4,6-Dichloro-5-fluoropyrimidine

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:

2005-05-20 to 2005-08-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

27 July 1995

GLP compliance:

yes

Limit test:

no

Test material

Test material information

1

Reference substance name:

-

EC Number:

446-560-3

EC Name:

-

Cas Number:

213265-83-9

Molecular formula:

C4HCl2FN2

IUPAC Name:

4,6-dichloro-5-fluoropyrimidine

Test animals

Species:

rat

Strain:

Wistar

Remarks:

(Hsd Cpb:WU)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 6 weeks
- Weight at study initiation: males: 139.8 g (mean); females: 122.5 g (mean)
- Fasting period before study:
- Housing: during the experimental period two or 3 animals each were placed
together in Makrolon® cages Type IV under controlled environmental conditions on low-dust wood granules (supplier: Ssniff Spezialdiaten Inc. Soest/Westfalen, Germany; manufacturer: J. Rettenmeier, Ellwangen-Holzmuhle, Germany). Deviating therefrom animals were kept individually in cages Type IIa during the FOB/MA investigations.
- Diet (e.g. ad libitum): ad libitum, fixed-formula standard diet (Provimiv Kliba 3883.0.15 supplied by Provimi Kliba SA, CH-4303 Kaiseraugst, Switzerland)
- Water (e.g. ad libitum): ad libitum, tap water
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-2
- Humidity (%): 55+/-5
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: May 20, 2005 To: End of in Life-Phase: Jun. 24, 2005

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: demineralized water / Cremophor EL (98% / 2%; v/v)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was formulated with demineralized water / Cremophor EL (98% / 2%; v/v) at the appropriate concentrations at room temperature and maximally used over the stability period determined by chemical analyses.

VEHICLE
- Concentration in vehicle: 0.1, 0.5, 2.5 mg/mL
- Amount of vehicle (if gavage): 10mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A UV-spectrometric method for quantifying the test item in liquid formulations was developed. For analytical investigations, representative samples from the test item formulation covering the test item concentration range used in the study, were taken.
These samples were diluted with a mixture of methanol and 0.4 % aqueous perchloric acid (4:1) and subsequently quantified by a spectrometric method with UV-detection; wave length: 262 nm. Standard solutions of the authentic test item were used for calibration. Linearity, Precision, Specificity and Accuracy of the analytical method were evaluated apart from this GLP-study and fulfil the predefined acceptance criteria. Additional system suitability tests in terms of specificity, precision and linearity indicated, that qualified analytical procedures were followed during the study.
Analysis of blank samples (0 mg/ml) revealed no measurable traces of test item.
In order to generate and process the analytical data a commercial available computerised system was used.
Hardware and operating system:
• UV 2401-PC Photospectrometer; Fa. Shimadzu
• Windows NT computer with UV-Probe software version 1.10

Duration of treatment / exposure:

30 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

25 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

5 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

0 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels used in this study were based on the results of a pilot study. During this study Wistar rats (2 males and 2 females per dose group) received the test substance in doses of 0, 5, 25,150 mg/kg for a period of two weeks. Animals of the high dose group died or had to be sacrificed in moribund condition. These animals showed clinical findings like piloerection, apathy, laboured breathing. No clinical findings were recorded for the animals of the other groups.
At 5 mg/kg body weight development was comparable to that of the control groups.
At 25 mg/kg body weight development was affected by the treatment (mainly during the first part of the study). At necropsy at the end of the study the stomach of males and females at 25 mg/kg was discolored and appeared thickened. Based on these results the following dose scheme was used for the present study (in agreement with the sponsor representative): 0,1, 5, 25 mg/kg.
Food intake at 25 mg/kg was lower in males and females during the first week.
- Rationale for animal assignment (if not random): Before the start of the study, male and female animals were assigned to the dose
groups. For that purpose they were placed singly at their arrival in cages in the order they were taken out of the boxes. Thereafter, the animal weights were determined and recorded as well as the position of the animal on the shelf. Animals with extremely high or low body weights as well as surplus animals were removed. Based on evenly distributed chance numbers especially generated for this study, the animals were allocated to their final cage number specified by the random list (whereby the first random number on the list gave the actual position of the animal to be moved into cage No. 1, and so on). The cages were then placed one after the other in shelves in the order of increasing numbers.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: daily

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before start and near the end i
- Dose groups that were examined: n the high dose group and the control

HAEMATOLOGY: Yes
- Time schedule for collection of blood: once at the experimental end
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: All

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: once at the experimental end
- Animals fasted: No
- How many animals: All

PLASMA/SERUM HORMONES/LIPIDS: No
-
URINALYSIS: Yes
- Time schedule for collection of urine: once day 23
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: yes, during urine collection


NEUROBEHAVIOURAL EXAMINATION: Yes / No / Not specified
- Time schedule for examinations: once day 29 or 30
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity / other:
- home cage observation: posture, piloerection, gait abnormalities, involuntary motor
movements, vocalization, others.
- observations during handling: ease of removing, reaction to being handled, muscle
tone, palpebral closure, lacrimation, nasal discharge, salivation, stains, others.
- open field observations: piloerection, respiratory abnormalities, posture,
involuntary motor movements, stereotypy, bizarre behavior, gait abnormalities, vocalization, arousal, rearing, defecation, urination, others.
- reflex/physiologic observations and measurements: approach response, touch response, auditory response, tail pinch response, righting reflex, pupil size, body temperature, grip strength, landing foot splay.

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical observations of the animals revealed no treatment-related findings at 1 or 5 mg/kg. At 25 mg/kg 3 males and 2 females showed increased salivation.
Furthermore, piloerection, emaciation, sunken flanks, poor general condition, reduced motility, hunched back and breathing sounds were observed for the male (No. 16) of this dose group, which had to be sacrificed in moribund condition. Tremor was observed once in the 25 mg/kg male No. 18.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Survival was not affected by the treatment up to 25 mg/kg in males and females.
Male No. 16 (25 mg/kg group) had to be sacrificed on day 21 of the study due to its moribund condition. Histopathology revealed that the lung showed granulocytic infiltration and foreign body granuloma formation. In the wall of the esophagus severe inflammation and necroses accompanied by acanthosis of the epithelium was observed. As these findings are indicative of gavage error the death of this animals is not considered to be test substance-related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight development was not retarded by the treatment with the test substance up to 5 mg/kg. At 25 mg/kg mean body weights of males were (on most days statistically) significantly lower compared to the control group. The differences reached up to 22% and were 7% at the end of the study. Although the differences in body weights between the 25 mg/kg female group and the control group were not statistically significant it can be seen, that the body weight development was retarded also in this group. The difference to control reached up to 9% and were 8% at the end of the study. The transient decrease in body weights on day 23 in male and female groups was caused by the urinary collection period during which animals received no food.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

At 25 mg/kg the food intake per animal was slightly lower compared to that of the
control group. Related to the body weight it was comparable to that of the control
group.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

These examinations gave no evidence for treatment-related effects on the eyes.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The hematological investigations resulted in a few values, which were statistically significantly different to control. However, these differences were not dose related and/or the difference to the respective control value was slight. A toxicological relevance is therefore not assumed. The hematological investigations gave no indication of a test substance related effect on blood coagulation up to 25 mg/kg.
Differential blood count revealed an increase in the number of neutrophils at 25
mg/kg in males and females.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Clinical laboratory tests [determination of enzyme activities, concentrations of substrates and electrolytes in peripheral blood revealed in males a slight decrease in concentrations of protein and albumin at 25 mg/kg. The significantly decreased creatinine (25 mg/kg) and glucose (all dose groups) concentrations in females were not considered to be of toxicological relevance as the differences were relatively slight, no individual value did exceed the -2s range of historical control values, a strict dose dependence was absent (glucose) and correlating findings (e.g. histopathological findings) were not observed.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Quantitative analyses of excreted volume, density, protein and creatinine showed that the protein excretion and the protein/creatinine quotient were slightly decreased at 25 mg/kg in males. The semi-quantitatively determined parameters (pH, blood, bilirubin, glucose, protein, ketone bodies and urobilinogen) were not relevantly changed by the treatment. The microscopy of sediment produced no remarkable findings.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

In summary, the investigations in the home cage, during handling and in the open
field as well as the determination of grip strength and landing foot splay showed no relevant signs or symptoms indicating evidence for a neurotoxic potential in rats exposed to the test substance. The activity determination over the entire 60 minute observation period did not reveal a relevant effect on motor (MA) and locomotor activity (LMA).
The results of the 10-minute intervals indicate that treated and control groups
reacted in a similar way and that there were no relevant differences in the first 10-minute intervals, which are regarded as best indicator of increases or decreases in activity before the animals habituate to a minimal level of activity.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Compared to the respective control group relative weights of livers at 25 mg/kg were 14% higher in males and 16% in females.
In 25 mg/kg females relative weights of hearts were significantly increased. The difference to control was 21%.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Gross findings related to the treatment were observed in the forestomach in male and female rats at 25 mg/kg. These were white coverings of the forestomach mucosa in four males and five females of the high dose group.
Furthermore, prostate and seminal vesicle were diminished in size in two high dose males. No further findings were observed which were regarded to be related to the treatment with the test substance.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At histopathology, squamous cell hyperplasia with hyperkeratosis in the forestomach was observed with incidences of 0/0/5/4 in males and 0/1/5/5 in
females. The severity score increased with the dose. At 25 mg/kg, hyperplasia and hyperkeratosis were accompanied by ulceration of the forestomach epithelium and chronic inflammation with giant cells of the underlying submucosa. These lesions were scored as grade 2 or 3 (slight to moderate).
Occasionally foreign bodies namely hair fragments were detectable within the
inflammatory lesions. The number of adipocytes was reduced in the femoral (males and females) and sternal (only females) bone marrow at 25 mg/kg. Concomitantly, granulopoiesis appeared to be increased in both femoral and sternal bone marrow in rats of either sex dosed at 25 mg/kg.
Corresponding to the reduced size of accessory sex glands seen in individual males at 25 mg/kg, prostate and seminal vesicles/coagulating glands were atrophic in males of this group. Testes and epididymides revealed no changes influenced by the treatment.
A variety of spontaneous changes was noted in control and treated animals with no indication of an effect of treatment. The spectrum of changes is mainly consistent with changes commonly encountered in laboratory rats of this age and strain.

Effect levels

1

Key result

Dose descriptor:

NOAEL

Remarks:

local

Effect level:

1 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other:

Remarks on result:

other: local NOAEL was determined based on local irritative effects on the forestomach mucosa

2

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

5 mg/kg bw/day (nominal)

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

presumably yes

Applicant's summary and conclusion

Conclusions:

Under the conditions described the systemic no-observed-adverse-effect level (NOAEL) was 5 mg/kg body weight for males and females.
With respect to the findings resulting from the local irritative effects on the forestomach mucosa a NOAEL could not be established in females. The changes in
the forestomach in a single female at 1 mg/kg is considered as a borderline case. In males the NOAEL for local toxicity was 1 mg/kg.

Executive summary:

In a subacute toxicity study according to OECD test guideline 407 4,6-dichlor-5-fluorpyrimidine (100 % a.i.)] was administered to 5 Wistar rats/sex/dose in demineralized water containing 2 % v/v Cremophor EL at dose levels of 0, 1, 5, 25 mg/kg bw/day.

At the highest dose of 25 mg/kg bw clinical signs. 3 males and 2 females showed increased salivation. At 25 mg/kg mean body weights of males were (on most days statistically) significantly lower compared to the control group. The differences reached up to 22% and were 7% at the end of the study. The differences in body weights between the 25 mg/kg female group and the control group were not statistically significant but the body weight development was retarded also in this group. The difference to control reached up to 9% and were 8% at the end of the study. Compared to the respective control group relative weights of livers at 25 mg/kg were 14% higher in males and 16% in females. In 25 mg/kg females relative weights of hearts were significantly increased. At histopathology, squamous cell hyperplasia with hyperkeratosis in the forestomach was observed with incidences of 0/0/5/4 in males and 0/1/5/5 in females. The severity score increased with the dose. The difference to control was 21%. At 25 mg/kg, hyperplasia and hyperkeratosis were accompanied by ulceration of the forestomach epithelium and chronic inflammation with giant cells of the underlying submucosa. These lesions were scored as grade 2 or 3 (slight to moderate).

Occasionally foreign bodies namely hair fragments were detectable within the inflammatory lesions. The number of adipocytes was reduced in the femoral (males and females) and sternal (only females) bone marrow at 25 mg/kg. Concomitantly, granulopoiesis appeared to be increased in both femoral and sternal bone marrow in rats of either sex dosed at 25 mg/kg. Based on these results the systemic NOAEL is 5 mg/kg bw for both males and females. With respect to the findings resulting from the local irritative effects on the forestomach mucosa a NOAEL could not be established in females. The changes in the forestomach in a single female at 1 mg/kg is considered as a borderline case. In males the NOAEL for local toxicity was 1 mg/kg.

 

This subacute toxicity study in the rat is acceptable and satisfies the guideline requirement for a subacute oral study in rats.