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Toxicological information

Genetic toxicity: in vitro

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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-conform study performed under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): EXP 3982 (N-2-hydroxyethylurea)
- Physical state: clear slightly yellow liquid
- Analytical purity: aqueous solution containing 57.58% hydroxyethyl urea
- Impurities (identity and concentrations): not reported
- Composition: aequeous solution containing 57.58 % hydroxyethyl urea
- Purity test date: not reported
- Lot/batch No.: not reported
- Expiration date of the lot/batch: 15 July 2001
- Storage condition of test material: at ambient temperature protected from freezing

Method

Target gene:
Histidine locus for Salmonella
Tryptophan locus for E. Coli
Species / strain
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100; E. coli: WP2 uvrA
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S9 mix
Test concentrations with justification for top dose:
The dosing solutions were adjusted to compensate for the purity (57.58%) of the test article. Doses were as follows:

Initial Toxicity-Mutation Assay, without S9: 2.5, 7.5, 25, 75, 200, 600, 1800, 5000 µg/plate
Initial Toxicity-Mutation Assay, with S9: 2.5, 7.5, 25, 75, 200, 600, 1800, 5000 µg/plate
Confirmatory Mutatgenicity Assay, without S9: 75, 200, 600, 1800, 5000 µg/plate
Confirmatory Mutatgenicity Assay, with S9: 75, 200, 600, 1800, 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: selected based on compatibility with the target cells and solubility of the test article (soluble in water at a maximum concentration of approximately 50 mg/mL, the highest concentration tested)
Controls
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: With S9: 2-amonoanthracene. Without S9: 2-nitrofluorene (TA98), Sodium azide (TA100, TA1535), 9-aminoacridine (TA1537), Methyl methanesulfonate (WP2 uvrA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 - 72 h

NUMBER OF REPLICATIONS:
Initial Toxicity-Mutation Assay: 2
Confirmatory Mutatgenicity Assay: 3

DETERMINATION OF CYTOTOXICITY
- Method: The condition of the bacterial background lawn was evaluated for evidence of test article toxicity by using a dissecting microscope.
Evaluation criteria:
For the test article to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test article. Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than three times the mean vehicle control value. Data sets for tester strains TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than two times the mean vehicle control value.
Statistics:
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.

Results and discussion

Test results
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100; E. coli: WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: soluble in water at a maximum concentration of approximately 50 mg/mL, the highest concentration tested
- Precipitation: no

RANGE-FINDING/SCREENING STUDIES: In the initial toxicity mutation assay, the maximum dose tested was 5000 µg per plate; this dose was achieved using a concentration of 50 mg/mL and a 100 µL plating aliquot. Neither precipitate nor appreciable toxicity was observed. Based on the findings of the toxicity assay, the maximal dose plated in the mutagenicity assay was 5000 µg per plate.

COMPARISON WITH HISTORICAL CONTROL DATA: The mean revertants per plate in the negative and positive controls were in the range of the historical control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY: No appreciable toxicity was observed in either assay.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, EXP 3982 (N-2-hydroxyethylurea) did not cause a positive response in the presence and absence of Aroclor-induced rat liver S9 mix.
Executive summary:

The test article, EXP 3982 (N-2-hydroxyethylurea), was tested under GLP in the Bacterial Reverse Mutation Assay using Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA in the presence and absence of Aroclor-induced rat liver S9 mix. The study followed the method outlined in OECD TG 471.

The assay was performed in two phases, using the plate incorporation method. The first phase, the preliminary toxicity-mutation assay, was used to establish the dose-range for the mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, the mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the test article. The dosing solutions were adjusted to compensate for the the concentration of hydroxyethyl urea (57.58%) in the test substance.

Water was selected as the solvent of choice based on compatibility with the target cells and solubility of the test article. The test article was soluble in water at a maximum concentration of approximately 50 mg/mL, the highest concentration tested.

In the initial toxicity-mutation assay, the maximum dose tested was 5000 µg per plate; this dose was achieved using a concentration of 50 mg/mL and a 100 µL plating aliquot. Neither precipitate nor appreciable toxicity was observed. Based on the findings of the toxicity-mutation assay, the maximum dose plated in the mutagenicity assay was 5000 µg per plate. In the confirmatory mutagenicity assay, no positive response was observed. Neither precipitate nor appreciable toxicity was observed.

Under the conditions of this study, test article EXP 3982 (N-2-hydroxyethylurea) was concluded to be negative in the Bacterial Reverse Mutation Assay.