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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 April 2009 to 29 October 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
1996
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
3-sec-[C15-18-(branched and linear)-alk-2-enyl]pyrrolidine-2,5-dione
EC Number:
701-350-3
Molecular formula:
Not possible to assign, UVCB
IUPAC Name:
3-sec-[C15-18-(branched and linear)-alk-2-enyl]pyrrolidine-2,5-dione
Test material form:
liquid
Details on test material:
- Appearance: brown liquid
- Storage: room temperature, in the dark

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Wistar Han HsdRccHan:WIST
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: approximately twelve weeks old
- Weight at study initiation: the males weighed 318 to 366 g and the females weighed 191 to 230 g
- Housing: Initially, all animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding. During the mating phase, 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.
- Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels except for mated females during gestation and lactation. Mated females were also given softwood flakes as bedding, throughout gestation and lactation.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 13 days

DETAILS OF FOOD AND WATER QUALITY:
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 CONDITIONS
- Temperature: 21 ± 2 °C
- Humidity: 55 ± 15 %
- Air changes: at least fifteen air changes per hour
- Photoperiod: low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
For the purpose of the study, the test material was prepared at the appropriate concentrations as a solution in arachis oil. The stability and homogeneity of the test material formulations were determined by Harlan Laboratories Ltd., Shardlow, UK Analytical Services. Results showed the formulations to be stable for at least fourteen days. Formulations were therefore prepared weekly and stored at approximately 4 °C in the dark.
Samples were taken of each test material formulation and analysed for concentration of test material at Harlan Laboratories Ltd., Shardlow, UK Analytical Services. The results indicate that the prepared formulations were within 10 % of the nominal concentration.

- VEHICLE
- Amount of vehicle: 4 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
METHOD OF ANALYSIS
- The concentration of test material in the test material formulations was determined by gas chromatography (GC) using an external standard technique.
- Samples: The test material formulations were extracted with methanol to give a final, theoretical test material concentration of approximately 1 mg/mL.
- Standards: Standard solutions of test material were prepared in methanol at a nominal concentration of 1 mg/mL containing the equivalent amount of vehicle as the sample solutions.
- Procedure: The standard and sample solutions were analysed by GC using the following conditions:
GC system: Agilent Technologies 5890, incorporating autosampler and workstation
Column: DB-1 (30 m x 0.32 mm id x 0.25 µm film)
Oven temperature program: initial: 100 °C for 0 mins, rate: 10 °C/min, final: 300 °C for 6 mins
Injection temperature: 300 °C
Flame ionisation detector temperature: 325 °C
Injection volume: 1 µL
Retention time: Profile of peaks from ~6 to 8 mins

- Homogeneity Determinations: The test material formulations were deemed homogeneous by visual inspection.
- Stability Determinations: The test material formulations were sampled and analysed initially and then after storage at approximately +4 °C in the dark for fourteen days.
- Verification of Test Material Formulation Concentrations: The test material formulations were sampled and analysed within three days of preparation.

RESULTS
- Stability of Test Material Formulations: 102 and 101 % of the initial concentration was found in the test material after storage for 14 days.
- Verification of Concentration of Weekly Test Material Formulation: 90 – 101 % of the nominal test material concentration was found.

METHOD VALIDATION
- Linearity: A range of standard solutions covering the concentration range 0 to 1.5056 mg/mL, were prepared containing the equivalent amount of vehicle as the low dose level sample solutions and analysed. The detector response was shown to be linear up to 1.5056 mg/mL.
- Specificity: The diluent solvent methanol and a blank Arachis Oil BP (control) were analysed. Analysis of the solvent and a blank Arachis Oil BP (control) produced no signal that interfered with the signal due to the test material.
- Accuracy: Samples of Arachis Oil BP were accurately fortified with known amounts of test material, and analysed. Mean recovery was 100 – 103 %. The analytical method has been considered to be sufficiently accurate for the purpose of this study. The test sample results have not been corrected for recovery.

CONCLUSION
- The analytical method has been satisfactorily validated in terms of linearity, specificity and accuracy for the purposes of the study.
Duration of treatment / exposure:
The animals were dosed for up to 54 days.
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
750 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: a preliminary 14-day repeated dose oral (gavage) range-finder study was conducted to establish the maximum tolerated dose level (up to 1000 mg/kg bw/day) and to provide information for selection of dose levels for use in the main OECD 422 study.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, up to thirty minutes after dosing, and one and five hours after dosing, during the working week. Animals were observed immediately before dosing, soon after dosing, and one hour after dosing at weekends (except for females during parturition where applicable). All observations were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Detailed individual clinical observations were performed for each animal using a purpose-built arena. The following parameters were observed: Gait, Tremors, Twitches, Convulsions, Bizarre/Abnormal/Stereotypic behaviour, Salivation, Pilo-erection, Exophthalmia, Lachrymation, Hyper/Hypothermia, Skin colour, Respiration, Palpebral closure, Urination, Defecation, Transfer arousal and Tail elevation.

BODY WEIGHT: Yes
- Individual bodyweights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until mating was evident. Bodyweights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post-partum.

FOOD CONSUMPTION: Yes
- During the maturation 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 Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum.

FOOD EFFICIENCY: Yes
- Food efficiency (the ratio of bodyweight change/dietary intake) was calculated retrospectively for males throughout the study period and for females during maturation and the first two weeks of gestation. Due to offspring growth and milk production, food efficiency could not be accurately calculated during the final week of gestation and during lactation.

WATER CONSUMPTION: Yes
- Water intake was observed daily by visual inspection of water bottles for any overt changes. Intergroup differences did not indicate any need for more formal gravimetric measurements.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Haematological and blood chemical investigations were performed on five males and five females selected from each test and control group on Day 14 (day prior to pairing) and blood chemical investigations were re-assessed for on five males on Day 42 and five females on Day 4 post partum from each test and control group. Blood samples were obtained from the lateral tail vein. Animals were not fasted prior to sampling.
- Haematology: The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant: 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) and Reticulocyte count (Retic): Methylene blue stained slides were prepared but reticulocytes were not assessed. Prothrombin time (CT) was assessed by 'lnnovin' and Activated partial thromboplastin time (APTT) was assessed by 'Actin FS' using samples collected into sodium citrate solution (0.11 mol/L).
- Clinical Chemistry: The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant: Urea, Glucose, Total protein (Tot.Prat.), Albumin, Albumin/Globulin (NG) ratio (by calculation), Sodium (Na+), Potassium (K+), Chloride (Cl-), Calcium (Ca++), Inorganic phosphorus (P), Aspartate aminotransferase (ASAT), Alanine aminotransferase (ALAT), Alkaline phosphate (AP), Creatinine (Creat), Total cholesterol (Chol) and Total bilirubin (Bili).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.
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 each day, 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/Hind-limb 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. 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. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988). The following parameters were observed: Grasp response, Vocalisation, Toe pinch, Tail pinch, Finger approach, Touch escape, Pupil reflex, Startle reflex and Blink reflex.

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43. Adult 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 sodium pentobarbitone. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 26 post coitum. For all 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). 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, removed from all surviving animals at terminal kill were dissected free from fat and weighed before fixation: Adrenals, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Spleen, Testes, Thymus and Thyroid.

HISTOPATHOLOGY: Yes
- Samples of the following tissues were preserved from all animals in buffered 10 % formalin except where indicated: Adrenals, Aorta (thoracic), Bone & bone marrow (femur including stifle joint), Bone & bone marrow (sternum), Brain (including cerebrum, cerebellum and pons), Caecum, Coagulation gland, Colon, Duodenum, Epididymides (preserved in Bouin's fluid then transferred to 70 % Industrial Methylated Spirits (IMS) approximately 48 hours later), Eyes (fixed in Davidson's fluid), Gross lesions, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs (with bronchi) (inflated to approximately normal inspiratory volume with buffered 10 % formalin before immersion in fixative), Lymph nodes (cervical and mesenteric), Mammary gland, Muscle (skeletal), Ovaries, Pancreas, Pituitary, Prostate, Oesophagus, 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 Bouin's fluid then transferred to 70 % Industrial Methylated Spirits (IMS) approximately 48 hours later), Thymus, Urinary bladder, Uterus/Cervix and Vagina.
- The tissues from five selected control and 750 mg/kg/day dose group animals and any animals which failed to achieve a pregnancy were prepared as paraffin blocks, sectioned at nominal thickness of 5 µm and stained with haematoxylin and eosin for subsequent microscopic examination. The tissues shown in bold from the remaining control and 750 mg/kg/day were also processed. In addition, sections of testes and epididymides from all control and 750 mg/kg/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined. Since there were indications of treatment-related changes in the liver, thyroid and thymus, examination was subsequently extended to include similarly prepared sections from five animals per sex from the low and intermediate 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:
REPRODUCTION SCREENING

Mating
- 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 the oestrous cycle 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.

Pregnancy and Parturition
- Each pregnant female was observed at approximately 08:30, 12:30 and 16:30 hours and around the period of expected parturition. Observations were carried out at approximately 08:30 and 12:30 hours at weekends and public holidays. The following was recorded for each female: date of pairing, date of mating, date and time of observed start of parturition and date and time of observed completion of parturition.

Litter Data
- On completion of parturition (Day 0 of 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. For each litter the following was recorded: Number of offspring born, Number of offspring alive recorded daily and reported on Day 1 and 4 post partum, Sex of offspring on Day 1 and 4 post partum, Clinical condition of offspring from birth to Day 5 post partum and Individual offspring and litter weights on Day 1 and 4 post partum.

Physical Development
- All live offspring were assessed for surface righting reflex on Day 1 post partum.


EVALUATION OF DATA
- Treatment of Data:
Data were processed to give group mean values and standard deviations where appropriate.
For bodyweights and food consumptions during gestation, group mean values were calculated using data from females which were observed to give birth to offspring.
For bodyweights and food consumptions during lactation, group mean values were calculated using data from females with live young at Day 5 of lactation.

REPRODUCTIVE INDICES
Mating Performance and Fertility
The following 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.
- Fertility Indices: For each group the following were calculated:
Mating Index (%) = (Number of animals mated / Number of animals paired) x 100
Pregnancy Index (%) = (Number of pregnant females/ number of animals mated) x 100
- Gestation and Parturition Data: The following parameters were calculated for 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 for observation of mating and the start of parturition.
Parturition Index: The following was calculated for each group. Parturition Index (%) = (Number of females delivering live offspring /number of pregnant females) x 100
- Litter Responses: The standard unit of assessment was considered to be the litter, therefore 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).
- Implantation Losses (%): Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter as follows:
%pre-implantation loss = [(number of corpora lutea – number of implantation 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: The following indices were calculated for each litter as follows:
Live Birth Index (%)= (Number of offspring alive on Day 1 / number of offspring born) x 100
Viability Index (%) = (number of offspring alive on day 4/ number of offspring alive on day 1) x 100
- Sex Ratio (% males): Sex ratio was calculated for each litter value on Day 1 and 4 post partum, using the following formula: (Number of male offspring / total number of offspring) x 100
Statistics:
The following parameters were subjected to statistical analysis: Quantitative functional performance data, Bodyweight and bodyweight change, Food consumption during gestation and lactation, Haematology, blood chemistry, absolute and bodyweight relative organ weights, Litter data, Sex ratio, Implantation losses and viability indices, Offspring bodyweight and bodyweight change and Offspring surface righting.
The following statistical procedures were used: 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: Kruskal­Wallis ANOVA and Mann-Whitney 'U' test.
The non-parametric methods were also used to analyse implantation loss, offspring sex ratio and landmark developmental markers.
Probability values (p) are presented as follows: p < 0.001 ***, p < 0.01 **, p < 0.05 * and p ≥ 0.05 (not significant).
Histopathology data were analysed using the following methods to determine significant differences between control and treatment groups for the individual sexes (excluding any not producing a pregnancy/litter):
Chi-squared analysis for differences in the incidence of lesions occurring with an overall frequency of 1 or greater.
Kruskal-Wallis one-way non-parametric analysis of variance for the comparison of severity grades for the more frequently observed graded conditions.
Probability values (P) were calculated as follows: P < 0.001 +++ --- ***, P < 0.01 ++ -- **, P < 0.05 + - *, P < 0.1 (+) (-) (*), P ≥ 0.01 N.S. (Not significant).
Plus (+) signs indicate positive differences from the control group and minus (-) signs indicate negative differences. Asterisks refer to overall differences between group variation which is non-directional.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
- No clinically observable signs of toxicity were detected.
- Episodes of increased salivation were detected in animals of either sex treated with 750 and 200 mg/kg/day together with sporadic incidents of noisy respiration evident in the 750 mg/kg/day males. Such findings are often observed following the oral administration of an unpleasant tasting test material formulation and are considered not to be indicative of systemic toxicity. Episodes of red/brown staining of the mouth were evident in animals of either sex treated with 200 and 750 mg/kg/day along with instances of generalised fur loss seen in the 750 mg/kg/day dose group. These findings are considered to be incidental and of no toxicological significance.
- Behavioural Assessments: Weekly open field arena observations did not reveal any treatment-related effects. All inter and intra group differences in urination, defecation and transfer arousal scores were considered to be a result of normal variation for rats of the strain and age used and were of no toxicological importance.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A reduction in bodyweight development was evident throughout the treatment period for males treated with 750 mg/kg/day, in comparison to controls. Statistically significant reductions in actual bodyweight were noted during Weeks 5 to 7 (P < 0.01 Week 5 and 6: P < 0.05 Week 7). Statistically significant reductions in weekly bodyweight change were evident throughout the treatment period (P < 0.01).
No such effect was evident in females treated with 750 mg/kg/day or animals of either sex treated with 200 or 50 mg/kg/day.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No adverse effect on dietary intake was detected for treated animals, in comparison to controls.
Food efficiency:
no effects observed
Description (incidence and severity):
No adverse effect on dietary intake was detected for treated animals, in comparison to controls.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No treatment-related intergroup differences in water intake were detected for treated animals when compared to controls.
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
There was no adverse effect on the haematological parameters measured for treated animals in comparison to controls.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant reduction in alkaline phosphatase and alanine aminotransferase levels (P < 0.01 : P < 0.05 respectively) was evident in males treated with 750 mg/kg/day during Day 14 in comparison to controls.
No such effect was evident in the female treatment groups.
A statistically significant reduction in alkaline phosphatase was seen in the 200 and 50 mg/kg/day males treatment groups (P < 0.05) during Day 14 in comparison to controls. In the absence of an effect on Day 42 or any histopathological correlates this finding is considered to be of no toxicological significance.
During Day 4 post partum a statistically significant increase in alanine aminotransferase levels (P < 0.01) was evident in the 750 mg/kg/day females. On Day 42 males treated with 750 mg/kg/day showed a statistically significant increase in albumin/globulin ratio and alanine aminotransferase levels (P < 0.01) with a statistically significant reduction in total protein, calcium and bilirubin levels (P < 0.05) in comparison to controls.
No such effect was evident in animals of either sex treated with 200 or 50 mg/kg/day.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional Performance Tests: There were no treatment-related changes in the functional performance test results from treated animals when compared to controls.
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.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant increase in liver weight (P < 0.01) both absolute and relative to terminal bodyweight was evident in animals of either sex treated with 750 mg/kg/day.
No such effects were evident in animals of either sex treated with 200 or 50 mg/kg/day.
A statistically significant increase in testes weights (P < 0.01) relative to terminal bodyweight was seen in the 750 mg/kg/day males. Males treated with 750 mg/kg/day also showed a statistically significant reduction in thymus weights (P < 0.01) both absolute and relative to terminal bodyweight. In the absence of any histopathogical correlates these findings are considered to be of no toxicological importance. A statistically significant increase in thyroid weights (P < 0.05) relative to terminal bodyweight was seen in the 200 mg/kg/day males. In the absence of a dose related response or any histopathological correlates this finding is considered to be of no toxicological significance. An increase in liver weight was evident in males only treated with 200 and 50 mg/kg/day. The statistical significance was only evident in the relative to terminal bodyweight measurement (P < 0.01 and P < 0.05). In the absence of any clinical chemistry findings to suggest an effect of treatment or any supporting histopathological correlates this finding is considered to be of no toxicological importance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related abnormalities were detected for treated adults in comparison to controls.
One 200 mg/kg/day male had no left testicle and epididymis and one 200 mg/kg/day female had epithelial sloughing of the glandular region of the stomach. Two control males had hydronephrosis, one on the left and the other of the right kidney. Red fluid in the bladder was also seen in one of these animals. In the absence of a dose related response or histopathological correlates these findings are considered to be of no toxicological importance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathogical examinations revealed the following treatment-related effects:

LIVER: Centrilobular hepatocyte enlargement was seen in relation to treatment for males only treated with 750 mg/kg/day. One male was also affected at 200 mg/kg/day but this was considered to be unrelated to treatment, since hepatocyte enlargement is seen occasionally among control animals as a spontaneous change.

THYROID GLAND: A higher incidence of follicular cell hypertrophy was seen among males only treated with 750 mg/kg/day, but not at any other dose level.

THYMUS: Higher grades of severity of lymphoid atrophy were seen among females only treated with 750 mg/kg/day. A similar effect was not seen among female rats in the remaining dose groups.


OTHER HISTOPATHOLOGY

The remaining histopathological changes seen among surviving control and intermediate dose animals were all considered to be spontaneous in origin and unrelated to treatment. The following conditions warrant specific mention:

ADRENAL GLANDS: Cortical vacuolation was seen in two high dose males and was of no toxicological significance in this investigation.

BONE MARROW: Adipose infiltration of the marrow is an indicator of changes in marrow cellularity and in this study there was no difference between control and treated groups.

ILEUM: Vacuolation of the lamina propria was seen for animals of either sex from the control and high dose groups. The marginally greater prevalence of the condition among high dose males was considered to be of no toxicological significance.

KIDNEYS: Globular accumulations of eosinophilic material, as a consequence of excessive accumulation of α2-microglobulin in renal proximal tubular epithelial cells, are occasionally encountered as a spontaneous change in male rats. Focal corticomedullary mineralisation is a commonly observed background condition among female rats and hydronephrosis was reported for four animals; this is considered to be a condition of congenital origin.

LIVER: Scattered mononuclear cell foci were observed in a few control and treated animals. Such are commonly observed in the rodent liver and are not indicative of any adverse condition at the severities encountered. Isolated instances of periportal pigment generalised hepatocyte enlargement and periportal lipid vacuolation were also seen.

LUNGS: A minimal severity of bronchus associated lymphoid tissue was reported for all control and high dose animals examined in the study and is not indicative of respiratory disease. A minimal severity of accumulations of alveolar macrophages was also observed for control and high dose animals; such are commonly observed in laboratory maintained rats of this age and are not suggestive of significant respiratory disease or an effect of treatment.

OESOPHAGUS: Inflammatory cell infiltrates in the peripheral musculature is a commonly observed change that is considered to be related to the physical trauma of gavage dosing.

SKELETAL MUSCLE: Mononuclear cell foci are commonly observed in the skeletal muscle of laboratory maintained rats and are of no toxicological significance at the incidences seen in this investigation.

SPLEEN: Extramedullary haemopoiesis is a normal background condition in the rat spleen and the severities observed were considered to be within normal limits. Post partum female rats frequently demonstrate elevated severities of splenic extramedullary haemopoiesis.


REPRODUCTIVE TRACT AND RELATED ORGANS

PITUITARY: No treatment-related changes were seen. Vacuolation of pars anterior cells is commonly observed, more especially among male rats.

TESTIS/EPIDIDYMIS: No treatment-related changes were seen. Minimal testicular atrophy was observed for two high dose males; this condition is seen occasionally as a spontaneous change in laboratory maintained rats of this age and was of no toxicological significance in this investigation.

SEMINAL VESICLES/COAGULATING GLAND: No treatment-related changes were seen.

PROSTATE: No treatment-related changes were seen. Interstitial chronic inflammatory cell infiltrates are a commonly observed background finding in laboratory maintained rats and prostatitis is occasionally seen as an associated change.

MAMMARY GLAND: Glandular hyperplasia was observed in the mammary tissue of the majority of females examined and is consistent with pregnancy and lactation.

OVARY: No treatment-related changes were seen.

UTERUS: Areas of haemorrhage and fibrosis were seen in the myometrium and adjacent connective tissue of the uterus in the majority of female animals examined from control and high dose groups. These conditions are consistent with normal post partum uterine changes in the rat.
All other morphological changes in the above and remaining tissues were those commonly observed in laboratory maintained rats of the age and strain employed and, since there were no differences in incidence or severity between control and treatment groups, all were considered to be without toxicological significance.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
REPRODUCTIVE PERFORMANCE
- Mating: No treatment-related effects were detected in mating performance. With the exception of one 750 mg/kg/day pair, all paired animals mated within five days of pairing.
- Fertility: All 200 and 50 mg/kg/day females were pregnant. One control female and one female treated with 750 mg/kg/day did not achieve pregnancy.
- Gestation Length: No treatment-related effects were detected in the length of gestation.
- Litter Responses: In total ten females from the 200 and 50 mg/kg/day dose groups and nine control and 750 mg/kg/day dose group females gave birth to a live litter and successfully reared young to Day 5 of age. The following assessment of litter response is based on all litters reared to termination on Day 5 of lactation/age.
- Offspring Litter Size and Viability: No significant differences were detected in sex ratio, litter sizes and viability for treated groups when compared to controls. Nine litters were evident for the 750 mg/kg/day and control groups at termination and ten litters were evident for the 200 and 50 mg/kg/day groups at termination.
- Offspring Growth and Development: There were no differences in litter weights or mean offspring bodyweights between control and treated animals. No obvious clinical signs of toxicity were detected.

OFFSPRING NECROPSY
- No treatment-related macroscopic abnormalities were detected for offspring dying during lactation or at termination on Day 5 post partum.
- One female treated with 750 mg/kg/day produced a litter with one pup which had a reddened right testis, whilst another female from this treatment group had one small pup.
- Three females treated with 200 mg/kg/day each produced a litter with a small pup. One of these litters also had a pup with no milk in the stomach. A further litter had a pup with a damaged tail.
- One female treated with 50 mg/kg/day produced a litter with one pup which had an open wound.
- Two control females each produced a litter with small pups one of these also had a pup with increased renal pelvic cavitation. Another litter had a pup with a wound on the top of its head and one with a reddened right testis.
- The effects observed in the offspring are considered to be incidental spontaneously occurring events of no toxicological significance.
Details on results:
DISCUSSION
The oral administration of the test material to rats for a period of up to 54 Days (including two weeks pre-mating, gestation and early lactation period for females) at dose levels of up to 750 mg/kg/day resulted in toxicologically significant effects at 750 mg/kg/day.
There were no clinically observable signs of toxicity detected in the treated animals in comparison to controls. A reduction in bodyweight development was evident in the 750 mg/kg/day males throughout the treatment period.
Laboratory investigations showed a slight reduction in alanine aminotransferase and alkaline phosphatase levels for the 750 mg/kg/day males during Day 14 in comparison to controls. Further blood chemical investigations performed on these animals during Day 42 revealed increases in albumin/globulin ratio and alanine aminotransferase levels with a reduction in total protein, calcium and bilirubin levels, in comparison to controls. Histopathological examination revealed liver changes identified as centrilobular hepatocyte enlargement seen in the 750 mg/kg/day males only. Organ weight data confirmed this finding with increased relative liver weight. Hepatocyte enlargement is commonly seen in the rodent liver following the oral administration of xenobiotics and in the absence of associated degenerative or inflammatory changes is regarded as adaptive in nature. Further histopathological changes were identified as thyroid changes, specifically higher incidence of follicular cell hypertrophy seen in the 750 mg/kg/day males only. This was probably a secondary response to the changes seen in the liver as thyroxine is ultimately excreted via the bile, having first been conjugated in the liver. Any induction of the enzymes involved would result in increased thyroxine excretion and compensatory TSH and thyroxine production resulting in the microscopic changes identified. Females treated with 750 mg/kg/day showed an increase in alanine aminotransferase on Day 4 post partum only and organ weight data confirmed this finding with increased absolute and relative liver weight. Histopathological examination of the 750 mg/kg/day females revealed thymus changes identified as higher grades of severity of lymphoid atrophy. Atrophy of the thymus gland is commonly seen among post partum females and although there was not a convincing effect on the incidence of atrophy between controls and those treated with 750 mg/kg/day an effect of treatment cannot be entirely excluded.
Pregnancy was achieved for ten females treated with 200 and 50 mg/kg/day and nine 750 mg/kg/day and control females. There were no treatment-related effects in gestation length, sex ratio, litter size, litter weight, mean offspring weight by sex, viability, clinical observations or developmental signs between the treatment and control animals.

Effect levels

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Key result
Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No observed adverse effects
Key result
Dose descriptor:
NOEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry
histopathology: non-neoplastic
organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

Table 1a: Bodyweights and Bodyweight Changes for Males - Group Mean Values

Dose Level (mg/kg/day)

Bodyweight (g) at Day

1

8

15

22

29

36

43

0 (Control)

Mean

335

348

362

369

380

393

401

SD

11

13

15

17

17

19

21

n

10

10

10

10

10

10

10

50

Mean

337

350

361

367

377

389

397

SD

9

11

9

8

9

11

11

n

10

10

10

10

10

10

10

200

Mean

338

349

362

368

378

392

399

SD

13

14

16

18

20

23

24

n

10

10

10

10

10

10

10

750

Mean

338

342

347

348

**358

**367

*374

SD

10

11

15

17

16

20

23

n

10

10

10

10

10

10

10

 

 Table 1b: Bodyweights and Bodyweight Changes for Males - Group Mean Values

Dose Level (mg/kg/day)

Bodyweight Change (g) During Week

1

2

3

4

5

6

0 (Control)

Mean

13

14 (27)

8 (34)

11 (45)

13 (58)

8 (66)

SD

5

4 (6)

3 (8)

3 (9)

3 (10)

5 (13)

n

10

10

10

10

10

10

50

Mean

13

12 (25)

5 (30)

11 (40)

12 (52)

9 (61)

SD

3

4 (3)

3 (4)

3 (4)

3 (5)

3 (7)

n

10

10

10

10

10

10

200

Mean

11

13 (24)

6 (30)

10 (40)

14 (54)

7 (61)

SD

4

5 (7)

5 (10)

3 (13)

4 (15)

3 (16)

n

10

10

10

10

10

10

750

Mean

**5

**5 (10)

**1 (10)

**10 (20)

**9 (29)

**7 (36)

SD

6

6 (6)

4 (8)

2 (9)

5 (12)

5 (15)

n

10

10

10

10

10

10

() = cumulative bodyweight change relative to Day 1

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study the no observable adverse effect level (NOAEL) for animals of either sex was considered to be 750 mg/kg/day. No such effects were detected at 200 mg/kg/day and the no observed effect level (NOEL) was considered to be 200 mg/kg/day.
Executive summary:

The repeated dose toxicity of the test material to rats was investigated in accordance with the standardised guideline OECD 422, under GLP conditions.

A Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test was performed.

The test material was administered by gavage to three groups each of ten male and ten female Wistar Han™:HsdRccHan™:WIST strain rats, for up to fifty-four consecutive days (including a two week maturation phase, pairing, gestation and early lactation for females), at dose levels of 750, 200 and 50 mg/kg/day. A control group of ten males and ten females was dosed with vehicle alone (arachis oil). Clinical signs, behavioural assessments, bodyweight development, food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated prior to mating and the blood chemistry was re-evaluated at termination on five selected males and females from each dose group. Pairing of 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 males from each dose group after the completion of the mating phase, and for five selected parental females from each dose group on Day 4 post partum.

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

The oral administration of the test material to rats for a period of up to fifty-four consecutive days at dose levels of up to 750 mg/kg/day resulted in toxicologically significant effects at 750 mg/kg/day. Effects on bodyweight development, blood chemistry, organ weights and liver changes identified as centrilobular hepatocyte enlargement and thyroid changes identified as follicular cell hypertrophy were evident in the 750 mg/kg/day males. These findings are considered to be adaptive in nature and not to represent "serious damage" to health. Females treated with 750 mg/kg/day showed changes in blood chemistry, organ weights and thymus changes identified as higher grades of severity of lymphoid atrophy. Atrophy of the thymus gland is commonly seen among post-partum females and in the absence of a convincing response of treatment this finding is not considered to be adverse.

Pregnancy was achieved for ten females treated with 200 and 50 mg/kg/day and nine 750 mg/kg/day and control females. There were no treatment-related effects in gestation length, sex ratio, litter size, litter weight, mean offspring weight by sex, viability, clinical observations or developmental signs between the treatment and control animals.

Under the conditions of this study the no observable adverse effect level (NOAEL) for animals of either sex was considered to be 750 mg/kg/day. No such effects were detected at 200 mg/kg/day and the no observed effect level (NOEL) was considered to be 200 mg/kg/day.