Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 July 2003 to 09 February 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: Colourless liquid
- Date received: 20 December 2002
- Storage condition of test material: Room tempeerature in the dark, over silica gel, under nitrogen

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
ANIMALS AND ANIMAL HUSBANDRY
- A sufficient number of male and female Sprague-Dawley Crl:CD (SD) BR strain rats were obtained from Charles River (UK) Limited, Margate, Kent.
- On receipt the animals were examined for signs of ill-health or injury.
- Animals were acclimatised for seven days during which time their health status was assessed.
- A total of fifty animals (twenty-five males and twenty-five females) were accepted into the study.
- At the start of treatment the males weighed 134 to 169g and the females weighed 131 to 161 g.
- All animals were approximately five to six weeks old.
- The animals were housed in groups of five by sex in polypropylene grid-floor cages suspended over trays lined with absorbent paper.
- Animals were allowed free access to food and water.
- A pelleted diet (Rodent 5LF2 (Certified) Diet, International Product Supplies Ltd., Northants, UK) was used.
- Mains drinking water was supplied fiom polycarbonate bottles attached to the cage.
- The diet and drinking water were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
- Environmental enrichment was provided in the form of wooden chew blocks (B & K Universal Ltd., Hull, UK) and cardboard fun tunnels (Datesand Ltd, Cheshire, UK).
- Animals were housed in an air-conditioned room within the Safepharm Barrier Maintained Rodent Facility.
- The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness.
- Environmental conditions were continuously monitored by a computerised system, and print-outs of hourly mean temperatures and humidities were included in the study records.
- Temperature and relative humidity controls were set to achieve target values of 21 ± 2°C and 55 ± 15% respectively. Occasional deviations fiom these targets were considered not to have affected the purpose or integrity of the study.
- The animals were randomly allocated to treatment groups using random letter tables 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. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: dried arachis oil BP
Details on oral exposure:
PREPARATION OF TEST MATERIAL
- The test material was prepared at the appropriate concentrations as a solution in dried Arachis oil BP. The homogeneity of the test material formulations were determined by Safepharm Analytical Laboratory.
- Formulations were prepared daily and stored at approximately +4 °C under nitrogen and over silica gel.

PROCEDURE
- Animals were allocated to treatment groups as shown in the table below.
- The test material was administered daily, for twenty-eight consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg/day of dried Arachis oil BP.
- The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at weekly intervals.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Representative samples were taken of each test material formulation and were analysed for concentration of the test item at Safepharm Analytical Laboratory.
- Results indicated that the prepared formulations were within acceptable limits for the purpose of the study.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
15 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
150 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
450 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
- Control: 5 male and 5 female
- Low (15 mg/kg/day): 5 males and 5 females
- Intermediate (150 mg/kg/day): 5 males and 5 females
- Intermediate II (450 mg/kg/day): 5 males and 5 females
- High (1000 mg/kg/day): 5 males and 5 females
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS
- All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing and one and five hours after dosing during the working week.
- Animals were observed immediately before dosing and one hour after dosing at weekends. All observations were recorded.

FUNCTIONAL OBSERVATIONS
- Prior to the start of treatment and on Days 6, 13,2 1 and 27, all animals were observed for signs of functional/behavioural toxicity.
- Functional performance tests were also performed on all animals during Week 4, together with an assessment of sensory reactivity to different stimuli.
- Observations were carried out from approximately two hours after dosing on each occasion.

BEHAVIOURAL ASSESSMENTS
- Detailed individual clinical observations were performed for each animal using a purpose built arena.
- The parameters observed were gait, tremors, twitches, convulsions, bizzare/abnormal/stereotypic behaviour, salivation, pilo-erection, exophthalmia, lachrymation, hyper/hypothermia, skin colour, respiration, palpebral closure, urination, defaecation, transfer arousal and tail elevation.

FUNCTIONAL PERFORMANCE TESTS
- Motor Activity: Twenty purpose built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The tests were performed at approximately the same time each day, under similar laboratory conditions. The evaluation period was sixteen hours for each animal. The percentage of time each animal was active and mobile was recorded for the overall sixteen 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 scoring system used is outlined in Appendix 1. The parameters observed were grasp response, vocalisation, toe pinch, tail pinch, finger approach, touch escape, pupil reflex, startle reflex and blink reflex.
- Startle reflex was measured using the ST1058 Startle Test Meter (Benwick Electronics). Each animal was placed on the force platform and allowed to settle. An audible tone was activated and any change in the force exerted by the animal on the force platform, as a result of startle induced tremor, was measured. The percentage average response, root of the mean square and peak response were calculated for each animal and two consecutive trials were performed.

BODYWEIGHT
- Individual bodyweights were recorded on Day 0 (the day before the start of treatment) and on Days 7, 14,21 and 28.
- Bodyweights were also recorded at terminal kill.

FOOD CONSUMPTION
- Food consumption was recorded for each cage group at weekly intervals throughout the study.

WATER CONSUMPTION
- Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.
Sacrifice and pathology:
PATHOLOGY
- 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.

ORGAN WEIGHTS
- Organs, removed ftom animals that were killed at the end of the study, were dissected free from fat and weighed before fixation.
- Organs subjected to fixation were adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes and thymus.

HISTOPATHOLOGY
- Samples of tissues were removed from all animals and preserved in buffered 10 % formalin.
- Tissues preserved were adrenals; aorta (thoracic); bone and bone marrow (femur including stifle joint); bone and bone marrow (sternum); brain (including cerebrum, cerebellum and pons); caecum; colon; duodenum; epididymides; eyes; gross lesions; heart; ileum; jejunum; kidneys; liver; lungs (with bronchi); lymph nodes (cervical and mesenteric); muscle (skeletal); oesophagus; ovaries; pancreas; pituitary; prostate; rectum; salivary glands (submaxillary); sciatic nerve; seminal vesicles; skin (hind limb); spinal cord (cervical); spleen; stomach; testes; thymus; thyroid/parathyroid; trachea; urinary bladder; uterus.
- Lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative.
- All tissues were despatched to Precision Histology International, One Eyed Lane, Weybread, Diss, Norfolk, UK for processing (Principal Investigator: J P Finn).
- Certain tissues from all control and 1000 mg/kg/day 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.
- Tissues prepared as paraffin blocks were adrenals; bone and bone marrow (sternum); brain (including cerebrum, cerebellum and pons); caecum; colon; duodenum; epididymides; gross lesions; heart; ileum; jejunum; kidneys; liver; lungs (with bronchi); lymph nodes (cervical and mesenteric); ovaries; prostate; rectum; sciatic nerve; seminal vesicles; spinal cord (cervical); spleen; stomach; testes; thymus; thyroid/parathyroid; trachea; urinary bladder; uterus.
- The liver and spllen from all 450, 150 and 15 mg/kg/day dose group animals were also processed.
- Since there were indications of treatment-related changes in the liver, thyroid glands and thymus, examination was subsequently extended to include similarly prepared sections of these tissues from all animals in other treatment groups.
- Microscopic examination was conducted by the Study Pathologist. All findings were entered into the ROELEE Pathology computerisation system for tabulation and report production.
Other examinations:
LABORATORY INVESTIGATIONS
- Haematological and blood chemical investigations were performed on all animals from each test and control group at the end of the study (Day 28).
- Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 29.
- Animals were not fasted prior to sampling.

HAEMATOLOGY
- Parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant.
- Parameters measured were haemoglobin (Hb); erythrocyte count (RBC); haematocrit (Hct); erythrocyte indices (mean corpuscular haemoglobin (MCH), mean corpuscular volume (MCV) and mean corpuscular haemoglobin concentration (MCHC); total leucocyte count (WBC); differential leucocyte count (neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos) and basophils (Bas)); platelet count (PLT); Reticulocyte count (Retic) in which cresyl blue stained slided were prepared but reticulocytes were not assessed.
- 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 (0.1 1 mol/L).

BLOOD CHEMISTRY
- Parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant.
- Parameters measured were urea, glucose, total protein (Tot Prot), albumin, albumin/globulin (A/G) ratio (by calculation), sodium (Na+), potassium (K+), chloride (Cl-), calcium (Ca++), inorganic phosphorus (P), aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), alkaline phosphatase (AP), creatinine (Creat), total cholesterol (Chol) and total bilirubin (Bili).
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), weekly bodyweight gain and quantitative functional performance and sensory reactivity data were assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (ANOVA) incorporating Levene’s test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett’s test. Where Levene’s test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney ‘U’ test.
- The haematology variable basophils was not analysed since consistently greater than 30% of the data were recorded as the same value.
- Probability values (p) are presented as p < 0.001; p < 0.01; p < 0.05 and p ≥ 0.05 (not significant).

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
see below
Mortality:
mortality observed, treatment-related
Description (incidence):
see below
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
see below
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
see below
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
see below
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
see below
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
see below
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
see below
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
see below
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see below
Histopathological findings: neoplastic:
no effects observed
Details on results:
MORTALITY
- There were no deaths during the study.

CLINICAL OBSERVATIONS
- A summary incidence of daily clinical observations is given in Table 1 and Table 2 (attached).
- Increased salivation was detected up to ten minutes after dosing (and on one occasion, up to one hour after dosing) in males and females treated with 1000 or 450 mg/kg/day from Days 2 and 3 respectively. Such observations are often reported following oral administration of an unpalatable or slightly irritant test material formulation, and in isolation are considered not to be indicative of systemic toxicity. Hunched posture and tiptoe gait were detected for one female treated with 1000 mg/kg/day on Day 27 but this was isolated and transient and possibly a reaction to the dosing procedure.
- A male treated with 450 mg/kg/day showed rearown staining around the eyes on Day 4 but this was transient and, in the absence of a dose-related response was considered to be incidental.
- No clinical signs were detected at 150 or 15 mg/kg/day.

FUNCTIONAL OBSERVATIONS
- A summary incidence of behavioural assessments is given in Table 3 and Table 4 (attached).
- Group mean functional test values and standard deviations are given in Table 5 and Table 6 (attached).
- A summary incidence of sensory reactivity assessments is given in Table 7 and Table 8 (attached) together with group mean startle reflex values and standard deviations.

BEHAVIOURAL ASSESSMENTS
- Behavioural assessment revealed no toxicologically significant changes in the parameters measured.
- Arena observations confirmed the clinical signs of hunched posture and tiptoe gait seen in a female treated with 1000 mg/kg/day.
- All remaining 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 treatment-related changes in the functional performance parameters measured.
- Statistical analysis of the data revealed no significant intergroup differences.

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 were of no toxicological importance.
- Statistical analysis of the quantitative data revealed no significant intergroup differences.

BODYWEIGHT
- Group mean weekly bodyweights and standard deviations are given in Table 9 and Table 10 (attached) and are presented graphically in Figure 1 and Figure 2 (attached).
- Group mean weekly bodyweight gains and standard deviations are given in Table 11 and Table 12 (attached, statistically significant differences are
indicated).
- A statistically significant reduction in bodyweight gain (p<0.001) was detected for 1000 mg/kg/day females during Week 3 of the study recovering thereafter. Interestingly, the female showing clinical signs failed to gain weight during this period. No adverse effect on bodyweight development was detected for 1000 mg/kg/day males or for animals of either sex treated at 450, 150 or 15 mg/kg/day.

FOOD CONSUMPTION
- Group mean weekly food consumptions are given in Table 13 and Table 14 (attached) and are presented graphically Figure 3 and Figure 4 (attached).
- Weekly food efficiencies are given in Table 15 and Table 16 (attached).
- Females treated with 1000 mg/kg/day showed a reduced dietary intake throughout the study period compared with that of controls. Food efficiency (the ratio of bodyweight gain to food consumption) was also reduced but this was confined to Week 3 only.
- No such effects were detected for males treated with 1000 mg/kg/day or for animals of either sex dosed at 450, 150 and 15 mg/kg/day.

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

HAEMATOLOGY
- Group mean values and standard deviations for test and control group animals are given in Table 17 and Table 18 (attached, statistically significant differences are indicated).
- Animals of either sex treated with 1000 mg/kg/day showed evidence of a microcytic hypochromic anaemia. Statistically significant reductions in haemoglobin, haematocrit, erythrocyte count, mean corpuscular volume and mean corpuscular haemoglobin were detected for the females whilst haematocrit and haemoglobin were reduced for the males. Many values were outside the respective normal ranges for rats of the strain and age used. A lmphocytopenia was also evident at this dose level with males and females showing reduced total leucocyte (p<0.01) and lymphocyte count (p<0.01) with values outside normally expected ranges. A statistically significant reduction in platelet count (p<0.05) was also detected at this dose level. This was confined to the females and all values were outside the normal range for rats of the strain and age used. Clotting (prothrombin) time was also affected with a statistically significant increase (p<0.01) in this parameter for the females treated with 1000 mg/kg/day compared with controls.
- There were no treatment-related changes detected in the haematological parameters measured for 450,150 or 15 mg/kg/day animals.

BLOOD CHEMISTRY
- Group mean values and standard deviations for test and control group animals are given in Table 19 and Table 20 (attached, statistically significant differences are indicated).
- A statistically significant reduction in total plasma protein (p<0.001) and increase in albumidglobulin ratio (p<0.01) was detected for 1000 mg/kg/day animals compared with controls. Plasma cholesterol was reduced in either sex (p<0.001) whilst reductions in inorganic phosphorus (p<0.01) and urea (p<0.001) were confined to males and a reduction in creatinine (p<0.001) was confined to the females at this dose level.
- Effects extended to males treated with 450 mg/kg/day with statistically significant reductions detected in total plasma protein, inorganic phosphorus (p<0.05) and cholesterol (p<0.01) compared with controls.
- Statistically significant reductions in plasma urea and cholesterol were detected for 450 and 1 50 mg/kg/day males and females respectively. The dose-response relationship was unconvincing but a relationship with treatment cannot be entirely discounted.
- No such effects were detected for animals of either sex treated with 15 mg/kg/day.

ORGAN WEIGHTS
- Group mean absolute and relative organ weights and standard deviations for test and control group animals are presented in Table 21 to Table 24 (attached, statistically significant differences are indicated).
- A statistically significant increase in liver weight, relative to bodyweight was detected for animals of either sex treated with 1000 mg/kg/day and for 450 mg/kg/day females compared with controls.
- All high dose values and one 450 mg/kg/day value were outside the respective normal ranges for rats of the strain and age used. Absolute liver weight reflected these increases and, although statistical significance was confined to the males, many values, including those of 450 mg/kg/day
females, were outside the normally expected ranges. Relative thymus weight was statistically significantly reduced for 1000 mg/kg/day animals. Female absolute thymus weight was similarly affected. An increase in relative kidney weight was also evident at 1000 mg/kg/day but statistical significance (and values outside the normal range) was confined to the males.
- No treatment-related organ weight changes were detected for 450 mg/kg/day males or for animals of either sex treated with 150 or 15 mg/kg/day.

NECROPSY
- A summary incidence of necropsy findings is given in Table 25 and Table 26 (attached).
- No treatment-related macroscopic abnormalities were detected at terminal kill.
- The incidental finding recorded for a male treated with 150 mg/kg/day, identified as dark foci on the caudal lobe of the lung, whilst showing no dose-related response, was consistent with a normally expected low incidence finding in laboratory maintained rats and was considered to be of no
toxicological importance.

HISTOPATHOLOGY
- A summary incidence of histopathological findings is given in Table 27 and Table 28 (attached).
- Treatment-related changes were observed in the liver, thyroids and thymus.
- Liver: Centrilobular hepatocyte enlargement was observed in relation to treatment for rats of either sex dosed at 1000 mg/kg/day or at 450 mg/kg/day. Isolated instances of the condition were also seen among treated animals from the 150 mg/kg/day and 1 Smg/kg/day treatment groups, but hepatocyte enlargement is occasionally encountered as a spontaneous entity in untreated rats thus the presence of the condition in these groups cannot be reliably associated with treatment. 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.
- Thyroids: Follicular cell hypertrophy was seen as a consequence of treatment for rats of either sex dosed at 1000 mg/kg/day, but not at any other treatment level.
- Thymus: Lymphoid atrophy was observed for female, but not for male rats dosed at 1000 mg/kg/day. Rats from the remaining dose levels were unaffected.
- 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.

Effect levels

open allclose all
Dose descriptor:
NOEL
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Dose descriptor:
NOAEL
Effect level:
450 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects related to dose treatment which are not considered to be adaptive responses.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

DISCUSSION

Repeated oral administration of test material, by gavage, at dose levels of up to 1000 mg/kg/day resulted in toxicologically significant effects at 1000 mg/kg/day and minor biochemical and adaptive changes at 450 and 150 mg/kg/day.

Animals treated with 1000 or 450 mg/kg/day showed increased salivation from Days 2 and 3 respectively and hunched posture and tiptoe gait were detected for one female on Day 27. Observations of this nature are often reported when the test material formulation is unpalatable or slightly irritant and are considered not to represent systemic toxicity. Bodyweight development and dietary intake were adversely affected for 1000 mg/kg/day females and haematological investigations revealed a microcytic hypochromic anaemia in either sex. A reduction in plasma phosphorus was detected but this was only evident in 1000 and 450 mg/kg/day males. Given the high concentration of phosphorus present in erythrocytes, this change may be associated with the anaemia. A marked lymphocytopenia was detected in either sex treated with 1000 mg/kg/day. Microscopic examination of thymus sections revealed lymphoid atrophy confined to 1000 mg/kg/&ay females but thymus weight was reduced in both sexes at this dose level. These findings indicate a treatment-related effect on imune fimction at the high dose. Females treated with 1000 mg/kg/day also showed a reduced platelet count aid increased clotting (prothrombin) time which may be associated with a general haemopoietic effect or with an adverse effect on the liver. Histopathological liver changes were identified as centrilobular hepatocyte enlargement but this condition 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. Liver weights were elevated at 1000 mg/kg/day and for 450 mg/kg/day females. Thyroid follicular cell hypertrophy was observed for animals of either sex treated with 1000 mg/kg/day. This may possibly be associated with the changes seen in the liver. Thyroxine is ultimately excreted via the bile, having first been conjugated in the liver. It is conceivable that conjugating hepatic enzymes may have been induced therefore increasing thyroxine excretion and stimulating compensatory TSH and thyroxine production resulting in the microscopic changes identified. Although liver changes appear to be adaptive, blood chemical investigations revealed a reduction in total plasma protein and increase in albumidglobulin ratio at 1000 mg/kg/day, the effect extending to 450 mg/kg/day males which may suggest some level of hepatic impairment. Although the reduction was in the globulin fraction and could equally be associated with the effect on immune function and a consequent reduced synthesis of immunoglobulins. A reduction in plasma cholesterol was also evident at the high dose and for 450 mg/kg/day males but this may have been associated with the anaemic condition of the animals.

The remaining treatment-related effects detected at 1000 mg/kg/day were confined to kidney weight which was elevated in either sex. Plasma urea was however, reduced for the males and creatinine was reduced for the females whereas increases in these parameters would be expected with an adverse effect on renal function. The aetiology of the kidney weight increase is therefore unclear.

No adverse effects of treatment were detected at 150 or 15 mg/kg/day although plasma urea and cholesterol were reduced in 150 mg/kg/day males and females respectively, albeit with an unconvincing dose-response relationship. The histopathological liver changes seen as isolated instances of centrilobular hepatocyte enlargement were considered to be spontaneous occurrences and, in isolation, did not represent an adverse health effect at these dose levels.

Applicant's summary and conclusion

Conclusions:
Oral administration of the test material to rats for a period of twenty-eight consecutive days at dose levels of up to 1000 mg/kg/day resulted in toxicologically significant effects at 1000 mg/kg/day and minor biochemical and adaptive changes at 450 and 150 mg/kg/day. No such
effects were demonstrated in animals treated with 15 mg/kg/day and the “No Observed Effect Level” (NOEL) was, therefore, considered to be 15 mg/kg/day.

Effects detected at dose levels of 150 and 450 mg/kg/day were confined to minor biochemical changes and, in the 450 mg/kg/day dose group, adaptive liver changes. These were not indicative of a serious adverse effect on health as defined by the European Labelling Guide, Commission
Directive 2001/59/EC. The ‘No Observed Adverse Effect Level (NOAEL) was, therefore, considered to be 450 mg/kg/day.
Executive summary:

GUIDELINE

The study was designed to investigate the systemic toxicity of the test material. It complies with the requirements for notification of a new chemical substance in the EC and follows the testing method described in Commission Directive 96/54/EC (Method B7) and OECD Guidelines for Testing of Chemicals No. 407 "Repeated Dose 28 Day Oral Toxicity Study in Rodents" (Adopted 27 July 1995).

METHODS

The test material was admiristered by gavage to four groups, each of five male and five female Sprague-Dawley Crl:CD (SD) IGS RR strain rats, for twenty-eight consecutive days, at dose levels of 15, 150,450 and 1000 mg/kg/day. A control group of five males and five females was dosed with vehicle alone (dried Arachis oil BP).

Clinical signs, functional observations, bodyweight development and food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for all animals at the end of the study.

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

RESULTS

Mortality: There were no deaths during the study.

Clinical Observations: Increased salivation was detected up to ten minutes after dosing in 1000 and 450 mg/kg/day animals from Days 2 and 3 respectively. Such observations are often reported following oral administration of an unpalatable or slightly irritant test material formulation, and in isolation are considered not to be indicative of systemic toxicity. Hunched posture and tiptoe gait were detected for one female treated with 1000 mg/kg/day on Day 27 but this was isolated and transient. No such findings were detected at 150 or 15 mg/kg/day.

Behavioural Assessment: No toxicologically significant effects were detected. Open field assessment confirmed the observations of hunched posture and tiptoe gait seen clinically in the 1000 mg/kg/day female.

Functional Performance Tests: No treatment-related effects were detected.

Sensory Reactivity Assessments: No treatment-related effects were detected.

Bodyweight: A statistically significant reduction in bodyweight gain was detected for 1000 mg/kg/day females during Week 3 of the study recovering thereafter. Bodyweight development was unaffected for 1000 mg/kg/day males or for animals of either sex treated at 450, 150 or 15 rng/kg/day.

Food Consumption: Females treated with 1000 mg/kg/day showed a reduced dietary intake throughout the study period. Food efficiency was also reduced but this was confined to Week 3 only. Animals treated at 450,150 and 15 mg/kg/day were unaffected.

Water Consumption: No intergroup differences were detected.

Haematology: Animals of either sex treated with 1000 mg/kg/day showed evidence of a microcytic hypochromic anaemia together with a lymphocytopenia. Clotting (prothrombin) time was elevated and platelet count was reduced for 1000 mg/kg/day females. No such changes were detected at the other dose levels.

Blood Chemistry: A statistically significant reduction in total plasma protein and increase in albumin/globulin ratio was detected for 1000 mg/kg/day animals. Plasma cholesterol was reduced in either sex whilst reductions in phosphate and urea were confined to males and a reduction in creatinine was confined to the females at this dose level. Effects extended to males treated with 450 mg/kg/day with statistically significant reductions detected in total plasma protein, cholesterol and inorganic phosphorus compared with controls. Statistically significant reductions were also apparent in plasma urea (males) and cholesterol (females) for animals treated with 450 or 150 mg/kg/day, although the dose response relationship was unconvincing. No such effects were detected at 15 mg/kg/day.

Organ Weights: A statistically significant increase in liver weight, relative to bodyweight was detected for animals of either sex treated with 1000 mg/kg/day and for 450 mg/kg/day females compared with controls; absolute liver weight reflected the increases seen at these dose levels. Relative thymus weight was statistically significantly reduced for 1 000 mg/kg/day animals whilst relative kidney weight was elevated (statistical significance confined to males). No treatmentrelated organ weight changes were detected at the other dose levels.

Necropsy: No treatment-related macroscopic abnormalities were detected at terminal kill.

Histopathology: Histopathological investigations revealed treatment-related liver, thyroid and thymus changes. Centrilobular hepatocyte enlargement was observed in relation to treatment for rats of either sex dosed at 1000 mg/kg/day or at 450 mg/kg/day. Isolated instances of the condition were also seen among treated animals from the 150 mg/kg/day and 15 mg/kg/day treatment groups, but hepatocyte enlargement is occasionally encountered as a spontaneous entity in untreated rats thus the presence of the condition in these groups cannot be reliably associated with treatment. 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. Thyroid follicular cell hypertrophy was seen as a consequence of treatment for rats of either sex dosed at 1000 mg/kg/day, but not at any other treatment level. Lymphoid atrophy was observed in the thymus for female, but not for male rats dosed at 1000 mg/kg/day. Rats from the remaining dose groups were unaffected.

CONCLUSION

Oral administration of the test material to rats for a period of twenty-eight consecutive days at dose levels of up to 1000 mg/kg/day resulted in toxicologically significant effects at 1000 mg/kg/day and minor biochemical and adaptive changes at 450 and 150 mg/kg/day. No such effects were demonstrated in animals treated with 15 mg/kg/day and the “No Observed Effect Level” (NOEL) was, therefore, considered to be 15 mg/kg/day.

Effects detected at dose levels of 150 and 450 mg/kg/day were confined to minor biochemical changes and, in the 450 mg/kg/day dose group, adaptive liver changes. These were not indicative of a serious adverse effect on health as defined by the European Labelling Guide, Commission Directive 2001/59/EC. The ‘No Observed Adverse Effect Level (NOAEL) was, therefore, considered to be 450 mg/kg/day.