Registration Dossier

Administrative data

Endpoint:
repeated dose toxicity: oral, other
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November - December 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Study was conducted at a GLP facility in accourdance with accepted guidance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
On Day 28 of the study no blood sample was received from animal number 38.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Test material form:
solid: bulk
Details on test material:
Buff-colored

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Sprague Dawley Crl:CD BR strain rats obtained from Charles River (UK) Limited, Margate, Kent.
Sex:
male/female
Details on test animals and environmental conditions:
At the start of the study animals were five to eight weeks. The weight variation did not exceed 4- 20% of the mean weight for either sex. Animals were housed in groups of five by sex in polypropylene cages with stainless steel mesh lids and grid bases, suspended over trays containing absorbent paper.

Rat and Mouse SQC Expanded Diet No. 1 (Special Diets Services Limited, Witham, Essex, UK) with batch analysis, and tap water ad libitum.
The diet and drinking water are routinely analysed and are considered not to contain any contaminant that could reasonably be expected to affect the purpose or integrity of the study.

Target temperature: 21 ± 2°C
Target humidity: 55 ± 15%
Lighting: Twelve hours of continuous artificial light in each twenty-four hour period.
Ventilation: At least fifteen air changes per hour.

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Once daily, by gavage, using a stainless steel dosing cannula attached to a graduated syringe for twenty-eight consecutive days.
Vehicle:
propylene glycol
Details on oral exposure:
The test material will be dissolved or suspended in a suitable vehicle weekly (subiect to confirmation of stability). Wherever possible, an aqueous formulation will be used, followed by consideration of formulation in vegetable oil (eg Arachis oil), then other specified vehicles. The method of preparation will be documented in the study records.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
The test material formulations were sampled and analysed within three days of preparation. The concentration of UK-114,958 in the test material formulations was determined by high performance liquid chromatography (HPLC) using an external standard technique.
Duration of treatment / exposure:
Not specified
Frequency of treatment:
Once daily for the duration of the study. Similar time each day wherever possib
Doses / concentrationsopen allclose all
Dose / conc.:
1.5 mg/kg bw/day (actual dose received)
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
vehicle alone
No. of animals per sex per dose:
Five animals
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Selection followed the results of a dose sighting study.
- Rationale for animal assignment: animals were selected at random and given a unique number within the study by ear punching.
Positive control:
None

Examinations

Observations and examinations performed and frequency:
Clinical Observations: All animals were examined for overt signs of toxicity, ill health or behavioural changes immediately before dosing and one and five hours after dosing.
Functional Observations: Priot to treatment on days 5, 12, 21 and 27 all animals were observed for signs of functional/behavioural toxicit. Sensory reactivity was tests were also performed on day 27.
Sacrifice and pathology:
On completionl animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
Organ Weight: The following organs removed from animals that were killed at the end of the study and observed for organ weight - Adrenals, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Spleen, Testes, Thymus
istopathology
Samples of tissues were removed from all animals for microscopic examination.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Animals of either sex treated with 150 mg/kg/day developed clinical signs from Day 2 onwards including fur loss, hunched posture, increased salivation and red/brown staining of the external body surface. By Day 18, all surviving animals appeared hunched whilst additional clinical signs were evident during the treatment period among isolated individuals including ataxia, pilo-erection, noisy respiration, occasional/prolonged body tremors and shuffling or splayed gait. Neither of the females that died during the study showed an appreciable deterioration in condition prior to death. The interim male decedent, however, appeared ataxic and hunched and showed pilo-erection, occasional/prolonged body tremors and splayed gait on the day prior to its death. A probable increase in urine micturation was also noted among animals of either sex at this dose level with cage tray-liners appearing saturated from Day 12 onwards; there was, however, no direct clinical evidence of diuresis.

There were no clinically observable signs of toxicity detected among animals of either sex treated with 15 or 1.5 mg/kg/day. Animals of either sex treated with 15 ring/kg/day showed increased salivation either before or approximately two minutes after dosing from Day 14 onwards.

Increased salivation is often observed around the time of dosing and is considered attributable to an unpleasant tasting or locally irritant test material formulation rather than an indication of systemic toxicity.

One female treated with 1.5 mg/kg/day showed red/brown staining of the fur on Day 28 of the study but such isolated findings are occasionally reported among animals housed in groups and are considered not to be toxicologically significant.
Mortality:
mortality observed, treatment-related
Description (incidence):
One female treated with 150 mg/kg/day was found dead at the start of Day 12 and a male from this treatment group was found dead at the start of Day 22.
A further female treated with 150 mg,/kg/day died on Day 28 but this occurred during blood sampling procedures and was considered to be in no way related to test material toxicity. There were no deaths among animals of either sex treated with 15 or 1.5 mg/kg/day.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Females treated with 150 mg/kg/day showed a statistically significant reduction in group mean bodyweight gain during Weeks 1 and 3 of the study. Males from this treatment group showed a reduced bodyweight gain during the first three weeks of treatment, although statistical significance was not achieved. Animals of either sex treated with 15 or 1.5 mg/kg/day showed similar bodyweight gains to control animals throughout the treatment period. Males treated with 15 or 1.5 mg/kg/day showed a statistically significant increase in bodyweight gain during Week 2 of the study but this is unlikely to represent an adverse effect on health and was disregarded.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Animals of either sex treated with 150 mg/kg/day showed a reduction in dietary intake during the first three weeks of treatment when compared with controls which extended to the final week of treatment among females at this dose level. Food efficiency (the ratio of bodyweight gain to dietary intake) was reduced among animals of either sex at this dose level during Weeks 1 and 3 of the study.

Animals of either sex treated with 15 or 1.5 mg/kg/day showed a similar dietary intake and food efficiency to control animals throughout the treatment period.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Quantitative measurement during the second half of the treatment period revealed an increase in water consumption for females treated with 150 mg/kg/day when compared with controls.
There was no effect on water intake detected for males treated with 150 mg/kg/day or among animals from the 15 or 1.5 mg/kg/day treatment groups.
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related changes in the blood chemical parameters measured. Statistical analysis of blood chemical data did not reveal any significant intergroup differences.
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Detailed open-field observations conducted during the study confirmed the clinically observable signs detected among animals at the 150 mg/kg/day dose level. Findings were noted from Week 1 onwards and included hunched posture, pilo-erection, noisy respiration, whole body tremors and spastic or splayed gait although behavioural changes were more evident among females.
There were no treatment-related behavioural differences detected between animals of either sex treated with 15 or 1.5 mg/kg/day and controls.

There were no treatment-related changes in the functional performance parameters measured. Statistical analysis of the data revealed no significant intergroup differences.
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Females treated with 150 mg/kg/day showed a statistically significant increase in liver weight, both absolute and relative to terminal bodyweight, when compared with controls.
Males treated with 150 mg/kg/day and animals of either sex from the remaining treatment groups showed no treatment-related changes in the organ weights measured.
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Description (incidence and severity):
Microscopic examination of bone marrow, lung and spleen sections revealed treatment-related changes among animals of either sex treated with 150 mg/kg/day. An increased severity of fatty infiltration in the bone marrow indicative of myeloid hypoplasia was observed among animals of either sex. A reduction in the thickness of the peripheral gastric musculature was also observed among two animals of either sex whilst females from this treatment group showed an increased incidence and severity of accumulations of alveolar macrophages.

No treatment-related microscopic abnormalities were detected among an of either sex treated with 15 or 1.5 mg/kg/day.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Details on results:
Repeated oral administration of the test material to rats by gavage for a period of up to twenty-eight consecutive days at dose levels of up to 150 mg/kg/day resulted in treatment-related adverse health effects at a dose level of 150 mg/kg/day. There werb no treatment-related changes detected among animals of either sex treated with 15 or 1.5 mg/kg/day

Effect levels

Dose descriptor:
NOEL
Effect level:
ca. 15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
150 mg/kg bw/day (nominal)
System:
other: respiratory, gastrointestinal track and bone marrow
Organ:
liver
bone marrow
spleen
lungs
other: Gastrointestinal system
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
The study was designed to investigate the systemic toxicity of the test material.
It complies with the requirements for notification of a new chemical substance in the EC and follows the testing method described in Commission Directive 96/54/EC (Method B7) and OECD Guidelines for Testing of Chemicals No. 407 "Repeated Dose 28 Day Oral Toxicity Study in Rodents" (Adopted 27 July 1995).
The test material was administered by gavage to three groups, each of five male and five female Sprague-Dawley Crl:CD®BR strain rats, for up to twenty-eight consecutive days, at dose levels of 1.5, 15 and 150 mg/kg/day. A 'control group of five males and five females was dosed with vehicle alone (Polyethylene glycol 400).

Clinical signs, functional observations, bodyweight development, food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for all animals at the end of the study.
All animals were subjected to a gross- necropsy examination and histopathological evaluation of a range of tissues was performed.

Repeated oral administration of the test material to rats by gavage for a period of up to twenty-eight consecutive days at dose levels of up to 150 mg/kg/day resulted in treatment-related adverse health effects at a dose level of 150 mg/kg/day. There werb no treatment-related changes detected among animals of either sex treated with 15 or 1.5 mg/kg/day.

The "No Observed Effect Level" (NOEL) was therefore considered to be 15 mg/kg/day.
Executive summary:

A Repeated Dose 28 Day Oral Toxicity Study in Rodents was performed on the test substance. The test was carried out in a GLP faclilty in accordance with OECD guideline No. 407. Data gathered during the study were used to determine the "No Observed Effect Level" (NOEL) for the substance. The NOEL for the test substance is 15 mg/kg/day.