Tetramethylene dimethacrylate

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:

1998-03-25 to 1998-05-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

draft version 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

29 December 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Tetramethylene dimethacrylate

EC Number:

218-218-1

EC Name:

Tetramethylene dimethacrylate

Cas Number:

2082-81-7

Molecular formula:

C12H18O4

IUPAC Name:

butane-1,4-diyl bis(2-methylacrylate)

Test material form:

liquid

Specific details on test material used for the study:

- Name of test material (as cited in study report): 1,4-Butanediol dimethacrylate

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann, Borchen, Germany
- Age at study initiation: no data
- Weight at study initiation: 26 – 36 g
- Assigned to test groups randomly: yes
- Fasting period before study: no data
- Housing: 5 animals of identical sex per cage
- Diet (e.g. ad libitum): pellet standard diet (Altromin)
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25°C
- Humidity (%): 55+/-10°C
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light):12/12

IN-LIFE DATES: no data

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: cotton seed oil
- Justification for choice of solvent/vehicle: relatively nontoxic to the animals
- Concentration of test material in vehicle:
- Amount of vehicle (if gavage or dermal): 16.7 mL/kg bw
- Lot/batch no. (if required): 27H0534
- Purity: no data

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
dilution in cotton seed oil; single standard volume of 16.7 mL/kg bw

Duration of treatment / exposure:

The animals received the test item once. Sampling of the bone marrow was carried out on animals 24 and 48 h after treatment.

Frequency of treatment:

single dose

Post exposure period:

The animals were sacrificed 24 and 48 hours after treatment.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Remarks:

24 h interval

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

24 h interval

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

Remarks:

24 h interval

Doses / concentrations 4

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

Remarks:

48 h interval

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s): appropriate reference mutagen
- Route of administration: single administration i.p.
- Doses / concentrations: 30 mg/kg bw in 0.9% NaCl (10 mL/kg bw)

Examinations

Tissues and cell types examined:

bone marrow erythrocytes (polychromatic erythrocytes)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
dose selection based on acute toxicity pre-test: at 2000 mg/kg bw no toxic signs were found two lower dose groups (200 and 1000 mg/kg bw) were selected for 24 h treatment interval

DETAILS OF SLIDE PREPARATION:
- removal of femura
- bone marrow was flushed out with 1 mL FCS followed by resuspension of cells and centrifugation at 1500 rpm for 10 min
- cells were resuspended in FCS and smeared on slides
- air drying of slides, staining with May-Grünwald/Giemsa
- at least one slide prepared from each bone marrow sample

METHOD OF ANALYSIS:
- 2000 polychromatic erythrocytes were analysed per animal for micronuclei
- ratio between polychromatic erythrocytes (PCE) and normochromatic erythrocytes (NCE) was determined to assess cytotoxicity (expressed as NCE per 1000 PCE)
- analysis carried out with coded slides

Evaluation criteria:

A test item is classified mutagenic if it induces either a statistically significant dose related increase in the number of micronucleated PCE or a reproducible statistically significant positive result for at least one test point.

Statistics:

non-parametric Mann-Whitney test

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg bw
- Solubility: no data
- Clinical signs of toxicity in test animals: after 1 hour reduced spontaneous activity
- Other: only pre-test for toxicity, no further analysis

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no induction of micronuclei over background (see table); the mean values of micronuclei observed after treatment with the test item (0.11% to 0.15%) were in the same range as the negative control groups (0.10% to 0.13%)
- Ratio of PCE/NCE (for Micronucleus assay): slight effect on PCE/NCE ration by treatment with test item at a concentration of 2000 mg/kg bw at 24 and 48 h indicating slight cytotoxic properties
- Statistical evaluation: no significance found for 1000 mg/kg bw (24 h), 2000 mg/kg bw (24 h), 2000 mg/kg bw (48 h); lowest dose of 200 mg/kg bw (24 h) not tested for significance, as values were equal to or lower than negative control

Any other information on results incl. tables

group	sex	mean MN/2000 PCE	%MN	relative to N.C.	PCE/NCE
N.C.	m	2	0.1	1.0	1000/808.2
P.C.	m	34.6	1.73	17.3	1000/1045.6
test item 2000mg/kg, 24 h	m	2.6	0.13	1.3	1000/995.2
test item 2000mg/kg, 48 h	m	3	0.15	1.5	1000/1011
test item 1000mg/kg, 24 h	m	2.4	0.12	1.2	1000/868
test item 200mg/kg, 24 h	m	2.2	0.11	1.1	1000/820.2
N.C.	f	2.6	0.13	1.0	1000/844.2
P.C.	f	38.8	1.94	14.9	1000/1055.8
test item 2000mg/kg, 24 h	f	2.4	0.12	0.9	1000/972.8
test item 2000mg/kg, 48 h	f	2.6	0.13	1.0	1000/1016.8
test item 1000mg/kg, 24 h	f	2.8	0.14	1.1	1000/884.4
test item 200mg/kg, 24 h	f	2.4	0.12	0.9	1000/828.6

N.C. = negative control

P.C. = positive control

m = male

f = female

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
In this study under the experimental conditions reported, 1,4-BDDMA did not induce micronuclei over background as determined by the micronucleus test in bone marrow cells of the mouse.

Executive summary:

In a NMRI mouse bone marrow micronucleus assay according to OECD guideline 474, draft version 1997, 5 animals per sex per dose were treated by oral gavage (single dose) with 1,4-BDDMA (93.9% a.i.) at doses of 0, 200, 1000 and 2000 mg/kg bw. Bone marrow cells were harvested at 24 h (200, 1000 and 2000 mg/kg dose groups) and 48 h (2000 mg/kg dose group) post-treatment. The vehicle was cotton seed oil.

There were no signs of toxicity during the study based on mortality or clinical signs. A slight effect on the ratio of polychromatic to normochromatic erythrocytes was observed in the 2000 mg/kg dose group 24 and 48 h post-treatment indicating a possible weak toxic effect to the bone marrow. There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time. 1,4-BDDMA was tested at an adequate dose evel which included the guideline limit dose of 2000 mg/kg bw. The positive control induced the appropriate response.

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