Registration Dossier

Toxicological information

Genetic toxicity: in vivo

Currently viewing:

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From January 01 to March 22, 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: in vivo micronucleus study

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Clear to slightly turbid yellowish liquid
Details on test material:
- Name of test material (as cited in study report): Trimethylolpropantrimethacrylate (TMPTMA)
- Physical state: Clear to slightly turbid yellowish liquid

- Storage condition of test material: At room temperature

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: HARLAN WINKELMAN, Borchen, Germany
- Age at study initiation: 23-33 g
- Assigned to test groups randomly: Yes
- Housing: Housed in a group of 5 mice/sex in Macaroon Type III cage
- Diet (e.g. ad libitum): Pelleted standard diet (ALTROMIN, Lage/Lippe, Germany)
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 55 ± 10
- Photoperiod (hours dark / hours light): 12 hours dark/12 hours light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: CMC (carboxymethyl cellulose)
- Justification for choice of solvent/vehicle: Nontoxic to animals
- Source: SIGMA, Germany
- Amount of vehicle (if gavage or dermal): 33.3 mL/kg bw
- Lot/batch no. (if required): 36H0738
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Test material was prepared and diluted with carboxymethylcellulose.
Duration of treatment / exposure:
24 hours (in all test groups) and 48 hours (in 2000 mg/kg bw group)
Frequency of treatment:
Once
Post exposure period:
Not applicable
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 200, 600 and 2000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
Five
Control animals:
yes, concurrent vehicle
Positive control(s):
CPA (cyclophosphamide)
- Source: SIGMA, Germany
- Analytical purity: At least 98%
- Route of administration: i.p.
- Doses / concentrations: 30 mg/kg bw
- Volume administered: 10 mL/kg bw
- Lot/batch no.: 087H0207

Examinations

Tissues and cell types examined:
- Femora bones were removed for marrow extraction and the prepared slides were examined for polychromatic erythrocytes (PCEs), normochromatic erythrocytes (NCEs) and total erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Dose selection was based on a preliminary range-finding test conducted on 3 mice/sex/dose at 2000 mg/kg bw. No toxic signs were recorded.

TREATMENT AND SAMPLING TIMES: Femora were removed for marrow extraction from single animals in each treatment and control group at 24 or 48 hours after exposure.

DETAILS OF SLIDE PREPARATION: Bone marrow cells extracted, preparations smeared on slides and air-dried. The slides were stained with May-Grunwald solution/ Giemsa and coded.

METHOD OF ANALYSIS: Slides were scanned using Olympus microscopes to determine the frequency of micronuclei in 2000 polychromatic erythrocytes (PCEs) per animal. In addition, PCE:NCE ratio was determined in the same sample and expressed as NCE/1000 PCEs.
Evaluation criteria:
- Criteria for a positive response: Detection of either a statistically significant dose related increase in the number of micronucleated PCEs or a reproducible statistically significant positive response for at least one of the test points.
- Test article that does not induce both of these responses is considered negative.
- Both biological and statistical significance should be considered together.
Statistics:
Statistical significance at the 5% level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Results and discussion

Test results
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:
RESULTS OF RANGE-FINDING STUDY
- Dose: 2000 mg/kg bw
- Clinical signs of toxicity in test animals: No

RESULTS OF DEFINITIVE STUDY
- Frequency of micronuclei in polychromatic erythrocytes and PCE:NCE ratio: See table 1

Any other information on results incl. tables

Table 1: Micronucleus Assay - summary table

 

Treatment 

Dose

Harvest time (hours)

% Micronucleated PCEs (mean of 2000 PCEs per animal)

Ratio PCE:NCE (mean)

males

females

males

females

all

small

all

small

Vehicle control

Carboxymethylcellulose 33.3 mL/kg bw

 24 

0.16

0.14

0.16

0.14

1.14

1.35

Positive control

Cyclophosphamide 30 mg/kg bw

 24

1.01

0.94

0.98

0.92

1.35

1.45

Test Article 

200 mg/kg bw

 24

0.18

0.13

0.14

0.11

1.19

0.99

600 mg/kg bw

 24

0.09

0.07

0.12

0.09

0.86

0.93

2000 mg/kg bw

 24

0.12

0.11

0.1

0.09

0.89

1.11

 48

0.16

0.16

0.12

0.12

0.58

1.19

Applicant's summary and conclusion

Conclusions:
Under the test conditions, trimethylolpropantrimethacrylate is not considered as mutagenic in the mouse bone marrow micronucleus test.
Executive summary:

In a bone marrow micronucleus test, performed according to OECD guideline 474 and in compliance with GLP, groups of NMRI mice (5/sex/dose) were given a single oral (gavage) dose of trimethylolpropantrimethacrylate in carboxymethylcellulose at doses of 0, 200, 600 and 2000 mg/kg bw. Bone marrow was extracted after 24 hours (in all test groups) or 48 hours (in 2000 mg/kg bw group) of exposure and the prepared slides were scanned to determine the frequency of micronuclei in 2000 polychromatic erythrocytes (PCEs) for each animal. In addition, PCE:NCE ratio was determined in the same sample and expressed as NCE/1000 PCEs. A preliminary range-finding test was also conducted on 3 mice/sex/dose at 2000 mg/kg bw in which no toxic signs were recorded.

 

No statistically significant increases in the frequency of micronucleated PCEs were observed at any dose levels. PCE:NCE ratio was slightly affected at a dose level of 2000 mg/kg bw at 24 and 48 hours indicating slight cytotoxicity of the test material. Positive control (cyclophosphamide, 30 mg/kg bw) induced the appropriate response.

 

Under the test conditions, trimethylolpropantrimethacrylate is not considered as mutagenic in the mouse bone marrow micronucleus test according to the criteria of the Annex VI to the Directive 67/548/EEC and CLP Regulation (EC) N° (1272-2008).