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Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information
Gene mutation in bacteria S. typhimurium TA 1535, TA 1537, TA 98, TA 100, E. coli WP2uvrA, with and without metabolic activation (Ames test): negative (GLP, OECD 471; Covance Lab, 2002) S. typhimurium TA 1535, TA, 1537, TA 98, TA 100, TA 102, with and without metabolic activation (Ames test): negative (GLP, OECD 471; BSL, 2001) HPRT-Test Chinese Hamster V79 Cells in vitro (HPRT) test: negative (GLP, OECD 476; BASF SE 2013) Cytogenicity in vivo Mouse, oral., up to limit doses, micronucleus test: negative (GLP, OECD 474; Rohm & Haas 2001)
Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2001-09-10 to 2001-10-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted 21 July 97,
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
Directive 92/69/EEC
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories (Kingston, N.Y.)
- Age at study initiation: 9 weeks
- Weight at study initiation: 30.8-39.9 g for males; 26.1-30.1 g for females
- Assigned to test groups randomly: yes, animals were assigned to treatment groups through the use of a computerized randomization procedure according to sex and body weight prior to administration of the test articles.
- Fasting period before study: yes, 3 h
- Diet: Purina Certified Rodent Chow 5002 C was available ad libitum
- Water: Water filtered through a reverse osmosis system was available ad libitum
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): 43-62
Route of administration:
oral: gavage
Vehicle:
- Vehicle used: water
- Justification for choice of solvent/vehicle: good solubility in water
Details on exposure:
The test article and the negative control were administered by gavage, in a single oral dose. For each treatment group and vehicle and positive control group, 5 male and 5 female animals were dosed per time point, with a volume of 10 mL/kg.
Duration of treatment / exposure:
24 and 48 hours
Frequency of treatment:
single treatment
Post exposure period:
none
Remarks:
Doses / Concentrations:
0, 200, 1000, 2000 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
For each treatment group and vehicle and positive control group, 5 male and 5 female animals were dosed per time point. In the high dose group, 2 additional animals per time point were dosed to account for the possibility of unexpected deaths.
Control animals:
yes, concurrent vehicle
Positive control(s):
The positive control MMC (at a dose of 2.0 mg/kg bw) was administered in a single dose by intraperitoneal (i.p.) injection since this is the accepted route for this substance.
Tissues and cell types examined:
- Clinical observations:Animals were observed for the presence of clinical signs during the treatment period and prior to sacrifice
- Organs examined at necropsy: Bone marrow was examined in each treatment and control group.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
The maximum concentration chosen for this study was a limit dose of 2000 mg/kg bw. In addition to this dose level, 1000 and 200 mg/kg bw of the test substance was tested.

TREATMENT AND SAMPLING TIMES:
Animals from test article and vehicle control groups were euthanized by cervical dislocation at approximately 24 and 48 hours after dosing. Animals from the positive control groups were euthanized 24 hours after dosing.

DETAILS OF SLIDE PREPARATION:
Animals were prepared for micronucleus evaluation as follows: The groin area was wetted with 70% ethanol in water. Both femurs were removed by making incisions at the hip joint and below the knee cap, and the knee cap was removed. The bone marrow was flushed into a 15-mL centrifuge tube, containing approximately 1 mL of Fetal Bovine Serum (FBS) using a 1-cc syringe fitted with a 25-gauge needle. The tubes were centrifuged at 120 x g for 5 minutes, and the supernatant was removed, leaving approximately 0.1 mL above the cell pellet. The cell pellet was re-suspended in the remaining serum until a homogeneous suspension was observed. A small drop of the cell suspension (approximately 10 µL) was placed on the unfrosted end of a clean microscope slide and spread along the length of the slide. The slides were air dried for at least 1 hour, and then fixed in methanol for 15 minutes and allowed to dry. The slides were stained with Acridirie Orange staining solution.

METHOD OF ANALYSIS:
Slides from at least five animals per sexldose group were observed when possible. Three slides were prepared per animal. Slides were coded and read blind in order to avoid bias on the part of the scorer. The slides were read using an epifluorescence microscope to illuminate the acridine orange stain. Slides were scanned for regions of suitable technical quality, where the cells were well spread, undamaged and well stained. These regions were normally located in a zone close to the middle of the smear. Staining was tan to faint grey in normochromatic erythrocytes (NCE) and bright orange in polychromatic erythrocytes (PCE). Micronuclei appeared bright green against an orange background in PCE and generally were round, although almond and ring-shaped micronuclei occasionally occur. Micronuclei have sharp borders and were usually between 1/20 and 115 the size of the PCE. The end point to be scored was the number of cells containing micronuclei (not the number of micronuclei per cell).
For each animal, a total of at least 1000 erythrocytes (polychromatic, referred to as PCE 1 and normochromatic) were recorded to calculate the PCEINCE ratio. For each animal, the remaining number of polychromatic erythrocytes were recorded to total at least 2000 (referred to as PCE 2) and were scored for the presence or absence of micronuclei. The frequency of micronucleated polychromatic erythrocytes (MN-PCE) and the PCE/NCE ratio were calculated on the basis of these data.
Evaluation criteria:
The criteria for a positive response was the detection of a statistically significant increase in micronucleated PCEs for at least one dose level, and a statistically significant dose-related response. A test article that did not induce both of these responses was considered negative. Statistical significance was not the only determinant of a positive response; the Study Director also considered the biological relevance of the results in the final evaluation.
Statistics:
Data were analyzed separately for male and female animals using a Statistical Analysis System (SAS), version 6.09 enhanced. An arcsine square root transformation was applied to the percent of micronucleated PCE's; all subsequent analyses for this parameter were conducted on transformed data. Initially, a three-way analysis of variance model was applied to the data to determine the significance of each main effect (sex, group, and day) and all two-way and three-way interaction effects. If significant interaction effects were identified, then the data were analyzed separately for each sex and/or day. Three independent single degree of freedom contrasts of the group means were used to test for trends in the group means and included an assessment of: 1) an overall effect of the test substance treatment relative to control, and 2) a linear dose-response trend among the test substance, and 3) a quadratic dose-response trend among the test substance. Additionally, pair-wise comparisons between each of the three test substance groups and the control group were made using Dunnett's t-test (Kirkland, 1989).
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Clinical Signs
No clinical signs of toxicity were observed in any of the mice treated with the test article or control articles.

Micronucleus Evaluation
The test article did not induce an increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow cells of male or female mice when compared to the vehicle control values. This was true for both the 24 and 48 hour time points. There was no statistically significant change in the polychromatic/normochromatic ratio at either 24 or 48 hours, which is indicative of the absence of cytotoxicity.
An increase in the frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow cells of male and female mice treated with 2.0 mg/kg of the positive control, mitomycin-C. When compared to the vehicle controls, the increase was greater than two-fold, indicating that the assay was sufficiently sensitive to detect induced cytogenetic damage.

CONCLUSION
Under the conditions of this study, the test substance was not mutagenic in the micronucleus assay in CD-1 mouse bone marrow cells.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

Valid experimental data were available to assess the genetic toxicity in vitro of ureido methylacrylate.

 

Gene mutation in bacteria

The ability to induce reverse mutations at the histidine locus in four strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537), and at the tryptophan locus in Escherichia coli tester strain WP2uvrA, in the presence or absence of an exogenous metabolic activation system (S9) containing mammalian microsomal enzymes was examined for ureido methacrylate (Covance Lab, 2002). A dose range finding assay was conducted on the test article using tester strains TA l00 and WP2uvrA (one plate per dose). Ten doses of test article, from 6.67 to 5000 μg/plate, were evaluated in the presence and absence of S9. Apparently normal growth was observed in both tester strains, and the test article was found to be freely soluble, at all doses evaluated with and without S9.

Based upon these results, ureido methacrylate was evaluated in the initial mutagenicity assay, in all five tester strains, at doses of 33.3, 100,333, 1000, 3330 and 5000 μg/plate with and without S9. All doses of the test article, as well as the concurrent positive and vehicle controls, were evaluated using three plates per dose. Apparently normal growth again was observed in all five tester strains, and the test article again was freely soluble, at all doses evaluated with and without S9. Revertant frequencies for all doses of

ureido methacrylate, in all tester strains with and without S9, approximated those observed in the concurrent vehicle control cultures.

The test article was re-evaluated in an independent confirmatory experiment under identical conditions, and similar results were observed. Apparently normal growth again was observed in all five tester strains, and the test article again was freely soluble, at all doses evaluated with and without S9. Increases in revertant frequencies, to approximately 2.0- to 2.7-fold control values, were observed in tester strain WP2uvrA at doses of 33.3 to 333 μg/plate without S9. However, these increases did not appear to be dose dependent, and all observed values were within acceptable negative control ranges. Revertant frequencies for all doses of ureido metharclate, in all other tester strainlS9 combinations, approximated control values. Thus, the slight increases observed in tester strainWP2uvrA without S9 in the confirmatory assay are considered to be a statistical aberration due to random fluctuation of the spontaneous revertant frequency. All positive and negative control values in both assays were within acceptable ranges, and all criteria for a valid study were met.

These results indicate that ureido methacrylate was negative in the Salmonella-Escherichia coli/Mammalian-Microsome Reverse Mutation Assay with a Confirmatory Assay under the conditions, and according to the criteria, of the test protocol.

A further high quality Ames test was performed as preincubation assay with Salmonella typhimurium TA1535, TA 1537, TA 98 and TA 100 and TA 102 with and without metabolic activation (Rohm & Haas 2001). Again, ureido methacrylate (purity unknown) showed no mutagenic potential.

Another high quality Ames test was performed as preincubation and plate incorporation assay with Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 with and without metabolic activation (RTC 2001). The test item ureido methacrylate (50 %) was examined for mutagenic activity by assaying for reverse mutation to histidine prototrophy in the prokaryotic organism Salmonella typhimurium. The tested concentrations were 5000, 2500, 1250, 625, and 313 µg/plate, based on active ingredient. Also in this test, ureido methacrylate showed no mutagenic potential.

HPRT-Test

The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours.The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The test concentrations was 250, 500, 1000, 2000 and 4000 µg/ml. The highest concentration of 4000 µg/mL was equal to approximately 10 mM. The test item was dissolved in water. No relevant toxic effects occurred up to the maximum concentration with and without metabolic activation following 4 hour treatment. After 24 hour treatment without metabolic activation minor cytotoxic effects were noted at the highest analysable concentration of 2000 µg/mL. At the next higher concentration of 4000 µg/mL exceedingly severe cytotoxic effects precluded analysis.

Ureidomethacrylate 50 % in water is considered to be non-mutagenic in this HPRT assay.

Cytogenicity in vivo

Ureido methacrylate was evaluated for its potential to induce chromosomal damage in vivo, as assessed by the micronucleus assay with mouse bone marrow cells. Adult CD-1 male and female mice (5 male and 5 female animals per group, except for the high dose group, which had 2 additional animals per time point) received a single oral dose of the test article at concentrations of 200, 1000 or 2000 mg/kg bw. Control animals received a single oral dose of distilled water (vehicle control), or an intraperitoneal injection of 2.0 mg/kg bw mitomycin-C (positive control) (MMC). Animals from test article and vehicle control groups were euthanized at 24 or 48 hours after treatment. Animals from the positive control group were euthanized 24 hours after treatment. Bone marrow slides were prepared and the frequency of micronucleated polychromatic erythrocytes was measured as an indicator of cytogenetic damage. For each animal, a total of at least 2000 polychromatic erythrocytes were scored for the presence or absence of micronuclei. In addition, the polychromatic erythrocyte1 normochromatic erythrocyte (PCENCE) ratio was measured to evaluate the cytotoxicity of the test agent.

The test article did not induce an increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow cells of male or female mice when compared to the vehicle controls. An increase in the frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow cells of male and female mice treated with 2.0 mg/kg bw of the positive control, MMC. When compared to the vehicle controls, the increase was greater than two-fold, indicating that the assay was sufficiently sensitive to detect induced cytogenetic damage. Under the conditions of this study, ureido methacrylate was not mutagenic in the micronucleus assay in CD-1 mouse bone marrow cells.


Justification for selection of genetic toxicity endpoint
GLP and guideline n vivo study.

Justification for classification or non-classification

Based on the available data, there is no indication for a mutagenic potential of the test substance. Thus, no classification is warranted according to 67/548/EEC and Regulation (EC) No 1272/2008 (GHS, CLP), respectively.

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