(7R)-7-(4-carboxybutanamido)cephalosporanate amidohydrolase (enzyme substance)

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

deoxyribose-phosphate aldolase EC 4.1.2.4.

Cas Number:

9026-97-5

Molecular formula:

Not applicable, please see remarks

IUPAC Name:

deoxyribose-phosphate aldolase EC 4.1.2.4.

Constituent 2

Reference substance name:

Protein as a constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available. See remarks.

IUPAC Name:

Protein as a constituent of enzyme deriving from the fermentation or extraction process

Constituent 3

Reference substance name:

Inorganic salts as a constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available. See remarks.

IUPAC Name:

Inorganic salts as a constituent of enzyme deriving from the fermentation or extraction process

Constituent 4

Reference substance name:

Carbohydrates constituent of enzyme deriving from the fermentation or extraction process.

Molecular formula:

Not available. See remarks.

IUPAC Name:

Carbohydrates constituent of enzyme deriving from the fermentation or extraction process.

Constituent 5

Reference substance name:

Lipids as a constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available. See remarks.

IUPAC Name:

Lipids as a constituent of enzyme deriving from the fermentation or extraction process

Test material form:

liquid

Remarks:

Tests based on dissolved material in the concentrate

Details on test material:

- Name of test material (as cited in study report): Aldolase (IUB 4.1.2.4)
- Substance type: enzyme
- Physical state: liquid
- Stability under test conditions: data not available
- Storage condition of test material: In freezer in the dark

Method

Species / strain

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

Controls

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.

Results and discussion

Test results

Test results 1

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

Test results 2

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'.

Any other information on results incl. tables

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 (***)

Applicant's summary and conclusion

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.