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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
There is an Ames bacterial reverse mutation assay available, and in addition an in-vitro mammalian cell gene mutation assay, the (L5178 TK+/-) mouse lymphoma assay and a mouse micronucleus test. All studies are Klimisch 1 and all are negative. The Ames and mouse micronucleus studies were carried out on a 57.58% solution of hydroxyethyl urea (EXP3982). The mouse lymphoma study was carried out a sample specially dried to give the solid hydroxyethyl urea.
Link to relevant study records
Reference
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
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine locus for Salmonella
Tryptophan locus for E. Coli
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)
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.
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'.
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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

There is an Ames bacterial reverse mutation assay available, this study included four strains of S. typhimurium, TA1535, TA1537, TA98 and TA100 and E. Coli WP2 uvrA. The study was negative with and without metabolic activation at the maximum concentration for the assay of 5000µg per plates.

Hydroxyethyl urea was also negative in the Mouse Lymphoma L5178Y TK +/- assay, where no toxicologically significant dose-related increases in the mutant frequency were seen at any dose level, either with or without metabolic activation. 

We do not have an in-vitro test for clastogenicity as there was already available an in-vivo mouse micronucleus assay. If the in-vitro assay had been preformed and had been positive then the in-vivo mouse micronucleus assay would have been required at Annex IX to clarify if there was any potential in-vivo. As the in-vivo mouse micronucleus study at the 2000mg/kg limit dose was negative it is not considered to be scientifically justified to perform the in-vitro assay as it will not have influence on the classification decision.

All three aspects of in vitro genetic toxicity testing are available covering gene mutation in bacterial and mammalian cells in-vitro and clastogenicity in in-vivo. In the absence of any adverse effects it is clear that hydroxyethyl urea is not genotoxic and there are no effects in vitro requiring clarifications with in vivo genetic toxicology testing.


Justification for selection of genetic toxicity endpoint
For the genetic toxicology end point it is difficult to select a single key study as there are three types of study, the Ames bacterial reverse mutation assay, which investigates the potential for mutations to DNA in bacteria, there is also an in-vitro mammalian cell gene mutation assay, the (L5178 TK+/-) mouse lymphoma assay. In addition there is an in-vivo mouse micronucleus assay by the oral gavage route. The Ames test study has been selected for the end point, but the others are all relevant.

Justification for classification or non-classification

There are three tests for genotoxicity available for hydroxyethyl urea, which are all Klimisch 1 validity. There is an Ames, bacterial reverse mutation studies, a Mouse lymphoma L5178Y (TK+/-) mammalian cell mutation assay and an in vivo mouse micronucleus test all were conducted to GLP and OECD guidelines.

These assays showed no evidence of mutagenicity or clastogenicity for Hydroxyethyl urea, CAS No 1320-51-0, therefore there is no data to indicate any requirement for classification for genetic toxicity under the EU CLP (GHS) criteria.