6-ethyl-5-fluoro-4(3H)-pyrimidone

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 1998-11-05 to 1998-12-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1995

Deviations:

yes

Remarks:

For recovery animals, bodyweight was not performed on Day 1 of recovery so weight change in the recovery period relate to the weight recorded on Day 28 of treatment which does not affect interpretation of the results or integrity of the study.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

6-ethyl-5-fluoro-4(3H)-pyrimidone

EC Number:

422-460-5

EC Name:

6-ethyl-5-fluoro-4(3H)-pyrimidone

Cas Number:

137234-87-8

Molecular formula:

C6 H7 F N2 O

IUPAC Name:

6-ethyl-5-fluoro-3,4-dihydropyrimidin-4-one

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Batch No.: 960509
Purity: 92.3%

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

(Crl:CD® BR)

Details on species / strain selection:

The albino rat was chosen as the test species as it has been shown to be a suitable model for this type of study and is the species recommended in the test guidelines. The strain of rat used Cri: CD BR was chosen on account of the availability of background data.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd, Margate, Kent, England
- Age at study initiation: 43 ± 2 days
- Weight at study initiation: 194-198 g for male, 162-166 g for female
- Fasting period before study: Not fasted
- Housing: caged in groups of five according to sex in metal cages with wire mesh floors. Each cage measured 35.8 cm wide, 53 cm deep and 25.7 cm high
- Diet: standard pelleted laboratory rodent diet, ad libitum
- Water: ad libitum
- Acclimation period: 15 day

DETAILS OF FOOD AND WATER QUALITY:
The batches of diet used for the study were analysed for nutrients, possible contaminants and microorganisms.
Results of the routine physical and chemical examination of drinking water at source, as conducted, usually weekly by the supplier, are made available to lab as quarterly summaries.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24.5
- Humidity (%): 38-60%
- Air changes (per hr): approximately 19 air changes per hou
- Photoperiod (hrs dark / hrs light): 12 hours artificial light (0700 - 1900 hours) in each 24-hour period

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

administered by oral gavage to rats using a syringe and rubber catheter

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared daily by serial dilution.
For the high concentration, the required quantity of the test substance was dissolved in distilled water and the low and intermediate concentrations were prepared by serial dilution starting with the high concentration.

VEHICLE
- Concentration in vehicle: 5, 15, 50 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The method of analysis for test item in aqueous solutions involved dilution using mobile phase and injection of the diluted test samples onto a high performance liquid chromatograph (HPLC) with ultra-violet detection. The amount of test item in the test samples was quantified by reference to a series of five calibration standards of known concentrations.
Analysis of formulations used in Weeks 1 and 3 of dosing showed acceptable accuracy of preparation

Duration of treatment / exposure:

28 day

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

150 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dosages were selected on the basis of a preliminary oral toxicity study performed at this laboratory and in reference to the key dosages relative to EEC labelling requirements. In this preliminary study, dosing for 7 days at 150, 300 or 500 mg/kg/day, showed no clear treatment-related effects, hence 500 mg/kg/day was considered suitable as a high dose for the Main study.
- Animal assignment: Seven days prior to the start of treatment each animal was weighed and 60 rats were randomly allocated to four groups, two groups (Groups 1 and 4) consisting of ten males and ten females and two groups (Groups 2 and 3) consisting of five males and five females
- Fasting period before blood sampling for clinical biochemistry: Food and water was withdrawn overnight prior to collection of samples. After completion of
collection of urine (approximately 16 hours duration) animals were allowed access to water for about 1 hour prior to blood samples being taken.

Examinations

Observations and examinations performed and frequency:

CLINICAL SIGNS: Yes
During the treatment period all animals were observed prior to dosing and at regular intervals following dosing each day. Any signs of ill health, behavioural change or toxicosis were recorded.
All animals were additionally checked early in each working day and again in the late afternoon to look for dead or moribund animals.

BODY WEIGHT: Yes
- Time schedule for examinations: All rats were weighed the week prior to commencement of treatment (Day -7). In the dosing phase they were weighed prior to dosing on the day of commencement of treatment (Day 1), weekly intervals from Day 1 to Day 22 and then on Day 28 (6 days after the previous weighing). In the recovery phase animals were weighed on the first day of recovery (R1), and then Day 8 (R8) and Day 14 (R14) of recovery. Animals were not weighed in the animal room on Day 29 or Day 43 (R15) as the bodyweight was liable to be affected by the overnight food deprivation for clinical pathology investigation purposes.

FOOD CONSUMPTION:
- The quantity of food consumed by each rat was recorded on a weekly basis. Food intake per rat (g/rat/week) was calculated using the total amount of food given to and left by each cage.

EFFICIENCY OF FOOD UTILISATION:
- Food conversion ratios were calculated, where possible, from the bodyweight and food consumption data as weight of food consumed per unit gain in bodyweight

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to termination (Week 5 = Day 29 and Week 7 = Day 15 of recovery).
- Anaesthetic used for blood collection: Yes (isoflurane/nitrous oxide anaesthesia)
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked: see "CLINICAL PATHOLOGY" attached in background material.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to termination (Week 5 = Day 29 and Week 7 = Day 15 of recovery).
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked: see "CLINICAL PATHOLOGY" attached in background material.

URINALYSIS: Yes
- Time schedule for collection of urine: Day 29 and Day 43 (Day 15 of recovery
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes
- Parameters checked: see "CLINICAL PATHOLOGY" attached in background material.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during the pre-dose period, in Week 4 of treatment and in Week 2 of recovery
- Dose groups that were examined: all groups
- A full functional observational battery and motor activity assessment were performed.

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see "CLINICAL PATHOLOGY" attached in background material.)
After receiving treatment for 28 days (5 per sex per group) or 28 days followed by a 14 day posttreatment observation period (5 per sex groups 1 and 4) all surviving animals were killed by carbon dioxide asphyxiation and subjected to the necropsy

HISTOPATHOLOGY: Yes (see "CLINICAL PATHOLOGY" attached in background material.)

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 (1952/1953) was used. This analysis was followed by the non-parametric equivalent of Williams' 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 +p 0.05) or 1% (**p 0.01 or ++p .0.01) level. It was not possible to perform statistical analysis on food consumption data as there was only one cage in Group 2 and 3 and recovery Groups 1 and 4.
Microscopic pathology results were analysed using Fisher's exact test.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no clinical signs considered to be attributable to treatment. On occasions in Week 4 of treatment, minor transient post-dose salivation, present for up to 1 hour, was seen for all animals receiving 500 mg/kg/day. This clinical signs is commonly seen in studies employing gavage dosing, especially at higher concentrations and is considered to be related to the taste of the formulation; buccal exposure when inserting or withdrawing the catheter is unavoidable.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Bodyweight gains for all treated groups over the treatment and recovery periods were comparable with controls.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption for all treated groups was generally comparable with controls over the treatment period (Weeks 1 - 4).

Food efficiency:

no effects observed

Description (incidence and severity):

Group mean food conversion ratios (FCR) for all treated male and female groups were comparable with controls over the treatment and recovery periods.

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

After 4 weeks of treatment, males and females receiving 500 mg/kg/day and females receiving 150 mg/kg/day showed higher group mean PT values compared with controls, dose-related in degree, with statistical significance being attained. Females receiving 500 mg/kg/day also showed higher APTT mean values compared with controls, though not statistically significant. These differences were not evident following the 2 week recovery period.
All other values, including bone marrow smears, were generally comparable with controls, with any differences considered to be due to natural variation and not an effect of treatment.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In week 4, higher group mean AP values compared with controls were seen for all treated females and also for males receiving 150 or 500 mg/kg,/day, differences dosage-related although with statistical significance being attained only for both sexes receiving 500 mg/kg/day.
Statistically significantly higher group mean GPT values were seen for both sexes receiving 500 mg/kg/day and GOT values for males receiving 150 or 500 mg/kg/day compared with controls.
Males receiving 500 mg/kg/day also showed statistically significantly higher group mean total protein and albumin values compared with controls.
Statistically significantly higher group mean phosphorus and lower chloride values compared with controls were also seen for both sexes receiving 500 mg/kg/day, with higher phosphorus also seen for females receiving 150 mg/kg/day.
For all these parameters, following the 2 week recovery period, group mean values for those animals that had been receiving 500 mg/kg/day were generally comparable with controls showing clear evidence of reversibility.
All other differences, although some are statistically significant, are considered to be due to natural variation and not an effect of treatment.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Higher group mean urinary volume compared with controls was seen for males and females receiving 500 mg/kg/day, statistically significant only for males. Similar differences were not present after 2 weeks of recovery for those animals previously receiving 500 mg/kg/day. Slightly higher group mean urinary volume at 50 and 150 mg/kg/day were considered to be due to individual variation and not due to treatment.
All other differences, some statistically significant, were considered to be due to natural variation and not an effect of treatment.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no behavioural changes that were considered indicative of neurotoxicity.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Males receiving 500 mg/kg/day showed higher group mean bodyweight adjusted and relative liver weights compared with controls at the end of treatment and at the end of 2 weeks without treatment, with the differences attaining statistical significance. There were no corroborative microscopic changes in the liver.
All other differences are considered to be due to natural variation and not an effect of treatment.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The macroscopic examination performed at termination revealed no lesions attributable to treatment with test item.
The incidence and distribution of all findings were considered to fall within the expected background range of macroscopic changes.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Kidneys - An increased incidence and degree of cortical tubular basophilia was noted in male and female rats of the 500 mg/kg/day group, killed at termination. This change was more severe in females than males. Luminal eosinophilic material in the cortical basophilic tubules was found in the majority of male and female rats receiving 500 mg/kg/day.
Following the 2-week recovery period, there were no findings in the kidneys that were considered to be related to treatment.

Lungs - At the terminal kill, there were alveolar macrophages containing brown pigment, haematoidin crystals and/or erythrocytes associated with foci of pneumonitis in a greater number of animals in the 500 mg/kg/day group than in the Control group. Hence, histopathological examination was extended to terminal rats of the 50 and 150 mg/kg/day groups, and recovery rats of the Control and 500 mg/kg/day groups. This change was then also noted in occasional animals of the 50 and 150 mg/kg/day groups, and in a small number of animals in the Control and 500 mg/kg/day recovery groups.
Since this minor finding is sometimes seen in untreated control rats in 4-week studies, and because of the similar incidences in the Control and 500 mg/kg/day groups at the recovery kill, it was considered unlikely that the statistically significant increased incidence (p<0.05) in terminal male rats receiving 500 mg/kg/day was related to treatment.

Effect levels

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

haematology

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Treatment-related effects, indicative of toxicity, were seen for both sexes at 500 mg/kg/day. The degree of toxicity was not marked, given that overall, effects were reversible after two weeks without treatment.
The parameters noted for both sexes at 150 mg/kg/day may have been related to treatment, though with no pathological changes detected this was uncertain. The degree of the differences was small too, indicated by reduced statistical significance. Hence, whilst not a NOEL (no observed effect level), it is considered this dosage is a NOAEL (no observed adverse effect level). For 50 mg/kg/day, the only parameter of note, AP for females, was, after consideration of the small degree of difference and inter-individual variation not considered to be treatment-related or biologically relevant and hence this dosage is a NOEL.

Executive summary:

This study was performed to assess the potential systemic toxicity of test item to CD rats based on OECD 407.

The test item was administered by oral gavage once daily to male and female rats for 28 consecutive days at dosage levels of 50, 150 or 500 mg/kg/day.

There were no deaths at any dosage. There were no treatment-related clinical signs observed and also no treatment-related effects to bodyweight, Food consumption and Efficiency of food utilisation.

There were no behavioural changes which were indicative of neurotoxicity.

Haematology: both sexes receiving 500 mg/kg/day and females receiving 150 mg/kg/day had increased PT clotting times, with females receiving 500 mg/kg/day also showing increased APTT clotting times. None of these changes were evident after two weeks without treatment.

Biochemistry: Females at all dosages and males receiving 150 or 500 mg/kg/day had increased AP. GPT was increased for both sexes receiving 500 mg/kg/day and GOT was increased for males receiving 150 or 500 mg/kg/day. Males receiving 500 mg/kg/day also showed increased total protein/albumin.

Other changes included increased phosphorus and reduced chloride for both sexes at 500 mg/kg/day and increased phosphorus for females receiving 150 mg/kg/day. After completion of 2 weeks without treatment, none of these changes were apparent.

Urinalysis: Increased urine volume was seen for males and females at 500 mg/kg/day. Similar changes were not evident after completion of 2 weeks without treatment.

Organ weights: Increased liver weight was seen for males receiving 500 mg/kg/day, the difference still being present at the end of recovery.

Macroscopic examination: There were no treatment-related effects.

Microscopic examination: In the kidneys of both sexes receiving 500 mg/kg/day, there was increased incidence and degree of cortical tubular basophilia and luminal eosinophilic material. This effect was not present after completion of two weeks without treatment.

Conclusion:

Treatment-related effects, indicative of toxicity, were seen for both sexes at 500 mg/kg/day. The degree of toxicity was not marked, given that overall, effects were reversible after two weeks without treatment.

The parameters noted for both sexes at 150 mg/kg/day may have been related to treatment, though with no pathological changes detected this was uncertain. The degree of the differences was small too, indicated by reduced statistical significance. Hence, whilst not a NOEL (no observed effect level), it is considered this dosage is a NOAEL (no observed adverse effect level). For 50 mg/kg/day, the only parameter of note, AP for females, was, after consideration of the small degree of difference and inter-individual variation not considered to be treatment-related or biologically relevant and hence this dosage is a NOEL.