2-isocyanatoethyl methacrylate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 September 1979 to 17 November 1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

The study predates the appropriate OECD test guideline, but was conducted in compliance with GLP. Nevertheless, the study was conducted similar to the OECD TG 482 with acceptable restrictions. The restrictions were that less cells were evaluated than demanded in the guideline.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1980

Report date:

1980

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)

Version / remarks:

(1986)

Deviations:

yes

Remarks:

(only 30 cells evaluated)

GLP compliance:

yes

Type of assay:

other: DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro

Test material

Test material information

Constituent 1

Reference substance name:

2-isocyanatoethyl methacrylate

EC Number:

250-284-7

EC Name:

2-isocyanatoethyl methacrylate

Cas Number:

30674-80-7

Molecular formula:

C7H9NO3

IUPAC Name:

2-isocyanatoethyl methacrylate

Test material form:

not specified

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

hepatocytes: primary hepatocytes from one male Sprague Dawley rat (8-12 weeks of age; approximately 221 g bw)

Details on mammalian cell type (if applicable):

- Type and identity of media: Williams Medium E supplemented with 10% foetal calf serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: not applicable
- Periodically checked for karyotype stability: not applicable
- Periodically "cleansed" against high spontaneous background: not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

not applicable

Metabolic activation system:

primary hepatocyte cultures have sufficient metabolic capacity

Test concentrations with justification for top dose:

10 and 1 mM, 100, 10, and 1 µM, and 100, 10, and 1 nM

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: cell culture medium
- Due to limited solubility of the positive control substance, cell culture medium additional supplemented with 0.1% (v/v) DMSO was used for the positive control.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

(cell culture medium with 0.1% DMSO with respect to the positive control)

True negative controls:

no

Positive controls:

yes

Remarks:

diluted in cell culture medium with 0.1% DMSO

Positive control substance:

2-acetylaminofluorene

Remarks:

10, 1, and 0.1 µM

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium containing 3H-Thymidine (10 µCi/mL) and dexamethasone (1 µM in 0.1% ethanol final concentration)

DURATION
- Exposure duration: 18 h
- Fixation time (start of exposure up to fixation of cells): 20 h

NUMBER OF REPLICATIONS: 1

NUMBER OF CELLS EVALUATED: 30

DETERMINATION OF CYTOTOXICITY
- Method: morphological examination (detachment and/or rounded granular appearence of the cells)

Evaluation criteria:

The net number of grains in treated cells were compared to the appropriate control. Net nuclear labelling is often observed in control cells, but rarely exceeds a mean of 5 grains per nucleus. Therefore, a mean of 6 or more grains per nucleus (this may vary with experimental conditions) and statistical significance from control is required for an unequivocal positive result.
In cases where nuclear labelling is very intense (approximately greater than 75 grains per nucleus) the results are reported as the best approximation of the number of grains present. In addition to statistical significance from control, a dose response relationship should exist.

Statistics:

The net number of grains in treated cells were compared to the appropriate control by analysis of variance and Dunnett's t-test, with statistical significance from control at p ≤ 0.05.

Results and discussion

Test results

Species / strain:

hepatocytes: primary hepatocytes from one male Sprague Dawley rat

Metabolic activation:

not applicable

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1 mM and above clearly cytotoxic

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- none stated in the study report

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- The test item was cytotoxic to the rat hepatocyte cultures at concentrations of 10 and 1 mM as indicated by the detachment and/or rounded granular appearence of the cells after chemical treatment, however, enough cells were present after processing for UDS evaluation. The appearence of the cultures improved with decreasing concentration of the test material so that cultures treated with 0.1 mM to 1 nM approached and matched the appearence of control cultures.

Any other information on results incl. tables

Tab. 1: The effects of the test item in the Rat Hepatocyte Unscheduled DNA Synthesis (UDS) Assay

Treatment	Concentration		Net nuclear grains* (mean±SD)
Negative control	-	-	-2±3
Test item	10 mM	1550 µg/mL	0±1 #
	1 mM	155 µg/mL	1±2 #
	100 µM	15.5 µg/mL	-3±4
	10µM	1.55 µg/mL	-2±3
	1 µM	0.155 µg/mL	-2±3
	100 nM	0.0155 µg/mL	-2±2
	10 nM	0.00155 µg/mL	-2±3
	1 nM	0.000155 µg/mL	-3±3
Positive control (2-AAF)**	10 µM	2.2 µg/mL	40±10***
	1 µM	0.22 µg/mL	40±12***
	100 nM	0.022 µg/mL	33±12***
Solvent control (with 0.1% DMSO)	-	-	-3±4

* net nuclear grains = nuclear grains-background cytoplasm grains

**2 -AAF = acetylaminofluorene

*** positive responses, 6 or more net nuclear grains and p ≤ 0.05

# treatment related cytotoxicity was observed

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative