reaction mass of isomers of: C7-9-alkyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

Between 27 January 2009 and 26 August 2009.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, without deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): OS162170N
- Molecular formula (if other than submission substance): C21H34O3
- Molecular weight (if other than submission substance): 334.491
- Smiles notation (if other than submission substance): Oc1c(C(C)(C)C)cc(cc1C(C)(C)C)CCC(=O)OCCCC
- InChl (if other than submission substance): 1S/C21H34O3/c1-8-9-12-24-18(22)11-10-15-13-16(20(2,3)4)19(23)17(14-15)21(5,6)7/h13-14,23H,8-12H2,1-7H3
- Structural formula attached as image file (if other than submission substance): see Fig.1
- Substance type: alkylated phenol
- Physical state: Amber coloured solid block
- Analytical purity: 98%
- Storage condition of test material: room temperature in the dark
- Other:

Test animals

Species:

rat

Strain:

other: Wistar HanTM:HsdRccHanTM:WIST strain rats

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Ltd, Blackthorn, Bicester, Oxon.
- Age at study initiation: six to eight weeks old
- Weight at study initiation: At the start of treatment the males weighed 184 to 219g, the females weighed 146 to 177g.
- Fasting period before study: no
- Housing: in groups of up to four by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

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

IN-LIFE DATES: From: To: between 05 March 2009 and 04 June 2009.

Administration / exposure

Route of administration:

other: Dietary

Vehicle:

unchanged (no vehicle)

Remarks:

untreated laboratory diet

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: not applicable. The test material was incorporated into the diet.

DIET PREPARATION
A known amount of test material was mixed with a small amount of basal laboratory diet for nineteen minutes at a constant speed, setting 1 in a Hobart QE200 mixer. This premix was then added to a larger amount of basal laboratory diet and mixed for a further thirty minutes at a constant speed, setting 1 in a Hobart H800 mixer. The stability and homogeneity of the test materials in the diet were determined by Harlan Laboratories Ltd, Shardlow, UK Analytical Laboratory. Results show the dietary admixtures to be stable for a period of at least six weeks.

- Rate of preparation of diet (frequency): Dietary admixtures were prepared prior to treatment at approximately monthly intervals.
- Mixing appropriate amounts with (Type of food): A ground diet (Rat and Mouse SQC Ground Diet No. 1, Special Diet Services, Dietex International
Limited, Witham, Essex, UK) was used.
- Storage temperature of food: The diet was stored in labelled, double blue plastic bags in labelled, covered plastic bins when not in use.
- Samples were taken of each dietary admixture and were analysed for homogeneity and concentration at Harlan Laboratories Ltd, Shardlow, UK Analytical Laboratory prior to use. The results indicate that the mean prepared dietary admixture concentrations were within acceptable limits of the nominal concentration.

VEHICLE
- No vehicle was used. The test material was administered by dietary admixture. Untreated laboratory diet served as control.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chemical Analysis of Dietary Admixtures
1 Summary
The concentration of OS162170N in the dietary admixtures was determined by high performance liquid chromatography (HPLC) using an external standard technique.
2 Samples
The dietary admixtures were extracted with acetonitrile to give a final, theoretical test material concentration of approximately 20 ppm.
3 Standards
Standard solutions of test material were prepared in acetonitrile at a nominal concentration of 20 ppm.
4 Procedure
The standard and sample solutions were analysed by HPLC.
5 Homogeneity Determinations
The dietary admixtures were sampled from the middle and two opposite sides in triplicate and analysed.
6 Stability Determinations
The dietary admixtures were sampled and analysed initially and then after storage at ambient temperature in the dark for six weeks.
7 Verification of Test Material Formulation Concentrations
The dietary admixtures were sampled and analysed within four days of preparation.
8 Conclusions: The results indicate that the mean prepared dietary admixture concentrations were within acceptable limits of the nominal concentration.

Duration of treatment / exposure:

The test material was administered continuously for ninety consecutive days, by dietary admixture.

Frequency of treatment:

Continuously for ninety consecutive days.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0 mg/kg/day active ingredient (basal laboratory diet, control)
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
39 mg/kg/day active ingredient
Basis:
nominal in diet

Doses / concentrations 3

Remarks:

Doses / Concentrations:
80 mg/kg/day active ingredient
Basis:
nominal in diet

Doses / concentrations 4

Remarks:

Doses / Concentrations:
186 mg/kg/day active ingredient
Basis:
nominal in diet

No. of animals per sex per dose:

10 /sex/dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Dose levels based upon a preliminary range finding study. The 21-day range finding study was performed with 2 rats/sex/group at diet concentration of 0, 200, 1200 and 2500 ppm (equivalent to a mean achieved dosage levels of 20, 100 and 250 mg/kg/day respectively) to provide a basis for selection of dose levels for the 90-day study. Control animals were treated in an identical manner with basal laboratory diet. The following results were obtained:
Based on this preliminary testing, the dose levels for the main 90-day study were chosen as:
High dose 2500 ppm, or 186 mg/kg/day
Intermediate dose 1200 ppm, or 90 mg/kg/day
Low dose 500 ppm, or 39 mg/kg/day
plus a control group treated with basal laboratory diet.

- 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: No satellite groups were formed
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random): Random

Positive control:

Not used

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily
- Cage side observations checked in table [No.?] were included. No.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily. All animals were examined for overt signs of toxicity, illhealth or behavioural change. All observations were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual bodyweights were recorded on Day 1 (prior the start of treatment) and at weekly intervals thereafter. Bodyweights were also recorded at terminal kill.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- 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: Yes
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

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).
- 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 "Innovin" and Activated partial thromboplastin time (APTT) was assessed by "Actin FS" 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 aminotransferase (ASAT), Alkaline phosphatase (AP), Sodium (Na+), Potassi um (K +), Chloride (CI-), Calcium (Ca+ +), Creatinine (Creat), Total cholesterol (Chol), Total bilirubin (Bili)

URINALYSIS: Not examined

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.
- Battery of functions tested:
Behavioural Assessments
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, lachrymation.

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 one hour for each animal. The time in seconds each animal was active and mobile was recorded for the overall one hour period and also during the final 20% of the 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 for 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 Blink reflex; Tail pinch Startle reflex; Finger approach.

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, Tongue, Trachea, Urinary bladder, Uterus.
All tissues from control and 2500 ppm 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.

Other examinations:

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, ovaries, brain, spleen, epididymides, testes, heart, thymus, kidneys, uterus, liver.

Statistics:

Data were processed to give group mean values and standard deviations where appropriate.
All data were summarised in tabular form. Where appropriate, quantitative data were analysed by the Provantis™ Tables and Statistics Module. For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA or ANCOVA and Bartlett’s test. The transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for nonparametric data. If no dose response was found, but the data showed non-homogeneity of means, the data were analysed by a stepwise Dunnett (parametric) or Steel (nonparametric) test to determine significant differences from the control group. Finally, if required, pair-wise tests were performed using the Student t-test (parametric) or the Mann - Whitney U test (non-parametric).
Probability values (P) are presented as follows:
P < 0.01 **
P < 0.05 *
p >0.05 (not significant)
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.
Probability values (P) are presented as follows:
P<0.001 +++ --- ***
P<0.01 ++ -- **
P<0.05 + - *
P<0.1 (+) (-) (*)
p>0.1 N.S. (not significant)
With plus signs indicating positive differences from the control group and minus signs indicating negative differences. Asterisks refer to overall between group variation which is non-directional.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

but not an adverse effect of treatment

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

reduced in highest dose group for females and males during week 11 and week 13, respectively

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

related to hepatocyte enlargement; an adaptive response in nature

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

increased liver weight: adaptive response to xenobiotics

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

but without toxicological importance

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

but not an adverse effect of treatment

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
No clinically observable signs of toxicity were detected in test or control animals throughout the study period.

Mortality:
There were no deaths during the study.

BODY WEIGHT AND WEIGHT GAIN
Females treated with 2500 ppm showed a reduction in bodyweight gain during Week 11 whilst males from this treatment group showed a reduction in bodyweight gain during the final week of treatment. No toxicological significant effects were detected in animals of either sex treated with 1200 or 500 ppm.
Males from all treatment groups showed a statistically significant reduction in bodyweight gain during Week 4. Males treated with 1200 ppm also showed a statistically significant reduction in bodyweight gain during Week 12. In the absence of a true dose-related response the intergroup differences were considered of no toxicological importance.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
There was no adverse effect on food consumption during the study period.

FOOD EFFICIENCY
There was no adverse effect on food consumption during the study period. Food efficiency (the ratio of bodyweight gain to dietary intake) was however reduced for females treated with 2500 ppm during Week 11 and for males treated with 2500 ppm during Week 13. No such effects were detected in animals of either sex treated with 1200 or 500 ppm.

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

OPHTHALMOSCOPIC EXAMINATION:
There were no treatment-related ocular effects. The incidental findings recorded were those normally encountered in laboratory maintained rats of this age and strain.

HAEMATOLOGY
There were no treatment-related changes in the haematological parameters measured.
Statistical analysis of the data did not reveal any significant intergroup differences.

CLINICAL CHEMISTRY
Females treated with 2500 and 1200 ppm showed a statistically significant increase in plasma bilirubin levels. No such effects were detected in males treated with 2500 or 1200 ppm or animals of either sex treated with 500 ppm.
The remaining statistically significant intergroup differences were considered to be of no toxicological importance. Females treated with 2500 ppm showed a statistically significant reduction in plasma glucose and a statistically significant increase in sodium concentration. In the absence of any associated changes the intergroup differences were considered of no toxicological importance.

URINALYSIS
Not examined

NEUROBEHAVIOUR
Behavioural Assessments
There were no treatment-related changes in behaviour detected during the weekly open field arena assessments. All inter and intra group differences in behavioural scores were considered to be a result of normal variation for rats of the strain and age used and were of no toxicological importance.

Functional Performance Tests
There were no toxicologically significant changes in the functional performance parameters measured.
Females from all treatment groups showed a statistically significant reduction in overall mobility. In the absence of a dose-related response or any supporting clinical observations to suggest an effect of neurotoxicity; this finding was considered to be of no toxicological significance.
Sensory Reactivity Assessments
There were no treatment-related changes in sensory reactivity.
All inter and intra group differences in sensory reactivity scores were considered to be a result of normal variation for rats of the strain and age used, and was of no toxicological importance.

ORGAN WEIGHTS
Males from all treatment groups and females treated with 2500 and 1200 ppm showed statistically significant increases in liver weight both absolute and relative to terminal bodyweight. No such effects were detected in females treated with 500 ppm.
Males treated with 2500 ppm showed a statistically significant reduction in epididymide weight both absolute and relative to terminal bodyweight. In the absence of any histological correlates the intergroup difference was considered not to be of any toxicological significance.

GROSS PATHOLOGY:
No toxicologically significant macroscopic abnormalities were detected.
One male treated with 2500 ppm had small testes and epididymides at necropsy whilst one female from this treatment group had a reddened thymus. One male treated with 500 ppm had reddened lungs. In the absence of any histological correlates the intergroup differences were considered not to be of any toxicological importance.

HISTOPATHOLOGY: NON-NEOPLASTIC
The following treatment-related changes were detected:
LIVER: Centrilobular hepatocyte enlargement was seen in relation to treatment for animals of either sex treated with 2500 ppm (P <0.001), 1200 ppm (P <0.001), or at 500 ppm (P <0.05-0.001).
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.

SPLEEN: Generally lower grades of severity of extramedullary haemopoiesis were seen for females only treated with 2500 ppm or at 1200 ppm. Although this did not attain statistical significance at either dose level, a relationship to treatment cannot be excluded.

THYROID GLAND: Follicular cell hypertrophy was seen as a consequence of treatment for animals of either sex treated with 2500ppm (statistically significant for males only P <0.001), and for males only treated with 1200 ppm (P <0.01) or at 500 ppm (P <0.05).

LUNGS: A higher incidence of groups of alveolar macrophages was seen for males treated with 2500 ppm (P <0.01) or at 1200 ppm (P <0.01) compared with the control group. Females were not similarly affected.

Groups of alveolar macrophages are seen occasionally as a spontaneous background change among laboratory maintained rats and the group distribution can be variable. However, a relationship to treatment in this investigation cannot be excluded.

BONE MARROW: A generally lower cellularity of the bone marrow, as evidenced by higher grades of severity of adipose infiltration, was observed for animals of either sex at all treatment levels, attaining statistical significance for animals of either sex treated with 2500 ppm (P <0.05) and for females only treated with 500 ppm (P <0.05). Although the response was not convincingly dose related, and there were no indications of changes in any haematological parameters, the possibility of a genuine effect of treatment on marrow cellularity cannot be ruled out.

All remaining morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed, and there were no differences in incidence or severity between control and treatment groups that were considered to be of toxicological significance.

HISTOPATHOLOGY: NEOPLASTIC (if applicable): No neoplastic lesions were observed

HISTORICAL CONTROL DATA (if applicable): No

OTHER FINDINGS: No

Effect levels

Dose descriptor:

NOAEL

Effect level:

2 500 ppm

Based on:

test mat.

Remarks:

equivalent to 186 mg/kg/day

Sex:

male/female

Basis for effect level:

other: The microscopic effects detected at all dietary concentrations, the minor bodyweight changes at 2500 ppm and minor blood chemical changes at 2500 and 1200 ppm were not considered to be an adverse effect of treatment

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

BODY WEIGHT AND WEIGHT GAIN

Group mean weekly bodyweights and standard deviations are given in Table 7 and are presented graphically in Figure 1.

Group mean weekly bodyweight gains and standard deviations are given in Table 8 (statistically significant differences are indicated).

 

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):

Group mean weekly food consumptions are given in Table 9 and are presented graphically in Figure 3 and Figure 4. Weekly food efficiencies are given in Table 10.

 

ORGAN WEIGHTS        

Group mean absolute and relative organ weights and standard deviations for test and control group animals are presented in Table 13 (statistically significant differences are indicated).

 

 

Applicant's summary and conclusion

Conclusions:

The oral administration of OS162170N to rats for a period of ninety consecutive days at dietary concentrations of 500, 1200 and 2500 ppm resulted in treatment-related effects in animals of either sex at all dose levels. A No Observed Effect Level (NOEL) has, therefore not been established.
The microscopic effects detected at all dietary concentrations, the minor bodyweight changes at 2500 ppm and minor blood chemical changes at 2500 and 1200 ppm were not considered to be an adverse effect of treatment. The “No Observed Adverse Effect Level” (NOAEL) was therefore considered to be 2500 ppm.

Executive summary:

Introduction.The study was designed to investigate the systemic toxicity of the test material and complies with the recommendations of the OECD guidelines for Testing of Chemicals No. 408 “Subchronic Oral Toxicity - Rodent: 90 Day Study”.

Methods.The test material was administered continuously by dietary admixture to three groups, each of ten male and ten female Wistar HanTM:HsdRccHanTM:WIST strain rats, for ninety consecutive days, at dietary concentrations of 500, 1200 and 2500 ppm (equivalent to a mean achieved dosage of 39, 90 and 186 mg/kg/day respectively). A further group of ten males and ten females was exposed to basal laboratory diet to serve

as a control.

Clinical signs, functional observations, bodyweight development, dietary intake and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for all animals at the end of the study. Ophthalmoscopic examination was also performed on control group and high dose animals before the start of treatment and during Week 12 of the study.

All animals were subjected to gross necropsy examination and a comprehensive histopathological evaluation of tissues was performed.

 

Results.

Mortality.There were no unscheduled deaths.

 

Clinical Observations.No clinically observable signs of toxicity were detected in test or control animals throughout the study period.

 

Behavioural Assessment.There were no toxicologically significant changes in the

behavioural parameters measured.

 

Functional Performance Tests.There were no toxicologically significant changes in the functional performance parameters measured.

 

Sensory Reactivity Assessments.There were no treatment-related changes in sensory reactivity.

 

Bodyweight.Females treated with 2500 ppm showed a reduction in bodyweight gain during Week 11 whilst males from this treatment group showed a reduction in bodyweight gain during the final week of treatment. No toxicological significant effects were detected in animals of either sex treated with 1200 or 500 ppm.

Males from all treatment groups showed a statistically significant reduction in bodyweight gain during Week 4. Males treated with 1200 ppm also showed a statistically significant reduction in bodyweight gain during Week 12. In the absence of a true dose-related response the intergroup differences were considered of no toxicological importance.

 

Food Consumption.No adverse effect on dietary intake was detected. Food efficiency was reduced however during Week 11 for females treated with 2500 ppm and during Week 13 for males treated with 2500 ppm. No such effects were detected in animals of either sex treated with 1200 or 500 ppm.

 

Water Consumption.No intergroup differences were detected.

 

Ophthalmoscopy:No treatment-related ocular effects were observed.

 

Haematology.No treatment-related effects were detected in the haematological parameters measured.

 

Blood Chemistry.Females treated with 2500 and 1200 ppm showed a statistically significant increase in plasma bilirubin. No such effects were detected in males treated with 2500 or 1200 ppm or animals of either sex treated with 500 ppm.

 

Organ Weights.Males from all treatment groups and females treated with 2500 and 1200 ppm showed a statistically significant increase in liver weight both absolute and relative to terminal bodyweight. No such effects were detected in females treated with 500 ppm.

 

Necropsy .There were no toxicologically significant macroscopic abnormalities detected.

 

Histopathology.The following treatment-related changes were detected:

 

LIVER:Centrilobular hepatocyte enlargement was seen in relation to treatment for animals of either sex treated with 2500 ppm (P <0.001), 1200 ppm (P <0.001), or at 500 ppm (P <0.05-0.001).

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.

 

SPLEEN:Generally lower grades of severity of extramedullary haemopoiesis were seen for females only treated with 2500 ppm or at 1200 ppm. Although this did not attain statistical significance at either dose level, a relationship to treatment cannot be excluded.

 

THYROID GLAND:Follicular cell hypertrophy was seen as a consequence of treatment for animals of either sex treated with 2500ppm (statistically significant for males only P <0.001), and for males only treated with 1200 ppm (P <0.01) or at 500 ppm (P <0.05).

LUNGS: A higher incidence of groups of alveolar macrophages was seen for males treated with 2500 ppm (P <0.01) or at 1200 ppm (P <0.01) compared with the control group. Females were not similarly affected.

Groups of alveolar macrophages are seen occasionally as a spontaneous background change among laboratory maintained rats and the group distribution can be variable.

However, a relationship to treatment in this investigation cannot be excluded.

 

BONE MARROW:A generally lower cellularity of the bone marrow, as evidenced by higher grades of severity of adipose infiltration, was observed for animals of either sex at all treatment levels, attaining statistical significance for animals of either sex treated with 2500 ppm (P <0.05) and for females only treated with 500 ppm (P <0.05). Although the response was not convincingly dose related, and there were no indications of changes in

any haematological parameters, the possibility of a genuine effect of treatment on marrow cellularity cannot be ruled out.

 

Conclusion.The oral administration of OS162170N to rats for a period of ninety consecutive days at dietary concentrations of 500, 1200 and 2500 ppm resulted in treatment-related effects in animals of either sex at all dose levels. A No Observed Effect Level (NOEL) has, therefore not been established.

The microscopic effects detected at all dietary concentration were not considered to be an adverse effect of treatment. The “No Observed Adverse Effect Level” (NOAEL) was therefore considered to be 2500 pp