Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study equivalent to OECD Guideline

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1989
Reference Type:
other: Preliminary Study
Title:
Unnamed
Year:
1988
Report Date:
1988
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report Date:
1990

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Principles of method if other than guideline:
The study was condueted in accordance with the agreed protocol (Toxicol protocol number FCH/2/C).
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): ST 01 C 88
- Physical state: pure active substance
- Physical state: colourless to pale yellow liquid
- Analytical purity: 98% minimum, mixture of two isomers
- Lot/batch No.: 537375 8801, lot 04
- Storage condition of test material: in the dark at room temperature

Test animals

Species:
rat
Strain:
other: Crl: CD(SD)BR strain (VAF plus)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Hargate, England.
- Age at study initiation: not precised in study report
- Weight at study initiation: males: 132-175g ; females: 111-148g
- Fasting period before study: yes
- Housing: The animals were housed in groups of 5, by sex, in grid bottomed polypropylene cages (pattern RC1, North Kent PIastics Iimited, Dartford, England) measuring 56 x 38 x 18cm suspended over cardboard lined excreta trays.
- Diet (e.g. ad libitum): certified, ad libitum excepted deprivation period required in the study guideline
- Water (e.g. ad libitum): certified, ad libitum excepted deprivation period required in the study guideline
- Acclimation period: 12 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-24
- Humidity (%): 28-67 (The 2% fall in relative humidity below the specified protocol lower limit of 30% occurred on one occasion only.)
- Air changes (per hr): not precised (air-conditioned room)
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 1988-04-20 To: 1988-05-18/19

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 1% aqueous hydroxypropylmethylcellulose
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
All animals were dosed at a constant volume of 10ml/kg bodyweight. Individual doses were adjusted weekly according to bodyweight.
Method and frequency of test article formulation: On day 1 duplicate sets of formulations were prepared by 2 slightly different methods. Known amounts of test article were measured out by both weight and volume. These were then added to known volumes of vehicle whilst being mixed with a Silverson mixer. Both sets of formulations were analysed and subsequently, from day 2, formulations were prepared on a volume/volume basis.
Formulations were prepared freshly each day.

DIET PREPARATION
- Rate of preparation of diet (frequency): each day, max. 2 hours before use


VEHICLE
- Justification for use and choice of vehicle (if other than water): test material not sufficiently soluble in water, 1% Hydroxypropylmethylcellulose is used as emulsifier/"drug carrier" to improve solubilisation of the test material.
- Amount of vehicle (if gavage): 1% in aqueous solution
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each formulation (including the control formulation) prepared on days 1 and 27, were analysed for test article. Method: An aliquot of dosing formulation was diluted with acetone and the resultant solution was analysed by gas chromatography. Detection was by FID and the external standard method was used for calibration and quantitation. Actual concentrations were between 94-130% of theoretical concentrations. Therefore, variance bettween nominal and actual concentration is acceptable.
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
daily for 4 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 25, 125, 625 mg/kg bw/day (m/f)
Basis:
other: nominal
No. of animals per sex per dose:
10 (See Table 1 in "Any other information on Mat. & Methods incl. Tables")
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
A preliminary range-finding study was performed to establish suitable dose level (up to 5000 mg/kg/day) of the test material following repeated oral administration to rats of the same strain and source, and to provide information for selection of dose levels for use in the 28 day oral toxicity phase. Four groups, each of 10 rats (five males and five females) were dosed with 0, 200, 1000 & 5000 mg/kg bw of ST 01 C 88. The test material was administered daily, for 7 consecutive days, by gavage. Control animals were treated in an identical manner with 1% aqueous hydroxypropylmethylcellulose alone . Animals were observed daily, bodyweights were recorded on days 1 and 8, and food consumption was recorded daily. The observations preformed were the following:
1. All animals given 5000mg/kg/day were either killed in extremis on day 2 or killed for humane reasons on day 3. There were no deaths at lower dose levels.
2. clinical observations: ataxia, lateral recumbency, prostration, lethargy, shallow respiration, piloerection, generalised deterioration in coat condition and fur staining. They were observed in animals given 1000 or 5000 mg/kg/day and were more prevalent and more severe at the higher dose level.
There were no treatment related clinical signs at 200 mg/kg/day.
3. Bodyweight: Terminal bodyweights of surviving animals were comparable to controls.
4. Food intake: The group mean total food intake of males given 1000mg/kg/day was slightly (10%) lower than the control value. Food intake of remaining survivors was similar to that of the controls.
5. Macroscopic observation/Necropsy: There were no treatment related macroscopic post mortem findings in animals killed after 2, 3 or 7 days of treatment. There were no organ weight data that could be directly compared with controls for animals given 5000mg/kg/day. There were no statistically significant treatment related changes in absolute or relative organ weights in males or females given 200 or 1000 mg/kg/day compared to controls.

In function of these observations, proposed dose levels for the subsequent 28 day oral (gavage) toxicity study in the rat are 0, 25, 125 and 625 mg/kg/day.

- Rationale for animal assignment (if not random): randomly (See Table 1 (Animal Assignement))
Positive control:
None

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day for 28 days


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily for 28 days


BODY WEIGHT: Yes
- Time schedule for examinations: Each animal was weighed on the first day of dosing, weekly thereafter and at necropsy.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):

The food consumed by each cage of animals was recorded weekly and group mean weekly intakes were calculated.

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: Yes / No / No data (Not required in OECD Guideline 407)

OPHTHALMOSCOPIC EXAMINATION: No (Not required in OECD Guideline 407)


HAEMATOLOGY: Yes
- Time schedule for collection of blood: during the final week of treatment (puncture of the retro-orbital sinus)
- Anaesthetic used for blood collection: Yes (light ether anaesthesia)
- Animals fasted: Yes (overnight)
- How many animals: all
- Parameters checked in table [No.?] were examined. See Table 2 Examinations Performed in "Any information on Material & Methods incl. Tables"


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during the final week of treatment (puncture of the retro-orbital sinus)
- Animals fasted: Yes (overnight)
- How many animals: all
- Parameters checked in table [No.?] were examined. See Table 2 Examinations Performed in "Any information on Material & Methods incl. Tables"


URINALYSIS: Yes
- Time schedule for collection of urine: during the final week of treatment
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes (overnight)
- Parameters checked in table [No.?] were examined. See Table 2 Examinations Performed in "Any information on Material & Methods incl. Tables"


NEUROBEHAVIOURAL EXAMINATION: No, according to first OECD Guideline 407 (This test performed in 1988 was performed according to the first OECD Guideline 407 that gives a few/no information about neurotoxicity and immunotoxicity)


OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Necropsies: All animals were weighed and then examined externally. A macroscopic examination was then performed by opening the cranial, thoracic and visceral cavities and observing the appearance of the tissues in situ. Abnormalities were recorded with details of location, colour, shape and size.
Organ weight: The following organs from all animals were weighed after trimming of fat and other contiguous tissue: adrenals, ovaries, thymus, kidneys, spleen, liver, testes.
Contralateral organs were weighed separately but combined weights only have been reported.

HISTOPATHOLOGY: Yes
The specified tissues (adrenals, liver, heart, spleen, kidneys from all control and high dose animals were dehydrated, wax embedded, cut at a nominal thickness of 5 microns, stained with haematoxylin and eosin and examined microscopically. Additional histopathology of the testes and bone (femur) were provided on a requirement from a regulatory authority.
Statistics:
Haematological and organ weight data were analysed by analysis of variance and, if a between groups difference significant at the 5% level occurred, by pairwise t-tests between the control and treatment groups.
Blood chemistry data were analysed by Kruskal-Wallis testing and if a between groups difference significant at the 5% level occurred, significant differences between the control group and the treatment group were determined using the Wilcoxon rank sum test.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY:
No deaths were recorded. Typical treatment related clinical signs were salivation after dosing and staining or wetting of the fur, noted in both sexes dosed at 625 mg/kg/day. One female given 625 mg/kg/day was found prostrate on one day towards the end of the study.

HEAMATOLOGY:
The mean red blood cell values (Hb, RBC, rCV) of male groups given 125 or 625mg/kg/day and of females given 625 mg/kg/day tended to be marginally (up to 6%) lower than control values. Although some of these differences were statistically significant, since they represent only minor variations within the expected ranges, they are considered to be of dubious biological significance.
The mean total leucocyte counts of treated female groups were statistically significantly lower, in an apparently dose related manner, than the control value. However, there were no shifts in the leucocyte differential count and all total leucocyte counts (individual and mean) were within the expected range. Accordingly, the significance of this observation is doubtful.
The variations in prothrombin time and partial thrompoblastin time noted are considered to be fortuitous, since individual and mean values were within expected ranges and since there were no definite trends with dose away from controls.

CLINICAL CHEMISTRY:
The mean plasma triglyceride levels of all male treated groups and of females given 125 or 625 mg/kg/day were moderately (up to about 50%) raised compared to control values. Most of the differences were statistically significant. However, all values were within the expected range and the differences did not show a linear response with dose.
Various other blood chemistry parameters, with values falling within the range for historical controls, showed statistically significant intergroup differences. Some of these apparent differences did not represent a trend with dose away from the controls (raised plasma calcium and lowered plasma chloride in females given 125 mg/kg/day only), others did not show the expected response, for diagnostic significance (lowered AST and bilirubin observed at 625 and also 125 mg/kg/day) and others failed both the above criteria for toxicological significance (lowered BUN-females given 25 mg/kg/day only, lowered alkaline phosphatase-males given 125 mg/kg/day only).

URINALYSIS:
The urine specimens of most males dosed at 625 mg/kg/day contained trace, small or moderate amounts of ketones. In contrast, with the exception of animal 16M (25 mg/kg/day) which had a trace response for ketones, ketones were not found in the specimens of other animals. Possibly, this response arises from breakdown products of the test substance.

ORGAN WEIGHTS:
The mean absolute and relative liver weights of both sexes dosed at 625 mg/kg/day were slightly (up to about 15%) higher than control valnes, these differences being statistically significant. However, the mean absolute and relative liver weights of males alone given 25 mg/kg/day were marginally (about 7%) but statistically significantly lower than the control value.
The mean absolute and relative adrenal weights of females given 625 mg/kg/day were moderately (20 and 23% respectively) and statistically significantly higher than control values.

GROSS PATHOLOGY:
In the testes only one case of bilateral and one of unilateral hypoplasia were seen in the highest dose group (625 mg/kg/day); all the controls were normal. However, such a low incidence of this common condition is unlikely to be of toxicological significance, particularly as one case was unilateral. The histopathological entities observed were recognised as those that occur commonly in this strain of rat maintained in this laboratory and were not significant. The minor variations in incidence of these changes between control and high dose animals were equally not considered to be significant.
The femur was included specifically for the bone marrow. This was examined for general features of the cellular population and proportion of cell types; no counts were undertaken. There was no evidence of any group related changes.

OTHER FINDINGS: None

Effect levels

Dose descriptor:
NOAEL
Effect level:
125 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion