Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 March 2014 to 31 July 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
see below
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: Amber coloured viscous liquid
- Analytical purity: 100 %
- Expiration date of the lot/batch: 17 March 2016
- Storage condition of test material: Room temperature in the dark

Test animals

Species:
rat
Strain:
other: Wistar Han
Details on test animals and environmental conditions:
ANIMALS AND ANIMAL HUSBANDRY
- A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK.
- On receipt the animals were examined for signs of ill-health or injury.
- The animals were acclimatised for eight days during which time their health status was assessed.
- A total of one hundred and sixteen animals (fifty eight males and fifty eight females) were accepted into the study.
- At the start of treatment the males weighed 296 to 352g, the females weighed 207 to 245g, and were approximately twelve weeks old.
- Initially, all animals were housed in groups of four or five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK).
- During the pairing phase, the non-recovery animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group.
- Following evidence of successful mating, the males were returned to their original cages.
- Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Recovery group animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and furnished with softwood flake bedding.
- Animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK.) was used.
- Mains drinking water was supplied from polycarbonate bottles attached to the cage.
- Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK) except
for paired animals and mated females during gestation and lactation.
- Mated females were also given softwood flakes, as bedding, throughout gestation and lactation.
- The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
- The animals were housed in a single air-conditioned room within the Harlan Laboratories Ltd., Shardlow, UK Barrier Maintained Rodent Facility.
- Rate of air exchange was at least fifteen 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 temperatures and humidities are
included in the study records.
- The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20 % respectively; there were no deviations from the target range for temperature and the deviations from the target range for relative humidity were considered not to have affected the purpose or integrity of the study; see deviations from Study Plan.
- The animals were randomly allocated to treatment groups using a stratified body weight randomisation procedure and the group mean body weights were then determined to ensure similarity between the treatment groups.
- Cage distribution within the holding rack was also randomised.
- The animals were uniquely identified within the study by an ear punching system routinely used by Harlan Laboratories.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
TEST ITEM PREPARATION
- The test item was prepared at the appropriate concentrations as a solution in Arachis oil BP.
- Stability and homogeneity of the test item formulations were determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services as part of this study. Results show the formulations to be stable for at least 21 days when stored at 4 °C in the dark.
- Formulations were therefore prepared in batches, normally sufficient to cover 14 days of dosing, which were then divided into daily aliquots and stored at approximately 4 °C in the dark.

PROCEDURE
- Animals were allocated to treatment groups as shown in the table (attached) where numbers in parentheses show the individual animal numbers allocated to each treatment group.
- The test item was administered daily 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 of Arachis oil BP.
- The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Representative Samples of the formulation were taken and analyzed for concentration of test item at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services.
- Results indicated that the prepared formulations were within 93% to 98% of the nominal concentration confirming the accuracy and suitability of the formulation procedure.
Details on mating procedure:
MATING
- Non-recovery animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days.
- Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina.
- A vaginal smear was prepared for each female and the stage of oestrus or the presence of sperm was recorded.
- The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages (unless required for additional pairing).
- Mated females were housed individually during the period of gestation and lactation.
Duration of treatment / exposure:
- 43 or 44 days for males
- Until 5 days post partum for females
- Recovery group males and females were maintained without treatment for a further 14 days after sacrifice of the non-recovery males
Frequency of treatment:
Daily
Duration of test:
Approximately six weeks
No. of animals per sex per dose:
- Control: 12 male and 12 female
- Recovery control: 5 male and 5 female
- Low (100 mg/kg bw/day): 12 male and 12 female
- Intermediate (250 mg/kg bw/day): 12 male and 12 female
- High (500 mg/kg bw day): 12 male and 12 female
- Recovery high: 5 male and 5 female
Control animals:
yes, concurrent vehicle
Details on study design:
CHRONOLOGICAL SEQUENCE OF STUDY

- Non-recovery dose groups
(i) Groups of twelve male and twelve female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.
(ii) Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioral toxicity.
(iii) On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
(iv) Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
(v) On completion of the pairing phase (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli.
(vi) Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Litter size, offspring weight and sex, surface righting and clinical signs were also recorded during this period.
(vii) At Day 4 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.
(viii) Urinalysis was performed on five randomly selected males from each dose group during the final week of dosing.
(ix) Blood samples were taken from five males from each dose group for hematological and blood chemical assessments on Day 42. The male dose groups were killed and examined macroscopically on Day 43 or Day 44.
(x) Blood samples were taken from five randomly selected females from each dose group for hematological and blood chemical assessment on Day 4 post partum. At Day 5 post partum, all females and surviving offspring were killed and examined macroscopically. Any female which did not produce a pregnancy was also killed and examined macroscopically.

- Recovery dose groups
(i) Groups of five male and five female rats were dosed according to dose group continuously up to the point of sacrifice of non-recovery males at which time treatment was discontinued.
(ii) The males and females were maintained without treatment for a further fourteen days.
(iii) Urinalysis was performed for all males during the final week of recovery.
(iv) Blood samples were taken for hematological and blood chemical assessment on Day 56.
(v) After fourteen days of recovery, males and females were killed and examined macroscopically on Day 57.

Examinations

Maternal examinations:
CLINICAL OBSERVATIONS
- All animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before dosing, up to thirty minutes after dosing, and one hour after dosing, during the working week (except for females during parturition where applicable).
- During the treatment-free period, recovery animals were observed daily. All observations were recorded.

FUNCTIONAL OBSERVATIONS
- Prior to the start of treatment and at weekly intervals thereafter, all non-recovery animals were observed for signs of functional/behavioral toxicity.
- Functional performance tests were also performed on five selected males and females from each non-recovery dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.

BEHAVIOURAL ASSESSMENTS
- Detailed individual clinical observations were performed for each animal using a purpose built arena. The test was developed from the methods used by Irwin (1968) and Moser et al (1988).
- Parameters observed were 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 and lachrymation.

FUNCTIONAL PERFORMANCE TESTS
- Motor Activity: 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 on each occasion (at least two hours after dosing), under similar laboratory conditions. The evaluation period was thirty minutes for each animal. The percentage of time each animal was active and mobile was recorded for the overall thirty minute period and also during the final 20% of the period (considered to be the asymptotic period, Reiter and Macphail, 1979).
- Forelimb/Hindlimb Grip Strength: An automated 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. The assessment was developed from the method employed by Meyer et al (1979).

SENSORY REACTIVITY
- Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. The assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988).
- Parameters observed were grasp response, touch escape, vocalization, pupil reflex, toe pinch, blink reflex, tail pinch, startle reflex and finger approach.

BODY WEIGHT
- Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum. Recovery animals were weighed on Day 1 (prior to dosing) and then weekly until termination.

FOOD CONSUMPTION
- During the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum. Weekly food consumptions were performed for each cage of recovery group animals throughout the study period.
- Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for non recovery males (except during the mating phase) and recovery group animals throughout the study period and for non-recovery females during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated for females, during gestation and lactation.

WATER CONSUMPTION
- Water intake was observed daily by visual inspection of water bottles for any overt changes.

LABORATORY INVESTIGATIONS
- Hematological and blood chemical investigations were performed on five males and five females selected from each non-recovery test and control group prior to termination (Day 42 for males and Day 4 post partum for females). In addition hematological and blood chemical investigations were performed on all recovery group animals after the fourteen day treatment free period at termination (Day 56). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were taken by cardiac puncture at termination. Animals were not fasted prior to sampling.
- Urinalytical investigations were performed on five non-recovery males from the control and each test group during the final week of treatment and on all recovery males during the final week of the recovery period. Urine samples were collected overnight by housing the rats in metabolisms cages. Animals were maintained under conditions of normal hydration during collection but without access to food.
- The following hematology parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant: hemoglobin (Hb), erythrocyte count (RBC), hematocrit (Hct), erythrocyte indices (mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC)), total leukocyte count (WBC), differential leukocyte count (neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos), basophils (Bas)), platelet count (PLT), reticulocyte count (Retic) with methylene blue stained slides prepared but reticulocytes not assessed.
- 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).
- The following blood chemistry parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant: urea Calcium (Ca++), glucose Inorganic phosphorus (P), total protein (Tot.Prot.), aspartate aminotransferase (ASAT), albumin Alanine aminotransferase (ALAT), albumin/Globulin (A/G) ratio (by calculation), alkaline phosphatase (AP), sodium (Na+) Creatinine (Creat), potassium (K+), total cholesterol (Chol), chloride (Cl-) , total bilirubin (Bili), bile acids.
- The following parameters were measured on collected urine: volume, ketones, specific gravity, bilirubin, pH, urobilinogen, protein, blood, glucose, appearance.

NECROPSY
- Adult non-recovery males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43 or Day 44. Adult non-recovery females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post
partum. Surviving offspring were terminated via intracardiac overdose of a suitable barbiturate agent. The female which failed to achieve pregnancy or produce a litter were killed on or after Day 25 post coitum.
- For all non-recovery females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964). The corpora lutea were also counted.
- Recovery group animals were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 57.
- All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

ORGAN WEIGHTS
- The following organs were dissected free from fat and weighed before fixation from five selected males and five selected females from each non-recovery dose group and from all recovery group animals: adrenals, prostate, brain, seminal vesicles, epididymides, spleen, heart, testes, kidneys, thymus, liver, thyroid (weighed post-fixation with parathyroid), ovaries, uterus (weighed with cervix), pituitary (post fixation).
- The prostate, seminal vesicles, epididymides, testes, ovaries, uterus (weighed with cervix) and pituitary (post fixation) were weighed from all remaining animals.

HISTOPATHOLOGY
- Samples of the following tissues were removed from five selected males and five selected females from each non-recovery dose group and recovery animals and preserved in buffered 10% formalin, except where stated: adrenals, aorta (thoracic), bone & bone marrow (femur including stifle joint), bone & bone marrow (sternum), brain (including cerebrum, cerebellum and pons), caecum, coagulating gland, colon, duodenum, epididymides (preserved in Modified Davidsons fluid), esophagus, eyes (fixed in Davidson's fluid), gross lesions, heart, ileum (including peyer's patches), jejunum, kidneys, liver, lungs with bronchi (lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixitive), lymph nodes (mandibular and mesenteric), mammary gland, muscle (skeletal), ovaries, pancreas, pituitary, prostate, rectum, salivary glands (submaxillary), sciatic nerve, seminal vesicles, skin (hind limb), spinal cord (cervical, mid-thoracic and lumbar), spleen, stomach, thyroid/parathyroid, trachea, testes (preserved in Modified Davidsons fluid), thymus, urinary bladder, uterus/cervix, vagina.
- The coagulating gland, epididymides (preserved in Modified Davidsons fluid), gross lesions, mammary gland, ovaries, pituitary, prostate, seminal vesicles, testes (preserved in Modified Davidsons fluid), uterus/cervix and vagina were preserved from all remaining animals.
- Tissues were dispatched to the Test Site (Propath UK Ltd, Willow Court, Netherwood Road, Rotherwas, Hereford, HR2 6JU) for processing (Principal Investigator: N Fower). The tissues from five selected non-recovery control and 500 mg/kg bw/day dose group animals, any animals dying during the study, and any animals which failed to mate or did not achieve a pregnancy were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with hematoxylin and eosin for subsequent microscopic examination. The tissues from the remaining control and 500 mg/kg bw/day animals and animals which did not achieve a pregnancy were also processed. In addition, sections of testes from all control and 500 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.
- Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell-or stage-specificity of testicular findings was noted.
- Following the results of the initial histopathological examinations of control and high dosage non-recovery animals, histopathology examinations were extended to include the liver, stomach, bladder and thyroid for both sexes and also, for females only, the spleen to the low and intermediate dosage groups and the control and high dosage recovery groups.
- Microscopic examination was conducted by the Study Pathologist (Wendy Henderson). A peer review was of the histopathology findings was performed by Peter Millar (Test site Peter Millar Associates Ltd. 3 Queen Charlotte Lane, Edinburgh EH6 6AY).
Ovaries and uterine content:
PREGNANCY AND PARTURITION
- Each pregnant female was observed at least three times a day (early morning, mid-day and as late as possible during the normal working day) around the period of expected parturition.
- Observations were carried out early morning and as late as possible at weekends and public holidays. The following was recorded for each female: (i) date of pairing (ii) date of mating (iii) date and time of observed start of parturition (iv) date and time of observed completion of parturition.
Fetal examinations:
LITTER DATA
- On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum. The following was recorded for each litter: (i) number of offspring born (ii) number of offspring alive recorded daily and reported on Days 1 and 4 post partum (iii) sex of offspring on Days 1 and 4 post partum (iv) clinical condition of offspring from birth to Day 5 post partum (v) individual offspring weights on Days 1 and 4 post partum (litter weights were calculated retrospectively from these data).

PHYSICAL DEVELOPMENT
- All live offspring were assessed for surface righting reflex on Day 1 post partum.
Statistics:
- Where considered appropriate, quantitative data was subjected to statistical analysis to detect the
significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05.
- Statistical analysis was performed on the following parameters: grip strength, motor activity, body weight, body weight change, food consumption during gestation and lactation, pre-coital interval, gestation length, litter size, litter weight, sex ratio, corpora lutea, implantation sites, implantation losses, viability indices, offspring body weight, offspring body weight change, offspring surface righting, hematology, blood chemistry, urine volume, urine specific gravity, absolute organ weights, body weight-relative organ weights (see below for further details of statistical methods).
- Probability values (p) are presented as p < 0.01, p < 0.05, p > 0.05 (not significant).
Indices:
MATING PERFORMANCE AND FERTILITY
- Mating performance and fertility parameters were calculated from the individual data during the mating period of the parental generation.
- Pre-coital interval calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.
- Mating index (%) calculated for each group = (number of animals mated / number of animals paired) x 100
- Pregnancy index (%) calculated for each group = (number of pregnant females / number of animals mated) x 100
- Gestation and parturition parameters were calculated from individual data during the gestation and parturition period of the parental generation.
- Gestation length calculated as the number of days of gestation including the day of observation of mating and the start of parturition.
- Parturition index (%) calculated for each group = (number of females delivering live offspring / number of pregnant females) x 100

LITTER RESPONSES
- Values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 5 of age).
- Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter.
- Pre-implantation loss (%) = [(number of corpora lutea – number of implanation sites) / number of corpora lutea] x 100
- Post-implantation loss (%) = [(number of implantation sites – total number of offspring born) / number of implantation sites] x 100
- Live birth and viability indices were calculated for each litter.
- Live birth index (%) = (number of offspring alive on Day 1 / number of offspring bron) x 100
- Viability index (%) = (number of offspring alive on Day 4 / number of offspring alive on Day 1) x 100
- Sex ratio (% males) was calculated for each litter on Days 1 and 4 post partum using the formula (number of males / total number of offspring) x 100

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
RESULTS
- An identification error occurred after the pairing phase of the study for Female 64 (250 mg/kg bw/day) and Female 88 (500 mg/kg bw/day) and these two females have generally been removed from subsequent assessment of results.

ADULT MORTALITY
- At 100 mg/kg bw/day, one male died during the blood sampling procedure on Day 42. No significant clinical signs had been apparent for this animal prior to this event and macroscopic necropsy examination and subsequent microscopic examination of the tissues did not indicate any underlying cause for this death. In the absence of any similar incidence at higher dosages, this death was considered to be incidental and unrelated to treatment.

ADULT CLINICAL OBSERVATIONS
- Clinical signs at 100, 250 and 500 mg/kg bw/day did not indicate any adverse effect of treatment.
- Treatment at 500 mg/kg bw/day was associated with increased post-dosing salivation in both sexes from Day 6, with all 17 males 17 and females animals being affected at this dosage at some stage of the study. Ten males and eight females additionally showed noisy respiration from Day 5 for males and Day 6 for females. Increased post-dosing salivation was also observed for five of the twelve males at 100 mg/kg bw/day and ten of the twelve males and eight of the twelve females at 250 mg/kg bw/day during the study but there was no evidence of noisy respiration at these lower dosages. Increased post-dosing salivation is frequently observed when a slightly unpalatable or irritant test item is administered via the oral gavage route and is generally considered to reflect distaste/slight irritancy of the dosing formulations rather than any systemic effect of the test item.

BEHAVIOURAL ASSESSMENTS
- There were no abnormal observations apparent during behavioural assessments and there was no consistent pattern in intergroup differences for behavioural assessment scores that indicated any adverse effect of treatment.

FUNCTIONAL PERFORMANCE TESTS
- There were no changes in functional performance considered to be related to treatment.
- For females at 250 and 500 mg/kg bw/day, higher values for hind limb grip strength during trial three attained statistical significance when compared with control but, in the absence of any statistically significant differences from control in previous assessments of grip strength for these animals, this finding was considered incidental and unrelated to treatment.

SENSORY REACTIVITY ASSESSMENTS
- There were no inter-group differences in sensory reactivity scores.

BODY WEIGHT OF MALES
- At 500 mg/kg bw/day both non-recovery and recovery males showed lower body weight gain during the first week of treatment, with differences for non-recovery males attaining statistical significance. For these non-recovery males, although differences from control occasionally attained statistical significance, there was generally recovery of body weight gain and overall body weight gain between Days 8 and 43 was similar to control. For recovery males, the recovery of body weight gain between Days 8 and 43 was not as clear and body weight gains were still frequently lower than the recovery control with differences occasionally attaining statistical significance. Following the cessation of treatment, overall body weight gain of males at 500 mg/kg bw/day during the recovery period was slightly higher than control. Comparison of body gain of non-recovery and recovery males at 500 mg/kg bw/day during Days 8 to 43 showed that the body weight gain for recovery animals was only slightly lower than their non-recovery counterparts, while for the control group the values for recovery control animals was clearly superior to the non-recovery control. The observed differences between the recovery animals at 500 mg/kg bw/day and control may, in part, reflect particularly good performance for the recovery control animals.
- At 250 mg/kg bw/day, lower body weight gain was also apparent for males during the first week of treatment, with differences from control attaining statistical significance. For the remaining treatment period, although differences from control occasionally attained statistical significance, there was generally recovery of body weight gain and overall body weight gain was considered to be similar to control.
- At 100 mg/kg bw/day, there was considered to be no adverse effect of treatment on body weight gain of males. Occasional statistically significance differences from control were observed but these were considered to reflect normal biological variation.

BODY WEIGHT OF FEMALES
- At 500 mg/kg bw/day, both non-recovery and recovery females showed lower body weight gain during the first week of treatment, with differences for non-recovery females attaining statistical significance. Thereafter there was recovery of body weight gain and no adverse effect of treatment was apparent for non-recovery females during gestation and lactation; overall body weight gains of recovery females during the treatment and recovery periods were also unaffected by treatment.
- At 100 and 250 mg/kg bw/day, body weight gain of females was unaffected by treatment throughout the pre-pairing, gestation and lactation phases of the study.

FOOD CONSUMPTION
- At 500 mg/kg bw/day, food consumption for both sexes was slightly lower than control during the first week of treatment. Thereafter food intake to termination was considered to be essentially similar to control and was unaffected by treatment; this included the gestation and lactation phases of the study for non-recovery females and recovery phases for both sexes.
- At 100 and 250 mg/kg bw/day, food consumption for both sexes was considered to be unaffected by treatment throughout the study. For females at 100 mg/kg bw/day, food intake was statistically significantly lower than control during the last week of gestation, but, in the absence of any similar effects at higher dosages, this finding was considered incidental and unrelated to treatment.

FOOD CONVERSION EFFICIENCY
- At 500 mg/kg bw/day, food conversion efficiency for both sexes was slightly lower than control during the first week of treatment. Thereafter, there were no consistent differences in food conversion efficiency from control for the remainder of the study that indicated an effect of treatment for either sex.
- At 100 and 250 mg/kg bw/day, intergroup differences in food conversion efficiency for both sexes during the pre-pairing phase and for males during the post-pairing phase of the study did not indicate any adverse effect of treatment.

WATER CONSUMPTION
- Visual inspection of water residues did not indicate any effect of treatment on water intake throughout the study at any of the dosages investigated.

MATING
- There were no treatment-related effects on mating performance at 100, 250 or 500 mg/kg bw/day, with all females mating within the first four days of pairing (i.e. at the first expected oestrus opportunity).

FERTILITY
- There were no treatment-related effects on fertility as assessed by the number of females achieving pregnancy at 100, 250 or 500 mg/kg bw/day.

GESTATION LENGTH
- Gestation length was unaffected by treatment at 100, 250 or 500 mg/kg bw/day with the majority of gestation lengths being between 21 and 23.5 days.
- At 500 mg/kg bw/day, one female showed a gestation length of 24 days; this female had only one implantation and, the elongation of gestation observed for this particular female, was considered to be due to this low litter size.

LITTER RESPONSES
- A total of 12, 12, 12 and 11 females achieved pregnancy at 0 (Control), 100, 250 and 500 mg/kg bw/day respectively and gave birth to a litter. Of these one female at 250 mg/kg bw/day and another at 500 mg/kg bw/day failed to maintain their litter to Day 4 post partum. One female at
250 mg/kg bw/day and another at 500 mg/kg bw/day have been excluded due to an identification error and the following assessment is generally based on the remaining 12, 12, 10 and 9 females that successfully reared offspring to Day 4 of age.

OFFSPRING LITTER SIZE, SEX RATIO AND VIABILITY
- There was considered to be no effect of maternal treatment on the corpora lutea count, preimplantation loss, numbers of implantations, post-implantation loss, litter size at birth/Day 1 and subsequent offspring survival to Day 4 of age at 100, 250 or 500 mg/kg bw/day. Sex ratio for the offspring was similar in all groups and did not indicate any selective effect of maternal treatment on survival for either sex.
- One female at 250 mg/kg bw/day and another female at 500 mg/kg bw/day both showed total litter loss post partum. Each of these females only had a single offspring in their litter and it is not unusual for females with such a small litter to fail to maintain their litter. In the absence of any effect on offspring survival for other litters at these dosages, these two litter losses were considered to be incidental and unrelated to treatment.

OFFSPRING GROWTH AND DEVELOPMENT
- At 500 mg/kg bw/day, offspring body weight for both sexes was lower than control at Day 1 of age with differences attaining statistical significance; however, litter weight was similar to control and the observed differences in offspring body weight may, in part, reflect a slightly higher litter size at this dosage. Body weight gain of the offspring to Day 4 of age was only marginally lower than control despite the slightly higher litter size; differences from control for offspring weight gain and mean body weight at Day 4 failed to attain statistical significance. Offspring performance during assessment of surface righting on Day 1 was slightly inferior to control but, again, differences from control failed to attain statistical significance.
- At 100 and 250 mg/kg bw/day, offspring body weight on Day 1 and subsequent body weight gain to Day 4, litter weights at Day 1 and Day 4 and offspring surface righting performance on Day 1 appeared to be unaffected by maternal treatment.

LITTER OBSERVATIONS
- Clinical signs observed for offspring during the study were typical for the age observed. Neither the incidence or distribution of these observations indicated any adverse effect of maternal treatment on offspring development at 100, 250 or 500 mg/kg bw/day.

HEMATOLOGY
- There was considered to be no adverse effect of treatment on hematology parameters for either sex at 100, 250 or 500 mg/kg bw/day.
- For females at 250 mg/kg bw/day and both sexes at 500 mg/kg bw/day lower hemoglobin at the end of the treatment period attained statistical significance when compared to control. Additionally for males at 500 mg/kg bw/day statistically significant lower mean cell hemoglobin values were also apparent. For both parameters, all individual values for these treated animals were within the historical control range and, while these hematology parameters may possibly have been influenced by altered liver function indicated by microscopic hepatic changes, at the level observed these differences were considered not to represent an adverse effect of treatment.
- At 500 mg/kg bw/day, higher neutrophil and platelet count and lower erythrocyte count for males at the end of the recovery period attained statistical significance when compared with control. All values were within the historical control range and, in the absence of any similar differences for these parameters at the end of treatment period, these findings were considered to be incidental and unrelated to treatment.
- For females at 500 mg/kg bw/day, platelet count was also statistically significantly higher than control at the end of the recovery period. This was principally due to one female with a particularly high platelet count; values for the remaining females were within the historical control range and in isolation this finding was considered to be of no toxicological significance.

BLOOD CHEMISTRY
- At 500 mg/kg bw/day, higher alanine aminotransferase and total cholesterol for males at the end of the treatment period attained statistical significance when compared to control; for alanine aminotransferase the majority of values exceeded the historical control range but for cholesterol only two values exceeded the historical range. For females at this dosage, statistically significantly higher aspartame aminotransferase and alanine aminotransferase levels, compared to control, were apparent at the end of the treatment period; the majority of individual values were within the historical control range. The increase in these blood chemistry parameters are consistent with the hepatic changes observed microscopically at this dosage.
- For males at 500 mg/kg bw/day, lower sodium levels at the end of the treatment period attained statistical significance. All individual values for treated animals were within the historical control range while this range was exceeded by two control values; this finding was considered unlikely to
be of any toxicological significance.
- For females at all dosages, higher albumin levels at the end of treatment period attained statistical significance compared to control, but mean values showed no dosage relationship. All individual values for treated females were within the historical control range and this finding was considered incidental and unrelated to treatment.
- At 500 mg/kg bw/day, statistically significant differences from control for blood chemistry parameters at the end of the recovery period were restricted to higher total cholesterol levels and lower billirubin levels for females. For total cholesterol all but one value were within the historical control range and all individual bilirubin values were within the historical control range. In the absence of any similar effect for females at the end of treatment these findings were considered to be incidental and unrelated to treatment.

URINALYSIS
- There was no obvious effect of treatment on urinalysis parameters for males at 100, 250 or 500 mg/kg bw/day during the last week of treatment or for males at 500 mg/kg bw/day during the last week of the recovery period.

NECROPSY
- Necropsy findings observed for offspring during the study were typical for the age observed and neither the incidence or distribution of these observations indicated any adverse effect of maternal treatment on offspring development at 100, 250 or 500 mg/kg bw/day.
- At 500 mg/kg bw/day, necropsy of adults at the end of the treatment period revealed raised areas in the nonglandular region of the stomach for four males and five females; three of these females also showed sloughing of the non-glandular and/or glandular region of the stomach.
- The type, incidence and distribution of other macroscopic findings observed at the end of the treatment period did not indicate any obvious effect of treatment at 100, 250 and 500 mg/kg bw/day. Unusually two control females showed sloughing of the glandular region of the stomach although this finding was not associated with any evidence of microscopic change.
- Macroscopic findings at the end of the recovery period did not reveal any obvious effects of treatment at 500 mg/kg bw/day. One control female and two females at 500 mg/kg bw/day showed reddened lungs (microscopic evaluation revealed agonal congestion/hemorrhage) during these examinations but, in the absence of any similar findings at the end of the treatment period, these findings were considered incidental and unrelated to previous treatment.

ORGAN WEIGHTS
- At 250 and 500 mg/kg bw/day, absolute and body weight-relative liver weights at the end of the treatment period were higher than control for both sexes, with differences attaining statistical significance. For this organ, body weight relative values are considered the more accurate
indicator of toxicological effect; at 500 mg/kg bw/day the majority of individual values for both sexes exceeded the historical control range but at 250 mg/kg bw/day all individual values for both sexes were within this historical range. At 500 mg/kg bw/day, at the end of the treatment-free
recovery period, only absolute and body weight-relative liver weights for females attained statistical significance when compared to control and all individual body weight-relative weights were within the historical control range.
- For males at 500 mg/kg bw/day, statistically significantly lower absolute and body weight-relative seminal vesicles weights were observed at the end of the treatment period, but only two individual absolute values and one body weight relative value were below the historical control range.
- For males at all dosages, higher absolute and body weight-relative pituitary weights at the end of the treatment period attained statistical significance when compared to control but differences showed no dosage relationship. For treated animals, absolute values for only one animal at 100 mg/kg bw/day and four animals at 250 mg/kg bw/day exceed the historical control range while values for three control animals were below this historical control range. In the absence of any supporting histopathological change, the observed differences were considered to represent low control values and were considered to be unrelated to treatment.
- At 500 mg/kg bw/day, absolute and body weight-relative spleen weights for males at the end of the recovery period attained statistical significance when compared with control but only values for one of the treated animal exceeded the historical control range. In the absence of any observed effects for this organ at the end of treatment, this finding was considered incidental and of no toxicological significance.
- At 500 mg/kg bw/day, higher absolute and body weight-relative thymus weights for females at the end of the recovery period attained statistical significance when compared with control and the majority of individual values for these treated animals exceeded the historical control range. In the absence of any observed effects for this organ at the end of treatment, this finding was considered unlikely to be of any toxicological significance.

HISTOPATHOLOGY
- Liver: Bile duct hyperplasia was present in 5/5 males and 3/5 females at 250 mg/kg bw/day and all five males and five females at 500 mg/kg bw/day. Periductal inflammation/fibrosis was present in 2/5 females at 250 mg/kg bw/day and 1/5 males and all five females at 500 mg/kg bw/day.
Periductal infiltration, mainly lymphocytic was present in 4/5 males and 2/5 females at 250 mg/kg bw/day. Centrilobular hypertrophy was present in 3/5 females at 250 mg/kg bw/day and 3/5 males at 500 mg/kg bw/day. At 500 mg/kg bw/day, 3/5 females also showed focal necrosis at a minimal level. After the recovery period the bile duct changes persisted in all five males and females although there appeared to be a slight reduction in severity; other changes had resolved.
- Stomach: Hyperplasia of the non-glandular epithelium (mild, moderate or marked) was noted in all five males and seven females at 500 mg/kg bw/day. This was still apparent after the treatment free period for 1/5 males and 3/5 females but had notably reduced in severity indicating partial recovery.
- Spleen: There was a minor increase in the severity of hemopoiesis in the spleen of all six females at 250 and all five females at 500 mg/kg bw/day compared with the control Group; this was not apparent at 100 mg/kg bw/day and had resolved after the recovery period.
- Thyroid Gland: Hypertrophy of the follicular epithelium was increased in 4/5 females, and marginally in 3/5 males, at 500 mg/kg bw/day. This had resolved after the recovery period. It was present in 2/5 Group 4 recovery males at a minimal level but this was considered not to be above a background level.
- Urinary Bladder: Mild, diffuse urothelial hyperplasia was present in 2/5 females at 250 mg/kg bw/day and 1/5 males and 3/5 females at 500 mg/kg bw/day. It was not present in animals at 100mg/kg/day and showed evidence of recovery with a reduction in incidence and severity (2/5 females affected) without complete resolution after the recovery period.
- Seminal vesicles: There was reduced secretion in the seminal vesicles of 1/12 males at 500mg/kg/day.
- All other microscopic observations were considered to be common background findings in rats of this age and strain.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

DISCUSSION

- At 500 mg/kg bw/day, increased salivation was observed for both sexes, with all 17 males and 17 females being affected, and noisy respiration was also being observed for ten males and eight females. These signs are suggestive of irritancy of the test item; a view supported by sloughing and/or raised areas in the stomach for four males and six females at necropsy and hyperplasia of the non-glandular epithelium of the stomach was observed microscopically for 5/5 males and 5/5 females at this dosage. Increased salivation was also observed for some males at 100 and the majority of animals at 250 mg/kg bw/day but there were no accompanying incidences of noisy respiration or macroscopic/microscopic stomach findings at these lower dosages.

- At 500 mg/kg bw/day, lower body weight gain with a concomitant reduction in food intake, compared to control, was apparent for both sexes during the first week of treatment. Subsequent body weight gain and food consumption to termination for both sexes was considered to be unaffected by treatment; the lower body weight gain observed for recovery males compared to control was considered to reflect particularly good performance by the control group and therefore was unrelated to treatment. Males receiving 250 mg/kg bw/day also showed lower body weight gain during the first week of the study but there was no obvious accompanying effect on food consumption. Overall these initial reductions in body weight and food consumption, although clearly related to treatment, were considered not to be adverse.

- There was no effect of treatment on mating, fertility or gestation at 100, 250 or 500 mg/kg bw/day. The single non-pregnant female at 500 mg/kg bw/day was considered to be incidental and unrelated to treatment. One female at 250 mg/kg bw/day and another female at 500 mg/kg bw/day both showed total litter loss post partum but each of these females only had a single offspring in their litter. It is not usual for females with such a small litter to fail to maintain their litter and, this is particularly the case at 500 mg/kg bw/day where it was associated with an extended gestation length. In the absence of any effect on implantation and pre- and post-natal offspring survival for other litters at these dosages, these litter losses were considered to be incidental and unrelated to treatment.

- The number of corpora lutea and implantations, pre- and post implantation loss, litter size, sex ratio and offspring survival were unaffected by treatment at 100, 250 or 500 mg/kg bw/day. At 500 mg/kg bw/day, offspring body weights were statistically significantly lower than control at Day 1 of age and surface righting performance was slightly inferior to control, although statistical significance was not attained. These findings are suggestive of slight immaturity of the offspring possibly due to delayed growth, however litter weight on Day 1 was similar to control and the findings observed may have been influenced by slightly higher litter size at this dosage, so an association with treatment was considered not to be proven. Subsequent body weight gain to Day 4 of age was unaffected by treatment and offspring body weight at Day 4 failed to attain statistical significance; in the absence of any effect on offspring survival or growth the lower Day 1 body weight was considered insufficient to represent an adverse effect of treatment.

- Statistically significantly lower haemoglobin was observed for females at 250 mg/kg bw/day and both sexes at 500 mg/kg bw/day at the end of treatment; males at 500 mg/kg bw/day also showed statistically significantly lower mean cell haemoglobin. An adaptive increase in hemopoiesis within the spleen was observed for females at 250 and 500 mg/kg bw/day. The differences in hematology parameters may have been influenced by altered liver function indicated by microscopic hepatic changes but, as all individual for treated animals were within the historical control range, they were considered unlikely to represent as adverse effect of treatment.

- At 500 mg/kg bw/day, alanine aminotransferase for males and aspartame aminotransferase and alanine aminotransferase levels for females were statistically significantly higher at the end of the treatment period. While the majority of individual male values for alanine aminotransferase exceeded the historical control range, most of the individual female values for aspartame aminotransferase and alanine aminotransferase were within this historical control range; however these blood chemistry changes were consistent with hepatic changes observed microscopically at this dosage. Additionally for males, higher total cholesterol levels also attained statistical significance; although the majority of values were within the historical control range these levels may have been influenced by altered liver metabolism.

- Microscopic evaluation of the liver at the end of the treatment period revealed bile duct hyperplasia in 5/5 males and 3/5 females at 250 mg/kg bw/day and all five males and five females at 500 mg/kg bw/day. Periductal inflammation/fibrosis was also present in 2/5 females at 250 mg/kg bw/day and 1/5 males and all five females at 500 mg/kg bw/day. These finding were still present for 5/5 males and 5/5 females at the end of the recovery period, although there was a slight reduction in severity. While these findings were considered to be advserse in nature for the rat, the toxicological significance of these findings for man is more equivocal; it is considered similar liver findings in other rat strains may not indicate any long term adverse health effect in man (Hailey et al). At 500 mg/kg bw/day, 3/5 females also showed focal necrosis at a minimal level; while this finding could be incidental, it may be a further indication of adverse effects of the test item.

- Centrilobular hypertrophy was also present in 3/5 females at 250 mg/kg bw/day and 3/5 males at 500 mg/kg bw/day. This finding was suggestive of a response to mixed function oxidase induction (Cattley et al., 2002); such as this particular change was considered to be an adaptive change of limited toxicological significance for man.

- These microscopic changes were accompanied by a statistically significant increase in liver weights compared with control for both sexes at 250 and 500 mg/kg bw/day, although all individual body weight relative values (considered the more accurate indicator of toxicological effect) at 250 mg/kg bw/day were within the historical control range. After the recovery period the microscopic changes had resolved with the exception of the bile duct findings which persisted with a slight reduction in severity. Higher female liver weights at the end of the treatment free period still attained statistical significance compared with control but all individual body weight-relative were within the historical control range.

- Histopathological evaluations also revealed incidences of diffuse urothelial hyperplasia in the urinary bladder for females 2/5 females at 250 mg/kg bw/day and 1/5 males and 3/5 females at 500 mg/kg bw/day. This may reflect a direct effect of the test item or an irritant effect of the test item or metabolite if this represents a route of excretion for the test item. As the cause is undetermined this finding is regarded as being an adverse effect of treatment. These changes showed a reduction in incidence and severity without complete resolution after the recovery period (2/5 females affected).

- Hypertrophy of the follicular epithelium of the thyroid was observed for 3/5 males and 4/5 females at 500 mg/kg bw/day. This finding was considered likely to be linked to hepatic enzyme induction leading to increased removal of thyroid hormones and is considered to be an adaptive response. These findings were considered to have resolved after the recovery period.

- For males at 500 mg/kg bw/day, statistically significantly lower absolute and body weight-relative seminal vesicles weights were observed at the end of the treatment period, but only two individual absolute values and one body weight relative value were below the historical control range. There was reduced secretion in the seminal vesicles of 1/12 males at 500 mg/kg bw/day but this male had previously successfully induced pregnancy in its female partner. Overall it was considered that an association with treatment and this finding for this reproductive organ was not proven.

- Overall, the most significant findings observed for the study were considered to be microscopic changes observed for the liver, stomach and urinary bladder. The changes observed for the stomach were considered to be the result of a direct irritant effect of the test item rather than

systemic toxicity and it is likely that this is also the case for the changes observed for the urinary bladder. It is possible that some of the histopathological changes observed for the urinary bladder and liver may also reflect irritancy of the test item or metabolite but the changes observed, particulary in the liver, cannot solely be explained by this course of action. In view of this, the observed changes in the liver, although they may have equivocal revelance to human health, preclude dosages of 250 and 500 mg/kg bw/day from representing a No Observed Adverse Effect Level. The NOAEL for systemic toxicity is therefore 100 mg/kg bw/day and this dosage probably also represent a No Observed Effect Level (NOEL).

- Offspring bodyweights were slightly lower than control on Day 1 but litter weights were unaffected as slightly larger litter size in combination with offspring weight resulted in approximately equal group mean values. As there was no impact on the survival or subsequent growth of the offspring, the lower offspring weight on Day 1 was considered not to represent an adverse effect of treatment. The No Observed Adverse Effect Level (NOAEL) for reproduction and the survival, growth and development of the offspring therefore was considered to be 500 mg/kg bw/day.

Applicant's summary and conclusion

Conclusions:
Treatment at 250 and 500 mg/kg bw/day was associated with adverse liver histopathology, although the toxicological significance of these findings for man is considered to be equivocal. The No Observed Adverse Effect Level (NOAEL) for systemic toxicity of the rat was considered to be 100 mg/kg bw/day. The No Observed Adverse Effect Level (NOAEL) for reproduction and the survival, growth and development of the offspring was considered to be 500 mg/kg bw/day.
Executive summary:

GUIDELINE

The study was designed to investigate the systemic toxicity and potential adverse effects of the test item on reproduction (including offspring development); it also assesses the ability of the animals to recover from any toxicity following the withdrawal of treatment. The study was designed to be compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test” (adopted 22 March 1996).

This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

METHODS

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han:RccHan:WIST strain rats, for approximately six weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 250 and 500 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Arachis oil BP) over the same treatment period. Two recovery groups, each of five males and five females, were treated with the high dose (500 mg/kg bw/day) or the vehicle alone for forty-two consecutive days and then maintained without treatment for a further fourteen days.

Clinical signs, behavioral assessments, body weight change and food and water consumption were monitored during the study.

Pairing of non-recovery animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex. Extensive functional observations were performed on five selected non-recovery males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 4 post partum. Urinalysis was performed on five non-recovery males per dose group during the final week of treatment and five non-recovery males and females from each dose group were selected for hematology and blood chemistry assessments prior to termination. Surviving adult non-recovery males were terminated on Day 43 or Day 44, with all females and offspring being killed on Day 5 post partum. Any female which did not produce a pregnancy was terminated on or after Day 25 post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed. Following forty-two days of treatment, recovery group animals were maintained without treatment for a further fourteen days. Urinalysis was performed on all recovery group males during the final week of the treatment period. In addition, hematological and blood chemical assessments were performed on all recovery group animals at the end of the treatment-free period. These animals were then subjected to a gross necropsy and histopathological examinations of selected tissues was performed.

RESULTS

Mortality: At 100 mg/kg bw/day, one male died during the blood sampling procedure on Day 42; this death was considered to be incidental and unrelated to treatment.

Clinical observations: Treatment at 500 mg/kg bw/day was associated with increased post-dosing salivation in all seventeen male and seventeen female; ten males and eight females also showed noisy respiration during the study. Increased post-dosing salivation was also observed for five males at 100 mg/kg bw/day and the twelve males and eight females at 250 mg/kg bw/day.

Behavioural assessment: There were no treatment-related changes in the behavioural parameters at 100, 250 or 500 mg/kg bw/day.

Functional performance tests: There were no changes in functional performance considered to be related to treatment at 100, 250 or 500 mg/kg bw/day.

Sensory reactivity assessments: There were no inter-group differences in sensory reactivity scores.

Body weight: At 500 mg/kg bw/day, males showed lower body weight gain during the first week of treatment, thereafter there was recovery of body weight gain although body weight gain still tended to be lower than control during the treatment period. Body weight gain of males was slightly superior to control during the recovery period. For females at 500 mg/kg bw/day, lower body weight gain was also observed during the first week of treatment. There was no subsequent effect of treatment on body weight gain during the remainder of the study, including gestation, lactation and recovery phases. At 250 mg/kg bw/day, males showed lower body weight gain during the first week of treatment; body weight gain was considered to be unaffected by treatment for the remaining treatment period and the recovery period. At both sexes at 100 mg/kg bw/day and females at 250 mg/kg bw/day, body weight gain was considered to be unaffected by treatment.

Food consumption: At 500 mg/kg bw/day, food consumption for both sexes was slightly lower than control during the first week of treatment; thereafter food intake was unaffected by treatment for the remainder of the study including gestation, lactation and recovery phases. At 100 and 250 mg/kg bw/day, food consumption for both sexes was considered to be unaffected by treatment throughout the study.

Food consumption efficiency: At 500 mg/kg bw/day, food conversion efficiency for both sexes was slightly lower than control during the first week of treatment. At 100 and 250 mg/kg bw/day, food conversion efficiency for both sexes was considered to be unaffected by treatment.

Water consumption: Water consumption for both sexes was unaffected by treatment at 100, 250 or 500 mg/kg bw/day.

Reproductive performance (mating): Mating performance was unaffected by treatment at 100, 250 or 500 mg/kg bw/day.

Reproductive performance (fertility): Fertility was unaffected by treatment at 100, 250 or 500 mg/kg bw/day.

Reproductive performance (gestation lengths): Gestation length was unaffected by treatment at 100, 250 or 500 mg/kg bw/day.

Litter responses (offspring litter size, sex ratio and viability): The number of corpora lutea and implantations, pre- and post implantation loss, litter size, sex ratio and offspring survival was unaffected by treatment at 100, 250 or 500 mg/kg bw/day.

Litter responses (offspring growth and development): At 500 mg/kg bw/day, offspring body weight for both sexes was lower than control at Day 1 of age; surface righting performance on Day 1, litter weights on Day 1 and 4 and body weight gain to Day 4 was considered to be unaffected by maternal treatment. At 100 and 250 mg/kg bw/day, offspring body weights, body weight gain, litter weights and surface righting performance on Day 1 was unaffected by maternal treatment. Clinical signs and necropsy findings observed for offspring did not indicate any effect of maternal treatment on offspring development at 100, 250 or 500 mg/kg bw/day.

Laboratory investigations: There was considered to be no adverse effect of treatment on hematology parameters for either sex at 100, 250 or 500 mg/kg bw/day.

Blood chemistry: At 500 mg/kg bw/day, males showed higher alanine aminotransferase and total cholesterol levels and females higher aspartame aminotransferase and alanine aminotransferase levels at the end of the treatment.

Urinalysis: Analysis of urinalysis parameters for males at 100, 250 or 500 mg/kg bw/day during treatment and for males at 500 mg/kg bw/day following recovery did not indicate any effect of treatment.

Necropsy: At 500 mg/kg bw/day, raised areas in the non-glandular region of the stomach were apparent for four males and five females at the end of the treatment period, with three of the females also showing sloughing of the non-glandular/glandular region of the stomach.

Organ weights: At 250 and 500 mg/kg bw/day, absolute and body weight-relative liver weights at the end of the treatment period were statistically significantly higher than control for both sexes at the end of treatment. For males at 500 mg/kg bw/day, statistically significantly lower absolute and body weight relative seminal vesicles weights were observed at the end of the treatment period, but only two individual absolute values and one body weight relative value were below the historical control range.

Histopathology (liver): Bile duct hyperplasia was observed for the majority of animals at 250 mg/kg bw/day and all animals at 500 mg/kg bw/day. Periductal inflammation/fibrosis was present for two females at 250 mg/kg bw/day and one male and all females at 500 mg/kg bw/day. Periductal infiltration, mainly lymphocytic, was present in the majority of males and two females at 250 mg/kg/day. Centrilobular hypertrophy was present for females at 250 mg/kg bw/day and males at 500 mg/kg bw/day. Females at 500 mg/kg bw/day showed focal necrosis at a minimal level. After the recovery period the bile duct changes persisted for both sexes with a slight reduction in severity; other changes had resolved.

Histopathology (stomach): Hyperplasia of the non-glandular epithelium (mild, moderate or marked) was observed at 500 mg/kg bw/day at the end of treatment. This was still apparent at notably reduced severity after the treatment free period indicating partial recovery.

Histopathology (spleen): There was a minor increase in the severity of hemopoiesis in the spleen of females at 250 and 500 mg/kg bw/day at the end of treatment; this had resolved after the recovery period.

Histopathology (thyroid gland): Hypertrophy of the follicular epithelium was increased for both sexes at 500 mg/kg bw/day at the end of treatment; this had resolved after the recovery period.

Histopathology (Urinary bladder): Mild, diffuse urothelial hyperplasia was present for two females at 250 mg/kg bw/day and one male and three females at 500 mg/kg bw/day at the end of treatment. Evidence of recovery, with a reduction in incidence and severity without complete resolution, was apparent after the recovery period.

CONCLUSION

Treatment at 250 and 500 mg/kg bw/day was associated with adverse liver histopathology, although the toxicological significance of these findings for man is considered to be equivocal. The No Observed Adverse Effect Level (NOAEL) for systemic toxicity of the rat was considered to be 100 mg/kg bw/day. The No Observed Adverse Effect Level (NOAEL) for reproduction and the survival, growth and development of the offspring was considered to be 500 mg/kg bw/day.