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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 March 2020 - 02 February 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
25th June 2018
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetra(isobutyl)thioperoxydicarbamic acid
EC Number:
221-312-5
EC Name:
Tetra(isobutyl)thioperoxydicarbamic acid
Cas Number:
3064-73-1
Molecular formula:
C18H36N2S4
IUPAC Name:
tetra(isobutyl)thioperoxydicarbamic acid
Test material form:
other: light-yellow powder
Specific details on test material used for the study:
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Willing New Materials Technology Co., Ltd; 23191022201
- Expiration date of the lot/batch: 21 October 2020
- Purity: 97%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25℃) protected from humidity (in a tightly closed container)

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source:Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633, Sulzfeld, Germany
- Age at study initiation:Young adult rats were 7 weeks old at start of dosing.
- Weight at study initiation:Males: 286 – 323 g, females: 192 – 251 g; did not exceed ± 20 % of the mean weight for each sex at onset of treatment.
- Housing: Type II and/or III polycarbonate cages with SAFE ¾ -S (produced by J. Rettenmaier & Söhne GmbH + Co.KG, Germany) Bedding for Laboratory Animals and nest building material (SAFE crinklets natural (produced by J. Rettenmaier & Söhne GmbH + Co.KG, Germany)) bedding and nesting. Rodents were housed up to 3 animals of the same sex and group/cage. Additional enrichment (GLP Mini Fun Tunnel, produced by LBS (Serving Biotechnology) Ltd., United Kingdom) was also used during the study.
- Diet (e.g. ad libitum) and water (e.g. ad libitum): :Animals received ssniff® SM R/M "Autoclavable complete diet for rats and mice –breeding and maintenance" (Batch no.: 232 58297, Expiry date: 30 June 2020) produced by ssniff Spezialdiäten GmbH (D-59494 Soest Germany), ad libitum, and tap water from municipal supply, as for human consumption from 500 mL bottles, ad libitum.
- Acclimation period:5 days (males), 6 days (females)

DETAILS OF FOOD AND WATER QUALITY:
The food was removed overnight prior to necropsy. The supplier provided analytical certificate for the batch used, which is archived with the raw data. Water quality control analysis was performed once every three months and microbiological assessment was 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 were archived with the raw data at Charles River Laboratories Hungary Kft.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):20.8 – 26.2 °C
- Humidity (%):24 – 70 %
- Air changes (per hr):15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light):12 hours daily, from 6.00 a.m. to 6.00 p.m.

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The test item formulation or vehicle were administered at a single dose to the animals by oral gavage.
Vehicle:
methylcellulose
Remarks:
1% (w/v) aqueous methylcellulose solution
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The dose formulations or vehicle were administered daily starting from Day 1 for 90 consecutive days by oral gavage, using a bulb tipped gastric feeding tube attached to a syringe. The formulations were stirred with a magnetic stirrer during dosing. A constant dose volume of 5 mL/kg bw/day was administered to all animals. The actual volumes to be administered were calculated and adjusted based on the most recent individual body weight. Control animals were treated concurrently with the vehicle only.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on the results of the previous experimental work (Test Facility study code 17/053-001P) and a short solubility test performed at the Test Facility, and the analytical method development (Charles River Laboratories Hungary Kft. study code 19/306-316ANE), 1% (w/v) methylcellulose solution (abbreviation: 1% MC) was selected as vehicle for this study.
- Concentration in vehicle: The test item was formulated in 1% methyl cellulose at the appropriate concentrations according to the dose levels selected.
- Lot/batch no. (if required): Batch number: 7105702
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 applied during the study (concentration range and storage conditions of the dose formulations pending use, according to analytical validation: Charles River Laboratories Hungary Kft. study code: 19/306-316AN). During the method validation 7 days of stability of the Test Item in the selected vehicle was obtained. The Test Item proved to be stable on room temperature in the concentration range of 10-210 mg/mL.

The Test Item was formulated in 1% methyl cellulose at the appropriate concentrations according to the dose levels selected, in the Pharmacy of Charles River Laboratories Hungary Kft. The formulations were prepared fresh prior to administration to animals.

Analysis of test item formulations for concentration and homogeneity was performed using a High-Performance Liquid Chromatography method with UV detection (HPLC-UV) at the Test Facility (19/306-316AN). Top, middle and bottom duplicate samples were taken from test item formulations four times during the study (during first, fifth, tenth and thirteenth week of the treatment of males), one set to analyse and one set as a back-up. Similarly, duplicate samples were taken from the middle of the vehicle control formulation for concentration measurement.

Acceptance criteria of the concentration analysis were set according to the analytical method validation, expected to be at 100 ± 15 % of the nominal concentration.

Acceptance criteria of the homogeneity was that the CV of replicates (top, middle and bottom of test item formulations) to be less than 10%.


Duration of treatment / exposure:
90 days
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control (Vehicle)
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Low dose
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Mid dose
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
High dose
No. of animals per sex per dose:
10 males and 10 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In a 14 day oral gavage dose finding test up to 1000 mg/kg bw/day, there were no clear adverse effects of test item treatment. Therefore the doses selected for the main test were 100, 300 and 1000 mg/kg bw/day. Refer to Supporting, RL1, rat/Willing, 2020/Repeated dose toxicity (DRF).003.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: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 weekly thereafter, in the morning hours (am) and once before necropsy.

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/self-mutilation, backward motion, abnormal vocalization, 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.

CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day). General clinical observations were made twice a day before and after treatment, considering the peak period of effects, if any, observed on Day 1 after dosing, at the beginning and towards the end of the working day as practical, in case no clinical changes should be noted. Only one general clinical observations were made on those days when detailed clinical observations were registered

BODY WEIGHT: Yes
- Time schedule for examinations:Body 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 weekly, including on Day 90, and prior to necropsy, fasted on Day 91

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
The 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 weekly from Day 8 with a precision of 1 g. Daily food consumption were calculated for reporting purpose.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule:Ophthalmoscopic examination was conducted in all animals before treatment (Day -1/-2), and in the Control group and High dose group animals during Week 13 (Day 87/88). Mydriasis was produced after instillation of eye drops "Cicloplegicedol" (10 mg/mL cyclopentolate hydrochloride) into the conjunctival sac. The evaluation was performed using an Omega 500 ophthalmoscope. As in Week 13 no treatment related alterations were found in the Control and High dose group animals, the remaining animals were not examined at termination.

HAEMATOLOGY: Yes
At the end of the treatment period, prior to scheduled necropsy on Day 91, haematology investigations were conducted in all animals. 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). The parameters evaluated in all animals are in the table below.

CLINICAL CHEMISTRY:
At the end of the treatment period, prior to scheduled necropsy on Day 91 clinical biochemistry investigations were conducted in all animals. After an overnight period of food deprivation of animals, 3 blood samples were collected by heart puncture under pentobarbital anaesthesia with one to obtain serum (in tubes with no anticoagulant). The parameters evaluated in all animals are in the table below.

URINALYSIS: Yes / No / Not specified
Food was withdrawn during the overnight urine collection. Urine collection was conducted from all surviving animals 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 parameters evaluated in all animals are in the table below.

NEUROBEHAVIOURAL EXAMINATION: Yes
Towards the end of the treatment period, during Week 13 (Day 84-86), 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.

OTHER: For thyroid hormone analysis, blood samples were taken from all animals at termination by sublingual venepuncture into tubes containing K3-EDTA as anticoagulant. The levels of the thyroid hormone (T3 and T4) and thyroid stimulating hormone (TSH) were determined respectively in plasma by LC-MS/MS or Luminex MAP® technology in Charles River Laboratories Evreux, France. For more details on the analytical procedure, refer to below..


Sacrifice and pathology:
GROSS PATHOLOGY: Yes.
Necropsy and macroscopic examination were performed on all animals, at the end of treatment period, on Day 91 (after the blood sample collection). The animals were euthanized by exsanguination under pentobarbital anaesthesia. After 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, but not stained and analysed.

The organs listed in the table below were trimmed of fat and weighed in all animals.

After the organ-weight measurement of testis and epididymis, one of each organ were collected for sperm evaluation as described below. Enumeration of homogenisation-resistant testicular spermatids and cauda epididymal sperm reserves were performed. In addition, sperm from the cauda epididymis were collected for determination of motility and sperm morphology. For measurement of sperm morphology, at least 200 sperm per sample were evaluated from fixed, wet preparations and classified as either normal or abnormal (fusion, isolated heads, misshapen heads and/or tails). For enumeration of spermatids, caudal epididymal reserves and morphology, samples from Control, Low, Mid and High-dose males were evaluated. Sperm motility was evaluated immediately after sacrifice. The percentage of progressively motile sperms was determined subjectively. The smears for morphology were prepared and stored for later analysis, as were the testes and epididymides for homogenisation-resistant sperm counts. Analyses on these parameters were done on the Control and High dose group.


HISTOPATHOLOGY: Yes.
On completion of the macroscopic examination the tissues and organs listed in the table below were retained from all animals. The retained tissues and organs were embedded in paraffin wax, sections were cut at 4 6 µm 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). In addition, any organs or tissues with macroscopic abnormalities (except minor changes) were subjected to histological examination from all groups.
Statistics:
The statistical evaluation of appropriate data was performed with the statistical program package of SAS 9.2 software package (within the validated Provantis system). The following decision tree was applied automatically for statistical evaluation of continuous numeric data.

The normality and heterogeneity of variance between groups were 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, Dunnett’s (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 is 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 is 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 were performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate.

For non-continuous data, the Cochran-Armitage test for trend was applied and the Chi-squared test was used for statistical differences relative to control.

For pathology data (macroscopic and microscopic data) the Cochran-Armitage test for trend was applied, then if appropriate, the Chi-squared test homogeneity test. If significance was plausible based on a user-defined value (0.05), a pairwise test of each treatment group versus the control group was made. If the group size was <5 then Fisher’s Exact Test was used, if the group sizes were bigger then the Chi-squared test was used; identifying differences of <0.05, <0.01 or <0.001 as appropriate.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test item related clinical signs were noted in any animals during the study. Summary tables are presented in Appendix 1.1.1.
Mortality:
no mortality observed
Description (incidence):
No animals were found dead during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant lower body weight (-11.2%, p<0.01) and body weight gain (-23.7 %, p<0.01) was seen in High dose males, with the difference between controls and High dose developing progressively during the study. A very similar trend was seen in High dose females but generally without statistical significance (-24.3% lower overall weight gain). When compared with historical control data, the concurrent control matched the historical mean for most of the study; the High dose treatment related effect in males and females was confirmed. The growth rate of body weight and body weight gain of the High dose males and females was below the historical control minimum value. Summary tables are presented in Appendices 1.2.1. and 1.2.2.

The Mid and Low dose groups were not affected.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no test item-related differences in the mean daily food consumption in all treated groups when compared to the controls. Summary tables are presented in Appendix 1.3.
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 treatment-related changes compared to pre-treatment were noted at ophthalmoscopy examination in males or females. Summary tables are presented in Appendix 1.1.2.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No clearly adverse treatment-related changes were observed in the haematology or coagulation parameters in males or females. High dose males had statistically significantly lower (p<0.01) RBC count, however the percentage difference was relatively low (<6%). No change was seen in female RBC count, however, mean values of all groups, including Controls, were slightly outside of the historical control range. Other statistically significant differences were considered to be incidental, were not related to dose and/or the recorded values were within the historical control ranges. These differences were considered not to reflect an adverse effect of the test item. Summary tables are given in Appendix 1.5.1.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The clinical chemistry parameters were considered not to show any clearly adverse effects in males or females. The observed statistically significant differences were not consistent between sexes, and did not correlate with any histopathology changes. These statistical differences were considered not to reflect an adverse effect of the test item. Summary tables are given in Appendix 1.5.2.
Urinalysis findings:
no effects observed
Description (incidence and severity):
The urine parameters were considered normal in all study groups. Summary tables are given in Appendix 1.5.3
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
At the functional observation battery (FOB) performed at the end of exposure (Week 13), there were no changes in animal behaviour, general physical condition, grip strength, foot splay, motor activity, or in the reactions to different type of stimuli in the control or treated groups. The locomotor activity of all groups showed a normal response, initially high then reducing to a plateau by about 30 minutes. Sporadic statistically significant at some of the intervals were not an indication of a treatment effect. Summary tables are presented in Appendices 1.4.1. – 1.4.4.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The terminal body weights of High Dose males were statistically significantly lower (11.6%; p<0.01) and the females were lower (9.1%) but not statistically significantly, compared to controls.

There were no test item-related statistically significant differences among groups in the weights of organs measured when compared to controls in the study. Statistically significant organ weight increases were recognized in the absolute (11.3%) and relative to body (27.1%) and brain (14.0%) weight of the kidney in High Dose males, but without microscopic relevance. The other statistically significantly increased organ weight changes in the High dose males and females (the relative organ weight to body of brain, spleen, epididymis, testis, prostate and pituitary in males and kidney, liver in females) without microscopic changes can be correlated with body weight loss in this dose group. The statistically significantly lower thymus and thyroid/parathyroid weight in High Dose males did not correlate to any microscopic changes and can be considered as incidental. The ovary weight (relative to body) increased by 25% compared with controls in Mid dose females and the absolute organ weight of the seminal vesicle decreased by 12.8% in Low dose males without microscopic changes and dose relation can be considered as incidental. In summary, the statistical differences in organ weights did not reflect an adverse effect of the test item. Most statistical differences were a result of the lower High dose body weight or were not part of a dose response. Other statistical differences were generally within the normal historical control range and without any histological change and are hence considered not to reflect a test item effect.

The only changes that are considered to be potentially related to the test item are statistically significant (p < 0.01) increased kidney weights (27% (outside historical control range) and 15% in High dose males and females respectively when adjusted for body weight) and liver weight in High dose females (20% when adjusted for body weight). However, there were no histopathological changes in these organs, indicating that these differences were common adaptive changes, not considered to be adverse. Summary data are presented in Appendix 1.7.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related macroscopic findings were detected at necropsy.Summary tables are presented in Appendix 1.6.

No treatment-related changes were observed in the sperm number, morphology or motility in any of the treated males. Summary tables are presented in Appendix 1.1.3

Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related microscopic findings were detected in any tissues or organs. Specifically, there were no microscopic findings in the male/female reproductive organs and in the thyroid/parathyroid system. The harderian metaplasia both in Control and High dose males with the same incidence and severity is a common background finding mainly in male rats. A few findings, without meaningful differences in severity and incidence between groups were considered to be incidental or a common background. Summary tables are presented in Appendix 1.8.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related differences in thyroid hormone concentrations (T3, TSH) were observed in males or females. Statistically significantly lower T4 values in High dose males were not considered as a test item-related effect, as there were no correlating histopathology finding and sperm sample analysis values were normal in all male groups. All groups were considered to be normal. Summary data are presented in Appendix 1.9.

Effect levels

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
body weight and weight gain

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

Dose formulation analysis

All test item formulations were shown to be homogeneous. The measured concentrations of WILLING TiBTD evaluated for each test item-dose group varied between 94% and 104%. No test item was detected in the control samples. These results were within the acceptable ranges (85% - 115%) and were considered suitable for the study purposes. Based on the study sample analysis, and the validation date, the concentration, homogeneity and stability of the dose formulations were considered to be fully satisfactory. The results of the dose formulation including concentration and homogeneity analysis are presented below.

Formulation occasion

Nominal concentration (mg/mL)

Measured concentration
(mg/mL)

Percentage of the nominal concentration
(%)

RSD
(%)

10 March 2020

Control

not detected

-

-

20

19.67 ± 0.257

98

1.2

60

60.85 ± 0.852

101

1.3

200

207.37 ± 2.49

104

1.1

07 April 2020

Control

not detected

-

-

20

19.23 ± 0.362

96

1.8

60

57.97 ± 0.660

97

1.1

200

200.73 ± 1.86

100

0.9

05 May 2020

Control

not detected

-

-

20

19.27 ± 0.307

96

1.5

60

58.15 ± 0.834

97

1.4

200

195.36 ± 2.08

98

1.0

02 June 2020

Control

not detected

-

-

20

18.74 ± 0.307

94

1.6

60

57.68 ± 1.87

96

3.1

200

194.21 ± 1.91

97

0.9

All formulations proved to be homogeneous. Acceptance criteria for homogeneity is that the relative standard deviation (RSD%) of the replicates must be less than 10%.

Applicant's summary and conclusion

Conclusions:
In conclusion, under the conditions of this study, the no observed adverse effect level (NOAEL) for systemic toxicity in male and female Wistar rats for WILLING TiBTD is considered to be 300 mg/kg bw/day (based on a reduction in body weight at the High dose level).
Executive summary:

In a repeated dose toxicity study (OECD 408/GLP), WILLING TiBTD (97%) was administered to Crl:WI Wistar rats (10/sex/dose) by gavage in 1% (w/v) aqueous methylcellulose at dose levels of 0, 100 (low dose), 300 (mid dose) and 1000 (high dose) mg/kg bw/day daily for 90 days.

All test item formulations were shown to be homogeneous and stable at room temperature. The measured concentrations of WILLING TiBTD evaluated for each test item-dose group varied between 94% and 104%.

A statistically significant lower body weight (-11.2%) and body weight gain (-23.7%) was seen in High dose males with the difference between controls and High dose appearing later in the study. The overall High dose weight gain was significantly affected in males. A similar trend was seen in High dose females (-24.3% lower overall weight gain) but generally without statistical significance. The High dose group growth rates were below the historical control minimums in both sexes; the lower growth rates were attributed to a treatment effect. The Mid and Low dose groups were not affected. There were no significant test item-related differences in the mean daily food consumption in treated groups when compared to the controls.

The functional observation battery (FOB) showed no changes in animal behaviour, general physical condition, grip strength, motor activity, or in the reactions to different type of stimuli in the control or test groups. No treatment-related changes were noted on ophthalmoscopy examination.

No clearly adverse treatment-related findings were seen in the clinical pathology parameters (haematology, clinical chemistry, urinalysis). High dose males had a statistically significant RBC count close to the historical control minimum; however, the percentage difference was relatively low (<6%). No change was seen in females in RBC count, however, mean values were slightly out of the historical control range (also in the Control group). Other statistically significant differences in haematology and clinical chemistry were considered to be incidental, were not related to dose and/or the recorded values were within the historical control ranges. These differences were considered not to reflect an adverse effect of the test item.

No adverse treatment-related macroscopic or microscopic findings were detected at necropsy. The statistical differences in organ weights did not reflect an adverse effect of the test item. Most statistical differences were a result of the lower High dose body weight or were not part of a dose response. The only changes that are considered to be potentially related to the test item are increased relative kidney weights in High dose males and females and relative liver weight in High dose females. However, there were no histopathological changes in these organs, indicating that these differences were common adaptive changes, not considered to be adverse.

No treatment-related changes were observed in the sperm number, morphology and motility in any of the treated males, or in thyroid hormone levels (T3, T4, TSH) in males or females. None of the required endpoints recommended for detection of endocrine activity per the guideline (organ weights, histopathology, serum/plasma biochemistry/hormone analysis) gave indications of concern.

In conclusion, under the conditions of this study, the no observed adverse effect level (NOAEL) for systemic toxicity in male and female Wistar rats for WILLING TiBTD is considered to be 300 mg/kg bw/day (based on a reduction in body weight at the High dose level).

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