Methyl tetrahydro-2-furancarboxylate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 18 January 1996 to 4 June 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study according to international guideline

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Methyl tetrahydro-2-furancarboxylate

EC Number:

420-670-1

EC Name:

Methyl tetrahydro-2-furancarboxylate

Cas Number:

37443-42-8

Molecular formula:

C6H10O3

IUPAC Name:

methyl oxolane-3-carboxylate

Test material form:

other: liquid

Details on test material:

Identity: Methfat
Chemical name: Methyl tetrahydro-2-furoate
Intended use: Pharmaceutical intermediate
Appearance: Clear colourless liquid
Storage conditions: Room temperature
Batch number: 0002
Expiry date: 7 November 1996
Purity: 99%
Date received: 17 November 1995

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

A total of 22 male and 22 female healthy CD rats of Sprague-Dawley origin (Crl:CD BR VAF PLUS) were received from Charles River (UK) Ltd., Margate, Kent, England on 10 January 1996.
The rats were 28 +/- 1 days old, in a weight range of 65 to 84 g on arrival. An eight day acclimatisation period was allowed between delivery of the animals and start of treatment.
The rats were initally caged, as far as possible, in groups of five according to sex in metal cages with wire mesh floors. A standard pelleted laboratory rodent diet (Special DIet Services Rat and Mouse Maintenance Diet) and drinking water were provided ad libitum.
The batches of diet used for the study had been analysed for nutrients, possible contaminants and micro-organisms.
Results of routine physical and chemical examination of drinking water at source, as conducted, usually weekly by the supplier, were made available to Huntingdon LifeSciences Ltd. as quarterly summaries.
Animal room temperature was controlled in the range 19-23°C and relative humidity was controlled in the range of 32 to 59% RH. These parameters were continuously monitored using a Kent Clearspan M105 7-day chart recorder. Air exchange was maintained at approximately 19 air changes per hour and lighting was controlled to provide 12 hours artificial light (0700-1900 hours) in each 24-hour period.
The health status of all animals was monitored, by daily observation throughout the acclimatisation period, to ensure that the rats selected for final assignement to the study were satisfactory.
Two days prior the start of the treatment each animal was weighed and forty rats were randomly allocated to four groups, each consisting of five males and five females. This allocation was carried out using a computer program, so that the weight distribution within each group was similar and the initial group mean bodyweights were approximately equalised.
Each rat was identified within each cage by ear-punch and individually by tail mark (tattoo).
Following the commencement of treatment spare animals were removed from the study. No further investigations were performed on these animals.
The cage (each contaning five rats) were distributed in batteries in such a manner that possible environmental influences arising from their spatial distribution were equilibrated, as far as possible, for all treatments.
Each cage was identified by a coloured label according to group. Each label displayed the study schedule number, cage number, sex, individual animal numbers and the initials of the study director and home office licensees.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: distilled water

Details on oral exposure:

The test substance was administered by oral gavage to rats using a syringe and rubber catheter at a dose volume of 10 ml/kg/day.

Duration of treatment / exposure:

28 days

Doses / concentrations

Remarks:

Doses / Concentrations:
0; 15; 150 and 1000 mg/kg/day
Basis:
other: see "Details on study design"

No. of animals per sex per dose:

5

Control animals:

yes

Details on study design:

Doses/concentration basis:
The high dosage was selected on the basis of available toxicity data and a preliminary oral toxicity investigation performed at this laboratory (Schedule number GLC/19). A dosage of 1000 mg/kg is the limit level for this study design. The intermediate and low dosage levels were selected on the basis of the key dosages relative to EEC labelling requirements.

Examinations

Observations and examinations performed and frequency:

Clinical signs
All animals were observed three times daily for signs of ill health, behavioural changes or toxicosis. Any observed changes were recorded.
All animals were checked early in each working day and again in the late afternoon to look for dead or moribund animals. This allowed a post mortem examination to be undertaken during the working part of that day. On Saturdays and Sundays a similar procedure was followed except that the final check was carried out at approximately mid-day.

Bodyweight
All rats were weighted prior to dosing on Day 1 (Week 0) and subsequently at weekly intervals throughout the study.

Food consumption
The quantity of food consumed in each cage was measured at weekly intervals throughout the study.

Water consomption
Daily monitoring by visual appraisal was maintained throughout the dosing period.

Sacrifice and pathology:

Clinical pathology - Removal of blood sample
Food was withdrawn overnight prior to collection of samples. Blood was withdrawn under light ether anaesthesia from the orbital sinus of all rats prior to termination (Week 4). Further removal of blood samples for re-analysis was carried out for individual animals.
Haematological and biochemical parameters were analysed.

Terminal studies
Termination
After 28 days of treatment (Day 29) all animals surviving treatment were killed by carbon dioxide asphyxiation and a complete autopsy undertaken. The order of sacrifice was determined using pre-set cage sequence. Specified organs were weighed and relevant tissue samples were fixed for microscopic examination.
Organ weight
The following organs from each animal were dissected free of fat and weighed: adrenals, brain, epididymides, kidneys, liver, ovaries, prostate, seminal vesicles, spleen, testes.

Statistics:

All statistical analyses were carried out separately for males and females using the individual animal as the basic experimental unit.

The following sequence of statistical tests were used for bodyweight gains, organ weight and clinical pathology data:
If the data consisted predominantly of one particular value (relative frequency of the mode exceeded 75%), the proportion of values different from the mode were analysed by Fisher's exact test followed by Mantel's test for a trend in proportions. Otherwise:
Bartlett's test was applied to test for heterogeneity of variance between treatments. If significant heterogeneity was found at the 1% level, a logarithmic transformation was tried to see if a more stable variance structure could be obtained.
If no significant heterogeneity was detected (or if a satisfactory transformation was found), a one-way analysis of variance was carried out followed by Williams' test for a dose related response.
If significant heterogeneity of variance was present and could not be removed by a logarithmic transformation, the Kruskal-Wallis analysis of ranks was used. This analysis was followed by the non-parametric equivalent of Williams' test (Shirley's test).
Covariate analysis of organ weight data (with final bodyweight as covariate) was also performed using adjusted weights for organs where a correlation between organ weight and bodyweight was established at the 10% level of significance.
Significant differences between control animals and those treated with the test substance were expressed at the 5% (P<0.05) or 1% (P<0.01) level.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no mortalities. There were no clinical signs throughout the treatment period that were considered to be of toxicological importance. Increased salivation following dosing was noted occasionally throughout the study for male and female rats receiving 1000 mg/kg/day. Increased salivation following dosing is commonly observed in orally dosed rat studies and is considered to be as a result of unpalatability of the test substance.

Mortality:

mortality observed, treatment-related

Description (incidence):

There were no mortalities. There were no clinical signs throughout the treatment period that were considered to be of toxicological importance. Increased salivation following dosing was noted occasionally throughout the study for male and female rats receiving 1000 mg/kg/day. Increased salivation following dosing is commonly observed in orally dosed rat studies and is considered to be as a result of unpalatability of the test substance.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

see "Details on results"

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was slightly lower than control for high dosage group male and female rats. Food consumption for remaining groups of treated rats was similar to that for controls.

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

no effects observed

Description (incidence and severity):

No differences from control to treated groups

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

see "Details on results"

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

see "Details on results"

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Lower than control male reproductive organ weight were recorded for male rats receiving 1000 mg/kg/day.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

see "Details on results"

Details on results:

Bodyweight
Bodyweight gains were statistically significantly lower than control for male and female rats from the high dosage group throughout the treatment period. The effect on bodyweight for these rats was progressive, with very low gains recorded in the final week of the study.
At 150 or 15 mg/kg/day lower than control bodyweight gains were recorded for female rats throughout the treatment period. Bodyweight gains for these females improved in the latter half of the study. The effect on bodyweight for female rats was dosage-related.
For male rats of the intermediate dosage group bodyweight gains were lower than control in the first half of the study but did not achieve statistical significance. Bodyweight gains for male rats treated at 15 mg/kg/day were similar to control.

Haematology
Neutrophils, lymphocytes, eosinophils, basophils, monocytes (males only), large unstained cells (LUC) and hence total white blood cell counts were statistically significantly lower than control for rats treated at 1000 mg/kg/day. At 150 mg/kg/day total white blood cell counts and LUC counts were also statistically significantly lower than control for female rats.
LUC counts were lower than control for males treated at 150 mg/kg/day and females treated at 15 mg/kg/day. However in the absence of an effect on total white blood cell count, these minor differences were not considered to be of toxicological importance.
Statistically significantly lower than control platelet levels were recorded for male and female rats receiving 1000 mg/kg/day.
Haemoglobin concentration (Hb), mean corpuscular haemoglobin concentration (MCHC), mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH) were slightly lower than control for male rats receiving 1000 mg/kg/day.
At the intermediate dosage of 150 mg/kg/day mean corpuscular haemoglobin concentration (MCHC) and mean corpuscular haemoglobin (MCH) were lower than control for male rats. However, in the absence of an effect on the main red cell parameters at this dosage, these minor differences from control were considered to have arisen by chance.
There were no further differences from control in haematological parameters measured.

Clinical chemistry
Statistically significantly lower than control alkaline phosphatase (AP) levels were recorded for male rats at 1000 mg/kg/day and for female rats treated at 1000 or 150 mg/kg/day.
For male rats treated at 1000 mg/kg/day and female rats receiving 1000 or 150 mg/kg/day globulin levels and hence total protein levels were statistically significantly lower than control. Hence, the A/G ratio for female rats treated at 1000 mg/kg/day was marginally higher than control. For male rats of the high dosage group lower than control albumin levels were recorded.
Minor differences from control for some electrolytes, namely lower sodium (males at 1000 mg/kg/day), lower calcium (males mg/kg/day), higher phosphorous (females at 1000 mg/kg/day) and lower chloride (females at 1000 mg/kg/day) achieved statistical significance. However, these differences from control were small and were not considered to be of toxicological importance.
Glucose levels were statistically significantly higher than control for male rats in all treatment groups. However, there was considerable individual variation and, in the absence of any dosage relationship, these differences from control were not considered to be of toxicological importance.

Macroscopic pathology
The macroscopic examination performed at termination revealed a reduction in the size of the testes (5/5 male rats), the epididymes (5/5 male rats) and the prostate (4/5 male rats) at 1000 mg/kg/day. Minimal contents of the seminal vesicles was observed in 3/5 male rats treated at 1000 mg/kg/day.

Microscopic pathology
Treatment related changes
Treatment related changes were seen in the male reproductive tract at the 1000 mg/kg/day dosage level. The principle change seen was a reduction in spermatogenesis or atrophy in the seminiferous tubules of the testes and the presence of abnormal and round spermatids in the epididymal tubule lumenae.
Some evidence of reduced amounts of prostatic and seminal vesicular colloid was also seen, with some acini/vesicles being not fully distended with colloid. However, this was also seen to a minor degree in a small proportion of control animals and is of doubtful significance, the amount of colloid reduction being considered, particularly in the seminal vesicles, to be within the normal background range for animals of this age. These histopathological changes explain the observations made at necropsy.
Incidental changes
All other histological changes seen were the normal range for rats of this age range and strain and were considered to be of no toxicological significance.

Effect levels

Dose descriptor:

NOAEL

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: NOAEL could not be established in this study

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The target organ for toxicity of the high dosage level of 1000 mg/kg/day was the male reproductive tract for methfat in the rat when administered orally for 28 days.
A no-observed-adverse-effect level (NOAEL) could not be established in this study.