any way DNA-reactive. Up to now no enzyme preparations have been demonstrated to be mutagenic in in vitro test systems (Pariza and Johnson et al., 2001). In fact, 102 bacterial mutagenicity tests (according to OECD 471 and 472) and 63 chromosome aberration/mutagenicity tests (OECD 473, 474 and 476) with enzymes preparations are available to date (Pariza and Johnson et al., 2001). Out of these tests, seven of the Ames and six of the chromosomal aberration tests were found to be false positive. The remaining 152 tests (95 Ames and 57 chromosome aberration) were all negative. The false positive results observed in the Ames tests were demonstrated to be due to the growth-enhancing effects of histidine in the enzyme preparations. The false positive results observed in the chromosome aberration tests were attributed to the following: 

 

-         Additional studies on the same endpoint are available, in different in vitro test systems, which all gave negative results.

-         The production of hydrogen peroxide by some enzymatic reactions, which is known to act as a clastogen.

-         No in vivo tests are available to confirm the positive in vitro tests. The main reason for this is because enzymes are metabolised in the body to amino-acids and ceases to exist.

 

These findings underscore the conclusion that testing enzymes preparations in traditional and genetically modified micro-organisms for genotoxicity is unnecessary for safety evaluation. Furthermore, mutagenicity in an in vivo test is a systemic effect, as the agent has to be transported to the target organ. Enzymes are hydrolysed like any protein before absorption in the body, therefore even if enzymes are mutagenic; its mutagenic activity will be lost before it can reach the appropriate cells.  

 

Reference:

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.







Justification for classification or non-classification