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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to other study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
Dose-Range-Finding study for OECD 407
GLP compliance:
yes
Limit test:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch number of test material: KL384
- Expiration date of the lot/batch: not specified
- Purity test date: not specified


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark
- Stability under storage conditions: not specified
- Stability under test conditions: not specified

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: mixed with the vehicle (1% w/v aqueous methylcellulose)
- Final dilution of a dissolved solid, stock liquid or gel: not specified

FORM AS APPLIED IN THE TEST (if different from that of starting material): mixed with the vehicle. Formulations were prepared freshly each day.
Species:
rat
Strain:
Sprague-Dawley
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: 28 ± 1 d (on arrival)
- Weight at study initiation: males: 124.83 g (mean); females: 125.6 g (mean)
- Fasting period before study: no
- Housing: 3 rats of the same sex per cage
- Diet: standard pelleted laboratory rodent diet ad libitum
- Water: drinking water ad libitum
- Acclimation period: 6 days
- Microbiological status when known: not specified
- Method of randomisation in assigning animals to test and control groups: One day prior to the start of treatment each animal was weighed and twelve rats were randomly allocated to two groups, each consisting of three males and three females. This allocation was carried out using a computer program, so that the weight distribution within each group was similar and the initial group mean bodyweights were approximately equalised.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5 - 22.0
- Humidity (%): 49 - 64
- Air changes (per hr): approx. 19
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 13 July 1994 (arrival date) To: not specified
Route of administration:
oral: gavage
Vehicle:
other: 1 % w/v aqueous methylcellulose
Details on oral exposure:
The high dosage was selected on the basis of available toxicity data, in particular an acute oral toxicity study (LD50 >2 g/kg/bodyweight).
Groups of six rats (three males and three females) were dosed as follows:

Group Cage label/colour code Treatment Dosages No. of rats Rat number
m f m f
1 White Control 1% MC# - 3 3 1-3 7-9
2 Red Test substance 1000 mg/kg bw 3 3 4-6 10-12


# 1 % w/v aqueous methylcellulose

The test substance was administered by oral gavage to rats of Group 2 using a syringe and rubber catheter at a dose volume of 10 ml/kg/day.
Control animals received the same dose volume of the vehicle alone, by oral gavage (1 % MC, 10 ml/kg/day).
Each animal received a constant dosage based on its most recently recorded bodyweight.
Animals were treated once daily for seven consecutive days.
Prior to dosing, the test substance formulations were mixed by inversion (x 20) and subsequent mixing using a magnetic stirrer occurred for a period of at least 10 minutes before dosing commenced.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
7d
Frequency of treatment:
once daily
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
3 sex / dose
Control animals:
yes, concurrent vehicle
Details on study design:
Clinical signs
All animals were observed five times on Day 1 and three times per day subsequently for signs of ill health, behavioural changes or toxicosis. Any observed changes were recorded. All animals were checked early in each working day and again in the late afternoon to look for dead or moribund animals. On Saturdays, Sundays and public holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.

Bodyweight
All rats were weighed prior to dosing on Days 1 (Week 0), 4 and 8.

Food consumption
The quantity of food consumed in each cage was measured at the end of the treatment period.

Water consumption
Daily monitoring by visual appraisal was maintained throughout the dosing period.
Positive control:
none
Observations and examinations performed and frequency:
Animals were observed five times on Day 1, three times per day subsequently for signs of ill health, behavioural changes or toxicosis. Any observed changes were recorded. All animals were checked early in each working day and again in the late afternoon to look for dead or moribund animals. On Saturdays, Sundays and public holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (All animals were subjected to a macroscopic examination which consisted of opening the thoracic and abdominal cavities.)

The liver, kidneys and spleen from each animal were dissected free of fat and weighed.
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:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
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:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Details on results:
The only clinical observation during the study was yellow-coloured faeces on the tray paper for all rats receiving 1000 mg/kg/day. This incidental finding was considered to be due to the presence of the test substance in the faeces and not a manifestation of toxicity.

Slightly lower than control bodyweight gains were recorded for male rats receiving treatment. However, this difference from control was small and all individual gains were satisfactory. This difference from control was considered to represent variation and not an effect of treatment. The bodyweight gains for the female rats were similar to controls.

The group mean liver weight of male rats receiving 1000 mg/kg/day was slightly lower than the controls, this was attributable to the lower terminalbodyweights recorded for these rats and was not considered to be an effect of treatment. The remaining organ weights were comparable to controls.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed.
Critical effects observed:
no
Conclusions:
The results of this seven-day preliminary study suggest that 1000 mg/kg/day was a suitable choice for the high dosage level for the subsequent four-week toxicity study with a two-week recovery period.
Executive summary:

The test substance was administered orally to a group of three male and three female rats at 1000 mg/kg/day (at a dosage volume of 10 ml/kg/day), formulated as a 10% w/v suspension in 1 % w/v aqueous methylcellulose (1 % MC). A further group of three male and three female rats received the vehicle (1 % MC) alone as a control.

Following seven consecutive days of treatment all animals were killed on Day 8. Clinical signs, bodyweight, food consumption, organ weight and macroscopic examinations were recorded for the study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report date:
1995

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
12 May 1981
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Test material form:
solid: particulate/powder
Details on test material:
- Appearance: Yellow powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch number of test material: KL384
- Expiration date of the lot/batch: not specified
- Purity test date: not specified


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark
- Stability under storage conditions: not specified
- Stability under test conditions: The chemical stability and homogeneity of test substance formulations were assessed prior to the start of treatment and concentration analyses of formulations prepared for administration on Days 1 and 22 were performed by the testing facility. The results confirm that specimen formulations were homogeneous and stable for a 4-hour period representing the maximum time from preparation to completion of dosing.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: mixed with the vehicle (1% w/v aqueous methylcellulose)
- Final dilution of a dissolved solid, stock liquid or gel: not specified

FORM AS APPLIED IN THE TEST (if different from that of starting material): mixed with the vehicle. Formulations were prepared freshly each day.

Test animals

Species:
rat
Strain:
Sprague-Dawley
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: 28 ± 1 d (on arrival)
- Weight at study initiation: males: 181 g (mean); females: 165.5 g (mean)
- Fasting period before study: no
- Housing: 5 rats of the same sex per cage
- Diet: standard pelleted laboratory rodent diet ad libitum
- Water: drinking water ad libitum
- Acclimation period: 13 days
- Microbiological status when known: not specified
- Method of randomisation in assigning animals to test and control groups: Four days prior to the start of treatment each animal was weighed and sixty rats were randomly allocated to four groups, two groups consisting of ten males and ten females and two groups consisting of five males and five females. This allocation was carried out using a computer program, so that the weight distribution within each group was similar and the initial group mean bodyweights were approximately equalised.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.5 - 25
- Humidity (%): 39 - 71
- Air changes (per hr): approx. 19
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 13 July 1994 (arrival date) To: not specified

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was weighed out and mixed with the vehicle (1% w/v aqueous methylcellulose) using a high shear homogeniser. For each concentration this process was repeated. Formulations were prepared freshly each day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The chemical stability and, homogeneity of test substance formulations were assessed prior to the start of treatment and, concentration analyses of formulations prepared for administration on Days 1 and 22 were performed by the testing facility.

The analytical results confirm that the doses were accurately formulated for Day 1 and Day 22 of the toxicity study. The results also confirm that specimen formulations were homogeneous and stable for a 4-hour period representing the maximum time from preparation to completion of dosing.
Duration of treatment / exposure:
28 d
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:
10 for high dose and control
5 for low and mid dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The high dosage was selected on the basis of available toxicity data and a preliminary oral toxicity investigation. A dosage of 1000 mg/kg/day is the limit level for this study design.
- Rationale for selecting satellite groups: not specified
- Post-exposure recovery period in satellite groups: Following the 4 week treatment period, five male and five female animals from Groups 1 and 4 (lowest animal numbers were selected) and surviving animals of Groups 2 and 3 were killed on Day 32 (post-treatment sacrifice). These rats were dosed until the day prior to sacrifice. The remaining animals of Groups 1 and 4 were retained for a 2 week post-treatment observation period and were killed on Day 46 (recovery sacrifice); these rats received their last dose on Day 28.
- Section schedule rationale (if not random): not specified
Positive control:
none

Examinations

Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: 3 x daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: Yes
- The quantity of food consumed in each cage was measured at weekly intervals throughout the study

FOOD EFFICIENCY: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of treatment period (day 30); for remaining rats after the 2 week recovery period (Day 44)
- Anaesthetic used for blood collection: Yes (light ether anaesthesia)
- Animals fasted: Yes (overnight)
- How many animals: five male and five female rats selected from vehicle control and high dose group and all rats of low and mid dose groups
- Parameters examined: Packed cell volume (PCV), Haemoglobin (Hb), Red blood cell count (RBC), Mean corpuscular haemoglobin concentration (MCHC), Mean corpuscular volume (MCV), Platelet count (Plts), Total white blood cell count (WBC), Thrombotest (TT), Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of treatment period (day 30); for remaining rats after the 2 week recovery period (Day 44)
- Animals fasted: Yes (overnight)
- How many animals: five male and five female rats selected from vehicle control and high dose group and all rats of low and mid dose groups
- Parameters examined: Glucose, Total protein, Albumin (Alb), Globulin (Glob), Albumin/Globulin ratio (A/G), Urea nitrogen (Urea Nitr), Creatinine, Alkaline phosphatase (AP), Glutamic-pyruvic transaminase (GPT), Glutamic-oxaloacetic transaminase (GOT), Gamma-glutamyltransferase (γ-GT), Total bilirubin (Bilirubin), Sodium (Na), Potassium (K), Calcium (Ca), Chloride (Cl), Inorganic phosphorus (P), Cholesterol (Chol), Triglycerides (Tri-glyc)

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of treatment period (day 30); for remaining rats after the 2 week recovery period (Day 44)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (overnight)
- Parameters examined: Volume (Vol), pH, protein (not determined for recovery male animals), Total reducing substances (TRS), Glucose, Ketones, Bile pigments, Urobilinogen, Haem pigments

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
adrenals*, aorta, brain (medullary, cerebellar and cortical sections), caecum, colon, duodenum, eyes (Davidson's fluid as fixative), femur (for bone and marrow sections) with joint Harderian gland, heart*, head (to preserve nasal cavity, paranasal, inuses, oral cavity, nasopharynx, middle ear, teeth, lachrymal gland and Zymbal's gland), ileum, jejunum, kidneys, larynx, liver*, lungs, lymph nodes (cervical and mesenteric*), mammary gland, oesophagus, ovaries, pancreas, pharynx, pimitary, prostate, rectum
salivary gland, sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord (cervical level), spleen*, stomach, sternum, testes including epididymides* (Bouins as fixative), thymus (where present), thyroid (with parathyroid), tongue, trachea, urinary bladder, uterus (with cervix), vagina, any macroscopically abnormal tissue*

* Tissues required for histopathological examination for rats from vehicle group and high dose group

HISTOPATHOLOGY: Yes
Statistics:
All statistical analyses were carried out separately for males and females using the individual animal as the basic experimental unit. Different sequence of statistical tests was used for bodyweight gains, food consumption (using weekly cage totals), organ weight and clinical pathology data.

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:
not examined
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:
no effects observed
Behaviour (functional findings):
not examined
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:
no effects observed
Details on results:
The only clinical observation during the treatment period of the study was yellow-coloured faeces on the cage tray paper under the cages of rats receiving 1000 mg/kg/day. This incidental finding was considered to be due to the presence of the test substance in the faeces and not a manifestation of toxicity.

After the recovery period, kidney weight (bodyweight adjusted) was statistically significantly lower than the control for male rats treated at 1000 mg/kg/day and higher than control adrenal weights were recorded for female rats treated at 1000 mg/kg/day. In the absence of any treatment-related effects on these organs at Week 5 these small differences from control were considered to reflect variation.

Effect levels

Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: No effects observed up to and including the highest tested dose.

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
It was concluded that 1000 mg/kg/day represents the no-observed effect level (NOEL) for the test substance in the rat.
Executive summary:

The substance was administered by oral gavage, once daily, to three groups of rats for a minimiun of twenty eight consecutive days, at dosage levels of 15, 150 or 1000 mg/kg/day. The test material was prepared as suspensions in 1% w/v aqueous methylcellulose (1% MC) at concentrations of 0.15, 1.5 or 10% w/v and was administered at a dosage volume of 10 ml/kg/day. Control animals received the vehicle (1% MC) alone at the same dose volume (10 ml/kg/day).

All rats of Groups 2 and 3 (15 and 150 mg/kg/day respectively) and five males and five females from each of Groups 1 and 4 (Control and 1000 mg/kg/day respectively) were killed following the fourweek treatment period (Day 32). The remaining animals (five males and five females from Groups 1 and 4) were retained for a two-week recovery period, following which, they were also killed (Day 46).

Bodyweights, food consumption and clinical observations were recorded during the study. Blood and urine samples were taken from all rats shortly prior to termination following the four-week treatment and two-week recovery periods. All animals were killed and subsequently examined macroscopically; specified tissues were then prepared for histopathological examination.

There were no changes seen in the parameters measured in this study namely clinical signs, bodyweights, food consumption, clinical pathology, organ weight analysis, macroscopic or microscopic pathology.