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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
other: mammalian bone marrow chromosome aberration test (migrated information)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
red powder
Batch # : 6718 Fass 10/20

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
PEG 400
Frequency of treatment:
three consecutive treatments at 24 hours intervals
Post exposure period:
24 hours after the last administration
Doses / concentrationsopen allclose all
Dose / conc.:
187.5 mg/kg bw/day (actual dose received)
Dose / conc.:
375 mg/kg bw/day (actual dose received)
Dose / conc.:
750 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
6
Control animals:
yes, concurrent vehicle
Positive control(s):
40 mg/kg bw cyclophosphamide

Examinations

Details of tissue and slide preparation:
Slides were prepared from the bone marrow preparations and stained with May-Grünwald/Giemsa.
Evaluation criteria:
At least 2000 PCEs per animal were evaluated for the number of polychromatic erythrocytes (PCE) with micronuclei (without knowledge of the dose group). In addition, the ratio between polychromatic and total erythrocytes per animal was determined. Ten animals per test group (5 males and 5 females) were evaluated as described above.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
only in the highest test dose of 750 mg/kg bw
Vehicle controls valid:
yes
Positive controls valid:
yes

Applicant's summary and conclusion

Conclusions:
Hydroxyethyl-2-nitro-p-toluidine was not mutagenic in the in vivo micronucleus test using NMRI mice after multiple oral administrations up to the maximum tolerated dose of 750 mg/kg bw.
Executive summary:

Hydroxyethyl-2-nitro-p-toluidine, dissolved in PEG 400, was administered once daily for three consecutive days to groups of six male and six female NMRI mice (supplied by RCC Ltd, Switzerland; 8-10 week old) by gavage of 187.5, 375 and 750 mg/kg bw in a total volume of 10 ml/kg bw. Dose selection was based on findings in the preliminary experiments for toxicity, in which 1000 and 750 mg/kg bw were administered to two female and two male mice each under the same treatment regimen. Treatment with 1000 mg/kg bw lead to excessive toxicity and death in both genders, whereas 750 mg/kg bw was considered to be a suitable high dose for the main experiment. Negative control groups received PEG 400 as described above and concurrent positive control groups received 40 mg/kg bw cyclophosphamide (CPA) dissolved in deionised water once 24 hours before sacrifice. Femoral bone marrow was sampled from mice after sacrifice (24 hours after the last dosing) for all dose groups and the vehicle control. Bone marrow of the concurrent positive control group animals was sampled 24 h after administration of the single dose. Slides were prepared from the bone marrow preparations and stained with May-Grünwald/Giemsa. At least 2000 PCEs per animal were evaluated for the number of polychromatic erythrocytes (PCE) with micronuclei (without knowledge of the dose group). In addition, the ratio between polychromatic and total erythrocytes per animal was determined. Ten animals per test group (5 males and 5 females) were evaluated as described above. The animals of the high dose group were examined for acute toxic symptoms at several time points within the first 24 hours of each administration. Stability and homogeneity data were not provided in the study. However, the dosing-solutions were prepared fresh each day immediately before administration and continuously stirred during the administration. Consequently, it can be assumed that the test item solutions were homogenous and stable for the required single application. In the first preliminary experiment, in which 1000 mg/kg bw was administered three times to 4 animals (2 males and 2 females), one female died within 24 h after the first dose and one male died within 4 hours after the second administration. After this fatality the experiment was terminated and the two remaining animals were humanely killed. At 750 mg/kg bw clear signs of toxicity (apathy, ruffled fur, eyelid closure, abdominal position and reduced spontaneous activity) were noted between 1 and 24 hours after all three administrations, but no cases of death occurred. Therefore 750 mg/kg bw was chosen as the top dose for the main experiment. In the main experiment at the highest test dose of 750 mg/kg bw, the signs of toxicity observed were identical to those of the preliminary experiment. An orange discolouring of the urine was noted within 24 hours after each administration in the high dose group. The ratio of PCEs among total erythrocytes was not affected by the test item at any dose tested. Hence, Hydroxyethyl-2-nitro-p-toluidine showed no clear cytotoxic effect in the bone marrow even if tested at clearly systemically toxic doses with multiple applications. However, the occurrence of discoloured urine together with the occurrence of pronounced systemic toxic effects clearly demonstrates that the substance was systemically distributed and bioavailable. This assumption is further supported by the observations of the subcronic toxicity studies and the results of an in vitro toxicokinetics study, which demonstrated a very good penetration of Hydroxyethyl-2-nitro-p-toluidine through the intestinal barrier. There was no statistically significant or biologically relevant increase in the number of micronuclei per 2000 PCEs in the mice of any of the Hydroxyethyl-2-nitro-p-toluidine treated groups versus the respective vehicle control groups. The positive control group (CPA) produced a clear and significant increase in micronucleated PCEs and the vehicle control was well within the range of historical control data of the performing laboratory. This demonstrates the validity and sensitivity of the test system.