Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
10th March 1988 to 7th April 1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study is conducted by a recognised test house, to in-house methods and in compliance with GLP. OECD compliance is referenced, although no specific guideline is quoted within the report.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1989

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
No recovery group was assessed
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Bontron E-84
- Substance type: Solid
- Physical state: White Powder
- Analytical purity: Not specified
- Impurities (identity and concentrations): Not specified
- Lot/batch No.: C-7672
- Stability under test conditions: Determined to be stable under conditions of the test by evaluation by HPLC and subsequent recovery.
- Storage condition of test material: At ambient temperatures, in non-continuous artificial light.

Test animals

Species:
rat
Strain:
other: Crl CD(SD)BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd, Margate, Kent, England
- Age at study initiation: 28 +/- 1 days old
- Weight at study initiation: 69 to 83g
- Fasting period before study:
- Housing: Caged in groups of five initially according to sex.
- Diet (e.g. ad libitum): Labsure LAD1 Diet, ad libitum. Analyses were made on all batches of diet used to establish levels of basic nutrients and of specified substances and microorganisms likely to be present in feed components and which, if in excess of specified amounts, might have had an undesirable effect on the test system. Although occasional slight deviation may have been permitted, all batches of diet conformed with the acceptable standard agreed by the study director and the Department of Quality Assurance at the test house.
- Water (e.g. ad libitum): Tap water, ad libitum. Results of routine physical and chemical examination of water at source (Grafham Final Water) as conducted usually weekly by the supplier, Anglian Water Authority, were made available to the laboratory as a quarterly summary report. Additionally, levels of specified substances known to be present from time to time in local water, and which, if present in excess of the recognised maxima recommended (for humans) might have had an undesirable effect on the test system, were determined in the tap water at approximately 6 monthly intervals.
- Acclimation period: 8 days from delivery until start of treatment


ENVIRONMENTAL CONDITIONS
- Temperature (°C): Controlled in the range of 20.4 to 22.7 °C
- Humidity (%): Uncontrolled; mean value of 45.2% by wet and dry bulb thermometer
- Air changes (per hr): Approximately 15 per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial light, per 24 hour period.


IN-LIFE DATES: From: 10th March 1988 To: 7th April 1988

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: methylcellulose
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The physical and chemical stability and homogeneity of suspensions of the test substance in 1% aqueous methylcellulose were assessed by the testing laboratories department of Analytical Chemistry prior to the start of treatment, and the results documented within the report. The test material was deemed to be stable as prepared. Concentration analyses of suspensions prepared for administration on Day 1 were similarly conducted by the analytical chemistry department.
The substance was formulated daily as 2.4, 0.6 and 0.15% w/v suspensions in 1% aqueous methylcellulose and administered orally to rats at dosage levels of 240, 60 and 15 mg/kg/day for 28 consecutive days. Control animals received doses of 1% aqueous methylcellulose alone.
The test substance formulations were magnetically stirred from the time of receipt from the Formulation Department until after administration of the final dose.
Animals were treated once daily, seven days per week for four weeks. Each animal received a constant dosage level based on its most recently recorded bodyweight.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Not specified, although history of experience of use anticipated to the reason.
- Concentration of vehicle: 1% aqueous methylcellulose
- Amount of vehicle (if gavage): 10ml/kg/day.
- Lot/batch no. (if required): Not specified
- Purity: Not specified.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The mean concentrations of the test substance in test suspensions prepared for Day 1 of dosing and the deviation of mean results from nominal values were assessed. Mean results were within 10% of nominal concentrations.
It was determined at concentrations of 0.5 and 30 mg/ml the test substance produced a homogenous suspension in 1% methylcellulose which can be maintained for up to one hour after preparation, while magnetically stirred and successfully resuspended after storage at ambient temperatures for four hours.
Procedural recovery data obtained during method validation and the determination of stability confirm good extraction efficiency of the substance from 1% methylcellulose. A mean recovery of 99.1% +/- 1.04 SD was obtained for 0.5 mg/ml and 100.4% +/- 1.50 SD for 30 mg/ml.
All analysis was carried out using HPLC, with a mobile phase of Methanol/0.05M aqueous ammonium acetate (70:30 v/v)
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily, 7 days per week
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
15 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
60 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
240 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
10 per dose group, 5 male, 5 female
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dosage levels of the test substance were selected on the basis of available toxicity data on the substance.

- Rationale for animal assignment (if not random):
Two days before the start of treatment, each animal was weighed and forty rats were randomly allocated to four groups, each consisting of five males and five females, using a computer program, so that the weight distribution within each group were approximately equalised.
On the day of commencement of treatment, spare animals were removed from the study. No further investigations were performed on these rats.
The cages, (each containing five rats) constituting each group were distributed in batteries in such a manner that possible environmental influences arising from their spatial distribution were equilibrated, as far as possible, for all treatments.

- Rationale for selecting satellite groups:
No satellite groups were used during the study.

- Post-exposure recovery period in satellite groups:
No satellite groups were used during the study.

- Section schedule rationale (if not random): Not specified.
Positive control:
None

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were checked early in each working day and again in the late afternoon to look for dead or moribund animals. At weekends and public holidays a similar procedure was followed, except that the final check was carried out at approximately mid day.
- Cage side observations results are not recorded.

DETAILED CLINICAL OBSERVATIONS: Not conducted.

BODY WEIGHT: Yes
- Time schedule for examinations: All rats were weighed prior to dosing and subsequently at weekly intervals throughout the study. Observations are recorded in Figures 2a and 2b, Table 1, Appendix 1, attached below.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Not applicable; gavage study

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 data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not applicable; gavage study

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Prior to termination
- Anaesthetic used for blood collection: Yes, light ether.
- Animals fasted: Yes. Food was withdrawn overnight prior to collection of samples
- How many animals: All on study
- Parameters checked in table 3, Appendix 3 (attached below) were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Prior to termination
- Animals fasted: Yes. Food was withdrawn overnight prior to collection of samples
- How many animals: All on study
- Parameters checked in table 4, Appendix 4 (attached below) were examined.

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data

OTHER: Organ weights.
Organs weights recorded at termination (liver, kidneys, adrenals, gonads) were analysed adjusting for final bodyweight as covariate as appropriate.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes – See table 6, Appendix 6 attached below.
HISTOPATHOLOGY: Yes – see table 7, Appendix 6 attached below.

Samples of the following tissues from all rats in the control and high dose groups were preserved in 10% buffered formalin for subsequent histopathalogical examination:

Adrenals
Heart
Kidneys
Liver
Spleen
Any other macroscopically abnormal tissue.

The following additional tissues were preserved but were not processed further:
Adrenals (intermediate and low dosage groups)
Aorta
Brain (medullary, cerebellar and cortical sections)
Caecum
Colon
Duodenum
Eyes (Davidsons fluid as fixative)
Heart (intermediate and low dosage groups)
Ileum
Jejunum
Kidneys (intermediate and low dosage groups)
Larynx
Liver (intermediate and low dosage groups)
Lungs
Lymph Nodes (cervical and mesenteric)
Mammary gland
Oesophagus
Ovaries
Pancreas
Pharynx
Pituitary
Prostate
Salivary gland
Aciatic nerve
Seminal vasicles
Skeletal muscle
Spleen (intermediate and low dosage groups)
Sternum (for bone and marrow section)
Stomach
Testes (including epididymis)
Thymus (with parathyroid)
Tongue
Trachea
Urinary bladder
Uterus
Vagina
Any other macroscopically abnormal tissue (intermediate and low dosage groups)

Fixed tissue samples required for microscopic examination were embedded in paraffin wax sections cut to 4 micrometres and stained with haematoxylin and eosin. Microscopic examinations were carried out on tissues as listed above from all rats of the control and high dosage groups.

Other examinations:
None
Statistics:
All statistical analyses were carried out separately for males and females.
Bodyweight data were analysed using weight gains.
The following sequence of statistical tests were used for bodyweight, organ weight and clinical pathology data:
i) If the data consisted primarily of one particular value (relative frequency of the mode exceeds 75%), the proportion of values different from the mode was analysed by appropriate methods. Otherwise:
ii) Bartletts Test was applied to test for heterogeneity of variance between treatments. Where significant (at the 1% level) heterogeneity was found, a logarithmic transformation was tried to see if a more stable variance structure could be obtained.
iii) If no significant heterogeneity was detected (or if a satisfactory transformation was found), a one-way analysis of variance was carried out. If significant heterogeneity of variance was present and could not be removed by a transformation, the Kruskal-Wallis analysis of ranks was used
iv) Except for pre-dose data, analyses of variance were followed by Students “t” test and Williams test for a dose related response, although only the one thought more appropriate for the response pattern observed was reported. The Kruskal-Wallis analyses were followed by the non-parametric equivalents of the “t” test and Williams test.
Where appropriate for organ weight data, analysis of covariance was used in place of analysis of variance in the above sequence. The final bodyweight was used as covariate in an attempt to allow for differences in bodyweight which might have influenced the organ weights.

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):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY (Appendix 6, attached below)
All rats survived the treatment period. There were no clinical signs of ill-health or behavioural changes that were considered to be related to treatment.

BODY WEIGHT AND WEIGHT GAIN (Figures 2a and 2b, Table 1, Appendix 1 attached below)
Bodyweight gains of each group of treated rats were similar to those of the controls throughout the study.

FOOD CONSUMPTION AND COMPOUND INTAKE ( Table 2, Appendix 2, attached below)
The quantity of food consumed by each cage of rats was measured at weekly intervals throughout the study. Food consumption was not disturbed by oral administration of the test material

FOOD EFFICIENCY – Not applicable, gavage study.


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study) – Not applicable, gavage study.

OPHTHALMOSCOPIC EXAMINATION – Not measured.

HAEMATOLOGY (Table 3, Appendix 3, attached below)
There were statistically significant differences in MCHC and MCV values for female rats treated at high dosage level and MCV values for females dosed at 60 mg/kg/day in comparison with the female controls (P<0.05 in all cases). However, the extent of the reductions of MCHC and/or MCV were insufficient to be considered toxicologically important.
Platelet counts for male and female rats treated at 240 mg/kg/day were greater than those for the corresponding controls. The intergroup difference was statistically significant in females only (P<0.05) and no individual values for males or females lay above the ranges expected for this parameter. Accordingly, this apparent increase of platelet counts were attributed to chance.
No other haematological parameter showed a statistically significant difference between control group and treated groups.


CLINICAL CHEMISTRY (Table 4, Appendix 4 listed below)
Albumin levels were lower and globulin levels were higher among male treated rats than among male controls. The differences from control values were statistically significant (P<0.01) only in respect of albumin levels in males dosed at 60 or 240 mg/kg/day. There was a corresponding decrease in A/G ration among treated male rats that attained statistical significance (P<0.05 or P<0.01) at all dosage levels. In view of the absence of any treatment-related changes of total protein in treated male rats, the intergroup differences of albumin, globulin and A/G ratio were insufficient to be considered toxicologically important.
AP values for three rats treated at the high dosage level clearly exceeded the upper limit of the range expected for rats of this age and strain (female AP levels (mU/ml): median178, 95percentile 285). Accordingly, the statistically significant increase of AP in females dosed at 240 mg/kg/day (P<0.05) could not be discounted as a possible treatment-related effect.
The apparent decrease of inorganic phosphorus levels in treated female rats was attributed to chance. The individual values for control females approached or slightly exceeded the upper limit for the range expected for this parameter.
No other apparent changes of biochemical parameters were considered to be of toxicological importance.


URINALYSIS – Not measured


NEUROBEHAVIOUR – Not measured


ORGAN WEIGHTS (Table 5, Appendix 5, attached below)
Organ weights were recorded at termination (liver, kidneys, adrenals, gonads) were analysed adjusting for final bodyweight as covariate as appropriate.
The liver weights of two female rats of the 240 mg/kg/day dose group were substantially higher than those of the controls; statistical analysis of adjusted weights revealing a significant increase for the group as a whole (P<0.01). No observations were made at microscopic examination which could account for these differences in organ weight.
The weights of the kidneys, adrenals and gonads of treated rats were similar to those of the control animals.


GROSS PATHOLOGY (Table 6, Appendix 6, attached below)
Macroscopic findings at termination were considered to be unrelated to treatment with the test substance.

HISTOPATHOLOGY: NON-NEOPLASTIC (Table 7, Appendix 6, attached below)
There were no changes that could be attributed to the administration of the test substance. In particular, there were no histopathological findings to account for the increased liver weights found in female rats dosed at 240 mg/kg/day.

HISTOPATHOLOGY: NEOPLASTIC (if applicable) – Not applicable


HISTORICAL CONTROL DATA (if applicable) – Not applicable


OTHER FINDINGS – None reported

Effect levels

Dose descriptor:
NOAEL
Effect level:
240 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: Highest dose level

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The substamce is deemed to be not harmful or toxic by sub-acute exposure at the doses selected for the study
Executive summary:

The test substance was exposed to male and female rates at three dose levels plus a control group with no test sample exposure daily over a period of 28 days. Clinical observations were recorded during the in-life phase followed by pathological and histopathology examination. No toxicologically significant reactions were noted at any of the test concentration dose levels. No classification is applicable.