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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15th July 1999 to 14th November 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
(adopted 27 July 1995)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
Commission Directive 96/54/EC
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection: 23rd March 1998 Date of signature: 21st July 1998
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Sponsor's identification: ST 03 C 99
Description: colourless liquid
Date received: 30 June 1999
Storage conditions; under nitrogen at room temperature in the dark

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd, Margate, Kent, United Kingdom.
- Age at study initiation: Approximately 5 to 7 weeks old.
- Weight at study initiation: 178 - 223 g (male) and 159 - 194 g (female).
- Fasting period before study: No data.
- Housing: The animals were housed in groups of 5 by sex in polypropylene grid-floor cages suspended over trays lined with absorbent paper.
- Diet (e.g. ad libitum): A pellet diet (Rat and Mouse SQC Expanded Diet No.1, Special Diets Services Limited, Witham, Essex, United Kingdom). Ad libitum.
- Water (e.g. ad libitum): Mains water. Ad libitum.
- Acclimation period: 11 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2ºC.
- Humidity (%): 55 ± 15%.
- Air changes (per hr): ≥ 15 changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours continuous light and 12 hours darkness.

IN-LIFE DATES: Day 0 (the day of dosing) to Day 28.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test material was prepared at 7.5, 75 and 500 mg/ml, as a solution in Arachis oil BP. The stability and homogeneity of the test material formulations were determined at the test laboratory. The formulations to be stable for at least 14 days. Formulations were therefore prepared weekly and stored at approximately 4ºC in the dark.

DIET PREPARATION
Not applicable.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The stability and homogeneity of the test material formulations were determined at the test laboratory. The samples used were test material extracted with acetonitrile with final theoretical concentration of approximately 0.01 mg/ml. The formulations were found to be stable for at least 14 days.
- Concentration in vehicle: 7.5, 75 and 500 mg/ml.
- Amount of vehicle (if gavage): 2 ml/kg.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Summary; The concentration of ST 03 C 99 in the test material formulations was determined by gas chromatography (GC) using an external standard technique.

Samples; The test material formulations were extracted with acetonitrile with final theoretical concentration of approximately 0.01 mg/ml.

Standards; Standard solutions of test material were prepared in acetonitrile at a nominal concentration of 0.01 mg/ml.

Procedure; The sample and standard solutions were analysed by GC.

Homogeneity determinations; It was apparent by visual inspection that the test material formulations were homogeneous.

Stability determinations; The test material formulations were sampled and analysed initially and then after storage at 4ºC in the dark for 14 days.

Verification of test material formulation concentrations; The test material formulations were sampled and analysed within 3 days of preparation.

Results; Mean concentration found was in the range of 90 – 102% of nominal concentrations. The results showed the formulation to be stable for at least 14 days.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were chosen based on the results of the range-finding study performed. See the summary of the range-finding study in 'any other information on materials and methods incl. tables'.

Positive control:
No

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS / DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Immediately before dosing, and 1 and 5 hours after dosing during the working week. Animals were observed immediately before dosing, and 1 hour after dosing at weekends.
- Cage side observations checked: Overt signs of toxicity, ill-health and behavioural change.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual bodyweights were recorded on Day 0 (the day before the sart of treatment) and on Day 7, 14, 21 and 28. Bodyweights were also recorded at terminal kill.

FOOD CONSUMPTION: Yes
Weekly food consumption was recorded for each cage group.

FOOD EFFICIENCY: Yes
- Food efficiency (group mean bodyweight gain / group mean food consumption) was calculated.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water intake was observed daily for each cage group by visual inspection of the water bottles for any overt changes.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
Haematological and blood chemical investigations were performed at the end of the study (Day 28). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 29.
- Anaesthetic used for blood collection: Not reported.
- Animals fasted: No.
- How many animals: All animals from each test and control group.
- Parameters checked:
(Haematology) Hb, RBC, Hct, MCH, MCV, MCHC, WBC, APTT, Neut, Lymph, Mono, Eos, Bas, CT and PLT.
(Chinical Chemistry) Urea, Glucose, Tot.Prot, Albumin, A/G, Na+, K+, Cl-, Ca++, P, ASAT, ALAT, AP, Creat, Chol and Bili.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
Behavioural assessment: Prior to the start of treatment and on Days 6, 13, 20 and 27.
Functional Performance Tests: On Days 24 and 27.
Sensory Reactivity: On Day 27.
- Dose groups that were examined: All animals.
- Parameters examined:
Behavioural assessment: Detailed individual clinical observations were performed for each animal using a purpose-built arena. The following parameters were observed:
Gait, hyper/hypothermia, tremors, skin colour, twitches, respiration, convulsions, palpebral closure, bizarre/abnormal/stereotypic behaviour, urination, salivation, defecation, pilo-erection, transfer arousal, exophthalmia, tail elevation and lachrymation.
Functional Performance Tests: Motor Activity. Twenty purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals of one sex were tested at each occasion and were randomly allocated to the activity monitors. The tests were performed at approximately the same time each day, under similar laboratory conditions. The evaluation period was sixteen hours for each animal. The percentage of time each animal was active and mobile was recorded for the overall time period and also during the final 20% of thetrial period (considered to be the asymptotic period).

Forelimb/Hindlimb Grip Strength. An automated grip strength meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required
to break the grip of each animal was made. Three consecutive trials were performed for each animal.

Sensory Reactivity: Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. The following parameters were observed:
Grasp response, touch escape, vocalisation, pupil reflex, toe pinch, startle reflex, tail pinch, blink reflex and finger approach.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals were killed by intravenous overdose of sodium pentobarbitone (Sagatal, 60 mglml; May and Baker Limited, Dagenham, Essex, United Kingdom), followed by exsanguination on completion of the dosing period. All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
Organ weights:
The following organs, removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation.
Adrenals, liver, brain, ovaries, epididymides, spleen, heart, testes, kidneys and thymus.

HISTOPATHOLOGY: Yes
Samples of the following tissues were preserved from all animals and preserved in buffered 10% formalin.
Adrenals, aorta (thoracic), bone & bone marrow (femur including stifle joint), bone & bone marrow (sternum), brain (including cerebrum, cerebellum and pons), caecum, colon, duodenum, epididymides, eyes, gross lesions, heart, ileum, jejunum, kidneys, liver, lungs (with bronchi. Lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative), lymph nodes (cervical and mesenteric), muscle (skeletal), oesophagus, ovaries, pancreas, pituitary, prostate, rectum, salivary glands (submaxillary), sciatic nerve, seminal vesicles, skin (hind limb), spinal cord (cervical), spleen, stomach, testes, thymus, thyroid/parathyroid, trachea, urinary bladder and uterus.

All tissues were despatched to the external facility for processing. The tissues (except aorta, bone & bone marrow, eyes, muscle, oesophagus, pancreas, pituitary, salivary glands and skin) from all control and 1000 mg/kg/day group animals were prepared as paraffin blocks, sectioned at nominal thickness of 5 μm and stained with haematoxylin and eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed together with the liver and spleen from all 15 and 150 mg/kg/day dose group animals.
Other examinations:
None
Statistics:
Haematology, blood chemistry and absolute and bodyweight relative organ weights, weekly bodyweight gain and quantitative functional performance and sensory reactivity data were assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (ANOVA) incorporating Levene’s test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett’s test. Where Levene’s test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney ‘U’ test.

Probability values (p) were calculated as follows:
p < 0.001 ***
p < 0.01 **
p < 0.05 *
p ≥ 0.05 (not significant)

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY: No death. No clinically observable signs of toxiicty detected.
Increased salivation was observed which often accompanies the oral administration of a test material formulation and is usually attributed to an unpleasant tasting or locally irritant formulation rather than an indication of systemic toxicity.

BODY WEIGHT AND WEIGHT GAIN: No adverse effects seen.
Females treated with 1000 mglkglday showed a slight but statistically significant reduction in bodyweight gain during Week 3 ofthe study When compared with controls. The level of significance achieved was minimal (p<0.05) and in view of the superior bodyweight gains noted at this dose level during the first half of the study, this isolated reduction was considered to be of no toxicological consequence.

FOOD CONSUMPTION AND FOOD EFFICIENCY: No adverse effects were detected.

WATER CONSUMPTION: No adverse effects were detected.

HAEMATOLOGY: No treatment-related changes were seen.

CLINICAL CHEMISTRY: No treatment-related changes were seen.
Males treated with 1000 mg/kg bw/day showed a statistically significant reduction in plasma glucose when compared with controls but in the absence of any degenerative liver changes or effects on food consumption, this reduction was considered to be of no toxicological importance. Males from this treatment group also showed an increase in plasma creatinine concentration when compared with controls. There was no concomitant change in plasma urea concentration amongst these animals nor any histopathological evidence to support a view of renal dysfunction and, in isolation, this intergroup difference was considered to be of no toxicological significance.
Females treated with 1000 mg/kg bw/day showed a statistically significant reduction in plasma potassium concentration together with an increase in plasma inorganic phosphorus concentration when compared with controls. In the absence of any imbalances in other plasma electrolytes or any evidence of microscopic renal change, these intergroup differences were considered to be of no toxicological importance.

NEUROBEHAVIOUR: There were no intergroup differences in sensory reactivity detected during the study.
Statistical analysis of functional performance revealed a significant increase in forelimb grip strength among 1000 mg/kg bw/day females when compared with controls but in the absence of any changes in the other functional performance parameters measured, this isolated intergroup difference was considered to be of no toxicological significance.

ORGAN WEIGHTS: No treatment-related changes were seen.

GROSS PATHOLOGY: No macroscopic abnormalities were seen.

HISTOPATHOLOGY: No treatment-related changes were seen.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no toxicologically significant effects were observed

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

none

Applicant's summary and conclusion

Conclusions:
Repeated oral administration of the test material, ST 03 C 99, to rats by gavage for a period of 28-consecutive days at dose levels of up to 1000 mg/kg bw/day produced no toxicologically significant effects in the parameters measured. The NOAEL for this substance was considered to be 1000 mg/kg bw/day.
Executive summary:

Introduction, materials and methods

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 CrI:CD®BR strain rats, for twenty-eight consecutive days, at dose levels of 15, 150 and 1000 mg/kg bw/day. A control group of five males and five females was dosed with vehicle alone (Arachis oil BP). Clinical signs, functional observations, bodyweight development and 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 selected tissues from 1000 mg/kg bw/day and control animals was performed.

Results

There were no deaths during the study. No clinically observable signs of toxicity were detected during the study.

No intergroup differences were observed during Behavioural Assessments, Functional Performance Tests and Sensory Reactivity Assessments.

No adverse effect on bodyweight development, food consumption and water intake was observed during the study.

No treatment-related haematological changes, organ weight changes, macroscopic abnormalities or microscopic abnormalities were detected.

Clinical Chemistry: High-dose males showed increased plasma creatinine and reduced plasma glucose, while the females showed reduced potassium and increased inorganic phosphorous. Apart from the difference in creatinine, the differences were not reflected in the lower dose groups or animals of the other sex. While not statistically significant, the creatinine level for high-dose females was higher than for the controls, and in all treated animals, very small non-significant increases relative to controls were seen. While there is an appearance of a dose-response for creatinine concentration, the differences are at most barely significant, and no corresponding change in plasma urea or kidney histopathology was seen to indicate kidney dysfunction.

Conclusions

Repeated oral administration of the test material, ST 03 C 99, to rats by gavage for a period of twenty-eight consecutive days at dose levels of up to 1000 mg/kg bw/day produced no toxicologically adverse significant changes in the parameters measured. The "No Observed Adverse Effect Level" (NOAEL) was therefore considered to be 1000 mg/kg bw/day.

This study is considered as acceptable and satisfies the requirement for sub-acute repeated dose toxicty endpoint.