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Description of key information

90-day repeat dose oral toxicity study combined with a neurotoxicity test, Kumar (2020)

Under the conditions of the study, the no observed-adverse-effect level (NOAEL) is 60 ppm in the diet (approximately 5.74 mg/kg/day) in females and 175 ppm in diet (14.96 mg/kg/day) in males.

28-day repeat dose oral toxicity study combined with a neurotoxicity test, Kumar (2019)

Under the conditions of this study, it is concluded that the no-observed adverse-effect level (NOAEL) is less than 175 ppm in the diet (approximately 20 mg/kg/day in males and 22 mg/kg/day in females).

Key value for chemical safety assessment

Effect on neurotoxicity: via oral route

Link to relevant study records
Reference
Endpoint:
neurotoxicity: sub-chronic oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3 July 2019 to 17 January 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 424 (Neurotoxicity Study in Rodents)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
25 June 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: In-house
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: 5 weeks (Males), 5 to 6 weeks (Females)
- Weight at study initiation (Day 1): 58.49 to 76.14 g (Males), 53.35 to 72.66 g (Females)
- Fasting period before study: No
- Housing: A maximum of two animals of the same sex were housed in a standard polypropylene cage (Size: L 430 × B 285 × H 150 mm) with a stainless steel mesh top grill having facilities for holding pelleted food and drinking water in a water bottle fitted with stainless steel sipper tube. Clean sterilised paddy husk was provided as bedding material. Animal enrichment (polycarbonate tunnels) were provided in cages where single animals were housed.
- Diet: Powder maintenance diet for rats and mice was provided ad libitum to the animals throughout the experimental period. Powder feed was provided in stainless steel feed hoppers.
- Water: Water was provided ad libitum throughout the acclimatisation and experimental period. Deep bore-well water passed through a Reverse Osmosis Unit was provided in plastic water bottles with stainless steel sipper tubes.
- Acclimation period: Healthy young adult animals were acclimatised to laboratory conditions for a period of five days (males) and 6 days (females).

DETAILS OF FOOD AND WATER QUALITY: The certificate of contaminant analysis provided with the feed showed that the product was compliant with the required specifications and quality requirements and was therefore suitable for use. The certificate of analysis that accompanied the water declared it to be accepted and released for use.

ENVIRONMENTAL CONDITIONS
- Temperature: 19.4 to 23.4 °C
- Humidity: 47 to 68 % (relative)
- Air changes (per hr): 12 to 15
- Photoperiod: 12 hrs of darkness / 12 hrs of light (fluorescent light)

IN-LIFE DATES
From: 11 July 2019 - start of acclimatisation
To: 15 October 2019 (males), 16 October 2019 (females) - necropsy date
Route of administration:
oral: feed
Vehicle:
acetone
Details on exposure:
DIET PREPARATION
The fortified diet was prepared and used within the stability period (determined experimentally to be 15 days). Eight kg each of fortified diet was prepared on Day 1 for the control (G1), low (G2), mid-I (G3), mid-II (G4), and high (G5) dose groups by adding 0, 80.0, 200.1, 480.1 and 1400.2 mg of the test material, respectively.
The required quantity of test material was weighed and initially solved in a small volume of acetone (1.4 mL). Then the solution was premixed with a small amount of feed in a stainless-steel container for 2 minutes. This premix was then added to remaining amount of feed in order to obtain the desired dietary concentration and mixed in a Ribbon Blender for 10 minutes.
The quantity of experimental diet prepared, and the amount of test material weighed, varied depending on the requirement. The left out formulated diet was sent for disposal. The fortified diets were stored in polyethylene bags which were kept in stainless steel drums in the study room at a temperature of 22 ± 3 °C and used.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test material was soluble in acetone and it also mixed well with the powdered diet.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Fortified diet analysis for homogeneity and dose concentration verification was conducted by the Analytical Chemistry Department of the testing facility during week 1, month 2, and month 3 of the treatment period.
For homogeneity and concentration verification analysis, feed samples were collected in duplicate from the top, middle and bottom layers of the container whereas the vehicle control feed sample (in duplicate) was collected only from the middle layer of container.
The collected samples were transferred to the Analytical Chemistry Department for concentration analysis. One set of each aliquot of each formulation was analysed. The second set of samples were discarded as the results obtained from the first set of samples were within the acceptance limits of ± 20 % of nominal concentrations with a Relative Standard Deviation (RSD) of < 20 % (2.91 to 4.74 %).
Homogeneity was evaluated from the top, middle and bottom layers of the collected test formulations. The mean concentrations from all three sampled layers were used for the dose concentration verification. The results of homogeneity test and dose concentration verification were within the acceptance limits of ± 20 % of the nominal concentrations with relative standard deviation < 20 % (0.30 to 5.11 %).

ANALYTICAL METHOD - ICP
- Instrument: ICP-OES (Perkin Elmer)
- Element analysed: Tin
- Mode: ICP
- Analyte wavelength: 189.927 nm
- Read Delay Time: 30 Sec
- View mode: Radial
- RF power: 1500 watts
- Pump flow rate: 1 mL/min
- Gas Flows: Plasma: 12 L/min; Auxiliary: 0.2 L/min; Nebulizer: 0.7 L/min
- Diluent: Milli-Q water and 2 % hydrochloric acid

PREPARATION OF SAMPLE SOLUTIONS
Digestion Procedure: The required quantity of each diet sample was weighed into a 250 mL stoppered conical flask; 10 mL of concentrated nitric acid was added and gently heated on a hot plate to initiate digestion, avoiding excessive frothing. The flask was removed from the hot plate. Immediately 10 mL of concentrated hydrochloric acid was added and heated gently on hot plate until the bumping from evolution of chlorine stopped. The heating was increased until the volume dropped to 10 mL, then 20 mL of Milli-Q water was added to the flask, cooled to room temperature and the volume was made up to 50 mL with Milli-Q water. The resulting solution was filtered and used for ICP-OES analysis.

FORMULAE
Conc. obtained (ppm) = (Conc. of Sample (ppm) × 50 × 100) / (Sample Weight taken (g) × Sample purity (19.56 %))

Recovery (%) = (Concentration obtained (ppm) / Nominal concentration (ppm)) x 100
Duration of treatment / exposure:
91 days
Frequency of treatment:
Continous in the diet
Dose / conc.:
0 ppm
Remarks:
Group 1 (G1) Vehicle
Dose / conc.:
10 ppm
Remarks:
Group 2 (G2) Low Dose
Dose / conc.:
25 ppm
Remarks:
Group 3 (G3) Mid Dose I
Dose / conc.:
60 ppm
Remarks:
Group 4 (G4) Mid Dose II
Dose / conc.:
175 ppm
Remarks:
Group 5 (G5) High Dose
No. of animals per sex per dose:
15 males and 15 females per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: A 28-day repeated dose toxicity study is available. The mortalities in the 28-day study suggest that the toxic response to the test material may be delayed in onset in a longer study. Due to this concern, and the findings in the 28-day study, a dose of 175 ppm is proposed as the high dose for this 90-day study. This dose is expected to represent the maximally tolerated dose over the anticipated time period of 90 days.
Taking into consideration the recommendation of the OECD 408 guideline for a dose level spacing between 2 and 4, the following lower dose levels are proposed: 60 ppm, 25 ppm and 10 ppm. Four treatment groups are proposed to ensure compliance with the test guideline. The 4-dose design is based on the demonstrated steepness of the dose-response and the concern for delayed toxicity observed from the 28-day study.
Specific toxicities are difficult to predict; however, 60 ppm is a dose in a range that would be expected to result in clinical signs and neuropathology without compromising the survival of the animals.
A dose of 25 ppm is in the range of the toxicity threshold yet could be a NOAEL for clinical signs and neuropathology.
A dose of 10 ppm is expected to be a clear NOAEL for the study.
- Rationale for animal assignment: The animals for the experiment were weighed and arranged in ascending order of their body weight. These body weight stratified rats were distributed to all experimental groups using a Microsoft Excel Spreadsheet, such that body weight variation of animals selected for the experiment did not exceed ± 20 % of the mean body weight of each sex (-15.62 to +12.63 % for males and -13.76 to +14.76 % for females). The grouping was done one day and two days for males and females respectively prior to the initiation of treatment. Body weight of the animals was analysed statistically for mean body weight to rule out the statistically significant difference between groups within each sex.
Observations and clinical examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed for clinical signs of toxicity and twice daily for mortality and morbidity throughout the experimental period.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical examinations were carried out on all animals prior to the test material administration and weekly thereafter. These observations were made outside the home cage in a standard arena.
Signs noted included but were not limited to, changes in skin, fur, eyes and mucous membranes, occurrence of secretions and excretions and autonomic activity such as lacrimation, piloerection, pupil size, unusual respiratory pattern and any unusual signs of urination or defecation. Responses with respect to body position, activity level and co-ordination of movement, as well as changes in gait, posture and reactivity to handling, placing or other environmental stimuli, as well as the presence of clonic or tonic movements, convulsions or tremors, stereotypies or bizarre behaviour or aggression were also observed.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual animal body weight was recorded on Day 1 prior to the test material administration and at weekly intervals of the experimental period. Fasting body weight of all the animals was recorded at terminal sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Cage wise feed consumption was measured at weekly intervals.
- Food consumption for each animal determined: Yes (Average feed intake per rat (g/rat/day) was calculated using the amount of feed offered, spillage and left over in each cage and the number of rats in each cage)
- Test material intake calculated: Yes

FOOD EFFICIENCY: Not examined

WATER CONSUMPTION: Not examined

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmological examination was performed on all animals before the start of treatment (during acclimatisation) and during week 13 for G1 and G5 group animals. No treatment related ocular changes were observed at high dose group animals, hence the examination was not extended to lower dose groups.
Neurobehavioural examinations performed and frequency:
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During acclimatisation (after grouping), and during weeks 2, 8 and 13 of the treatment period.
- Dose groups that were examined: All groups (the first 10 animals/sex/group)
- Battery of functions tested: The following Neurological/Functional observations were performed:
a) Home Cage Observations: Animals in the cage were observed for convulsions, tremors and palpebral closure and the scores were recorded.
b) Handling Observations: The animals were observed for the ease of removal from the cage and ease of handling. During handling observation, the animals were observed for resistance, lacrimation, red and crusty deposits around eyes, nose and mouth, salivation, fur appearance, piloerection, palpebral closure, respiratory character, eye prominence and muscle tone.
c) Open Field Observations: Animals were placed in an open field arena for 2 minutes and observed for their mobility, gait, arousal, rearing, urination, defecation, stereotypies and excessive grooming.
d) Sensory Observations: Animals were observed for startle response, touch response, pupil response, response to nociceptive stimuli and righting reflex.
e) Neuromuscular Observations: As a part of neuromuscular observations, the animals were dropped from a height of approximately 30 cm with painted paws to record the Hind Limb Foot Splay.
f) Physiological Observation (Rectal temperature): Physiological temperature of the animals was recorded using a calibrated digital thermometer.
g) Motor Activity Assessment: Assessment of motor activity of the animals for 10 minutes was carried out using an actimeter.
h) Limb Grip Strength Assessment: Assessment of hind limb and fore limb grip strength were carried out using a grip strength meter.
Sacrifice and (histo)pathology:
> TERMINAL PROCEDURES FOR THE FIRST 5 RATS/SEX/GROUP

PERFUSION AND FIXATION PROCEDURE
The first 5 rats/sex from each group were subjected to perfusion fixation. Deep surgical anaesthesia with sodium thiopentone at 40 mg/kg body weight injected intraperitoneally was achieved before the procedure began. The animals were perfused with 0.9 % normal saline first to flush the blood from the circulatory system, and then perfused with 10 % neutral buffered formalin for in situ fixation.

TISSUE COLLECTION
After in situ fixation the following organs and tissues were collected, trimmed of adherent tissue as appropriate and preserved in 10 % neutral buffered formalin: brain, spinal cord along with nerve fibres, eyes with optic nerve, sciatic nerve, tibial nerve, and calf muscles.

HISTOPATHOLOGY OF PERFUSED TISSUES
The examined areas included the forebrain, the centre of the cerebrum, including a section through the hippocampus, the midbrain, the cerebellum, the pons, the medulla oblongata, the eye with optic nerve and retina, the spinal cord at the cervical and lumbar swellings, the dorsal root ganglia, the dorsal and ventral root fibres, the proximal sciatic nerve, the proximal tibial nerve (at the knee) and the tibial nerve calf muscle branches. The spinal cord and peripheral nerve sections included both transverse and longitudinal sections.
A stepwise examination of tissue samples was done in which sections from the high dose group were first compared with those of the vehicle control group. Neuropathological alterations were observed in the brain of a few high dose group animals. Hence, brain samples from the mid and low dose group animals were examined sequentially. In addition to haematoxylin and eosin staining, the brain, spinal cord, tibial nerve and sciatic nerve were stained with silver nitrate and luxol fast blue stains for evaluation.
Other examinations:
HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected on experimental Day 92.
- Anaesthetic used for blood collection: Yes. Blood samples were collected from retro-orbital plexus puncture method under mild Isoflurane anaesthesia with the help of a fine capillary tube.
- Animals fasted: Yes. The animals were fasted overnight before blood collection. Water was provided ad libitum during the fasting period.
- How many animals: Blood samples were collected from all rats (except the first 5 rats/sex) separately into the tubes containing K2-EDTA for haematology and sodium citrate tubes for Prothrombin time and activated partial thromboplastin time parameters.
- Parameters checked included: haemoglobin concentration (HGB), haematocrit (HCT), erythrocyte count (RBC), total Leukocyte Count (WBC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelet count (PLT), mean platelet volume (MPV), reticulocyte count, absolute reticulocyte count, differential leucocytes count (DLC), and absolute differential leucocytes count (DLC) (all estimated using Advia Hematology System 2120 (Siemens Limited)), and prothrombin time (PT), and activated partial thromboplastin time (APTT) (estimated by coagulation analyser (from Tulip Diagnostics (p) Ltd., India). The plasma was separated by centrifuging the blood samples 1500 rpm for 15 minutes for determining the PT and APTT parameters.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected on experimental Day 92.
- Animals fasted: Yes. The animals were fasted overnight before blood collection. Water was provided ad libitum during fasting period.
- How many animals: Blood samples were collected from all rats (except the first 5 rats/sex) separately into the tubes containing sodium heparin.
- Parameters checked included: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), cholinesterase, total protein, albumin, total bilirubin, glucose, total cholesterol, creatinine, urea, triglycerides, phosphorous, calcium, high density lipoprotein (HDL), low density lipoprotein (LDL), blood urea nitrogen (BUN-calculated), globulin (calculated), and albumin/globulin ratio (calculated) (all parameters analysed using an “Rx Daytona+ Clinical Chemistry Analyser” from Randox Laboratories). Sodium, potassium and chloride were estimated using Easylyte plus Na/K/Cl analyser (Medica Corporation). The plasma was separated by centrifuging the blood samples at 5000 rpm for 10 minutes for determining the clinical chemistry parameters.

URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected from all the rats (except first 5 rats/sex) on experimental Day 92.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes. Animals were fasted overnight. Water was provided ad libitum during this period.
- Parameters checked included: blood, bilirubin, urobilinogen, ketones, protein, glucose, and leucocytes (all analysed using a “DIRUI H-500” from Dirui Industrial Company Ltd.). The volume of urine collected (mL), appearance and the colour were examined by physical evaluation. In addition, nitrite, pH and specific gravity was also analysed.
After analysis of the above parameters, the urine was subjected for centrifugation at 1500 rpm for 3 minutes. Then the urine was submitted for microscopic examination for urinary sediments.

OTHER:
- Vaginal Smear Examination: Vaginal smear was examined for all the group females on the day of sacrifice.
- Assessment of Thyroid Hormones T3 (Triiodothyronine), T4 (Thyroxine) and Thyroid Stimulating Hormone (TSH): Blood samples for estimation of T3, T4 and TSH were collected at termination, at the same time when samples were collected for haematology and clinical chemistry (except for the first 5 rats/sex). The collected blood samples were centrifuged at 5000 rpm for 10 minutes, the serum was separated and stored frozen (-80 °C) until estimation. The analysis was performed using commercially available ELISA kits procured from Cusabiotech Company. Before analysing the samples, the standards for each batch of kits were checked to confirm they were working properly, as per the kits’ instructions.

> TERMINAL PROCEDURES FOR THE LAST 10 RATS/SEX/GROUP

GROSS PATHOLOGY: Yes
At the end of treatment period all animals were fasted overnight (water allowed), subjected to blood sample collection and on the same day (day 92), weighed and then subjected to necropsy. Animals were euthanised by using excess CO2 anaesthesia followed by exsanguination. A gross pathological examination was carried out for each rat. Necropsy included an examination of external surfaces, external orifices, abdominal, thoracic and cranial cavities, organs and tissues. All these animals were sacrificed as per random numbers and complete gross pathological examination was performed.

ORGAN WEIGHTS: Yes
On the day of scheduled necropsy, the organs in the following list were collected from all animals and were trimmed of adherent tissue and fat as appropriate and weighed wet as soon as possible to avoid drying: kidneys*, adrenals*, spleen, heart, liver, thymus, brain, lungs, testes*/ovaries*, uterus/epididymis*, thyroid and parathyroid**, prostate + seminal vesicles with coagulating gland, and pituitary gland**.
*Paired organs were weighed together
**: weighed after fixation
Organ weight ratio was calculated against fasting body weight.

HISTOPATHOLOGY: Yes
The following organs and tissues were collected, and trimmed of adherent tissue, as appropriate, from the last 10 animals/sex from each group. All the tissues mentioned below, from all animals were preserved in 10 % v/v Neutral Buffered Formalin except testes and eyes which were preserved in modified Davidson’s fixative.
Eyes, ovaries, brain, lungs, spinal cord, prostate gland, stomach, oesophagus, duodenum, pharynx, jejunum, urinary bladder, ileum with Peyer’s patches, mesenteric lymph nodes, caecum, mandibular lymph nodes, colon, sciatic nerve, rectum, bone marrow smear, liver, epididymis, kidneys, aorta (thoracic), adrenals, skin (with mammary glands in males and females), spleen, sternum, heart, skeletal muscle, thymus, thyroid, seminal vesicles with coagulating gland, parathyroid, pancreas, vagina, uterus and cervix, trachea, testes, salivary gland, tongue, nasal cavity, femur (femoro-tibial joint), pituitary, and harderian gland.
Histopathological examination was conducted on all the preserved organs/tissues from the vehicle control and high dose animals. All gross lesions, organs and tissue samples were processed, embedded in paraffin, cut at a thickness of 4-6 micrometres and stained with haematoxylin and eosin.
Testes were sectioned at a thickness of 3-4 micrometres; in addition to haematoxylin and eosin stain, Periodic acid–Schiff and haematoxylin stain was used for spermatogenesis evaluation.
A detailed qualitative examination of the testes was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen or any cell- or stage-specificity of testicular findings.

BONE MARROW SMEAR EXAMINATION
Bone marrow smear (from one femur) was prepared for all the animals at the time of necropsy (the bone marrow smears were fixed in methanol and stained with May-Grunwald-Giemsa) but not evaluated as it was not warranted by haematology findings.
Statistics:
The data were subjected to statistical analysis. The computer printout of the data was verified with the original raw data. After verification, the data were subjected to statistical analysis using SPSS software, version 22. Body weight, percent change in body weight with respect to day 1, feed consumption, absolute organ weights, organ weight relative to body weight, haematological and clinical chemistry parameters, urinalysis parameters (urine volume, pH, specific gravity and urobilinogen), FOB parameters (rearing, urination, defecation, excessive grooming, body temperature, movement count, hindlimb foot splay and grip strength) and thyroid hormone levels were subjected to statistical analysis. One-way ANOVA followed by Dunnett’s post-test was done for the different treatment groups in comparison with the control group data. All analysis and comparisons were evaluated at the 95 % level of confidence (P < 0.05).
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs of toxicity at all the tested dose levels in either sex. The detailed clinical examination of animals did not reveal any treatment related changes up to the highest dose tested in either sex.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
None of the animals died during the study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment related changes were observed in mean body weight and body weight gain (percent change in body weight with respect to Day 1) in all the tested dose groups. Statistically significant lower mean body weights were noted in females at the mid dose I (G3, 25 ppm) on days 57, 64, 71, 78, 85 and 91. Mean body weights were decreased by 7 to 8 % when compared to controls. These were considered incidental and not related to treatment due to the small magnitude and the lack of any dose dependency.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No treatment related changes in feed consumption were noted in all the tested dose groups in either sex. A single incidence of statistically significant lower feed consumption was noted in G5 males on week 5 which is considered incidental due to its single occurrence.
Test material intake was as follows:
Males: 0.86, 2.12, 5.15 and 14.96 mg/kg/day for the low dose (G2, 10 ppm), mid dose I (G3, 25 ppm), mid dose II (G4, 60 ppm), and high dose (G5, 175 ppm) groups, respectively.
Females: 0.94, 2.54, 5.74 and 16.73 mg/kg/day for the low dose (G2, 10 ppm), mid dose I (G3, 25 ppm), mid dose II (G4, 60 ppm), and high dose (G5, 175 ppm) groups, respectively.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No ophthalmological abnormalities were noted.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The following statistically significant differences were noted in haematology parameters: in males, an increase in platelets (G3, G5); in females, there was an increase in total erythrocyte counts (G5) and haematocrit (G5).
The variations noted are considered incidental due to lack of dose responsiveness.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The following statistically significant differences in clinical chemistry parameters were noted: in males, increases in triglycerides (G5), albumin (G3, G4, G5), calcium (G3, G4, G5), phosphorous (G4), A/G ratio (G5) and decrease in sodium (G3).
In females, decreases in urea (G2, G3), creatinine (G2 to G5), BUN (G2, G3) and potassium (G2, G5) were observed.
Increases in albumin, calcium and A/G ratio in males and a decrease in creatinine in females are considered incidental in the absence of histopathology changes in high dose animals (G5 - 175 ppm). All other variations noted are considered incidental in the absence of dose responsiveness and to be due to random biological variation.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically adverse treatment related changes in urinary parameters were noted. Statistically significant increases in urine volume (G5M, G3F, G4F and G5F) were noted.
Urine volumes (mean ± SD) in G1M, G2M, G3M, G4M and G5M were 5.7 ± 0.7, 5.6 ± 0.4, 5.4 ± 0.7, 5.7 ± 0.6, and 6.9 ± 0.9* mL, respectively (* Statistically significant at p<0.05).
Urine volumes (mean ± SD) in G1F, G2F, G3F, G4F and G5F were 5.7 ± 0.6, 5.6 ± 0.8, 7.2 ± 1.0*, 7.0 ± 1.7*, and 8.5 ± 0.8* mL, respectively (* Statistically significant at p<0.05).
The slight increase in urine volume is considered incidental in the absence of any infection or histopathology changes in the kidneys of high dose animals. Also, no record of significant urination was noted during the detailed clinical examination or functional examination tests.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related changes noted in neurological/functional examination battery test and motor activity. The following statistically significant differences were noted during different measurement time points:
a) Pre-dose: increase in hind limb foot splay in G2 males; decrease in hind limb foot splay in G3, G4 and G5 females and decrease in urination in G3 females.
b) Week 2: increase in hind limb foot splay in G2 and G3 males and decrease in rearing in all treated females (G2, G3, G4, G5).
c) Week 8: decrease in rearing in G3, G4 and G5 males, and decrease in hind limb foot splay in G3 females.
d) Week 13: decrease in grooming in G2 males, forelimb grip strength in G5 males, increase in movement counts in G3, G4, G5 males.
The decrease in rearing in treated females during week 2 was likely due to an abnormally high mean value (12.2) and individual rearing counts values (≥10 in 8 out of 10 animals) of female animals of the control group rather than an effect of treatment. The decrease in rearing in males during week 8 is considered incidental and not adverse as none of the other exploratory behaviour was affected, and all individual values were comparable between the treated and the control group animals. The increase in movement counts (6 to 13 %) is considered incidental as the increase was minimal and there were no other adverse neuromuscular changes noted.
All the other variations recorded were not associated with changes in other neurological parameters or clinical signs or were inconsistent between different measurement time points or lacked dose responsiveness, hence, they were considered as incidental and not related to treatment.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
The following statistically significant changes were noted:
- Absolute organ weights: increased spleen weight in G4M, increased heart weight in G2F, increased liver weight in G2F, increased pituitary weight in G2F; decreased testes weight in G2M, decreased epididymis weight in G2M, decreased heart weight in G3M, decreased brain weight in G3M, decreased prostate + seminal vesicles with coagulating gland weight in G4M and G5M, decreased lungs weight in G2M, decreased pituitary weight in G2M, decreased ovaries weight in G5F.
- Organ weights relative to body weight: increased spleen weight in G4M, increased uterus weight in G5F, increased pituitary weight in G2F; decreased heart weight in G3M, decreased prostate + seminal vesicles with coagulating gland weight in G4M, decreased lungs weight in G2M, decreased pituitary weight in G2M, decreased thyroid and parathyroid weight in G2F.
All the differences observed in organ weights are considered as incidental and not related to treatment in the absence of microscopic changes in the high dose group and also due to lack of dose responsiveness.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No gross pathological findings were noted in the study.
Neuropathological findings:
effects observed, treatment-related
Description (incidence and severity):
Microscopically, test material-related changes were observed in the brain of three female rats in the high dose group (175 ppm). Histologically, changes consisted of neuronal necrosis characterised by cytoplasmic shrinkage with intense eosinophilia (red neuron) accompanied by karyorrhexis or karyolysis of nucleus in the piriform cortex, cornu ammonis (CA1) and dentate gyrus of hippocampus. Brains from lower dose groups revealed normal histology.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Some histopathological findings such as mononuclear cells infiltrate in kidney, prostate, salivary glands and Harderian gland, concretion in prostate and basophilic tubules in kidney were randomly distributed across the groups and were considered as incidental findings commonly observed in the rat.
The presence of an ectopic thymus in the thyroid gland is a congenital lesion and it does not have any relationship to treatment.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
- Vaginal Smear Examination: Vaginal smear examination revealed normal stages of oestrous cyclicity in all the group females.
- Thyroid Hormones - T3 (Triiodothyronine), T4 (Thyroxine) and Thyroid Stimulating Hormone (TSH): No treatment related changes in thyroid hormones (T3, T4 and TSH) were noted. A statistically significant increase in T3 in G2M, and decreases in T3 in G3M, G4M, G5M, G3F, G4F and G5F were noted. Statistically significant increases in T4 in G4M, G2F and G4F were noted.
In the absence of any dose responsiveness or any associated macroscopic or microscopic changes in the thyroid of animals of the G5 group (high dose-175 ppm), the observed changes are considered as incidental.
Key result
Dose descriptor:
NOAEL
Effect level:
60 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
neuropathology
Remarks on result:
other: (ca. 5.74 mg/kg/day)
Key result
Dose descriptor:
NOAEL
Effect level:
175 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: no treatment-related effects
Remarks on result:
other: (ca. 14.96 mg/kg/day)
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
60 ppm
System:
central nervous system
Organ:
brain
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
not specified

Summary of body weights, g (mean ± SD), n = 15

Group and Sex (Dose, ppm in diet)

Day

G1M

(0)

G2M

(10)

G3M

(25)

G4M

(60)

G5M

(175)

G1F

(0)

G2F

(10)

G3F

(25)

G4F

(60)

G5F

(175)

1

67.47 ± 4.90

66.77 ± 4.85

66.76 ± 4.73

66.75 ± 5.13

67.41 ± 4.28

63.60 ± 4.80

63.97 ± 5.19

63.95 ± 5.26

64.53 ± 5.02

64.25 ± 4.93

8

99.37 ± 8.60

96.22 ± 12.30

99.41 ± 6.46

99.72 ± 4.80

97.56 ± 8.80

93.53 ± 6.69

93.35 ± 5.75

93.38 ± 4.97

93.21 ± 4.88

93.45 ± 4.70

15

131.02 ± 9.31

129.20 ± 12.13

131.13 ± 8.32

132.85 ± 4.71

129.87 ± 9.37

130.18 ± 7.75

130.95 ± 11.97

126.36 ± 14.95

127.91 ± 16.07

125.48 ± 8.35

22

154.46 ± 15.13

154.14 ± 15.74

154.66 ± 11.39

153.81 ± 7.74

152.16 ± 11.82

138.20 ± 9.55

137.85 ± 12.48

134.75 ± 16.29

135.88 ± 16.51

134.49 ± 9.06

29

162.50 ± 16.42

161.85 ± 16.01

161.62 ± 11.20

161.42 ± 9.46

159.10 ± 11.94

144.68 ± 9.52

144.32 ± 12.40

141.22 ± 15.55

142.36 ± 16.68

140.96 ± 8.65

36

201.08 ± 15.55

210.84 ± 22.66

212.55 ± 16.40

211.63 ± 19.32

211.46 ± 18.41

184.50 ± 10.43

184.06 ± 15.63

176.65 ± 17.50

177.47 ± 20.22

179.15 ± 15.60

43

243.40 ± 22.13

260.03 ± 20.13

257.80 ± 13.23

247.35 ± 26.79

249.05 ± 23.25

198.27 ± 10.43

198.16 ± 16.28

184.42 ± 18.12

192.08 ± 20.92

191.69 ± 16.20

50

275.09 ± 27.70

287.45 ± 23.15

289.31 ± 17.93

278.21 ± 32.91

279.38 ± 29.51

209.13 ± 12.14

208.86 ± 16.59

194.09 ± 18.05

199.63 ± 21.05

198.69 ± 16.91

57

294.87 ± 29.94

306.66 ± 22.80

308.73 ± 17.24

293.13 ± 32.80

298.15 ± 27.60

221.80 ± 14.26

223.92 ± 19.20

205.56 ± 17.25*

214.46 ± 19.53

214.64 ± 15.88

64

312.73 ± 32.74

325.05 ± 28.56

324.20 ± 18.95

310.34 ± 36.15

317.69 ± 31.39

226.98 ± 14.32

229.61 ± 18.54

210.86 ± 17.00*

219.03 ± 19.84

220.07 ± 15.12

71

331.93 ± 38.29

339.58 ± 31.84

339.45 ± 20.69

332.50 ± 36.24

338.42 ± 35.05

235.31 ± 16.07

238.79 ± 16.75

217.93 ± 16.68*

225.56 ± 19.89

226.56 ± 16.73

78

348.52 ± 38.40

355.69 ± 30.37

353.60 ± 20.78

345.35 ± 37.43

350.77 ± 34.74

245.42 ± 17.17

247.53 ± 16.20

226.13 ± 16.61*

234.12 ± 19.83

234.88 ± 16.99

85

365.76 ± 42.44

370.85 ± 32.35

371.98 ± 23.29

367.64 ± 41.92

374.75 ± 39.33

252.36 ± 18.61

260.12 ± 16.73

231.93 ± 16.05*

241.83 ± 20.07

240.51 ± 18.16

91

379.54 ± 43.10

384.40 ± 33.09

384.19 ± 24.23

381.38 ± 42.26

387.83 ± 41.19

258.20 ± 19.23

265.99 ± 16.60

237.19 ± 16.17*

246.94 ± 20.07

245.70 ± 18.05

G: Group

M: Male

F: Female

*Statistically significant (p < 0.05)

Summary of average feed consumption, g/rat/day (mean ± SD), n = 8

Group and Sex (Dose, ppm in diet)

Week

G1M

(0)

G2M

(10)

G3M

(25)

G4M

(60)

G5M

(175)

G1F

(0)

G2F

(10)

G3F

(25)

G4F

(60)

G5F

(175)

1

15.59 ± 2.21

15.76 ± 2.04

15.01 ± 1.46

15.15 ± 1.03

15.21 ± 0.98

13.31 ± 1.25

13.97 ± 0.55

13.43 ± 0.74

13.68 ± 0.37

13.20 ± 1.08

2

16.61 ± 1.23

16.68 ± 0.46

17.16 ± 1.17

16.18 ± 1.02

16.55 ± 1.41

15.12 ± 1.26

15.07 ± 0.94

15.03 ± 1.08

15.41 ± 0.95

15.23 ± 0.97

3

17.28 ± 0.60

17.73 ± 0.57

17.97 ± 0.63

17.67 ± 1.05

17.52 ± 2.10

15.11 ± 1.26

15.06 ± 0.93

15.04 ± 1.08

15.39 ± 0.92

15.25 ± 0.96

4

18.22 ± 0.96

18.72 ± 0.66

18.77 ± 1.00

18.87 ± 0.81

18.48 ± 1.12

15.93 ± 0.96

16.06 ± 1.09

16.05 ± 1.00

16.04 ± 1.09

15.94 ± 0.74

5

19.49 ± 0.64

18.50 ± 1.48

18.43 ± 1.00

18.69 ± 0.82

17.87 ± 1.46*

16.70 ± 1.20

16.99 ± 0.69

16.95 ± 0.87

17.01 ± 1.34

17.02 ± 0.91

6

20.53 ± 0.70

20.10 ± 1.08

19.92 ± 0.91

20.02 ± 1.31

19.44 ± 1.11

16.98 ± 1.06

17.01 ± 1.12

17.18 ± 1.21

17.04 ± 0.82

16.99 ± 1.24

7

21.29 ± 0.56

21.32 ± 0.83

20.82 ± 1.32

21.04 ± 0.85

21.23 ± 1.69

17.14 ± 1.24

17.84 ± 1.69

18.03 ± 1.94

17.13 ± 1.06

17.22 ± 1.35

8

22.45 ± 0.94

22.02 ± 0.90

21.85 ± 1.19

21.83 ± 1.09

21.82 ± 1.61

17.66 ± 1.30

18.39 ± 1.65

18.69 ± 2.18

17.52 ± 1.19

17.75 ± 1.43

9

22.94 ± 1.03

23.49 ± 1.00

23.01 ± 0.78

22.73 ± 1.34

23.10 ± 1.29

18.19 ± 1.75

18.65 ± 1.61

18.95 ± 2.18

17.80 ± 1.35

18.06 ± 1.53

10

23.56 ± 0.81

24.08 ± 0.98

23.74 ± 1.18

23.61 ± 1.48

23.94 ± 1.14

18.70 ± 1.64

19.19 ± 1.45

19.43 ± 1.79

18.41 ± 1.28

18.57 ± 1.46

11

24.19 ± 0.81

24.71 ± 0.78

24.35 ± 0.98

23.83 ± 1.58

24.23 ± 1.04

19.19 ± 1.56

19.51 ± 1.44

19.65 ± 1.61

18.73 ± 1.29

18.92 ± 1.26

12

24.75 ± 0.91

25.28 ± 0.78

24.84 ± 0.83

24.33 ± 1.47

24.60 ± 0.74

19.61 ± 1.59

19.74 ± 1.54

19.82 ± 1.50

19.15 ± 1.33

19.39 ± 1.29

13

25.15 ± 0.92

25.59 ± 0.85

25.26 ± 0.96

24.64 ± 1.32

24.95 ± 0.63

20.12 ± 1.84

20.39 ± 1.61

19.99 ± 1.38

19.63 ± 1.44

19.51 ± 1.23

G: Group

M: Male

F: Female

*Statistically significant (p < 0.05)

Summary of haematology findings for selected parameters (mean ± SD), n = 10

Group and Sex (Dose, ppm in diet)

Parameter

G1M

(0)

G2M

(10)

G3M

(25)

G4M

(60)

G5M

(175)

G1F

(0)

G2F

(10)

G3F

(25)

G4F

(60)

G5F

(175)

Total Erythrocyte Count (RBC) (10^6 cells/µL)

7.74 ± 0.88

8.27 ± 0.25

8.06 ± 0.37

7.89 ± 0.40

8.18 ± 0.47

7.14 ± 0.50

7.23 ± 0.90

7.45 ± 0.42

6.67 ± 1.03

7.94 ± 0.44*

Haematocrit (HCT) (%)

44.66 ± 4.57

46.73 ± 1.00

45.98 ± 1.66

46.11 ± 1.91

47.50 ± 2.70

41.68 ± 2.68

41.67 ± 4.59

42.94 ± 2.09

40.34 ± 3.57

45.44 ± 2.36*

Platelet Count (PLT) (10^3 cells/µL)

700.30 ± 124.15

801.80 ± 74.58

851.80 ± 88.55*

809.10 ± 119.23

829.80 ± 74.77*

869.20 ± 122.86

836.90 ± 148.61

828.70 ± 90.58

713.20 ± 195.85

856.30 ± 124.89

G: Group

M: Male

F: Female

*Statistically significant (p < 0.05)

Summary of clinical chemistry findings for selected parameters (mean ± SD), n = 10

Group and Sex (Dose, ppm in diet)

Parameter

G1M

(0)

G2M

(10)

G3M

(25)

G4M

(60)

G5M

(175)

G1F

(0)

G2F

(10)

G3F

(25)

G4F

(60)

G5F

(175)

Urea (mg/dL)

45.44 ± 7.99

39.77 ± 5.59

39.22 ± 6.61

40.43 ± 8.83

40.53 ± 7.14

37.23 ± 6.22

30.11 ± 3.63*

31.90 ± 4.53*

34.47 ± 2.67

33.43 ± 4.73

Creatinine (CRE) (mg/dL)

0.56 ± 0.03

0.55 ± 0.02

0.56 ± 0.04

0.54 ± 0.03

0.55 ± 0.04

0.63 ± 0.03

0.56 ± 0.05*

0.56 ± 0.04*

0.56 ± 0.05*

0.56 ± 0.03*

Triglycerides (TRI) (mg/dL)

36.30 ± 12.01

47.50 ± 17.70

44.20 ± 15.80

47.40 ± 15.61

60.70 ± 28.80*

31.50 ± 11.34

34.50 ± 7.85

31.20 ± 14.97

35.10 ± 9.65

39.60 ± 10.85

Albumin (ALB) (g/dL)

3.11 ± 0.23

3.26 ± 0.14

3.36 ± 0.14*

3.35 ± 0.16*

3.39 ± 0.09*

3.54 ± 0.23

3.44 ± 0.19

3.54 ± 0.23

3.45 ± 0.23

3.57 ± 0.22

Calcium (CAL) (mg/dL)

9.35 ± 0.29

9.52 ± 0.23

9.76 ± 0.25*

9.96 ± 0.23*

9.70 ± 0.23*

9.41 ± 0.44

9.24 ± 0.40

9.55 ± 0.30

9.66 ± 0.36

9.43 ± 0.31

Phosphorus (PHO) (mg/dL)

5.19 ± 0.67

5.34 ± 0.44

5.50 ± 0.41

6.61 ± 1.47*

5.90 ± 0.46

4.45 ± 0.33

4.19 ± 0.77

4.34 ± 0.47

5.22 ± 1.08

4.47 ± 0.50

Albumin/Globulin Ratio (A/G Ratio)

0.98 ± 0.11

1.05 ± 0.10

1.04 ± 0.10

1.08 ± 0.09

1.13 ± 0.08*

1.10 ± 0.11

1.06 ± 0.08

1.13 ± 0.12

1.13 ± 0.10

1.12 ± 0.10

Blood Urea Nitrogen (BUN) (mg/dL)

21.21 ± 3.73

18.56 ± 2.61

18.30 ± 3.08

18.87 ± 4.12

18.91 ± 3.33

17.37 ± 2.91

14.05 ± 1.69*

14.89 ± 2.11*

16.09 ± 1.25

15.60 ± 2.20

Sodium (Na) (mmol/L)

148.58 ± 1.25

147.66 ± 1.37

146.61 ± 1.77*

148.01 ± 0.39

147.82 ± 1.23

146.23 ± 1.03

146.26 ± 1.28

146.94 ± 1.10

147.19 ± 0.85

146.96 ± 1.02

Potassium (K) (mmol/L)

3.75 ± 0.28

3.66 ± 0.21

3.41 ± 0.47

3.47 ± 0.30

3.69 ± 0.23

3.45 ± 0.22

3.17 ± 0.22*

3.34 ± 0.19

3.53 ± 0.26

3.11 ± 0.20*

G: Group

M: Male

F: Female

* Statistically significant (p < 0.05)

Summary of Functional Observational Battery findings for selected parameters and selected time points (mean ± SD), n = 10

Group and Sex (Dose, ppm in diet)

Parameter

Time point

G1M (0)

G2M (10)

G3M (25)

G4M (60)

G5M (175)

G1F (0)

G2F (10)

G3F (25)

G4F (60)

G5F (175)

Number of Urination

Pre-dose

1.9 ± 1.2

1.5 ± 1.1

1.6 ± 1.0

1.4 ± 1.3

1.3 ± 0.9

2.0 ± 0.9

1.3 ± 0.9

0.9 ± 0.9*

1.1 ± 0.9

1.1 ± 0.7

Number of Rearing

Week 2

5.8 ± 1.8

5.8 ± 1.8

5.7 ± 1.9

6.1 ± 1.9

6.1 ± 1.9

12.2 ± 3.9

6.4 ± 1.3*

5.9 ± 1.7*

6.0 ± 1.2*

5.6 ± 1.3*

Week 8

7.6 ± 1.5

7.2 ± 1.3

6.1 ± 1.0*

6.1 ± 1.0*

5.9 ± 0.9*

5.5 ± 1.8

4.8 ± 2.5

4.3 ± 1.8

4.4 ± 2.0

7.4 ± 2.5

Number of Grooming

Week 13

3.1 ± 0.7

2.3 ± 0.8*

2.9 ± 0.6

2.8 ± 0.8

2.9 ± 0.6

3.0 ± 0.7

2.9 ± 0.6

3.2 ± 0.8

3.1 ± 0.6

2.9 ± 0.6

Hind limb foot splay (cm)

Pre-dose

4.9 ± 1.3

6.2 ± 0.5*

5.3 ± 0.5

5.6 ± 0.7

5.5 ± 0.8

6.6 ± 0.5

6.3 ± 0.2

5.6 ± 0.6*

5.6 ± 0.6*

5.4 ± 0.4*

Week 2

5.7 ± 1.6

6.9 ± 0.7*

7.2 ± 0.4*

6.8 ± 0.3

6.9 ± 1.4

6.6 ± 0.4

6.7 ± 0.5

6.5 ± 0.5

6.4 ± 0.2

6.5 ± 0.3

Week 8

8.4 ± 0.8

8.6 ± 0.9

8.6 ± 1.0

8.5 ± 1.0

8.5 ± 1.1

7.7 ± 0.9

6.7 ± 0.9

6.1 ± 0.4*

6.8 ± 1.0

7.3 ± 1.0

Movement counts

Week 13

1963.1 ± 57.1

1985.2 ± 104.1

2087.2 ± 52.8*

2215.1 ± 116.6*

2126.0 ± 73.8*

2370.1 ± 211.1

2242.7 ± 275.2

2300.6 ± 78.3

2341.1 ± 46.90

2276.8 ± 76.2

Fore limb grip strength (kgf)

Week 13

1.537 ± 0.033

1.530 ± 0.025

1.518 ± 0.025

1.509 ± 0.027

1.488 ± 0.032*

1.385 ± 0.048

1.374 ± 0.038

1.382 ± 0.036

1.387 ± 0.045

1.380 ± 0.027

G: Group

M: Male

F: Female

* Statistically significant (p < 0.05)

Summary of selected thyroid hormone parameters (mean ± SD), n = 10

Group and Sex (Dose, ppm in diet)

G1M

(0)

G2M

(10)

G3M

(25)

G4M

(60)

G5M

(175)

G1F

(0)

G2F

(10)

G3F

(25)

G4F

(60)

G5F

(175)

Serum T3 Levels (ng/mL)

2.983 ± 0.174

3.174 ± 0.066*

2.379 ± 0.061*

2.291 ± 0.086*

2.339 ± 0.292*

3.307 ± 0.160

3.318 ± 0.110

2.416 ± 0.074*

2.436 ± 0.086*

2.288 ± 0.136*

Serum T4 Levels (ng/mL)

79.130 ± 10.154

90.481 ± 24.948

69.956 ± 3.743

109.255 ± 23.075*

86.814 ± 6.853

74.518 ± 7.289

114.344 ± 49.994*

71.209 ± 7.225

109.267 ± 20.808*

90.534 ± 7.103

G: Group

M: Male

F: Female

*Statistically significant (p < 0.05)

Selected organ weights (g) and selected organ weights relative to fasting body weights (%) (mean ± SD), n = 10

Group and Sex (Dose, ppm in diet)

Organ

G1M

(0)

G2M

(10)

G3M

(25)

G4M

(60)

G5M

(175)

G1F

(0)

G2F

(10)

G3F

(25)

G4F

(60)

G5F

(175)

Fasting body weight (g)

359.51 ± 36.91

355.72 ± 30.04

359.57 ± 22.67

369.15 ± 40.25

357.18 ± 31.37

238.20 ± 19.13

255.27 ± 13.39

218.27 ± 13.43

227.34 ± 23.71

229.75 ± 18.97

Spleen - absolute (g)

0.7660 ± 0.1259

0.6696 ± 0.0645

0.7684 ± 0.2546

1.1217 ± 0.3454*

0.7313 ± 0.0768

0.5104 ± 0.1000

0.4953 ± 0.0945

0.4670 ± 0.0518

0.4955 ± 0.1525

0.4457 ± 0.0705

Spleen - relative (%)

0.2160 ± 0.0489

0.1889 ± 0.0191

0.2147 ± 0.0749

0.3030 ± 0.0840*

0.2059 ± 0.0258

0.2148 ± 0.0424

0.1945 ± 0.0387

0.2144 ± 0.0253

0.2229 ± 0.0833

0.1949 ± 0.0332

Testes - absolute (g)

3.3322 ± 0.2061

3.1336 ± 0.1385*

3.5220 ± 0.1873

3.3057 ± 0.2017

3.3899 ± 0.1258

- - - - -

Epididymides - absolute (g)

1.5976 ± 0.1261

1.4003 ± 0.0907*

1.5952 ± 0.2162

1.5750 ± 0.1720

1.5280 ± 0.0922

- - - - -

Ovaries - absolute (g)

- - - - -

0.1442 ± 0.0153

0.1476 ± 0.0286

0.1370 ± 0.0147

0.1403 ± 0.0226

0.1133 ± 0.0146*

Uterus - relative (%)

- - - - -

0.2556 ± 0.0412

0.2341 ± 0.0287

0.2974 ± 0.0587

0.2519 ± 0.0743

0.3346 ± 0.1073*

Heart - absolute (g)

1.4470 ± 0.0758

1.4274 ± 0.0777

1.2782 ± 0.1491*

1.4463 ± 0.0913

1.4343 ± 0.0716

0.9143 ± 0.0720

1.0383 ± 0.0695*

0.8786 ± 0.0695

0.9003 ± 0.0788

0.8739 ± 0.0568

Heart - relative (%)

0.4067 ± 0.0501

0.4036 ± 0.0391

0.3553 ± 0.0333*

0.3958 ± 0.0483

0.4048 ± 0.0450

0.3871 ± 0.0510

0.4079 ± 0.0365

0.4037 ± 0.0385

0.4003 ± 0.0598

0.3819 ± 0.0306

Brain - absolute (g)

2.0836 ± 0.0978

2.0426 ± 0.0849

1.9574 ± 0.1825*

2.1579 ± 0.0822

2.0953 ± 0.0478

1.9206 ± 0.1204

1.9742 ± 0.0698

1.8942 ± 0.0575

1.8668 ± 0.0595

1.8777 ± 0.1033

Liver - absolute (g)

13.5232 ± 1.3533

13.3248 ± 1.1089

13.0066 ± 1.0951

14.0392 ± 1.3746

13.4491 ± 0.6115

7.6240 ± 0.8038

8.6382 ± 0.5745*

7.7299 ± 0.9692

8.0259 ± 1.0397

7.4752 ± 1.0024

Prostate+Seminal vesicles with coagulating glands (PSC) - absolute (g)

3.7101 ± 0.4151

3.4878 ± 0.2865

3.7128 ± 0.5596

3.0538 ± 0.3124*

3.1411 ± 0.3866*

- - - - -

Prostate+Seminal vesicles with coagulating glands (PSC) - relative (%)

1.0465 ± 0.2012

0.9857 ± 0.1073

1.0310 ± 0.1257

0.8379 ± 0.1408*

0.8863 ± 0.1362

- - - - -

Lungs - absolute (g)

2.9231 ± 0.6863

2.1693 ± 0.1805*

2.7268 ± 0.8388

3.0516 ± 0.4917

2.7660 ± 0.5824

1.7479 ± 0.1490

1.8831 ± 0.1932

1.8569 ± 0.2307

1.7222 ± 0.2896

1.8532 ± 0.3523

Lungs - relative (%)

0.8247 ± 0.2268

0.6134 ± 0.0678*

0.7665 ± 0.2598

0.8327 ± 0.1442

0.7754 ± 0.1532

0.7382 ± 0.0867

0.7384 ± 0.0751

0.8508 ± 0.0942

0.7730 ± 0.1936

0.8017 ± 0.1031

Thyroid with parathyroid - relative (%)

0.0038 ± 0.0012

0.0041 ± 0.0008

0.0041 ± 0.0016

0.0033 ± 0.0008

0.0040 ± 0.0007

0.0078 ± 0.0015

0.0058 ± 0.0010*

0.0070 ± 0.0009

0.0067 ± 0.0022

0.0073 ± 0.0018

Pituitary gland - absolute (g)

0.0462 ± 0.0153

0.0304 ± 0.038*

0.0491 ± 0.0175

0.0395 ± 0.0099

0.0381 ± 0.0043

0.0325 ± 0.0073

0.0525 ± 0.0033*

0.0365 ± 0.0072

0.0267 ± 0.0059

0.0382 ± 0.0050

Pituitary gland - relative (%)

0.0131 ± 0.0053

0.0086 ± 0.0013*

0.0137 ± 0.0050

0.0109 ± 0.0032

0.0107 ± 0.0014

0.0138 ± 0.0037

0.0207 ­ 0.0022*

0.0168 ± 0.0035

0.0118 ± 0.0025

0.0168 ± 0.0031

G: Group

M: Male

F: Female

* Statistically significant (p < 0.05)

Summary of test material-related histopathological findings

Sex

Males

Females

Group

G1

G2

G3

G4

G5

G1

G2

G3

G4

G5

No of animals

5*+10#

5*+10#

5*+10#

5*+10#

5*+10#

5*+10#

5*+10#

5*+10#

5*+10#

5*+10#

Number examined

15

15

15

15

15

15

15

15

15

15

Within normal limits

15

15

15

15

15

15

15

15

15

12

Brain - Cerebrum; Piriform Cortex; Neuronal necrosis

Mild

0

0

0

0

0

0

0

0

0

2

Brain - Hippocampus; Dentate gyrus; Neuronal necrosis

Minimal

0

0

0

0

0

0

0

0

0

1

Brain - Hippocampus; CA1; Neuronal necrosis

Minimal

0

0

0

0

0

0

0

0

0

1

* animals subjected to in situ perfusion fixation

# animals whose organs/tissues were fixed by immersion

Summary of spontaneous or incidental histopathological findings

Group no.

G1

G5

G1

G5

Dose (ppm in diet)

0

175

0

175

Sex

Male

Female

No. of animals observed

5*+10#

5*+10#

5*+10#

5*+10#

ORGANS AND OBSERVATIONS/SEVERITY

KIDNEYS

No. examined

10

10

10

10

Interstitium, Mononuclear cells infiltrate; unilateral

Minimal

1

0

1

0

Basophilic tubules; unilateral

Mild

0

1

0

0

Urothelium; Mononuclear cells infiltration; bilateral

Minimal

0

0

0

1

SALIVARY GLANDS

No. examined

10

10

10

10

Periductal Mononuclear cells infiltrate

Minimal

0

1

0

0

PROSTATE GLAND

No. examined

10

10

NA

NA

Interstitium, Mononuclear cells infiltrate

Minimal

2

0

NA

NA

Mild

1

0

NA

NA

Concretions

Minimal

1

0

NA

NA

HARDERIAN GLAND

No. examined

10

10

10

10

Interstitium, Mononuclear cells infiltrate; unilateral

Minimal

0

0

1

1

THYROID

No. examined

10

10

10

10

Ectopic Tissue: Thymus

Not graded

2

0

1

0

* animals subjected to in situ perfusion fixation

# animals whose organs/tissues were fixed by immersion

NA: Not applicable

Conclusions:
Under the conditions of the study, the no observed-adverse-effect level (NOAEL) is 60 ppm in the diet (approximately 5.74 mg/kg/day) in females and 175 ppm in diet (14.96 mg/kg/day) in males.
Executive summary:

The repeated dose toxicity and neurotoxicity of the test material was investigated in a study which was conducted in accordance with the standardised guidelines OECD 408 and OECD 424, and under GLP conditions.

During the study, the test material was administered to groups of 15 male and 15 female Sprague-Dawley rats for a period of 91 consecutive days, by the oral (dietary route). Animals in groups G1, G2, G3, G4 and G5 received test material fortified diets at the doses of 0 (vehicle (acetone) control), 10, 25, 60, and 175 ppm in diet, respectively.

All animals were observed once daily for clinical signs of toxicity and twice daily for mortality. Detailed clinical examination, body weight and feed consumption measurements were conducted weekly. Ophthalmoscopic examination was performed during acclimatisation on all the animals and during week 13 for G1 and G5 group animals. A Neurological/Functional observational battery test was performed on the first 10 animals/sex/group once prior to the test material exposure (during acclimatisation, after grouping), and during weeks 2, 8 and 13 of the treatment period.

At the end of treatment period, on day 91 all animals were fasted overnight before necropsy. The first 5 rats/sex from each group were subjected to perfusion fixation method. The brain, spinal cord along with nerve fibres, eyes with optic nerve, sciatic nerve, tibial nerve and calf muscles were collected and preserved. From the last 10 rats/sex from each group, blood and urine samples were collected and analysed; animals were then subjected to detailed necropsy and specified organs were collected, weighed and preserved. The tissues/organs from animals of the high dose group (G5) and the control group (G1) were subjected to histopathological examination. Further, the brain from animals of the lower dose groups was evaluated because of microscopic changes in the high dose group.

The test material fortified diets at 10, 25 and 3000 ppm were stable for 15 days at room temperature. The test material fortified diets were prepared and used within the stability period of 15 days. Homogeneity and dose concentration verifications were carried out during week 1, month 2 and month 3 of the treatment period. All results obtained were considered acceptable as the mean results were within the range of 80 to 120 % to the nominal concentrations with relative standard deviations (RSD) of < 20 %.

No clinical signs of toxicity and mortality were observed in all tested dose groups up to 175 ppm (G5) in either sex. No treatment related changes were observed in mean body weight, body weight gain (percent change in body weight with respect to Day 1) and feed consumption in all tested dose groups up to 175 ppm (G5) in either sex. No ocular abnormalities were noted. No treatment related changes were noted in the neurological/functional observation battery test in all the tested dose groups.

No adverse treatment related changes were noted in haematology, clinical chemistry, coagulation and urinalysis parameters. No adverse treatment related changes were observed in thyroid hormones (T3, T4 and TSH). No treatment related changes were noted in fasting body weights, organ weights and their ratios.

No treatment related gross pathological changes were observed in any of the tested dose group animals.

Microscopically, test material-related changes were observed in the brain of three female rats of the high dose group (175 ppm). Histologically, changes consisted of neuronal necrosis characterised by cytoplasmic shrinkage with intense eosinophilia (red neuron) accompanied by karyorrhexis or karyolysis of nucleus in the piriform cortex, cornu ammonis (CA1) and dentate gyrus of hippocampus. Brains from male animals and female animals in the lower dose groups revealed normal histology.

Under the conditions of the study, the no observed-adverse-effect level (NOAEL) is 60 ppm in the diet (approximately 5.74 mg/kg/day) in females and 175 ppm in diet (14.96 mg/kg/day) in males.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
5.74 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The 28-day and 90-day neurotoxicity studies conducted by Kumar (2019 and 2020, respectively) were carried out on the registered substance itself in accordance with a standardised guideline under GLP conditions. The quality of the database is therefore considered to be high.

Effect on neurotoxicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Effect on neurotoxicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Following receipt of a Decision on a Testing Proposal the registrant has undertaken two repeated dose neurotoxicity studies. Initially conducted was a 28-day neurotoxicity study, also acting as a range-finder (OECD 424) and then followed a 90-day repeat dose oral toxicity study combined with a neurotoxicity test (OECD 408 and 424). As neurotoxicity is a known endpoint of concern for methyltin substances the two repeated dose studies proposed reflect this and were performed to specifically evaluate the neurotoxicity endpoint.


 


28-day repeat dose oral toxicity study combined with a neurotoxicity test, Kumar (2019)


A study was conducted to assess the neurotoxic (neurobehavioral and/or neurological abnormalities) effects of the test material in accordance with the standardised guideline OECD 424 under GLP conditions.


During the study, the test material was administered for a period of 29 consecutive days by the oral (dietary) route to Sprague Dawley rats. The doses were 0, 175, 500 and 700 ppm and each dose was administered to 10 rats per sex.


All animals were observed once daily for clinical signs and twice daily for mortality. Detailed clinical examination, body weight and feed consumption measurements were conducted weekly. Ophthalmoscopic examination was performed during acclimatisation on all animals, and during week 4 in all surviving animals. Neurological/Functional observational battery tests were performed once prior to treatment and during week 4 for all control and low dose group animals. At the end of treatment period, all surviving animals were sacrificed and subjected to gross pathological examination. The first 5 surviving rats/sex from each group were subjected to the perfusion fixation method.


No clinical signs of toxicity and mortality were observed in the control and low dose (175 ppm) group animals. Six out of 10 males and 5 out of 10 females of the mid dose (500 ppm) group died or were sacrificed before study termination. All males and females of the high dose (700 ppm) group died or were sacrificed before study termination. No treatment related changes were observed in mean body weight, body weight gain (percent change in body weight with respect to Day 1) and feed consumption in the low dose group. Treatment resulted in significant, markedly lower mean body weight, body weight gain and feed consumption in the mid and high dose groups.


No ocular abnormalities were noted. No treatment related changes were noted in the neurological/functional observation battery test in the low dose group animals (175 ppm).


Treatment related gross pathological lesions were observed in thymus and spleen of animals found dead or sacrificed moribund.


Microscopic evaluation revealed treatment related changes in the brain such as neuronal necrosis in different sites (granular cell layer of the olfactory bulb, piriform cortex and entorhinal cortex of the cerebrum, CA1, CA2, CA3 and dentate gyrus of hippocampus and Purkinje cells of the cerebellum). Neuronal necrosis was observed in all animals surviving to termination of the 500 ppm group. In 2/10 animals of the 175 ppm group the lesions were limited to minimal neuronal necrosis of the piriform cortex. Necrotic neurons showed cytoplasmic shrinkage with intense eosinophilia (red dead) accompanied by karyorrhexis or karyolysis of nucleus.


Histopathology of gross lesions of 500 ppm dose group animals sacrificed at termination of the study showed atrophy of thymus and spleen.


Analysis showed the diets to be stable and that the concentrations were within ± 20 % of nominal with relative standard deviations ranging between 0.21 and 2.67 %.


In conclusion, administration of the test material resulted in excessive toxicity at 500 and 700 ppm, manifested by clinical signs of toxicity and mortalities, severe reduction in body weights and feed consumption, and gross changes of thymus and spleen. Neuronal necrosis in the brain was noted at 500 ppm (9/9 animals; all surviving animals) and 175 ppm (2/10 animals) at histopathology evaluation. Under the conditions of this study, the no-observed adverse-effect level (NOAEL) was determined to be less than 175 ppm in the diet (approximately 20 mg/kg/day in males and 22 mg/kg/day in females).


 


90-day repeat dose oral toxicity study combined with a neurotoxicity test, Kumar (2020)


The repeated dose toxicity and neurotoxicity of the test material was investigated in a study which was conducted in accordance with the standardised guidelines OECD 408 and OECD 424, and under GLP conditions.


During the study, the test material was administered to groups of 15 male and 15 female Sprague-Dawley rats for a period of 91 consecutive days, by the oral (dietary route). Animals in groups G1, G2, G3, G4 and G5 received test material fortified diets at the doses of 0 (vehicle (acetone) control), 10, 25, 60, and 175 ppm in diet, respectively.


All animals were observed once daily for clinical signs of toxicity and twice daily for mortality. Detailed clinical examination, body weight and feed consumption measurements were conducted weekly. Ophthalmoscopic examination was performed during acclimatisation on all the animals and during week 13 for G1 and G5 group animals. A Neurological/Functional observational battery test was performed on the first 10 animals/sex/group once prior to the test material exposure (during acclimatisation, after grouping), and during weeks 2, 8 and 13 of the treatment period.


At the end of treatment period, on day 91 all animals were fasted overnight before necropsy. The first 5 rats/sex from each group were subjected to perfusion fixation method. The brain, spinal cord along with nerve fibres, eyes with optic nerve, sciatic nerve, tibial nerve and calf muscles were collected and preserved. From the last 10 rats/sex from each group, blood and urine samples were collected and analysed; animals were then subjected to detailed necropsy and specified organs were collected, weighed and preserved. The tissues/organs from animals of the high dose group (G5) and the control group (G1) were subjected to histopathological examination. Further, the brain from animals of the lower dose groups was evaluated because of microscopic changes in the high dose group.


The test material fortified diets at 10, 25 and 3000 ppm were stable for 15 days at room temperature. The test material fortified diets were prepared and used within the stability period of 15 days. Homogeneity and dose concentration verifications were carried out during week 1, month 2 and month 3 of the treatment period. All results obtained were considered acceptable as the mean results were within the range of 80 to 120 % to the nominal concentrations with relative standard deviations (RSD) of < 20 %.


No clinical signs of toxicity and mortality were observed in all tested dose groups up to 175 ppm (G5) in either sex. No treatment related changes were observed in mean body weight, body weight gain (percent change in body weight with respect to Day 1) and feed consumption in all tested dose groups up to 175 ppm (G5) in either sex. No ocular abnormalities were noted. No treatment related changes were noted in the neurological/functional observation battery test in all the tested dose groups.


No adverse treatment related changes were noted in haematology, clinical chemistry, coagulation and urinalysis parameters. No adverse treatment related changes were observed in thyroid hormones (T3, T4 and TSH). No treatment related changes were noted in fasting body weights, organ weights and their ratios.


No treatment related gross pathological changes were observed in any of the tested dose group animals.


Microscopically, test material-related changes were observed in the brain of three female rats of the high dose group (175 ppm). Histologically, changes consisted of neuronal necrosis characterised by cytoplasmic shrinkage with intense eosinophilia (red neuron) accompanied by karyorrhexis or karyolysis of nucleus in the piriform cortex, cornu ammonis (CA1) and dentate gyrus of hippocampus. Brains from male animals and female animals in the lower dose groups revealed normal histology.


Under the conditions of the study, the no observed-adverse-effect level (NOAEL) is 60 ppm in the diet (approximately 5.74 mg/kg/day) in females and 175 ppm in diet (14.96 mg/kg/day) in males.

Justification for classification or non-classification

The key data from the available 90-day toxicity combined with neurotoxicity study provides a no-observed adverse-effect level (NOAEL) of 60 ppm (approximately 5.74 mg/kg/day) in females and 175 ppm (14.96 mg/kg/day) in males, triggering classification with respect to repeated dose toxicity as STOT RE Category 2 (H373: May cause damage to nervous system and immune system through prolonged or repeated exposure).

However, in accordance with Annex VI, Regulation (EC) No 1272/2008, the substance has a harmonised classification with respect to repeated dose toxicity as STOT RE Category 1 (H372: Causes damage to nervous system and immune system through prolonged or repeated exposure).