Reaction mass of (2,4,6-trioxo-1,3,5-triazine-1,3,5(2H,4H,6H)-triyl)tri-2,1-ethanediyl triacrylate and 2-Propenoic acid, 1,1'-[[dihydro-5-(2-hydroxyethyl)-2,4,6-trioxo-1,3,5-triazine-1,3(2H,4H)-diyl]di-2,1-ethanediyl] ester

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:

From December 08, 2005 to June 08, 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study is according to OECD Guideline 474, EU Method B.12, OPPTS 870.5395 and Japanese guidelines in compliance with the Principles of Good Laboratory Practice.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

; requirements of the Japanese Government under the revised Chemical Substance Law (1986)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

2-ethyl-2-[[(1-oxoallyl)oxy]methyl]-1,3-propanediyl diacrylate

EC Number:

239-701-3

EC Name:

2-ethyl-2-[[(1-oxoallyl)oxy]methyl]-1,3-propanediyl diacrylate

Cas Number:

15625-89-5

Molecular formula:

C15H20O6

IUPAC Name:

2,2-bis(prop-2-enoyloxymethyl)butyl prop-2-enoate

Constituent 2

Reference substance name:

Trimethylolpropane Triacrylate (TMPTA)

IUPAC Name:

Trimethylolpropane Triacrylate (TMPTA)

Details on test material:

- Name of test material (as cited in study report): Trimethylolpropane triacrylate (TMPTA)
- Physical state: Colorless viscous liquid
- Analytical purity (mixed isomers): 87.9 wt %
- Purity test date: 2004-07-13
- Lot/batch No.: 07415FZ4
- Storage condition of test material: Away from light and at 4°C
- Viscosity: 118 mPa.s
- Acid value: 0.8 mg KOH/g

Test animals

Species:

mouse

Strain:

other: Swiss Ico: OF1 (IOPS Caw)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, 1'Arbresle, France
- Age at study initiation: 6 wk
- Weight at study initiation: 32.3 g for males (ranging from 28.4 to 35.2 g) and 23.9 g for females (ranging from 21.0 to 26.5 g).
- Assigned to test groups randomly: Yes
- Housing: Housed by groups in polycarbonate cages
- Diet (e.g. ad libitum): A04 C pelleted maintenance diet, ad libitum
- Water (e.g. ad libitum): FG Millipore membrane filtered water, ad libitum
- Acclimation period: at least 5 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C,
- Humidity (%): 30- 70 %
- Air changes (per h): 12 cycles/h
- Photoperiod (h dark / h light): 12 h/12 h


IN-LIFE DATES: From: 2005-12-13 to: 27-01-27

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Corn oil
- Concentration of test material in vehicle: Preliminary tests - 175 and 200 mg/mL; Main test - 43.75, 50, 87.5, 100, 175 and 200 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw
- Lot/batch no. (if required): 015K0115

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Preliminary tests - The test item was dissolved in the vehicle in order to achieve the concentrations of 175 and 200 mg/mL and then homogenized using a magnetic stirrer. The resulting formulations were light yellow limpid solutions. Using a treatment volume of 10 mL/kg bw, the target doses were 1750 and 2000 mg/kg bw.

Main test - The test item was dissolved in the vehicle in order to achieve the concentrations of 43.75, 50, 87.5, 100, 175 and 200 mg/mL and then homogenized using a magnetic stirrer. Using a treatment volume of 10 mL/kg bw, the target doses were 437.5, 500, 875, 1000, 1750 and 2000 mg/kg bw.

The preparations were made immediately before use.

Duration of treatment / exposure:

Single treatment

Frequency of treatment:

Single treatment

Post exposure period:

24 h (control, low, intermediate and high dose groups); 48 h (for control and high dose groups)

Doses / concentrations

Remarks:

Doses / Concentrations:
437.5, 875 and 1750 mg/kg bw (for males) or 500, 1000 and 2000 mg/kg bw (for females)
Basis:
nominal conc.

No. of animals per sex per dose:

5 animals/sex/dose (with exception of 8 animals/sex/high dose for 48 h sampling time)

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Route of administration: Oral
- Doses / concentrations: 50 mg/kg bw

Examinations

Tissues and cell types examined:

Bone marrow cells of mice

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Preliminary test was performed on groups of 6 animals (3 males and 3 females). The initial preliminary test indicated that males were the more sensitive sex. Additional preliminary testing was conducted on a group of 3 male mice to determine the high dose for this sex. Clinical signs and any mortality were recorded for a period of 48 h after each preliminary dosing.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): 24 or 48 h following dosing

DETAILS OF SLIDE PREPARATION: The femurs of the animals were removed and the bone marrow was flushed out using fetal calf serum. After
centrifugation, the supernatant was removed and the cells in the sediment were resuspended by shaking. A drop of this cell suspension was placed and spread on a slide. The slides were air-dried and stained with Giemsa. The slides were coded so that the scorer was unaware of the treatment group of the slide under evaluation ("blind" scoring).

METHOD OF ANALYSIS:
For each animal, the number of the micronucleated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes; the polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).

Evaluation criteria:

- Statistically significant increase in the frequency of MPE when compared to the vehicle control group was required for a positive finding;
- Reference to historical data, or other considerations of biological relevance was also taken into account in the evaluation of data obtained

Statistics:

- Normality and homogeneity of variances was tested using a Kolmogorov Smirnov test and a Bartlett test.
- Student t-test (2 groups) or a one-way analysis of variance (≥ 3 groups) followed by a Dunnett test (if necessary): If normality and homogeneity of variances were demonstrated.
- Mann/Whitney test (2 groups) or a Kruskall Wallis test (≥ 3 groups) followed by a Dunn test (if necessary).: If normality or homogeneity of variances was not demonstrated.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Only in males (piloerection at 875 mg/kg bw; 2 deaths and piloerection in surviving animal at 1750 mg/kg bw)

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Clinical signs of toxicity in test animals:
- Neither mortality nor clinical signs were noted in the 3 treated females; Piloerection and ocular secretion or soiled urogenital area were noted in 2 surviving males.
- Confirmatory assay was performed using 3 males at 2000 mg/kg bw in order to confirm the previous findings. Two males were found dead 24 and 48 h following treatment. The surviving male showed piloerection.
- Based on these results, an additional test was performed using 3 males at 1750 mg/kg bw. Only piloerection was noted in males given 1750 mg/kg bw in this second preliminary test and no mortality was induced.
- For males, since toxic effects were observed, the choice of the top dose was based on the level of toxicity, such that a higher dose was expected to induce lethality. Therefore, 1750 mg/kg bw was selected as the top dose for the main test.
For females, since no toxic effects were observed, 2000 mg/kg bw was selected as the top dose for the main test.

RESULTS OF DEFINITIVE STUDY
- Mean values of MPE as well as the PE/NE ratios in the groups treated with the test item, were equivalent to those of the vehicle control group, for both harvest times.
- All frequencies of micronucleated cells (MPE) obtained in treated animals were clearly within or consistent with the historical range.

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative
Trimethylolpropane triacrylate (TMPTA) was considered to be non-genotoxic in micronucleus test in the mouse.

Executive summary:

A study was performed to investigate the potential of Trimethylolpropane triacrylate (TMPTA) to induce micronuclei in bone marrow cells of mice. The study was performed according to guidelines (OECD 474, Commission Directive No. B12, OPPTS 870.5395 and Japanese guidelines) and in compliance with the Principles of Good Laboratory Practice. 

 

A preliminary toxicity test was performed to define the highest possible dose to be used for the cytogenetic study. In the main study, four groups of five male and five female Swiss Ico: OF1 (IOPS Caw) mice received a single oral administration of the test material at the following doses: 437.5, 875 and 1750 mg/kg bw (for males) or 500, 1000 and 2000 mg/kg bw (for females). Two groups of five males and five females received the vehicle (corn oil) acted as the control groups. One group of five males and five females received Cyclophosphamide (positive control) once by the oral route at a dose of 50 mg/kg bw. The animals were killed 24 (control, low, intermediate and high dose groups) or 48 h (control and high dose groups) after treatment. The animals of the positive control group were killed 24 h after treatment. Bone marrow smears were then prepared. For each animal, the number of the micronucleated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes. The polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE). 

In males, no clinical signs and no mortality attributed to the treatment were observed in controls and 437.5 mg/kg bw group. At 875 mg/kg bw, piloerection was noted and at 1750 mg/kg bw, two males were found dead 24 h following the treatment and piloerection was noted at the same time in the surviving males. In females, no clinical signs and no mortality attributed to the treatment were observed in animals given 0, 500, 1000 or 2000 mg/kg bw.

Mean values of MPE as well as the PE/NE ratios in the treated group were equivalent to those of the control group, for both harvest times. All frequencies of micronucleated cells (MPE) obtained in treated animals were clearly within or consistent with the historical vehicle control range. Cyclophosphamide induced a significant increase in the frequency of MPE, demonstrating the sensitivity of the test system under the experimental conditions of this study.

In conclusion trimethylolpropane triacrylate (TMPTA) was considered to be non-mutagenic in the micronucleus test.