Propanoic acid, 2-hydroxy-2-methyl-, 2-propen-1-yl 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

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: MITI (March 31, 1987), No. 303, (Sho-62)

Deviations:

yes

Principles of method if other than guideline:

A mixture of CMC (0.5% v/v) and Tween 80 (0.1% v/v) was used as vehicle for the test substance.
The dose used in the study were 50, 100 and 200 mg/kg body weight.
During husbandry the relative himidity (20 - 49%) fell below the accepted lower limit of 30% for a few hours.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

prop-2-en-1-yl 2-hydroxy-2-methylpropanoate

EC Number:

700-908-3

Cas Number:

19444-21-4

Molecular formula:

C7H12O3

IUPAC Name:

prop-2-en-1-yl 2-hydroxy-2-methylpropanoate

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Animal Farm of IFFA CREDO, 79592 L'Arbresle, France
- Age at study initiation: 7 - 8 weeks
- Weight at study initiation: 23 - 27 g (males), 29 - 34 g (females)
- Assigned to test groups randomly: The animals were randon selected
- Fasting period before study:
- Housing: Animals housed in groups of 5 and marked with colur pens
- Diet (e.g. ad libitum): Ad libitum (pelleted, certified standard diet)
- Water (e.g. ad libitum): Ad libitum (tap water)
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5 °C
- Humidity (%): 20-49 % rH
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12/12 hours

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Carboxymethylcellulose (CMC), 0.5% in water + 0.1% Tween 80.

- Justification for choice of solvent/vehicle: Found to be best suited to yieldng an applicable suspension at the dose level of 200 mg/kg.

- Concentration of test material in vehicle: Sampling time 24 hours: 50mg/kg, 100 mg/kg and 200mg/kg per 10 ml/kg p.o.
Sampling time 48 hours: 200 mg/kg per 10 ml/kg p.o.

- Amount of vehicle (gavage): 10 ml/kg body weight

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Carboxymethylcellulose (CMC), 0.5% in water + 0.1% Tween 80 was found to be best suited to yieldng an applicable suspension at the dose level of 200 mg/kg.

Duration of treatment / exposure:

Single dose - oral by stomach tube

Frequency of treatment:

Single dose

Post exposure period:

24 hour and 48 huurs

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
50mg/kg, 100 mg/kg and 200mg/kg per 10 ml/kg p.o.
Basis:
analytical conc.
24 hour

Doses / concentrations 2

Remarks:

Doses / Concentrations:
200 mg/kg per 10 ml/kg p.o.
Basis:
analytical conc.
48 hour

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Positive control: Cyclophospamide, 64 mg/kg (ENDOXAN, Asta, Geramny), dissolved in bidistiled water.

Examinations

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Prior to the mutagenicity test the maximum tolerated dose (TD) of the test substance to be applied was determined in a tolerability test. The MTD is defined as the highest dose, which induces significant toxicity but no lethality. The experiments were performed stepwise. The highest dose tested was 200 mg/kg. In each step one male and one female animal were treated. The observation period was three days. The dose, which was finally selected as the MTD, was confirmed by treating an additional pair of mice.
Signs of toxicity were recorded hourly for the first few hours after application and once the following days.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
The test substance was administered once by gavage to groups of five male and five female mice at doses of 50 mg/kg, 100 mg/kg and 200 mg/kg. Additional groups of animals were treated with the vehicle alone or with the positive control cyclophosphamaide (CPA) at a dose of 64 mg/kg.
From the high dose group and from negative control group animals were sacrificed 24 and 48 hours thereafter. From the intermediate and low dosage group and from the positive control group animals were sacrificed 24 hours after application.

DETAILS OF SLIDE PREPARATION:
The animals were sacrificed by carbon dioxide asphyxiation. Bone marrow was harvested in fetal calf serum from the shafts of both femurs, centrifuged and resuspended in fetal calf serum. Smears prepared from this suspension were stained with May-Grünwald/Giemsa solution and mounted.

METHOD OF ANALYSIS:
Femoral bone marrow cells were prepared and polychromatic erythrocytes were scored for micronuclei.

Evaluation criteria:

Micronuclei are uniform, darkly stained, more or less round bodies in the cytoplasm of erythrocytes. Inclusions which are reflective, improperly shaped or stained, or which are not in the focal plain of the cells are judged to be artifacts and are not scored as micronuclei. Cells containing more than one micronucleus are only counted once.
Prior to analysis the slides were coded. The slides of five animals/sex/dose, showing good differentiation between mature and polychromatic erythrocytes, were scored by a laboratory technician. The incidences of micronucleated polychromatic erythrocytes (MNPCE) among at least 2000 polychromatic erythrocytes (PCE), and the ratio of PCE to normochromatic erythroyctes (NCE) among a total of at least 1000 erythrocytes was determined for each slide.

Statistics:

The significance of differences between control and treated groups was assessed by the Chi-Squared-Contingency-Test (F=1, p<0.05).

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY:
In a first step of the tolerability test, the dose of 2000 mg/kg of CA 2215 A (Intermediate of CGA 276854) was administered to one female and one male animal. Both animals died with two hours after administration, showing hunched posture. In a second step, one female and one male were dosed with 800 mg/kg of the test substance. Again, both animals died within two hours, showing hunched posture. In a third step, one female and one male were treated with 320 mg/kg. The female survived the treatment, the male was found dead the day after treatment; creeping movement was noted some hours after administration. In a fourth step another pair of mice were treated with 200 mg/kg of the test substance. Both animals survived without signs of toxicity. In step five, a female was treated with 500 mg/kg and died within eight hours after administration. Creeping movement and miosis were observed some hours after administration.
In consequence, the confirmatory experiment was carried out by dosing ome male with 200 mg/kg and one female with 320 mg/kg of the test substance. Both animals survived the treatment without showing symptoms of toxicity.
Based on the these results th doses of 200 mg/kg (males) and 320 mg/kg (females) were chosen as he highest doses to be administered in the micronucleus test.

Micronucleus test:
The test was performed with male mice and the doses of 50, 100 and 200 mg/kg at the 24 hour sampling time and the high dose (200 mg/kg) only at the 48 hour sampling time. The female mice initially were treated with 80, 160 and 320 mg/kg at the 24 hour sampling time and the high dose (320 mg/kg) at the 48 hours sampling time. Since in the female groups dosed with 320 mg/kg, ten of thirteen animals were found dead the day after administration, a repeat experiment with female mice was performed with doses of 50, 100, 200 mg/kg (24 hour sampling time) and 200 mg/kg (48 hour sampling time). A negative and a positive control were carried out I addition.
No toxicity symptoms occurred at any dose and sampling time.

At both sampling times (24 and 48 hours) there was no statistically significant increase in the number of micronucleated polychromatic erythrocytes in the animals treated with the respective doses of CA 2215 A (Intermediate of CGA 276854) as compared with the negative control animals.
Refer to table 1
CA 2215 A (ntermediate of CGA 276854) was devoid of any clastogenic/aneugenic activity in this study.

Any other information on results incl. tables

Table 1

Overall mean percentages of micronucleated PCEs
Treatment time	High dose (HD) 200 mg/kg	Intermediate dose (ID) 100 mg/kg	Low dose (LD) 50 mg/kg	Positive control (CPA)	Negative control (Vehicle)
24 hours	0.04	0.03	0.02	1.96	0.02
48 hours	0.02	---	---	---	0.02

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
It is concluded that under the given experimental conditions no evidence for clastogenic or aneugenic effects was obtained in mice treated with CA 2215 A.

Executive summary:

CA 2215 A (Intermediate of CGA 276854), identified as a liquid, 99.0% purity, batch no. P.608005 was investigated for clastogenic (and/or aneugenic) effects on mouse bone marrow cellsin-vivo. The test substance was administered once by gavage to groups of five male and five female mice at doses of 50 mg/kg, 100 mg/kg and 200 mg/kg. Additional groups of animals were treated with the vehicle alone or with the positive control cyclophosphamaide (CPA) at a dose of 64 mg/kg. From the high dose group and from negative control group animals were sacrificed 24 and 48 hours thereafter. From the intermediate and low dosage group and from the positive control group animals were sacrificed 24 hours after application. Subsequently femoral bone marrow cells were prepared and polychromatic erythrocytes were scored for micronuclei. 

No sighs of toxicity were observed with any animal from all dose groups at both sampling times of 24and 48 hours. 

In all dosage groups assessed at different periods post treatment, no statistically significant increase in the number of micronucleated polychromatic erythrocytes was observed when compared with the respective negative control group. 

It is concluded that under the given experimental conditions no evidence for clastogenic or aneugenic effects was obtained in mice treated with CA2215 A.