2-Propenoic acid, 2-isocyanatoethyl ester

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 Jan - 06 Mar 2008

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted in 1997

Deviations:

yes

Remarks:

please refer to principle of method if other than guideline

Principles of method if other than guideline:

only 200 erythrocytes counted for the proportion of immature among total erythrocytes, only 2000 erythrocytes per animal scored for micronuclei, no analytical determination of formulations, no rationale for route of administration, number of micronucleated erythrocytes not given separately for each animal

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

482-140-6

EC Name:

-

Cas Number:

13641-96-8

Molecular formula:

Hill formula: C6 H7 N O3 CAS formula: C6 H7 N O3

IUPAC Name:

2-isocyanatoethyl prop-2-enoate

Test animals

Species:

mouse

Strain:

CD-1

Details on species / strain selection:

Crlj:CD1 (ICR) SPF

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Japan Inc., Hino, Japan
- Age at study initiation: 7 weeks
- Weight at study initiation: average of 35.5 g (33.7 - 37.6 g) and 26.0 g (24.7 - 28.2 g) for males and females, respectively, in the preliminary test; average of 35.9 g (33.7 - 37.9 g) for males in the micronucleus test
- Assigned to test groups randomly: yes
- Housing: Groups of three animals were housed in polycarbonate cages with flat bottom with bedding
- Diet: MF pellets (Oriental Yeast Co. Ltd.), ad libitum
- Water: water from Hita City Water Supply (chloride addition water), ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.4 - 23.8
- Humidity (%): 52.0 - 58.5
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 29 Jan 2008 To: 07 Feb 2008 (Micronucleus test)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle/solvent used: olive oil as the substance is oil-soluble
- Concentration of test material in vehicle:
First administration of the preliminary test: 200 mg/mL solution
Second administration of the preliminary test: 25.0 mg/mL solution
First and second administration of the micronucleus test: 12.5 mg/mL solution

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
First administration of the preliminary test:
2.0 g of the test substance was weighed, olive oil was added and the test substance was dissolved using a tube mixer to make 10 mL of a 200 mg/mL solution. A portion of this solution was serially diluted with olive oil to make 100, 50.0, 25.0, 12.5 and 6.25 mg/mL solutions.

Second administration of the preliminary test:
0.25 g of the test substance was weighed, olive oil was added and the test substance was dissolved using a tube mixer to male 10 mL of a 25.0 mg/mL solution. A portion of this solution was serially diluted with olive oil to make 12.5 and 6.25 mg/mL solutions.

First and second administration of the micronucleus test:
0.25 g of the test substance was weighed, olive oil was added and the test substance was dissolved using a tube mixer to male 20 mL of a 12.5 mg/mL solution. A portion of this solution was serially diluted with olive oil to make 6.25, 3.13 and 1.56 mg/mL solutions.

The test substance solutions were prepared each administration day, kept in the dark at room temperature and used within 1.5 h. The test substance was administered twice with a 24-h interval by oral gavage. Administration was carried out at 10 mL/kg bw based on the body weight on the administration day.

Duration of treatment / exposure:

not applicable

Frequency of treatment:

The test substance was administered twice with a 24-h interval.

Post exposure period:

24 h

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

15.6 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

31.3 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

62.5 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

125 mg/kg bw/day

No. of animals per sex per dose:

preliminary test: 3
micronucleus test: 6 males were dosed and 5 males were evaluated

Control animals:

yes, concurrent vehicle

Positive control(s):

Mitomycin C (MMC)
- Justification for choice of positive control(s): recommended in OECD guideline 474 and the background data in the testing facility were abundant
- Route of administration and doses/concentrations: The positive control was intraperitoneally administered at 10 mL/kg bw based on the body weight on the administration day.

Examinations

Tissues and cell types examined:

bone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The dose selection was based on a preliminary test. Groups of 3 animals/sex/dose were treated with either the vehicle or test substance by oral gavage. 62.5, 125, 250, 500, 1000 and 2000 mg/kg bw/day of the test substance was administered, which was repeated twice with a 24-h interval. Animals were observed for 24 h after each administration. At 250 mg/kg bw/day and above, all animals died within 24 h after the second administration. The maximum tolerance dose (MTD) of the test substance was estimated at 125 mg/kg bw/day for male and female mice. There was no sex difference observed. Therefore, male mice were used in the micronucleus test. The doses selected for the micronucleus test were 125, 62.5, 31.3 and 15.6 mg/kg bw/day.

TREATMENT AND SAMPLING TIMES:
The test substance (15.6, 31.3, 62.5 and 125 mg/kg bw/day) was administered twice in an 24-h interval by oral gavage (10 mL/kg bw). 24 h after the second administration, animals were euthanized by cervical dislocation and the femoral bone marrow was prepared.

DETAILS OF SLIDE PREPARATION:
The femur was removed and the bone marrow cells were collected with approximately 0.8 mL of a heat-inactivated fetal bovine serum into a centrifuge tube. After centrifugation, a small amount of cell suspension was smeared on slide glass. Smears were air-dried and fixed with methanol. Subsequently, smears were stained with 3% Gisma solution and treated with 0.004% citrate aqueous solution. 5 animals were evaluated and two specimens were prepare per animal.

METHOD OF ANALYSIS:
All specimens were observed in a blinded manner microscopically. The specimens of the negative and positive control and three doses of the test substance were evaluated for the incidence of micronucleus. The evaluated doses of the test substance were 31.3, 62.5 and 125 mg/kg bw/day.

Incidence of micronuclei:
2000 polychromatic erythrocytes (PCE) per animal (1000 PCE per specimen) were observed and the incidence of the PCE with micronuclei (micronucleated polychromatic erythrocytes = MNPCE) per PCE was calculated.

Growth inhibition of bone marrow cells:
200 total erythrocytes (TE) per animal (100 TE per specimen) were observed and the ratio of PCE per TE was calculated.

Evaluation criteria:

Validity of the test:
The study was considered to be valid, when the means of the MNPCE/PCE in the negative control and the positive control groups were within the range of the background data in the testing facility (negative control group: mean ± 3 S.D., positive control group: mean ± 3 S.D.).

Assessment criteria:
The results was judged positive, if the MNPCE/PCE increased significantly comparing to the negative control group and a dose dependency was exhibited.

Statistics:

Incidence of micronucleus:
The conditional binomial test (Kastenbaum and Bowman) was performed in order to compare the MNPCE/PCE in the negative control group with that in each treatment group and the positive control group. The conditional binomial test was conducted at upper-tailed significance level of 5% and 1%. In case of a significant increase, Cochran-Armitage trend test was conducted for dose-dependency analysis.

Growth inhibition of bone marrow cells:
Data were tested for homogeneity of variance by Bartlett's test. Because the variance were homogeneous, William's test was performed. Dunnett's test was performed, because the Williams' test did not reveal significant differences. Data from the negative and positive control were tested by F test for homogeneity of variance between groups. Because the data were homogeneous, Student's t test was performed to compare the mean in the negative control group with the mean of the positive control group. Significance levels of the tests were 5% for Bartlett's, both sides of the 5% for Williams' test , 5% for the F test and both sides of 5% and 1% for Student's t test.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 62.5 - 2000 mg/kg bw/day
- Mortality: At doses of 250 mg/kg bw/day and above, all animals except one male given 250 mg/kg bw/day died before the second administration. One male in the 250 mg/kg bw/day dose group died within 1 h after the second administration.
- Clinical signs of toxicity in test animals:
No clinical signs were observed in animals given the vehicle control or 62.5 mg/kg bw/day of the test substance until 24 h after the second administration. At 125 mg/kg bw/day one male and one female mice showed decreased spontaneous locomotion until 1 h after the second administration. At higher doses all animals showed clinical signs including decreased spontaneous locomotion, decreased respiratory rate, prone position, subnormal temperature, tremor, clonic convulsion, lateral position and staggering gait.

RESULTS OF DEFINITIVE STUDY
- Mortality: No animals died during the treatment.
- Clinical signs: No animals showed clinical signs until 24 h after the second administration.
- Body weight: There were no treatment-related changes in body weights in any of the treatment groups when compared to the control animals.
- Induction of micronuclei: The means of MNPCE/PCE in the negative and positive controls were 0.08% and 7.82%, respectively. The means if the MNPCE/PCE at 31.3, 62.5 and 125 mg/kg bw/day of the test substance were 0.06, 0.11 and 0.11%, respectively. Therefore, no significant increases were noted in any of the treatment groups compared to the negative control. The positive control caused a significant increase in MNPCE/PCE compared to the vehicle control animals.
- Ratio of PCE/TE: The means of the PCE/TE of the negative and positive control were 55.5% and 46.8%, respectively. The means of the PCE/TE at 31.3, 62.5 and 125 mg/kg bw/day were 53.2, 58.6 and 53.4%, respectively. Thus, no significant changes were noted in any of the treatment groups compared to the vehicle control group.
- Historical control data: Historical control data for the negative and positive control were provided. Both the MNPCE/PCE for the negative and positive control fell within the range of the historical control, accordingly the assay is considered valid.

For details, please refer to table 1 and 2 under any other information on results incl. tables.

Any other information on results incl. tables

Table 1: Summary of micronucleus results in male mice

Test group	Dose (mg/kg bw/day)	Sampling time after the last administration (h)	Animal number	PCE/TE^a) (%)	MNPCE/PCE^b)  (%)
Vehicle control (olive oil)	0	24	101	46.5	0.20
			102	65.5	0.05
			103	48.0	0.05
			104	60.0	0.05
			105	57.5	0.05
		Mean ± S.D.		55.5 ± 8.09	0.08 ± 0.067
		Max./Min.		65.5/46.5	0.20/0.05
Test substance	31.5	24	113	64.0	0.00
			114	45.5	0.05
			115	57.0	0.05
			116	53.5	0.10
			117	46.0	0.10
		Mean ± S.D.		53.2 ± 7.78	0.06 ± 0.042
		Max./Min.		64.0/45.5	0.10/0.00
	62.5	24	119	68.5	0.10
			120	56.5	0.10
			121	51.0	0.10
			122	57.5	0.20
			123	59.5	0.05
		Mean ± S.D.		58.6 ± 6.37	0.11 ± 0.055
		Max./Min.		68.5/51.0	0.20/0.05
	125	24	125	47.5	0.05
			126	60.0	0.15
			127	61.5	0.15
			128	56.5	0.25
			129	41.5	0.05
		Mean ± S.D.		53.4 ± 8.59	0.11 ± 0.055
		Max./Min.		61.5/41.5	0.15/0.05
Positive control (MMC)	2	24	131	34.5	6.55
			132	47.0	9.30
			133	60.5	7.50
			134	43.5	6.75
			135	48.5	9.00
		Mean ± S.D.		46.8 ± 9.39	7.82 ± 1.269**
		Max./Min.		60.5/34.5	9.33/6.55

PCE: polychromatic erythrocytes; TE: total erythrocytes; MNPCE: polychromatic erythrocytes with micronucleus;

MMC: mitomycin C; S.D.: standard deviation

a) observed for 200 erythrocytes per animal

b) observed for 2000 polychromatic erythrocytes per animal

** significantly different compared to the negative control at p≤ 0.01 (Kastenbaum abd Bowman)

Table 2: Historical control data

Historical control data	Incidence (%)			Number of tests
	PCE/TE	MNPCE/PCE
	Mean ± S.D. (%)	Mean ± S.D. (%)	Range (%)
Negative control	49.8 ± 4.00	0.11 ± 0.032	0.01 – 0.21 (mean ± 3 S.D.)	20
Positive control	40.2 ± 5.25	6.95 ± 1.142	3.52 – 10.38 (mean ± 3 S.D.)	20

PCE: polychromatic erythrocytes; TE: total erythrocytes; MNPCE: polychromatic erythrocytes with micronucleus;

S.D.: standard deviation

Background data of at least 20 groups in the testing facility

Positive control: single intraperitoneally dose of 2 mg/kg bw mitomycin C (MMC)

Applicant's summary and conclusion

Conclusions:

Based on the results of the in vivo micronucleus test, there were no indications of a clastogenic effect after oral treatment of the mouse with the test substance.