Coumarin

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1979

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Groups of 24 Mice of CD1 strain (12 female, 12 male) were fed with 300ppm, 1000ppm and 3000ppm of coumarin respectively for 13 weeks respectively. control animals were given withour coumarin in the diet. After 13 weeks, the animals were killed. Gorss necropsies performed, and all lesions examined microscopically.

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

Coumarin

EC Number:

202-086-7

EC Name:

Coumarin

Cas Number:

91-64-5

Molecular formula:

C9H6O2

IUPAC Name:

2H-chromen-2-one

Test material form:

solid: crystalline

Details on test material:

-Name of test material: Coumarin
-Substance type: Pure substance
-Physical state: White crystalline powder
-Analytical purity: not known
-Lot/batch No.: Batch no. CA 78300-01
-Storage condition of test material: stored at room temperature, in a laboratory fume cupboard untile use.

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test animals
-Source: Charles River Laboratories (UK), Manston, Kent, England
-Age at study initiation: 24 days
-Weight at study initiation: 2g
-Hosing: The animals were housed four to a cage in opaque, polypropylene cages(North Kent Plastics Ltd.)
-Diet: Spratts Laboratory Diet No.2 (low fat)
-Water: free access to tap water
-Acclimation period: 7 days
Environmental conditions:
-Temperature: 22℃
- Humidity: 50%
- Lighting control: 12 hours light and 12 hours dark per 24 hours.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: Spratts Laboratory Diet No.2 (low fat)

Details on oral exposure:

Coumarin was administered by admixture with the diet. The required dietary concentrations were obtained by the dilution of a premix prepared each week. Control animals received normal (untreated) diet.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A sample of all test diets fed during weeks 1 and 13 week taken at the time the diets were mixed and dispatched to HRC, Department of Analytical Chemistry. The sample were analysed for:
Concentrations of Coumarin in the test diest; Homogeneity of mixing, Stability of Coumarin in the diet; A validation of the analytical procedure used.
A sample of diet (10g), obtained by standard riffling techniques, was weighed directly into an extraction thimble and extracted(Soxhlet) with methanol(100ml) for 2 hours. On cooling, the methanol extract was diluted volumetrically to 250ml.
This solution was further diluted with methanol to produce a solution, containing coumarin in the concentration range 5-15μg/ml, suitable for analysis by the HPLC.
Results obtained for week 1 samples were within minus 15% of the nominal concentration. Results obtained for week 13 samples were in the range, minus 20-25% of nominal concentrations. This greater deviation from nominal, for the week 13 diets, was probably a result of losses occurring, during both blending of the test diet and prior to solvent extraction, at the higher ambient temperatures experienced at week 13.
Results of analysis of homogeneity samples indicated satisfactory mixing, and stability of the test compound in diet was confirmed during 14 days storage at ambient temperature in the animal rooms.
Procedural recovery values obtained during method validation and concurrently with the determination of stability and analysis of the blended samples indicated that the method was satisfactory for analyzing coumarin in diet over the concentration 100-5000ppm

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Daily, 7days each week

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 300, 1000, 3000 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

12 male and 12 female per dose

Control animals:

yes, concurrent no treatment

Details on study design:

The object of this study, performed at the Huntingdon Research Centre, England, was to determine a suitable high dietary level of Coumarin for a
long term study in mice.
Treatment levels used in this study were based on a 4-week preliminary study carried out at the Huntingdon Research Centre.

Positive control:

No data

Examinations

Observations and examinations performed and frequency:

Clinical findings: no signs of reaction
Mortalities: no deaths.
Food consumption: food consumption by females receiving 3000 or 1000ppm was marginally lower than that of the controls.
Growth: Growth of males receiving 3000 ppm was marginally inferior to that of the controls.
Food utilisation efficiency: Associated in part with the effect upon growth rate, inferior food conversion efficiency was noted for males receiving 3000ppm.

Other examinations:

None

Statistics:

The following parameters were analyzed statistically: mean food consumption, mean bodyweight change, mean organ weight.
Mean values of all dose groups were compared to the mean value for the control group at each time interval.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

Clinical findings: there were no overt signs of reaction to treatment.
Mortalities: There were no deaths.
Food consumption: Probably as a result of slight unpalatability, food consumption by females receiving 3000 or 1000 ppm was marginally lower than that of he controls. Females receiving 300 ppm and males of all treated groups were not thus affected.
Growth: Growth of males receving 3000 ppm was marginally inferior to that of the controls. Growth of treated females and of males receiving 1000 or 300 ppm was not impaired by treatment.
Food utilization efficiency: Associated in part with the effect upon growth rate, inferior food conversion efficiency was noted for males receiving
3000ppm. Efficiency of food utilization by the other treated groups was essentially similar to that of the controls.
Macroscopic pathology: There were no macroscopic findings that were considered to be related to treatment.
Organ weight analysis: Liver weights in treated males were marginally higher than those of the controls although liver weights in treated females were not thus affected. Ulterus weights of females treated with 3000ppm or 1000ppm although it should be noted that histopathological examination did not reveal any changes at the cellular level. There were no other intergroup differences in organ weights that were considered to be related to
treatment.
Histopathological findings: An increase in the incidence and severity of vacuolation of centrilobular hepatocytes was seen armong female mice
receiving 3000ppm, although males receiving this level of treatment did not show this change. There were no other changes in mice receiving 3000 ppm that were considered to be related to treatment.

Effect levels

Dose descriptor:

NOAEL

Effect level:

> 138.3 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

female

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The only evidence ot toxicity was marginal effect on growth among males receiving 3000ppm and an effect on the livers of females receiving 3000ppm. NOAEL corresponding to 1000ppm expressed as 138.3 mg/kg bw in females.

Executive summary:

Introduction:

The object of this study, performed at the Huntingdon Research Centre, England, was to determine a suitable high dietary of coumarin for a long term study in mice. Treatment levels used in this study were based on a 4-week preliminary study carried out at the Huntingdon Research Centre. This report presents all the data obtained in the study.

Result and discussion:

Clinical finding, food consumption, growth and food utilization efficiency ware examined during the testing. At termination, macroscopic pathology, organ weight analysis and histopathological findings were examined.

Conclusion:

The only evidence of toxicity was marginal effect on growth among males receiving 3000ppm and an effect on the livers of females receiving 3000ppm. NOAEL corresponding to 1000ppm expressed as 138.3 mg/kg bw in females.