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Toxicological information

Genetic toxicity: in vivo

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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
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
Propylidynetrimethyl trimethacrylate
EC Number:
221-950-4
EC Name:
Propylidynetrimethyl trimethacrylate
Cas Number:
3290-92-4
Molecular formula:
C18H26O6
IUPAC Name:
2,2-bis[(methacryloyloxy)methyl]butyl methacrylate (non-preferred name)
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 or test system 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.