3,3'-methylenebis[5-methyloxazolidine]

Genetic toxicity: in vivo

Administrative data

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:

Sept 24, 2001 to July 25, 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

No details about the purity of the test substance (responsibility of the sponsor, minor restriction). Contradiction between the description of the results of the preliminary study on page 15 (no effects at 500 mg/kg bw) and page 19/Appendix 3 (100% mortality at 500 mg/kg bw) of the report. No differentiation between structural and numerical chromosome aberration (minor restriction).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

CONTRAM™ MBO (1/6120) / Bakzid

IUPAC Name:

CONTRAM™ MBO (1/6120) / Bakzid

Details on test material:

- Name of test material (as cited in study report): 3,3'-methylenebis[5-methyl- oxazolidine]
- Density: 1,0668 (20°C)
- Substance type: Formaldehyde releaser
- Physical state: Clear colourless liquid
- Analytical purity: Content of formaldehyde 42,28%; 1% in LM at 20°C (no further data)
- Composition of test material, percentage of components: Reaction product from paraformaldehyde and 2 hydroxypropylamine (ratio of 3:2)
- Purity test date: Sept 10, 2001
- Lot/batch No.: 24773
- Storage condition of test material: At room temperature in tightly closed container
- Other: No purity/stability data available with the performing Laboratory

Test animals

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

Crl:NMRI BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland GmbH, D 97633 Sulzfeld
- Age at study initiation: 29 to 30 days
- Weight at study initiation: males 20-25 g and females 19-23 g at administration
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: 5 per sex and cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 15
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From Octber 17, 2001 to January 25, 2002

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Corn oil
- Justification for choice of solvent/vehicle: to obtain a homogeneous suspension
- Concentration of test material in vehicle: 0, 0.15, 0.5, and 1.5%
- Amount of vehicle (if gavage): 20 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was suspended at the required concentartions in corn oil

Duration of treatment / exposure:

Exposure: 24 hours for all dose levels including vehicle control; 48 hours for vehicle control, positive control and high dose group

Frequency of treatment:

One single oral dosing

Post exposure period:

24 h (all dose levels including vehicle controls or 48 h (vehicle and positive control and high dose level only)

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 30, 100, 300 mg/kg bw plus positive control
Basis:
nominal conc.

No. of animals per sex per dose:

5 males and 5 females

Control animals:

yes, concurrent vehicle

Positive control(s):

Positive control substance: cyclophosphamide
- Justification for choice of positive control(s): To confirm the sensitivity of the test system
- Route of administration: Intraperitoneal
- Doses / concentrations: 27 mg/kg bw

Examinations

Tissues and cell types examined:

Erythrocytes in bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Preliminary dose range finding study.At ≥ 500 mg/kg bw all animals died ≤ 60 minutes after application. At 250 mg/kg bw abnormal clinical signs: moderately reduced motility, slight to moderate ataxia, slightly reduced muscle tone, slight to moderate dyspnoea. Hence 300 mg/kg bw was considered to be the maximum tolerated dose (MTD).

DETAILS OF SLIDE PREPARATION: Femora were excised and the bone marrow was flushed out and centrifuged. The supernatant was removed and sediment was suspended in a drop of calf serum. Then a smear was prepared, dried and fixed wit methanol. Cells were stained with Mayers Haemalum.

METHOD OF ANALYSIS: By microscopic analysis with coded slides, random analysis

Evaluation criteria:

The test item was considered positive if:
- a statistically significant increase in the frequency of micronucleated PCE occurred for at least on dose at one kill time

- the frequence of micronucleated PCE at such a point exceeded the historical control range

- corrobating evidence was obtained, for example, increased but statistically insignificant frequencies or micronucleated PCE at other doses or kill times or dose reponse profiles

Statistics:

Chi-square test corrected for continuity according to Yates

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

At 300 mg/kg bw slightly to moderately reduced motility, slight ataxia, slightly reduced muscle tone, and slight dyspnoea 15 minutes to 3 hours after administration

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 250, 500, 1000 and 2000 mg/kg bw; single oral dose (gavage)
- Solubility: Suspension in corn oil
- Clinical signs of toxicity in test animals: At ≥ 500 mg/kg bw all animals died ≤ 60 minutes after application. At 250 mg/kg bw abnormal clinical signs: moderately reduced motility, slight to moderate ataxia, slightly reduced muscle tone, slight to moderate dyspnoea


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): No significant differences to control
- Ratio of PCE/NCE (for Micronucleus assay): No significant differences to control
- Appropriateness of dose levels and route: Dose levels and route were appropriate as the clinical signs at the MTD (300 mg/kg bw) such as slightly to moderately reduced motility, slight ataxia, slightly reduced muscle tone, and slight dyspnoea 15 minutes to 3 hours after administration indicated systemic toxicity without mortality.
The quotient polychromatic/normochromatic erythrocytes (per 1000 erythrocytes) in the bone marrow was not altered compared to the concurrent control or historical controls of the same laboratory indicating no cytotoxic effects at the MTD.
- Statistical evaluation: see Table

Any other information on results incl. tables

Mouse bone marrow micronucleus assay

Treatment, dose in mg/kg bw	Number of cells scored	Sampling time	Ratio polychro-matic/normochromatic erythrocytes	Polychromatic cells with micronuclei per 1000 cells
Males
0	2000	24 h	0.65+-0.14	2.1+-1.3
30	2000	24 h	0.64+-1.12	2.1+-0.5
100	2000	24 h	0.71+-0.16	2.5+-1.1
300	2000	24 h	0.68+-0.08	1.5+-0.5
0	2000	48 h	0.76+-0.19	1.7+-1.3
300	2000	48 h	0.55+-0.05	1.9+-0.5
Positive control	2000	24 h	0.71+-0.18	12.2+-3.3*
Historical vehicle control			0.65 (range 0.29-1.16)	2.34 (range 0.9-3.9)
Females
0	2000	24 h	0.56+-0.10	2.2+-0.6
30	2000	24 h	0.53+-0.07	1.9+-1.4
100	2000	24 h	0.72+-0.07	2.6+-1.6
300	2000	24 h	0.69+-0.07	1.6+-0.4
0	2000	48 h	0.63+-0.25	0.7+-0.8
300	2000	48 h	0.64+-0.07	1.4+-1.1
Positive control	2000	24 h	0.69+-0.14	10.3+-4.7*
Historical vehicle control			0.72 (range 0.32-1.09)	2.46 (range 0.8-3.7)
Mean values and standard deviations are given of 5 males or 5 females. *:statistical significant

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
The concurrent negative and positive controls are valid. Under the conditions of this assay the test substance did not induce a statistical significant increase in the number of micronuclei at a dose level up to 300 mg/kg bw, the maximum tolerated dose. Therefore the tes item had no chromosome mutagenic activity under the conditions of the study.

Executive summary:

Study performed according to OECD guideline 474, mouse bone marrow micronucleus test; 5m + 5f per dose and sampling time received via gavage 0, 30, 100, 300 mg/kg bw (0, 0.15, 0.5, and 1.5% in corn oil); cytotoxicity and clastogenicity in bone marrow measured 24 or 48 h after single application. No mutagenic effect observed, as there was no substance-related increase in micronuclei in any treatment group.