2-ethyl-2-[[(1-oxoallyl)oxy]methyl]-1,3-propanediyl diacrylate; 2,2-bis(acryloyloxymethyl)butyl acrylate; trimethylolpropane triacrylate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From October 13 to December 20, 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was conducted according to OECD Guideline 473, EU Method B.10, OPPTS 870.5375 and Japanese guidelines in compliance with the Principles of Good Laboratory Practice.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

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:

- Physical state: colorless liquid
- Analytical purity: 84.6%
- Impurities: Water - 0.05%
- Lot No.: 00885GD4
- Purity test date: April 16, 2004
- Storage condition of test material: Stored at room temperature and away from light from receipt to 29 October 2004 and at 4°C (instead of room temperature) since that date

Method

Target gene:

Not applicable

Species / strain

Species / strain / cell type:

lymphocytes: primary cell cultures from human peripheral blood

Details on mammalian cell type (if applicable):

They have a stable karyotype with 46 chromosomes and an average cell cycle time of 12-14 h.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction consists of Aroclor 1254 treated rat liver microsomal fraction prepared and preserved in sterile tubes at -80°C. S9 mix was prepared at +4°C immediately before use and maintained at this temperature until added to the culture medium.

Test concentrations with justification for top dose:

Preliminary experiment:
23.1, 45.7, 92.6, 185, 370, 740, 1480 and 2960 μg/mL for the preliminary experiment both with and without S9 mix

Main experiment:
1) 0.78, 1.56, 3.13, 6.25, 12.5, 18.75, 25 and 37.5 μg/mL for the first experiment without S9 mix
2) 1.56, 3.13, 6.25, 12.5, 18.75, 25, 37.5 and 50 μg/mL for the first experiment with S9 mix
3) 3.13, 6.25, 9.38, 12.5, 18.75 and 28.13 μg/mL, for the second experiment without S9 mix
4) 9.38, 18.75, 28.13, 37.5, 50 and 75 μg/mL, for the second experiment with S9 mix

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethylsulfoxide (DMSO), batch nos. K32136150327 and K32311850346 (Merck Clévenot, Chelles, France)

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Migrated to IUCLID6: 3 μg/mL, without S9 mix

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Migrated to IUCLID6: 12.5 or 25 μg/mL, with S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: In RPMI medium containing fetal calf serum, penicillin, L-glutamine, Streptomycin and Phytohaemagglutinin


DURATION
- Incubation period of culture without test material: 48 h at 37°C
- Exposure duration: 3 h
- Expression time (cells in growth medium): 17 h
- Selection time (if incubation with a selection agent): 1.5 h
- Fixation time (start of exposure up to fixation or harvest of cells): 20 h


SELECTION AGENT (mutation assays): Colcemid (10 μg/mL)
SPINDLE INHIBITOR (cytogenetic assays): Not applicable
STAIN Used: Giemsa


NUMBER OF REPLICATIONS: Duplicate cultures in both the experiments


NUMBER OF CELLS EVALUATED:
Cytotoxicity - 1000 cells per culture.
Chromosomal aberration - Analysis of 200 metaphases/concentration (with 44 to 46 chromosomes) was made, with 100 metaphases/culture, whenever possible. Only 50 metaphases/culture were analyzed when at least 10% of the cells with structural chromosome aberrations were observed.


DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index (number of cells in mitosis/1000 cells examined)

Evaluation criteria:

A reproducible, concentration-related, and statistically significant increase in the frequency of cells with structural chromosome aberrations for at least one of the concentrations and one of the two harvest times was considered as a positive result. Reference to historical data or other considerations of biological relevance, was also taken into account in the evaluation of the findings, if needed.

Statistics:

For each test and for each harvest time, the frequency of cells with structural chromosome aberrations (excluding gaps) in treated cultures was compared to that of the vehicle control cultures. If necessary, the results were compared using the χ2 test, in which p = 0.05 was used as the lowest level of significance.

Results and discussion

Test results

Species / strain:

lymphocytes: Primary cell culture of human peripheral blood

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Observed at all conc. of the preliminary experiment and observed in both first and second experiments at conc. ≥ 12.5 μg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Additional information on chromosomal aberration:

With metabolic activation-
In the first experiment, a statistically significant increase in the frequency of cells with structural chromosomal aberrations was noted at 18.75 μg/mL (46% versus 1.5% for the vehicle control, p < 0.001).
In the second experiment, statistically significant and concentration-related increases in the frequency of cells with structural chromosomal aberrations were noted at concentrations ≥ 9.38 μg/mL (6, 10.7 and 30% at the concentrations of 9.38, p < 0.01 ; 12.5, p < 0.001 and 18.75 μg/mL, p < 0.001, respectively, versus 0.5% for the vehicle control).

Without metabolic activation-
In the first experiment, statistically significant and concentration-related increases in the frequency of cells with structural chromosomal aberrations were noted at concentrations of 37.5 and 50 μg/mL (21 and 53%, respectively versus 1% for the vehicle control, p < 0.001).
In the second experiment, statistically significant increases in the frequency of cells with structural chromosomal aberrations, without any clear evidence of a concentration-relationship, were noted at concentrations of 28.13 and 50 μg/mL (4.5%, p < 0.05 and 19%, p < 0.001, respectively, versus 1% for the vehicle control).



TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In the culture medium, the concentration of 2963.2 μg/mL showed a slight to moderate precipitate. At this concentration, the pH was approximately 7.1 and the osmolality equal to 337 mOsm/kg H2O. However, this concentration was rejected following severe cytotoxicity.


RANGE-FINDING/SCREENING STUDIES:
The preliminary study was with a treatment volume of 27.5 μL/5.5 mL culture medium and the treatment-levels were 23.1, 45.7, 92.6, 185, 370,740, 1480 and 2960 μg/mL both with and without S9 mix. High toxicity was observed at all concentrations resulting in rejection of the experiment.


ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxicity occurred at 90% for the lowest concentration and 100% for the other concentrations in the preliminary study. In view of the severe toxiciobserved, the first experiment was repeated at lower concentrations ranging from 0.78 to 37.5 μg/mL. The concentrations in the second experiment ranged from 3.13 to 28.13 μg/mL. Cytotoxicity ranged from 45 to 100% in the first experiment and 26 to 83% in the second experiment at concentrations ≥ 12.5 μg/mL.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Validity criteria followed:

This study was considered valid since the following criteria were met:

• the frequency of cells with structural chromosome aberrations in the vehicle controls was consistent with CIT historical data,

• The frequency of cells with structural chromosome aberrations in the positive control tests was significantly higher than that of the vehicle controls and consistent with CIT historical data.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive

Trimethylolpropane triacrylate induced chromosome aberrations in cultured human lymphocytes, both with and without metabolic activation.

Executive summary:

An in vitro mammalian cell chromosomal aberration study was carried out with trimethylolpropane triacrylate in accordance with OECD TG 473, EU Method B.10, Japanese guidelines and EPA OPPTS method 870.5375.

A preliminary experiment was carried out with trimethylolpropane triacrylate 23.1, 45.7, 92.6, 185, 370, 740, 1480 and 2960 μg/mL both with and without S9 mix. In the main experiment, primary cell cultured human peripheral blood from two donors were incubated with Trimethylolpropane Triacrylate at the following concentrations:

1) 0.78, 1.56, 3.13, 6.25, 12.5, 18.75, 25 and 37.5 μg/mL without S9 mix (first experiment);

2) 1.56, 3.13, 6.25, 12.5, 18.75, 25, 37.5 and 50 μg/mL with S9 mix (first experiment);

3) 3.13, 6.25, 9.38, 12.5, 18.75 and 28.13 μg/mL without S9 mix (second experiment);

4) 9.38, 18.75, 28.13, 37.5, 50 and 75 μg/mL with S9 mix (second experiment).

Cytotoxicity was observed at all concentrations of the preliminary experiment. Hence this study was rejected. Cytotoxicity ranged from 45 to 100% in the first experiment and 26 to 83% in the second experiment at concentrations ≥ 12.5 μg/mL.

Statistically significant and concentration-related increases in the frequency of cells with structural chromosomal aberrations were noted at concentrations ≥ 9.38 μg/mL with metabolic activation. Statistically significant increases in the frequency of cells with structural chromosomal aberrations were noted at concentrations at and above 28.13 μg/mL.

In conclusion trimethylolpropane triacrylate induced chromosome aberrations in cultured human lymphocytes both with and without metabolic activation.