2-Deoxyribose 5-phosphate aldolase

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Aldolase enzyme concentrate

IUPAC Name:

Aldolase enzyme concentrate

Constituent 2

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.

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: not indicated
- Storage condition of test material: In freezer in the dark

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

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

Controls

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

Results and discussion

Test results

Test results 1

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

Test results 2

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'.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.