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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 March 1998 to 15 December 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Cross-reference
Reason / purpose:
reference to same study

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 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
Occasionally minor deviations from standard conditions required for room temperature/humidity occurred, but were considered not to have affected the purpose or integrity
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: amber viscous liquid
- Analytical purity: no data
- Storage condition of test material: ambient conditions 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) Limited, Margate, Kent, UK
- Age at study initiation: ca. six to seven weeks
- Weight at study initiation: Males: 168 - 224 g, females: 151 - 192 g
- Fasting period before study: no
- Housing: groups of four by sex in polypropylene gridfloor cages suspended over trays lined with absorbent paper.
- Diet (e.g. ad libitum): Pelleted diet ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2°C
- Humidity (%): 55 ± 15%
- Air changes (per hr): at least 15 per hour.
- Photoperiod (hrs dark / hrs light): 12h light/12h dark

IN-LIFE DATES: From: 1998-03-05 To: 1998-12-15

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test material was prepared at the appropriate concentrations as a solution in Arachis oil BP. The stability of the test material formulations was determined by Safepharm Analytical Laboratory. Results show the formulations to be stable for at least fourteen days. Formulations were therefore prepared weekly and stored at approximately +4C in the dark.
Samples were taken of each test material formulation and were analysed for concentration of the test substance at Safepharm Analytical Laboratory. The results indicate that the prepared formulations were within 10% of the nominal concentration.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Unknown solubility in water
- Concentration in vehicle: 0, 25, 125, 500 mg/mL
- Amount of vehicle (if gavage): 2 mL
- Lot/batch no. (if required): N o data available
- Purity: No data available
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
HPLC using an external standard.

Samples and standards prepared in methanol to give a concentration of 0.5 mg/mL

HPLC system: Hewlett Packard 1050
Column: Hypersil C18 (150 x 4.6 id)
Mobile phase: Eluent A - water and Eluent B - methanol
At time 0 - 10% B; 14 mins - 100% B and 26 mins - 100% B
Flowrate: 1.5 mL/min
UV detector wavelength: 240 nm
Injection volume: 25 µL
Retention time: 14 to 15 mins

Stability determination: analysed initally and after 14 days in the dark at +4°C.
Stability: 92-96% of initial concentration after 14 days

Formulation concentrations: sampled and analysed within three days of preparation
Formulation: 93 - 109% of nominal
Duration of treatment / exposure:
90-days, consecutive
Frequency of treatment:
One dose per day
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
50 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
250 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10 animals/sex/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 (0, 150, 500 and 1000 mg/kg/day; 2 ml/kg/day in Arachis
oil BP). The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test material, and the results of the study are believed to be of value in predicting the likely toxicity of the test material to man. The test material was administered daily, for fourteen consecutive days, by gavage.

- Rationale for animal assignment (if not random): The animals were randomly allocated to treatment groups using a stratified bodyweight randomisation procedure and the group mean bodyweights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomised.
- Rationale for selecting satellite groups: NA.
- Post-exposure recovery period in satellite groups: no data available.
- Section schedule rationale (if not random): Random
Positive control:
None

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were observed immediately before dosing and one hour after dosing at weekends and on public holidays. During the treatment-free period, animals were observed twice daily, morning and afternoon (once daily at weekends and on public holidays). All observations were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were observed immediately before dosing and one hour after dosing at weekends and on public holidays. During the treatment-free period, animals were observed twice daily, morning and afternoon (once daily at weekends and on public holidays). All observations were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual bodyweights were recorded on Day 0 (the day before the start of treatment) and at weekly intervals thereafter. Bodyweights were also recorded at terminal kill.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. Food consumption was recorded for each cage group at weekly intervals throughout the study.

- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No.

FOOD EFFICIENCY: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): water intake was observed for each cage group by visual inspection of the water bottles for any overt changes.
Time schedule for examinations: daily.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: The eyes of all control and high dose animals were examined pre-treatment and before termination of treatment (during Week 12). Examinations included observation of the anterior structures of the eye, pupillary and corneal blink reflex following pupil dilation with 0.5% Tropicamide solution.
- Dose groups that were examined: all control and high dose animals.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Haematological investigations were performed on all animals from each test and control group at the end of the study (Day 90). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 91.
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: all animals from each test and control group
- Parameters were examined:
Haemoglobin (Hb), Erythrocyte count (RBC); Haematocrit (Hct); Erythrocyte indices mean corpuscular haemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC); Total leucocyte count (WBC); Differential leucocyte count: neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos), basophils (Bas); Platelet count (PLT); Prothrombin time (CT) was assessed by ’Hepato Quick’ and Activated partial thromboplastin time (APTT) was assessed by ’Preci Clot’ using samples collected into sodium citrate solution (0.11 mol/l).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood chemical investigations were performed on all animals from each test and control group at the end of the study (Day 90). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 91.
- Animals fasted: No
- How many animals: all animals from each test and control group
- Parameters were examined: Urea, Inorganic phosphorus (P), GIucose, Total protein (Tot.Prot.) AIbumin, Alanine aminotransferase (ALAT), Albumin/Globulin (NG) ratio (by calculation), Aspartate ami notransferase (ASAT), Alkaline phosphatase (AP), Sodium (Na+), Potassi um (K +), Chloride (CI-), Calcium (Ca+ +), Creatinine (Creat), Total cholesterol (Chol), Total bilirubin (Bili)

URINALYSIS: No.

NEUROBEHAVIOURAL EXAMINATION: Yes.
- Time schedule for examinations: Prior to the start of treatment and at weekly intervals thereafter.
- Dose groups that were examined: all animals were observed for signs of functional/ behavioural toxicity.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
On completion of the dosing period all animals were killed by intravenous overdose of
sodium pentobarbitone followed by exsanguination.
All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

HISTOPATHOLOGY: Yes
Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin:
Adrenals, Aorta (thoracic), Bone & bone marrow (femur including stifle joint), Bone & bone marrow (sternum), Brain, Caecum, Colon, Duodenum, Epididymides, Eyes, Gross lesions, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs (with bronchi), Lymph nodes (cervical and mesenteric), Mammary glands, Muscle (skeletal), Oesophagus, Seminal vesicles, Skin (hind limb), Spinal cord (cervical, mid-thoracic and lumbar), , Spleen, Stomach, Testes (with epididymides), Thymus, Thyroid/parathyroid, Tougue, Trachea, Urinary bladder, Uterus.
All tissues from control and 1000 mg/kg/d dose 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 lesion was also processed.
Since there were indications of treatment-related liver, spleen, thyroid, lung and bone marrow changes, examination was subsequently extended to include similarly prepared sections from all animals in the other treatment groups.
Microscopic examination was conducted by the Study Pathologist. All findings were entered into the ROELEE Pathology computerization system for tabulation and report production.
Statistics:
Data were processed to give group mean values and standard deviations where appropriate.
Haematological, blood chemical, organ weight (absolute and relative to terminal bodyweight) and weekly bodyweight gain 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.
Following statistical analysis of MCHC among control and test animals, statistical significance was assigned to females from the 50 mg/kg/day treatment group in comparison with controls. The difference in mean and standard deviation between these two groups was negligible and the results of this analysis were rejected for contravening the function of Dunnett's pairwise comparison test. A single t-test was performed to compare the control female MCHC values with those of each treatment group in turn and the results of this test were reported accordingly.
The haematology variable basophils was not analysed since consistently greater than 30% of the data were recorded as the same value.
Histopathology data were analysed using the following methods to determine significant differences between control and treatment groups for the individual sexes.
Chi squared analysis for differences in the incidence of lesions occurring with an overall frequency of 1 or greater.
Kruskal-Wallis one way non-parametric analysis of variance for the comparison of severity grades for the more frequently observed graded conditions.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
effects observed, treatment-related
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
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:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
The clinically observable signs detected throughout the study were considered to be without toxicological importance.

Mortality:
There were no deaths during the study.

BODY WEIGHT AND WEIGHT GAIN
A statistically significant reduction in bodyweight gain was detected for 1000 mg/kg/day males during Weeks 2 and 10 when compared with that of controls. Bodyweight development in these animals was unaffected throughout the rest of the study period.
No adverse effect on bodyweight gain was detected for 1000 mg/kg/day females or for animals of either sex treated with 250 or 50 mg/kg/day.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
A slight reduction in dietary intake was detected for 1000 mg/kg/day males over
the first three weeks of treatment. Food efficiency was also slightly reduced during Week 10, corresponding to the bodyweight reduction seen at this time.
No adverse effect on food consumption or efficiency was detected for 1000 mg/kg/day females or for animals of either sex treated with 250 or 50 mg/kg/day throughout the study period.

WATER CONSUMPTION
Daily visual inspection of water bottles revealed no intergroup differences.

OPHTHALMOSCOPIC EXAMINATION:
There were no treatment-related ocular effects.
The incidental abnormalities recorded for two control males were consistent with occasionally encountered findings in laboratory maintained rats of the strain and age employed.
HAEMATOLOGY
Statistically significant reductions in haemoglobin, haematocrit, mean
corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration were detected for 1000 mg/kg/day females when compared with controls.
No such changes were detected for 1000 mg/kg/day males or for animals of either sex treated at the remaining dose levels.
The statistically significant reductions in haemoglobin and MCH in 1000 and/or 250 mg/kg/day males were probably attributable to slightly higher than normal control values and, in the absence of effects on erythrocyte numbers and the fact that the majority of individual values were within the respective normal ranges, the intergroup differences were considered to be of no toxicological significance.

CLINICAL CHEMISTRY
A statistically significant reduction in plasma cholesterol was detected for both males and females treated with 1000 mg/kg/day when compared with controls. Many values from this and the other dose groups, including controls, were outside the respective normal ranges and, in the absence of any other changes in the blood chemical parameters measured, this finding was considered to be of dubious toxicological significance.
There were no other toxicologically significant changes in the blood chemical parameters measured.
The statistically significant increases in plasma albumin, albumin/globulin ratio and plasma calcium and sodium concentration all involved individual values within the respective normal ranges and, in isolation, all were regarded as incidental. The remaining intergroup differences were confined to a reduction in male 50 and 250 mg/kg/day alkaline phosphatase and female 1000 mg/kg/day alanine aminotransferase but reductions in these parameters cannot be regarded as toxicologically significant.

URINALYSIS
No data.

NEUROBEHAVIOUR
Behavioural Assessments
There were no treatment-related changes in behaviour detected.

Functional Performance Tests
No data.

ORGAN WEIGHTS
Females treated with 1000 mg/kg/day showed a statistically significant increase in liver and kidney weight, both absolute and relative to bodyweight, when compared with controls. All individual relative liver weights and three kidney weights were outside the respective normal ranges for rats of the strain and age used. The effect extended to 250 mg/kg/day females with increases in relative liver and kidney weight detected although statistical significance was only achieved for liver weight at this dose level.
No adverse organ weight changes were detected for 1000 mg/kg/day males or for animals of either sex treated with 250 or 50 mg/kg/day.
An increase in male 1000 mg/kg/day relative brain weight was detected but, in view of the reduced terminal bodyweight seen in these animals and the absence of any histopathological correlates, the increase was considered to be incidental. Relative adrenal and ovary weights were statistically significantly elevated in 1000 mg/kg/day females compared to those of controls. The level of significance was minimal (p<0.05) and, in the absence of any histopathological evidence to suggest an adverse effect in these organs, the increases were considered to be without toxicological importance.
The remaining statistically significant intergroup differences detected for the male treatment groups were confined to absolute weights and, in the absence of any similar changes in the respective relative weights, were considered to be fortuitous.

GROSS PATHOLOGY:
No treatment-related macroscopic abnormalities were detected.

HISTOPATHOLOGY: NON-NEOPLASTIC
Liver: Five females treated with 1000 mg/kg/day showed centrilobular hepatocyte enlargement in relation to treatment (p < 0.05). Hepatocyte enlargement is commonly observed in the rodent liver following the administration of xenobiotics and, in the absence of associated inflammatory or degenerative changes, is generally considered to be adaptive in nature. The condition was not observed amongst males, or amongst females from the remaining dose levels.
Kidneys: Five females dosed at 1000 mg/kg/day and one control female exhibited epithelial hypertrophy of inner cortical tubules. Although not quite attaining statistical significance, this minimal change was considered to be related to treatment at 1000 mg/kg/day for females, but was not associated with any degenerative changes. Such an effect of treatment was not observed for males or for females from the 250 or 50 mg/kg/day dose groups.
Thyroids: An increased incidence of follicular cell hypertrophy (p < 0.05), occasionally with associated colloid depletion, was observed in relation to treatment for females dosed at 1000 mg/kg/day. A similar effect was not observed for males or amongst females from the remaining treatment levels.
lungs: The incidence of groups of alveolar macrophages was increased in relation to treatment for females treated with 1000 rng/kg/day (pMesenteric lymph nodes: Sinus histiocytosis was observed for animals of either sex treated with 1000 mg/kg/day (p < 0.001 1. Histiocytes were generally foamy in appearance. Other lymphoid tissues including spleen, thymus, cervical lymph nodes, and Peyer’s patches were not affected and the condition did not involve animals from the remaining treatment groups.
Uterus: Although the incidence of uterine dilatation was increased for females dosed at 1000 mg/kg/day (p
HISTOPATHOLOGY: NEOPLASTIC (if applicable): NA.
HISTORICAL CONTROL DATA (if applicable): NA.
OTHER FINDINGS

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Justification please see Attachment file named "EC 294-785-9 NOAEL after 90d oral tx.pdf"
Dose descriptor:
NOEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No changes detected in any animal at this dose level.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

DISCUSSION

The administration of OS63217M by oral gavage for a period of ninety consecutive days resulted in treatment-related changes at dose levels of 1000 and 250 mg/kg/day.

A slight reduction in bodyweight development and food consumption and efficiency were observed for 1000 mg/kg/day males during the study but these were transient changes and did not persist past Week 10. No adverse effect on bodyweight gain or dietary intake was detected for 1000 mg/kg/day females, but haematological investigations revealed a mild hypochromic anaemia in this sex at this dose level. Absolute and relative liver weight were elevated for 1000 mg/kg/day females and microscopic examination of liver sections revealed changes identified as centrilobular hepatocyte enlargement for five out of the ten females treated at this dose level. This morphological change is often seen in the rodent liver following treatment with xenobiotics and is associated with induction of microsomal enzymes. In the absence of any associated degenerative or inflammatory changes, this is considered to be an adaptive response. Microscopic examination of thyroid sections revealed an increased incidence of follicular cell hypertrophy, occasionally with associated colloid depletion, for 1000 mg/kg/day females. One possible explanation for these changes is a secondary response to the changes occurring in the liver. It is possible that glucuronyltransferase may have been induced in response to treatment, thereby increasing thyroxine excretion and stimulating a compensatory thyroid response. There were no convincing changes in the blood chemical paramters measured, although plasma cholesterol was slightly reduced for animals of either sex treated with 1000 mg/kg/day.

 

Further treatment-related changes were also seen in females with increases in both absolute and relative kidney weight detected at 1000 mg/kg/day. Histopathologically five females exhibited epithelial hypertrophy of inner cortical tubules but there were no other findings indicative of an adverse effect on renal function at this dose level. Microscopic lung changes were also observed, identified as an increased incidence of alveolar macrophages and microscopic examination of mesenteric lymph nodes revealed histiocytosis at 1000 mg/kg/day. It is unclear whether these findings are related but the histiocytosis was probably a normal physiological consequence of infiltrating macrophages being unable to degrade their phagocytosed contents. There were no similar'findings for the other lymphatic tissues examined.

 

Treatment-related changes extended into the 250 mg/kg/day dose group with females showing a dose-related increase in relative liver and kidney weight. Although statistical significance was either absent or minimal (p < 0.05), individual values were raised compared with controls. In the absence of any histopathological correlates, however, this was considered not to represent an adverse health effect.

No treatment-related changes were detected in any of the parameters measured for 250 mg/kg/day males or for animals of either sex treated with 50 mg/kg/day.

Applicant's summary and conclusion

Conclusions:
Oral administration of the test material to rats for a period of ninety consecutive days at dose levels of up to 1000 mg/kg/day resulted in treatment-related changes at 1000 amongst animals of either sex and at 250 mglkglday amongst females only. No such changes were detected in animals treated with 50 mg/kg/day and for this reason the "No Observed Effect Level" (NOEL) was considered to be 50 mg/kg/day.
The effects detected at 250 mg/kg/day, however, were confined to marginal liver and kidney weight increases amongst females only and in the absence of any supporting histopathological correlates, these were considered not to represent an adverse health effect. NOAEL was suggested to be 250 mg/kg/day.
Executive summary:

Test Guidance

OECD 408 Guideline study

Method and Material

Rats (10 per sex per dose) were dosed by oral gavage for 90 consecutive days at dose levels of 0 (control: vehicle), 50, 250 and 1000 mg/kg bw/day test material in arachis oil. Clinical signs, 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. Opthalmoscopic examination was also performed on control group and high dose animals. All animals were subject to gross necrospy examination and a comprehensive histopathological evaluation of tissues was performed.

Results

Treatment-related changes were observed in all animals at the 1000 mg/kg bw/day dose level. Effects noted in females at the 250 mg/kg bw/day dose level were considered not to be adverse therefore the NOAEL is considered to be 250 mg/kg bw/day. The NOEL for both sexes is 50 mg/kg bw/day.

Conclusion

In accordance with EU CLP Regulation (EC) No. 1272/2008, classification of this substance is not required for sub-chronic repeat oral exposure.