Dibutyl fumarate

Endpoint summary

Description of key information

There were no effects of treatment up to 500 mg/kg bw/day on clinical signs, body weight/food intake or haematology. Effects in clinical chemistry, organ weights and histopathology were observed in the 100 and/or 500 mg/kg/day groups related to liver or kidneys. The hepatic effects including centrilobular hepatocellular hypertrophy were considered to be adaptive and not adverse. The kidney effects included tubular degeneration seen in the 500 mg/kg/day group in both sexes, and in one male at 100 mg/kg/day group were considered to be adverse. In conclusion, the NOAEL for systemic effects of DIBUTYLFUMARATE administered by oral gavage to Wistar rats for 90 consecutive days is considered to be 50 mg/kg bw/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 March 2016 to 29 June 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD No.: 408 “Repeated Dose 90-day Oral Toxicity Study in Rodents” (1998)

Deviations:

no

Remarks:

No signification deviations other than a typographical error in in the Study Plan in the Histopathology Section. This deviation is considered not to adversely affect the results or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Specific details on test material used for the study:

Name: DIBUTYLFUMARATEBatch/Lot Number: LEDB2D3051CAS Number: 105-75-9Chemical name: 2-Butenedioic acid (E)-, dibutyl esterDescription: Clear colourless liquidMolecular formula: C12H20O4Molecular weight: 228.29Purity: 99.2%Manufacture Date: June 2015Expiry Date: 31 December 2016Storage conditions: Refrigerator (2-8°C), protected from lightSafety Precautions: Routine safety precautions (lab coat, gloves, safety glasses and face mask) for unknown materials were applied to assure personnel health and safety.Hazards: Sensitising to skin, dangerous for the environment.

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Species and strain: Crl: WI Wistar ratsSource: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633 SulzfeldJustification of species/strain: The rat is regarded as suitable species for toxicology studies. Wistar rat as a rodent is one of the standard strains for repeat-dose toxicity studies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species and strain: Crl: WI Wistar ratsSource: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633 SulzfeldHygienic level: SPF at arrival, standard laboratory conditions during the studyNumber of groups: 4 groups; one control and 3 test item-treated groupsNumber of animals: 10 animals/group/sex: 40 male and 40 female rats, and 5 spare animals/sexAge of animals: Young adult rats (7 weeks) at start of treatment.Body weight: Not exceed ± 20% of the mean weight for each sex at onset of treatment males: 267-294 g, females: 176-206 gAnimal receipt: 24 March 2016Acclimation period: At least 6 daysHusbandryAnimal health: Only healthy animals were used for the test, as certified by the staff Veterinarian. Females were nulliparous and non-pregnant.Room number: 508Cage type: Type II and/or III polycarbonateBedding: Lignocel® (produced by J. Rettenmaier & Söhne GmbH + Co.KG, Germany) Bedding for Laboratory Animals and nest building material (ARBOCEL crinklets natural (produced by J. Rettenmaier & Söhne GmbH + Co.KG, Germany)) were available to animals during the study.Light: artificial lighting 12 hours daily, from 6.00 a.m. to 6.00 p.m.Temperature: 19.4 – 23.9°CRelative humidity: 35 - 67 %Ventilation: 15-20 air exchanges/hourHousing/Enrichment: Rodents were housed 2 animals of the same sex and group/cage. Group housing allows social interaction and the deep wood sawdust bedding allows digging and other normal rodent activities.The temperature and relative humidity were recorded twice daily during the study.Diet and water supplyAnimals received ssniff® SM R/M "Autoclavable complete diet for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany, ad libitum, and tap water from municipal supply, as for human consumption from 500 mL bottle, ad libitum. The food were removed overnight prior to necropsy.Water quality control analysis is performed once every three months and microbiological assessment is performed monthly by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A.u.36, Hungary). The quality control results will be archived with the raw data at CiToxLAB Hungary Ltd.Animal assignment to the study, randomisation and identificationEach animal was identified by a number unique within the study, written with indelible ink on the tail and cross-referenced to the Animal Master File at CiToxLAB Hungary Ltd.The animal number consists of 4 digits, the first digit being the group number, the second is 0 for the males and 5 for the females and the last 2 stand for the animal number within the group.The cages were identified by cards holding information about study code, sex, dose group, cage number and individual animal numbers.During the acclimation period, the animals were assigned to their respective dose groups by randomization based on body weights. Animals were randomly allocated to the control and dose groups based on the most recent actual body weight; the software PROVANTIS v.9 was used in order to verify homogeneity/variation among/within groups. Males and females were randomized separately.

Route of administration:

oral: gavage

Details on route of administration:

The oral route was selected as being the most direct and controlled way to expose the animals for detection of potential toxic effects of the test item, it was considered suitable to provide the exposure required for this toxicology study.

Vehicle:

corn oil

Details on oral exposure:

Corn oil was selected as vehicle for this study in agreement with the Sponsor.Name: Corn oilLot/Batch number: MKBV2080V / A0342691 / A0348063Manufacturer: SIGMA-ALDRICH / Acros Organics / Acros OrganicsExpiry/Retest Date: 31 March 2017 / 31 January 2018 / 31 May 2018Storage: Room temperatureFormulation and analysis of formulationThe test item was formulated in the vehicle, as a visibly stable homogenous formulation at the appropriate concentrations according to the dose level and volume selected, in the Pharmacy of CiToxLAB Hungary Ltd. Formulations were prepared freshly prior to administration to animals or at the appropriate frequency to allow their use according to stability assessment results.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability of the test item in the vehicle was assessed in the conditions employed on thestudy (concentration range and storage conditions of the dose formulations pending use, according to CiToxLAB study code 15/260-316AN).Analysis of test item formulations for concentration and homogeneity were performed in the Test Facility. Top, middle and bottom duplicate samples were taken from test item formulations at four times (during the first, fifth, ninth and last week of the study), one set to analyze and one set as a back-up. Similarly, one sample was taken in duplicate from the Group 1 (control) solution for concentration measurements. Any sample not required for analysis was discarded following acceptance of the results by the Study Director and/or Analytics.No test item was detected in the control solution samples. All formulations were shown to be homogeneous. All formulations were found to be in the range of 97 to 108% of nominal concentrations.

Duration of treatment / exposure:

90 consecutive days

Frequency of treatment:

The dose formulations were administered daily

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Dose / conc.:

100 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 animals/group/sex

Control animals:

yes, concurrent vehicle

Details on study design:

Rationale for dose-selection and route of administrationThe doses were selected by the Sponsor based on available information and results of “DIBUTYLFUMARATE: A 14-Day Dose Range Finding Toxicity Study by Oral Gavage in Wistar Rats (non-GLP)” [CiToxLAB study code 15/260-101PE].In this DRF study diuresis was observed in the high (from Day 5 until termination) and mid dose group (from Day 10 until termination) in both sexes. Enlarged livers and kidneys were also observed only in the high dose in both sexes, plasma proteins were also increased; these changes were not considered to be life-threatening.Based on these findings, the doses for the 90-day repeated dose study were selected by the Sponsor (500, 100, 50 mg/kg bw/day). However due to the specific observation of diuresis, measurement of water consumption and an additional urinalysis on the seventh week was added to the standard protocol.

Positive control:

Not required in this study.

Observations and examinations performed and frequency:

Mortality and clinical observationsAnimals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day).General clinical observations were made at least once a day at approximately the same time with minor variations as practical.Detailed clinical observations were made on all animals outside the home cage in a standard arena before the first treatment (on Day 1 male/female) and at least weekly thereafter, in the morning hours (am).Observations were performed on the skin, fur, eyes, eyeballs and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system (tremor, convulsion, muscular contractions, etc.), somatomotor activity and behaviour pattern (changes in exploratory behaviour, ordinary behaviour including changes in grooming, headshaking, gyration, etc., abnormal behaviour such as autophagia/ selfmutilation, backward motion, abnormal vocalization, etc., aggression, etc.), motor coordination, ambulatory abnormalities, changes in body position and posture (ex. hunchback posture, etc), gait, or response to handling and to environmental stimulation.Particular attention was directed to observations for tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.Neurological assessment (Functional Observation Battery)Towards the end of the treatment period, during Week 12, all animals were examined in the functional observation battery, including measurements of the landing foot splay, fore/hind grip strength and motor activity assessment.Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity were conducted and the general physical condition and behaviour of animals were tested. A modified Irwin test was performed.A detailed assessment for neurotoxicity effects were made on the basis of these measurements (Irwin, S.: Comprehensive Observational Assessment: Ia. A systematic, Quantitative procedure for Assessing the Behavioral and Physiologic State of the Mouse, Psychopharmacologia (Berl) 13 222-257 1968).Parameters such as, body position, locomotor activity, respiration rate, respiration type, piloerection, head searching compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and vocalisation were evaluated.To measure the landing foot splay, the hind paws of the rat were painted with ink and the rat was dropped from a horizontal position onto the appropriate record sheet covering the examination table. The distance between the two resulting ink spots was measured. The fore paws of the rat were painted for any possible additional measurements.Fore/hind grip strength measurements were conducted using a grip strength meter (Model GS3, Bioseb, Chaville, France), an instrument designed to quantify objectively rodent muscular strength, in order to identify and assess quantitatively any potential effect of test item. The rats were held appropriately such that the fore limbs were allowed to grip the support bar and pulled back until they released the bar; the device measured the maximum grip strength. This was performed 3 times for each animal on each test day. The procedure was repeated with the hind limbs with the appropriate grip support. The results were tabulated with individual and mean data.Motor activity assessment was conducted using Automatic Monitoring System of rat locomotor activity SMART v. 2.5 (Harvard Apparatus, Germany). Locomotor activity was monitored by placing each animal individually into an open-field for 1 hour observation time, when DVD recording of movement was made. Recording was made for a duration of at least 30 min, under dim-light and undisturbed conditions. The DVD was analysed with “SMART” software after all recordings are made to produce the appropriate parameters. At the first instance the data from the high dose and control groups were evaluated for distance travelled in 5 minute segments; since no treatment related changes were observed in the high dose group the mid and low dose data was not processed. The data from the 5 minute segments was presented graphically with the intention of showing plateau activity in controls, and comparing the treatment group.OphthalmologyOphthalmoscopy was conducted in all animals before treatment (Day -1/-2), and in the control group and high dose group animals, during Week 12 (Day 84/83). Mydriasis was produced after instillation of eye drops "Humapent" (0.5% tropicamide) (Batch number: 8680415, Expiry date: April 2017) into the conjunctival sac. The evaluation was performed using a Gowlland® ophthalmoscope.Body weight measurementsBody weights were recorded with a precision of 1 g at randomisation (pre-treatment period), on the first day of treatment (Day 1, prior to start of treatment), then at least weekly, including on Day 90, and prior to necropsy, fasted on Day 91.Food consumption measurementsThe determination of food consumption was performed for all groups once a week.The food was measured on Day 1 then the remaining, non-consumed food was weighed at least weekly from Day 8 with a precision of 1 g. Weekly food consumption was calculated for reporting purposes.Water consumption measurementThe water consumption was measured from start of the treatment until termination at least twice a week. The average water consumption is reported on a weekly basis.

Sacrifice and pathology:

CLINICAL PATHOLOGYAt the end of the treatment period, prior to scheduled necropsy on Day 91, clinical pathology investigations (haematology, coagulation, clinical biochemistry and urinalysis) were conducted in all animals. Additionally, urinalysis was also conducted at the half-time of the study on Week 7. Food was withdrawn during the overnight urine collection.After an overnight period of food deprivation of animals, 3 blood samples were collected by heart puncture under pentobarbital anaesthesia, for haematology (in tubes with K3-EDTA as anticoagulant, 1.6 mg/mL blood), for blood clotting times (in tubes with sodium citrate as anticoagulant) and one to obtain serum (in tubes with no anticoagulant) for clinical chemistry.Haematology and blood clotting timesBlood smears were prepared for all animals but not examined, as the standard haematology evaluation was considered to be adequate.The following parameters were evaluated in all animals: Reported in table form – See “Any other information” for details.Clinical chemistryThe following parameters were evaluated in all animals: Reported in table form – See “Any other information” for details.UrinalysisUrine collection was conducted over approximately 16 hours, during an overnight period of food deprivation of animals, which was placed in metabolic cages.The evaluation of the urine samples was performed by observation (e.g. colour, appearance) or test strips as applicable. The following parameters were evaluated in all animals: Reported in table form – See “Any other information” for details.PATHOLOGYTerminal proceduresNecropsy and macroscopic examination were performed on all animals, at the end of treatment period, on Day 91 (after the sample collection for clinical pathology evaluation). The animals were euthanized by exsanguination under pentobarbital anaesthesia.Macroscopic evaluationAfter exsanguination the external appearance was examined, all orifices, and the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate.In addition, bone marrow smears from the femur of each animal were prepared at necropsy. The smears were fixed, then stained but not analysed. The smears will be stored/archived at CiToxLAB Hungary Ltd.Organ weight measurementsPaired organs were weighed together. Absolute organ weights were measured, and relative organ weights to the body and brain weights were calculated and reported.The following organs were trimmed of fat and weighed in all animals: Reported in table for, - See “Any other information” for details.HistopathologyOn completion of the macroscopic examination the following tissues and organs were retained from all animals. Reported in table form – See “Any other information” for details. The eyes with the optic nerves and testes with epididymides were retained in modified Davidson’s fixative, all other organs in 10% buffered formalin solution.The retained tissues and organs were embedded in paraffin wax, sections were cut at 4-6μ by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope. Full histopathology was performed in Groups 1 (Control) and 4 (High dose) plus liver and kidneys from Mid an Low dose groups.

Other examinations:

Examination of vaginal smearsPrior to necropsy, the oestrus cycle of all females was determined by taking vaginal smears, which was prepared and stained with 1% aqueous methylene blue solution. The smear was examined with a light microscope, in order to provide information regarding the stage of oestrus cycle at the time of sacrifice and assist in histological evaluation of oestrogen sensitive tissues.

Statistics:

Group mean and standard deviation were calculated for numerical data. Statistical analysis was performed using SAS 9.2 (built in Provantis System).The following decision tree was automatically applied within the validated Provantis system for statistical evaluation of numeric data:The normality and heterogeneity of variance between groups was checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests showed no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test was carried out. If the obtained result was positive, Dunett (Multiple Range) test was used to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01 as appropriate. This parametric analysis it the better option when the normality and heterogeneity assumptions implicit in the tests are adequate.If either of the Shapiro-Wilk or Levene tests showed significance on the data, then the ANOVA type approach was not valid and a non-parametric analysis was required. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons was performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no toxicologically significant or test-item related clinical findings during the study. However the following incidental observations were made:- Thin fur in one male in the control group and one female in the mid dose.- Scar in one female in the mid dose group.- Swollen hind limbs of one male in the high dose group.

Mortality:

no mortality observed

Description (incidence):

There was no mortality during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no effects on bodyweight at any dose levels.The mean body weights of males and females were comparable to the controls throughout the treatment period. The overall body weight gains (Days 1-90) of males and females were similar to the controls.Minor difference to control was recorded in body weight gain, on one occasion attaining statistical significance (p<0.05): lower mean body weight gain was noticed between Day 85 and 90 in females in the mid dose, which was without any biological significance.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

In both sexes the food consumption was comparable to the controls throughout the treatment period. The total and mean food consumption (Days 1-90) of females were similar to the controls and in males was statistically higher than control (p<0.01). Minor statistically significances were seen in the mid and low dose groups, which was without any biological significance. The food intake of the high dose males was statistically higher than control from the third week, but the values were all well within the normal range for this strain of rat in our laboratory, hence this difference is not considered to reflect a treatment related effect.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

The water consumption compared to the control did not show biologically significant differences or consistent dose-dependent changes in either sex. In some periods statistically significant increases or decreases were observed but without a dose dependency. A statistically higher water intake for the total period (Days 1-90) was observed in the low dose males, but with no dose relationship.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No test item related changes compared to pre-treatment were noted at ophthalmoscopy examination.

Haematological findings:

no effects observed

Description (incidence and severity):

Haematology parameters evaluated at the completion of the 90-day treatment period did not show signs of toxicity. Variations were noted in a few parameters in the treated animals, on two occasions attaining statistical significance.In females, the Monocyte percentage (MONO%) was statistically (p<0.05) higher in the mid dose group than the control.In males, the Haemoglobin concentration (HGB) and Haematocrit (Hct) was statistically (p<0.01) higher in the low dose group, and the Mean Corpuscular (erythrocyte) Haemoglobin Concentration (MCHC) was statistically (p<0.05) lower in the high dose group than the control.These minor statistical differences were considered to be unrelated to treatment and of no toxicological significance.Coagulation parameters: No effects were noted on the coagulation parameters evaluated, which were considered to be related to the test item.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant (p<0.01) increased Cholesterol, Total protein and Albumin values were observed in both sexes, and Albumin/Globulin ratio A/G ratio in females at the high dose (p<0.01). Also a statisitcally significantly (p<0.05) increased albumin value was observed in the mid dose males. The mean mid dose group value is well within the normal control range and is not considered to be an adverse effect of treatment. At the high dose, the effects listed are considered to be treatment related.The Creatinine concentration in both sexes in the high dose was statistically (p<0.05) lower than the control values, however the mean vales in the high dose group were within the historical control range (males: 35.2-79 μmol/L; females: 43.8-85.4 μmol/L). The relationship between these changes and the test item treatment is considered to be equivocal.A few other clinical chemistry parameters were occasionally statistically significant, i.e. in females lower Urea concentration in the mid and high dose (p<0.01 and p<0.05, respectively, without a dose-dependent response); in males slightly decreased Chloride concentration in the low dose (p<0.05) and higher Bile acid concentration in the high dose (p<0.01), but the individual values were very variable and all were well within the normal range. Based on the expected normal range from historic data, the isolated incidence and/or the lack of a consistent dose response, these variations were regarded as incidental and not to be treatment related, therefore considered have no toxicological significance.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

No effects were noted on the urinalysis parameters evaluated, which were considered to be related to the test item.The urinalysis parameters monitored on Week 7 and at the end of treatment (Day 91) period were comparable to the controls in all test treated groups. Variations occurred and consisted of minor differences to controls, these were unrelated to treatment and within the normal range.Urine sediment analysis in the groups showed similar results, no treatment related effect was noted.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no treatment related effects.There were no toxicologically significant changes in the animal behaviour, general physical condition, in the reactions to different type of stimuli in the control or treated groups.There was no effect of treatment noted during the assessment of landing foot splay, grip strength or motor activity.Slightly lower values were noted in grip strength tests for fore limb in males in the mid dose. The difference was statistically significant (p<0.05). This change was inconsistent, isolated and not dose dependent and was considered to be normal, without toxicological significance.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Liver weights (absolute and relative) were higher than control in both sexes at the high dose (p<0.01; by about 30-38% in males and 15-19% in females). The similar statistical significance was seen in male liver weights in the mid dose group (14-23%). Mid dose females and Low dose both sexes were not statistically different to controls.Kidney weights were increased when compared to controls with statistical significance in absolute and relative to body and brain weights in both sexes at the high dose (p<0.01; by about 25-33% in males and 30-35% in females,). In females, increased kidney weights were also noted in the mid dose group (by 9% absolute weight, p<0.05 and approximately 11% related brain weight, p<0.01). Mid dose males and Low dose both sexes were not statistically different to controls.Higher liver and kidney weights in the high dose for both sexes and the mid dose in females were considered to be treatment related effects.There were no other toxicologically significant differences among groups in the weights of other organs measured when compared to controls.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Test item-related macroscopic changes were observed in the liver and kidney of the high dose males: Enlargement of the liver in 4/10 males and enlarged kidneys in 1/10male rat were seen at necropsy.No test item-related findings were noted in the high dose females.Other changes were incidental or a common background.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item treatment-related microscopic findings were noted in the liver and kidney. In the liver, centrilobular hepatocellular hypertrophy was observed. Degeneration of thecortical tubules and proteinaceous tubule casts were described in the kidneys. These changes correlated with increase of absolute weight or when relative to body and brain weights in affected mid and high dose animals.LiverHepatocellular hypertrophy was diagnosed as enlarged hepatocytes with centrilobular pattern of distribution. The severity and incidence were influenced by dose.In the low dose no treatment-related changes were detected. In the mid dose minimal or slight centrilobular hepatocellular hypertrophy was noted in 3/10 males and slight in 1/10 female. In the high dose hepatocellular hypertrophy in 10/10 males and 8/10 females was seen. Intensity ranged from minimal to moderate (mostly minimal/slight).Treatment-related centrilobular hepatocellular hypertrophy was regarded as adaptive non-adverse response to the administered test item. Hypertrophy was detected at the Mid and High Dose levels in both genders and manifested as enlargement of the liver in several High Dose males at necropsy. Clear dose dependency in both incidence and severity was concluded.KidneyDegeneration tubuleMostly bilateral, focal/multifocal tubular degeneration of cortical tubule epithelial cells with vacuolation of the cytoplasm and/or pyknosis of the nuclei were characteristic features observed by light microscopy.In the low dose no treatment-related changes were recorded. In the mid dose minimal bilateral focal degeneration occurred in 1/10 male. No degeneration change was seen in the females. In the high dose degeneration ranged from minimal to moderate severity (more frequently minimal/slight). Moderate degree was only recorded in one High Dose male. The incidence was slightly higher in the males compared with females, 9/10 males versus 5/10 female rats.CastThere was evidence of notable increased presence of proteinaceous tubular casts (uniform inclusions occupying tubular lumina of cortex/corticomedullary junction) in Mid and High Dose male’s groups compared to controls including 6/10 Mid and 7/10 High Dose. In the females, casts were present at the Mid (2/10) and High Dose levels (6/10). No casts were noted in Control and Low Dose females.In the low dose minimal unilateral/bilateral focal/multifocal casts were observed in 4/10 males. There was no meaningful difference in the incidence of recorded casts when compared to controls (2/10). The appearance and severity were the same as in controls. Therefore, it was concluded that the frequency of the casts at this level was not effected by treatment and considered to be similar to this noted in control kidneys. In the mid dose with exception of slight severity in one male, minimal unilateral/bilateral focal/multifocal casts filled tubules in 5/10 males and 2/10 females. In the high dose minimal to moderate (mainly minimal/slight) casts were noted in 7/10 males and 6/10 females.Treatment-related tubular degeneration altered kidneys of the males and females at the High Dose level. Only one male had affected kidneys with tubular degeneration at the Mid Dose level. Slightly higher incidence of degeneration was observed in the males when compared to females from the High Dose group. Degeneration was considered to be non-specific entity that may be either reversible or irreversible, this change was considered as a treatment related adverse change. In addition, there was evidence of treatment-related notable increased presence of proteinaceous tubular casts in Mid and High Dose male’s groups comparing to controls. In the females, casts were present in the Mid and High Dose groups. The casts were considered to be non-adverse microscopic observation. Renal changes corresponded with increase of absolute weight or when relative to body and brain weights when comparing with controls.Other changes were considered to be without relation to the treatment.

Histopathological findings: neoplastic:

not specified

Other effects:

no effects observed

Description (incidence and severity):

EXAMINATION OF VAGINAL SMEARSThere were no test item related changes in the animal oestrus cycle evaluated prior to necropsy and the animals showed the normal distribution of the oestrus phases.

Key result

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

Critical effects observed:

not specified

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

System:

other: digestive system, urinary system and endocrine system

Organ:

kidney

liver

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Summary of Clinical Observations

Observation Type: Routine	Male
From Day 1 (Start Date) to 90 (Start Date)	0 mg/kg bw/day	50 mg/kg bw/day	100 mg/kg bw/day	500 mg/kg bw/day
Normal Fur thin, Right Cheek	10 1	10 0	10 0	10 0
Observation Type: Routine	Female
From Day 1 (Start Date) to 90 (Start Date)	0 mg/kg bw/day	50 mg/kg bw/day	100 mg/kg bw/day	500 mg/kg bw/day
Normal Fur thin, Thorax dorsal area Scar, Thorax dorsal area, 1-2 cm	10 0 0	10 0 0	10 1 1	10 0 0
Observation Type: Detailed	Male
From Day 1 (Start Date) to 91 (Start Date)	0 mg/kg bw/day	50 mg/kg bw/day	100 mg/kg bw/day	500 mg/kg bw/day
Normal Fur thin, Right Cheek	10 1	10 0	10 0	10 0
Observation Type: Detailed	Female
From Day 1 (Start Date) to 91 (Start Date)	0 mg/kg bw/day	50 mg/kg bw/day	100 mg/kg bw/day	500 mg/kg bw/day
Normal Fur thin, Thorax dorsal area Scar, Thorax dorsal area, 1-2 cm	10 0 0	10 0 0	10 1 1	10 0 0

 

Clinical Chemistry

Some of the parameters attaining statistical significance are summarised in the following table:

Summary of some clinical chemistry parameters
		Male
		Total protein	Albumin	Creatinine	Bile acid	Cholesterol
Control	Mean	55.71	29.62	63.53	13.614	1.497
Low Dose	Mean	57.24	31.15	60.65	15410	1.669
(50 mg/kg bw)	Difference (%)	2.7	5.2	-4.5	13.2	11.5
Mid dose	Mean	58.37	31.54*	61.9	18.890	1.731
(100 mg/kg bw)	Difference (%)	4.8	6.5	-2.6	38.8	15.6
High dose	Mean	59.99**	32.43**	56.09*	21.852**	2.185**
(500 mg/kg bw)	Difference (%)	7.7	9.5	-11.7	60.5	46.0
		Female
		Total protein	Albumin	Creatinine	Bile acid	Cholesterol
Control	Mean	59.55	34.67	63.19	17.650	1.700
Low dose	Mean	59.48	35.26	64.40	20.320	1.828
(50 mg/kg bw)	Difference (%)	-0.1	1.7	1.9	15.1	7.5
Mid dose	Mean	59.98	35.21	57.86	19.453	1.944
(100 mg/kg bw)	Difference (%)	0.7	1.6	-8.4	10.2	14.4
High dose	Mean	64.62**	40.23**	55.54*	19.093	2.228**
(500 mg/kg bw)	Difference (%)	8.5	16.0	-12.1	8.2	31.1

Notes:

1. * = Significant (P<0.05, Dunnett 2 Sided Test)

2. ** = Significant (p<0.01, Dunnett 2 Sided Test)

 

Organ Weights

The liver and kidney weights are summarised in the following table:

LIVER       Terminal body weight (g)	Dose (mg/kg bw/day)
	Control	50	100	500	Control	50	100	500
	Males				Females
	531.5	545.3	557.1	558.6	273.6	274.3	275.6	277.7
Liver weight Absolute (g)                Differences %	14.055	15.787   12.3	16.842**   19.8	19.268**   37.1	8.045	7.888   -2.0	8.679   7.9	9.363**   16.4
Liver weight Body weight relative (%)                Differences %	2.64	2.89   9.4	3.01**   14.0	3.44**   30.1	2.94	2.87   -2.1	3.15   7.2	3.37**   15.0
Liver weight Brain relative (%)                Differences %	627.77	694.59   12.4	760.04**   23.0	853.75**   38.2	395.13	387.09   -2.0	437.56   10.7	469.44**   18.8

 

KIDNEY       Terminal body weight (g)	Dose (mg/kg bw/day)
	Control	50	100	500	Control	50	100	500
	Males				Females
	531.5	545.3	557.1	558.6	273.6	274.3	275.6	277.7
Kidney weight Absolute (g)                Differences %	3.158	3.373   6.8	3.490   10.5	4.170**   32.0	1.733	1.742   0.5	1.883*   8.7	2.290**   32.1
Kidney weight Body weight relative (%)                Differences %	0.59	0.62   4.4	0.63   5.6	0.75**   25.5	0.63	0.64   0.2	0.69   8.2	0.83**   30.4
Kidney weight Brain relative (%)                Differences %	138.60	148.23   7.0	157.17   13.4	184.85**   33.4	85.07	85.33   0.3	94.78**   11.4	114.91**   35.1

Notes:

1. * = Significant (p<0.05, Dunnett 2 Sided Test)

2. ** = Significant (p<0.01, Dunnett 2 Sided Test)

 

Summary of Necropsy Data

Removal Reason: Terminal Euthanasia	Males				Females
	0 mg/kg bw/day	50 mg/kg bw/day	100 mg/kg bw/day	500 mg/kg bw/day	0 mg/kg bw day	50 mg/kg bw/day	100 mg/kg bw/day	500 mg/kg bw/day
Number of Animals:	10	10	10	10	10	10	10	10
Number of Completed Animals:	10	10	10	10	10	10	10	10
AORTA ABDOMINAL Submitted Normal	10 10	10 10	10 10	10 10	10 10	10 10	10 10	10 10
ADRENAL GLAND Submitted Normal	10 10	10 10	10 10	10 10	10 10	10 10	10 10	10 10
AORTA THORACIC Submitted Normal	10 10	10 10	10 10	10 10	10 10	10 10	10 10	10 10
BRAIN Submitted Normal	10 10	10 10	10 10	10 10	10 10	10 10	10 10	10 10
JEJUNUM Submitted Normal	10 10	10 10	10 10	10 10	10 10	10 10	10 10	10 10
DUODENUM Submitted Normal	10 10	10 10	10 10	10 10	10 10	10 10	10 10	10 10
ILEUM Submitted Normal	10 10	10 10	10 10	10 10	10 10	10 10	10 10	10 10
EYE AND OPTIC NERVE Submitted Normal	10 10	10 10	10 10	10 10	10 10	10 10	10 10	10 10
CECUM Submitted Normal	10 10	10 10	10 10	10 10	10 10	10 10	10 10	10 10
COLON Submitted Normal	10 10	10 10	10 10	10 10	10 10	10 10	10 10	10 10
EPIDIDYMIS Submitted Normal	10 10	10 10	10 10	10 10	0 -	0 -	0 -	0 -
ESOPHAGUS Submitted Normal Perforation; thoratic	10 10 0	10 9 1	10 10 0	10 10 -	10 10 -	10 10 -	10 10 -	10 10 -
FEMUR & MARROW Submitted Normal	10 10	10 10	10 10	10 10	10 10	10 10	10 10	10 10
FUR Thin, right, cheek	1	-	-	-	-	-	-	-
HARDERIAN GLAND Submitted Normal	10 10	10 10	10 10	10 10	10 10	10 10	10 10	10 10
HEART Submitted Normal	10 10	10 10	10 10	10 10	10 10	10 10	10 10	10 10
KIDNEY Submitted Normal Enlargement, bilateral	10 10 0	10 10 0	10 10 0	10 9 1	10 10 -	10 10 -	10 10 -	10 10 -
LACRIMAL GLAND Submitted Normal	10 10	10 10	10 10	10 10	10 10	10 10	10 10	10 10
LIVER Submitted Norma; Enlargement; all lobes	10 10 0	10 10 0	10 9 1	10 6 4	10 10 -	10 10 -	10 10 -	10 10 -
LUNGS Submitted Normal Discoloration; dark red, diffuse, all lobes	10 10 0	10 9 1	10 10 0	10 10 0	10 10 -	10 10 -	10 10 -	10 10 -
LYMPH NODE, MANDIBULAR Submitted Normal	10 10	10 10	10 10	10 10	10 10	10 10	10 10	10 10
LYMPH NODE, MESENTERIC Submitted Normal	10 10	10 10	10 10	10 10	10 10	10 10	10 10	10 10
OVARY Submitted Normal	0 -	0 -	0 -	0 -	10 10	10 10	10 10	10 10
OVIDUCT Submitted Normal	0 -	0 -	0 -	0 -	10 10	10 10	10 10	10 10
PANCREAS Submitted Normal	10 10	10 10	10 10	10 10	10 10	10 10	10 10	10 10
PITUITARY Submitted Normal	10 10	10 10	10 10	10 10	10 10	10 10	10 10	10 10
PROSTATE Submitted Normal	10 10	10 10	10 10	10 10	0 -	0 -	0 -	0 -
RECTUM Submitted Normal	10 10	10 10	10 10	10 10	10 10	10 10	10 10	10 10
SALIVARY GLAND Submitted Normal	10 10	10 10	10 10	10 10	10 10	10 10	10 10	10 10
SCIATIC NERVE Submitted Normal	10 10	10 10	10 10	10 10	10 10	10 10	10 10	10 10
SEMINAL VESICLE WITH COAGULATING GLAND Submitted Normal	10 10	10 10	10 10	10 10	0 -	0 -	0 -	0 -
SKELETAL MUSCLE Submitted Normal	10 10	10 10	10 10	10 10	10 10	10 10	10 10	10 10
SPINAL CORD Submitted Normal	10 10	10 10	10 10	10 10	10 10	10 10	10 10	10 10
SPLEEN Submitted Normal	10 10	10 10	10 10	10 10	10 10	10 10	10 10	10 10
STERNUM & MARROW Submitted Normal	10 10	10 10	10 10	10 10	10 10	10 10	10 10	10 10
STOMACH Submitted Normal	10 10	10 10	10 10	10 10	10 10	10 10	10 10	10 10
TESTIS Submitted Normal	10 10	10 10	10 10	10 10	0 -	0 -	0 -	0 -
THORACIC CAVITY Material; yellow	-	1	-	-	-	-	-	-
THYMUS Submitted Normal	10 10	10 10	10 10	10 10	10 10	10 10	10 10	10 10
TONGUE Submitted Normal	10 10	10 10	10 10	10 10	10 10	10 10	10 10	10 10
THYROID GLAND + PARATHYROID GLAND Submitted Normal	10 10	10 10	10 10	10 10	10 10	10 10	10 10	10 10
TRACHEA Submitted Normal	10 10	10 10	10 10	10 10	10 10	10 10	10 10	10 10
URINARY BLADDER Submitted Normal	10 10	10 10	10 10	10 10	10 10	10 10	10 10	10 10
UTERINE CERVIX + BODY + HORN Submitted Normal Dilation; body; horn	0 - -	0 - -	0 - -	0 - -	10 8 2	10 9 1	10 10 0	10 10 0
VAGINA Submitted Normal	0 -	0 -	0 -	0 -	10 10	10 10	10 10	10 10
LYMPH NODE, LUNG-ASSOCIATED Submitted Enlargement	0 -	1 1	0 -	0 -	0 -	0 -	0 -	0 -
SKIN/SUBCUTIS WITH MAMMARY GLAND AREA Submitted Normal	10 10	10 10	10 10	10 10	10 10	10 10	10 10	10 10

 

 

Summary of Histophatalogy Data

Removal Reason: Terminal Euthanasia	Males				Females
	0 mg/kg bw/day	50 mg/kg bw/day	100 mg/kg bw/day	500 mg/kg bw/day	0 mg/kg bw day	50 mg/kg bw/day	100 mg/kg bw/day	500 mg/kg bw/day
Number of Animals:	10	10	10	10	10	10	10	10
Number of Completed Animals:	10	10	10	10	10	10	10	10
AORTA ABDOMINAL Examined Normal	10 10	0 -	0 -	10 10	10 10	0 -	0 -	10 10
ADRENAL GLAND Examined Normal	10 10	0 -	0 -	10 10	10 10	0 -	0 -	10 10
AORTA THORACIC Examined Normal	10 10	0 -	0 -	10 10	10 10	0 -	0 -	10 10
BRAIN Examined Normal Inflammation; mixed cell; unilateral, focal                Minimal	10 10 0 0	0 - - -	0 - - -	10 9 1 1	10 10 - -	0 - - -	0 - - -	10 10 - -
JEJUNUM Examined Normal	10 10	0 -	0 -	10 10	10 10	0 -	0 -	10 10
ILEUM Examined Normal	10 10	0 -	0 -	10 10	10 10	0 -	0 -	10 10
DUODENUM Examined Normal	10 10	0 -	0 -	10 10	10 10	0 -	0 0	10 10
EYE AND OPTIC NERVE Examined Normal	10 10	0 -	0 -	10 10	10 10	0 -	0 -	10 10
CECUM Examined Normal	10 10	0 -	0 -	10 10	10 10	0 -	0 -	10 10
COLON Examined Normal	10 10	0 -	0 -	10 10	10 10	0 -	0 -	10 10
EPIDIDYMIS Examined Normal	10 10	0 -	0 -	10 10	0 -	0 -	0 -	0 -
ESOPHAGUS Submitted Normal Perforation; mucosa; submucosa; focal                Minimal	10 10 0 0	0 - - -	0 - - -	10 9 1 1	10 10 - -	0 - - -	0 - - -	10 10 - -
FEMUR & MARROW Examined Normal Adipocyte, Bone Marrow, Increased                Minimal	10 10 0 0	0 - - -	0 - - -	10 9 1 1	10 10 - -	0 - - -	0 - - -	10 10 - -
HARDERIAN GLAND Examined Normal	10 10	0 -	0 -	10 10	10 10	0 -	0 -	10 10
HEART Examined Normal Degeneration; apex; focal                Slight Fibrosis; myocardium; ventricle; left, focal                Minimal	10 9 1 1 0 0	0 - - - - -	0 - - - - -	10 9 0 0 1 1	10 10 - - - -	0 - - - - -	0 - - - - -	10 10 - - - -
KIDNEY Examined Normal Cast; cortex; corticomedullary junction; proteinaceous, bilateral, multifocal                Minimal                Slight                Moderate Cast; cortex; corticomedullary junction; proteinaceous, left, multifocal                Slight Cast; cortex; corticomedullary junction; proteinaceous, right focal                Minimal Cast; cortex; tubule, proteinaceous, bilateral, multifocal                Minimal                Slight Cast; cortex; tubule, proteinaceous, left, focal                Minimal Cast; cortex; tubule; proteinaceous, right, focal                minimal Cast; cortex; tubule, proteinaceous, right, multifocal                Minimal Cast; corticomedullary junction; tubule; proteinaceous, right, multifocal                Minimal Cast; corticomedullary junction; proteinaceous, left, multifocal                minimal Basophilia; tubule; left, focal                Minimal Basophilia; tubule; right, focal                Minimal Degeneration, Tubule; cortex; bilateral, multifocal                Minimal                Slight                Moderate Degeneration, Tubule; cortex; bilateral, focal                minimal Degeneration, Tubule; cortex; left, focal                Minimal Degeneration, Tubule; cortex; right, focal                minimal Mineralization; pelvis; bilateral                Slight Pyelitis; bilateral                Slight Infiltrate, Inflammatory Cell; pelvis; bilateral                Slight	10 7 0   0 0 0 0   0 0   0 1 1 0 1 1 0 0 0 0 0   0 0   0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0	10 6 2   2 0 0 0   0 0   0 0 0 0 1 1 0 0 0 0 1   1 0   0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0	10 4 2   1 1 0 0   0 1   1 1 1 0 0 0 0 0 1 1 1   1 0   0 1 1 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0	10 1 4   1 2 1 1   1 0   0 1 0 1 0 0 0 0 1 1 0   0 0   0 0 0 0 0 8 4 3 1 1 1 0 0 0 0 0 0 0 0 0 0	10 8 0   0 0 0 0   0 0   0 0 0 0 0 0 0 0 0 0 0   0 0   0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1	10 10 0   0 0 0 0   0 0   0 0 0 0 0 0 0 0 0 0 0   0 0   0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0	10 8 0   0 0 0 0   0 0   0 0 0 0 0 0 1 1 0 0 0   0 0   0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0	10 3 6   3 3 0 0   0 0   0 0 0 0 0 0 0 0 0 0 0   0 0   0 0 0 0 0 3 2 1 0 1 1 0 0 1 1 0 0 0 0 0 0
LACRIMAL GLAND Examined Normal	10 10	0 -	0 -	10 10	10 10	0 -	0 -	10 10
LIVER Examined Normal Vacuolation, Hepatocellular; centrilobular                Minimal                Slight Hypertrophy, Hepatocellular; centrilobular                Minimal                Slight                Moderate	10 5 5 4 1 0 0 0 0	10 5 5 4 1 0 0 0 0	10 3 5 1 4 3 1 2 0	10 0 2 2 0 10 3 5 2	10 10 0 0 0 0 0 0 0	10 10 0 0 0 0 0 0 0	10 9 0 0 0 1 0 1 0	10 2 0 0 0 8 7 1 0
LUNGS Examined Normal Congestion; diffuse                Minimal Inflammation; pleura; subpleura; chronic, focal                Slight Alveolar Macrophage Aggregation; foamy, multifocal                Slight	10 9 0 0 0 0 1 1	1 0 1 1 1 1 0 0	0 - - - - - - -	10 10 0 0 0 0 0 0	10 10 0 0 0 0 0 0	0 - - - - - - -	0 - - - - - - -	10 10 0 0 0 0 0 0
LYMPH NODE, MANDIBULAR Examined Normal	10 10	0 -	0 -	10 10	10 10	0 -	0 -	10 10
LYMPH NODE, MESENTERIC Examined Normal	10 10	0 -	0 -	10 10	10 10	0 -	0 -	10 10
OVARY Examined Normal	0 -	0 -	0 -	0 -	10 10	0 -	0 -	10 10
OVIDUCT Examined Normal	0 -	0 -	0 -	0 -	10 10	0 -	0 -	10 10
PANCREAS Examined Normal	10 10	0 -	0 -	10 10	10 10	0 -	0 -	10 10
PITUITARY Examined Normal	10 10	0 -	0 -	10 10	10 10	0 -	0 -	10 10
PROSTATE Examined Normal Inflammation; dorsolateral lobe; mixed cell; diffuse                Slight	10 9 1 1	0 - - -	0 - - -	10 10 0 0	0 - - -	0 - - -	0 - - -	0 - - -
RECTUM Examined Normal	10 10	0 -	0 -	10 10	10 10	0 -	0 -	10 10
SALIVARY GLAND Examined Normal	10 10	0 -	0 -	10 10	10 10	0 -	0 -	10 10
SCIATIC NERVE Examined Normal Degeneration, Axonal; focal                Minimal	10 10 0 0	0 - - -	0 - - -	10 7 3 3	10 8 2 2	0 - - -	0 - - -	10 10 0 0
SEMINAL VESICLE WITH COAGULATING GLAND Examined Normal	10 10	0 -	0 -	10 10	0 -	0 -	0 -	0 -
SKELETAL MUSCLE Examined Normal	10 10	0 -	0 -	10 10	10 10	0 -	0 -	10 10
SPINAL CORD Examined Normal	10 10	0 -	0 -	10 10	10 10	0 -	0 -	10 10
SPLEEN Examined Normal Hematopoiesis, Extramedullary                Minimal                Slight	10 5 5 3 2	0 - - - -	0 - - - -	10 4 6 6 0	10 6 4 4 0	0 - - - -	0 - - - -	10 4 6 6 0
STERNUM & MARROW Examined Normal	10 10	0 -	0 -	10 10	10 10	0 -	0 -	10 10
STOMACH Examined Normal	10 10	0 -	0 -	10 10	10 10	0 -	0 -	10 10
TESTIS Examined Normal	10 10	0 -	0 -	10 10	0 -	0 -	0 -	0 -
THYMUS Examined Normal	10 10	0 -	0 -	10 10	10 10	0 -	0 -	10 10
TONGUE Examined Normal	10 10	0 -	0 -	10 10	10 10	0 -	0 -	10 10
THYROID GLAND + PARATHYROID GLAND Examined Normal	10 10	0 -	0 -	10 10	10 10	0 -	0 -	10 10
TRACHEA Examined Normal	10 10	0 -	0 -	10 10	10 10	0 -	0 -	10 10
URINARY BLADDER Examined Normal Inflammation; mixed cell; diffuse                Slight	10 10 0 0	0 - - -	0 - - -	10 10 0 0	10 9 1 1	0 - - -	0 - - -	10 10 0 0
UTERINE CERVIX + BODY + HORN Examined Normal Proestrus; bilateral	0 - -	0 - -	0 - -	0 - -	10 8 2	1 0 1	0 - -	10 10 0
VAGINA Examined Normal	0 -	0 -	0 -	0 -	10 10	0 -	0 -	10 10
LYMPH NODE, LUNG-ASSOCIATED Examined Accumulation, Foamy Macrophage; bilateral                Slight	0 - -	1 1 1	0 - -	0 - -	0 - -	0 - -	0 - -	0 - -
SKIN/SUBCUTIS WITH MAMMARY GLAND AREA Examined Normal	10 10	0 -	0 -	10 10	10 10	0 -	0 -	10 10

 

Conclusions:

There were no effects of treatment up to 500 mg/kg bw/day on clinical signs, body weight/food intake or haematology. Effects in clinical chemistry, organ weights and histopathology were observed in the 100 and/or 500 mg/kg/day groups related to liver or kidneys. The hepatic effects including centrilobular hepatocellular hypertrophy were considered to be adaptive and not adverse. The kidney effects included tubular degeneration seen in the 500 mg/kg/day group in both sexes, and in one male at 100 mg/kg/day group were considered to be adverse.In conclusion, the NOAEL for systemic effects of DIBUTYLFUMARATE administered by oral gavage to Wistar rats for 90 consecutive days is considered to be 50 mg/kg bw/day.

Executive summary:

The 90-day study was performed to obtain information on the toxicity of DIBUTYLFUMARATE when administered oral gavage to Wistar rats at 4 dose levels of 0, 50, 100 and 500 mg/kg bodyweight, daily for 90 days.

 

Analysis of test item formulations for concentration and homogeneity was performed four times during the treatment period (during the first, fifth, ninth and last week of treatment) using a validated GC-FID method. No test item was detected in the control solution samples. All formulations were shown to be homogeneous. All formulations were found to be in the range of 97 to 108% of nominal concentrations.

 

The examinations included clinical signs, mortality, body weights, food consumption, water consumption, ophthalmoscopy, neurological assessment including functional observation battery (FOB), measurements of the landing foot splay and grip strength, clinical pathology, vaginal smears, gross pathology, organ weights and histopathology (only control and high doses plus liver and kidneys from mid an low dose groups).

 

Results

There was no mortality and no toxicologically significant or test-item related clinical findings during the study.

There were no toxicologically significant changes in the animal behaviour, general physical condition, in the reactions to different type of stimuli in the control or treated groups. There was no effect of treatment noted during the assessment of landing foot splay, grip strength or motor activity.

No test item related changes compared to pre-treatment were noted at ophthalmoscopy examination.

There were no effects on bodyweight at any dose levels and the food consumption was comparable to the controls throughout the treatment period.

The water consumption compared to the control did not show biologically significant differences or consistent dose-dependent changes in either sex.

Haematology parameters evaluated at the completion of the 90-day treatment period did not show signs of toxicity.

Statistically significant increased Cholesterol, Total protein and Albumin values were observed in both sexes, and Albumin/Globulin ratio A/G ratio in females at the high dose. Also a statistically significantly increased Albumin value was observed in the mid dose males. The mean mid dose group value is well within the normal control range and is not considered to be an adverse effect of treatment. At the high dose, the effects listed are considered to be treatment related.

The Creatinine concentration in both sexes in the high dose was statistically lower than the control values, however the mean vales in the high dose group were within the historical control range. The relationship between these changes and the test item treatment is considered to be equivocal.

No effects were noted on the urinalysis parameters evaluated, which were considered to be related to the test item.

There were no test item related changes in the animal oestrus cycle evaluated prior to necropsy and the animals showed the normal distribution of the oestrus phases.

 

At necropsy test item-related macroscopic changes were observed in the liver and kidney of the high dose males: enlargement of the liver in 4/10 males and enlarged kidneys in 1/10 male rat were seen at necropsy. No test item-related findings were noted in the high dose females.

Liver weights (absolute and relative) were higher than control in both sexes at the high dose (by about 30-38% in males and 15-19% in females). Similar statistical significance was seen in male liver weights in the mid dose group (14-23%).

Kidney weights were increased when compared to controls with statistical significance in absolute and relative to body and brain weights in both sexes at the high dose (by about 25-33% in males and 30-35% in females,). In females, increased kidney weights were also noted in the mid dose group (by 9% absolute weight, and approximately 11% related brain weight).

 

Test item treatment-related microscopic findings were noted in the liver and kidney. In the liver, centrilobular hepatocellular hypertrophy was observed. Degeneration of the cortical tubules and proteinaceous tubule casts were described in the kidneys. These changes correlated with increase of absolute weight or when relative to body and brain weights in affected mid and high dose animals.

Treatment-related centrilobular hepatocellular hypertrophy was regarded as an adaptive non-adverse response to the administered test item in Mid and High dose animals of both sexes.

Treatment-related kidney tubular degeneration in both sexes at the High Dose level, and in only one male at the Mid Dose level, was observed. In addition, there was evidence of treatment-related increased incidence of proteinaceous tubular casts in Mid and High Dose in males and females. The tubular degeneration was considered to be non-specific adverse change, the casts were considered to be a non-adverse microscopic observation.

 

Conclusion

There were no effects of treatment up to 500 mg/kg bw/day on clinical signs, body weight/food intake or haematology. Effects in clinical chemistry, organ weights and histopathology were observed in the 100 and/or 500 mg/kg/day groups related to liver or kidneys. The hepatic effects including centrilobular hepatocellular hypertrophy were considered to be adaptive and not adverse. The kidney effects included tubular degeneration seen in the 500 mg/kg/day group in both sexes, and in one male at 100 mg/kg/day group were considered to be adverse.

 

In conclusion, the NOAEL for systemic effects of DIBUTYLFUMARATE administered by oral gavage to Wistar rats for 90 consecutive days is considered to be 50 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

GLP study

System:

urinary

Organ:

kidney

Justification for classification or non-classification

The NOAEL for systemic effects of DIBUTYLFUMARATE administered by oral gavage to Wistar rats for 90 consecutive days is 50 mg/kg bw/day, leading to a classification of STOT RE 2.