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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Remarks:
REPEATED DOSE 90-DAY ORAL TOXICITY STUDY
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03-Feb-2016 to 06-Dec-2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline

Test animals

Species:
rat
Strain:
other: CD® / Crl:CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS:
- Strain: Rat / CD® / Crl:CD(SD)
- Number and sex: 100 animals (50 males and 50 females)
- Breeder: Charles River Laboratories
- Body weigth: Males 262.6 - 306.3 g, Females 195.3 - 228.1 g
- Age: 60 days at 1st administration
- Adaptation period: 6 days

ENVIRONMENTAL CONDITIONS:
- Diet: a certified commercial diet (ssniff® R/M-H V1534) ad libitum
- Water: tab water ad libitum
- Housing singly in MAKROLON cages (type III plus)
- Temperature: 22°C ± 3°C
- Rel. Humidity: 55% ± 15%
- Light/dark period: 12/12h

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
Frequency of administration: Once daily for 90 consecutive days
Administration volume: 10 mL/kg b.w./day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration immediately after preparation and at study termination: 93.4% - 109.5%
Stability (left at room temperature for 8 h or 24 h): 107.7% - 109.0%
Homogeneity at start of administration, during administration; and before administration to the last animal: 107.7% - 109.7%

The results indicate that all test item formulations were correctly prepared, were very homogeneous, and were stable for at least 24 hours.
Duration of treatment / exposure:
Duration of study:
6 adaptation days
90 test days
31 recovery days for scheduled animals
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Vehicle control
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
Low dose
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Remarks:
Intermediate dose
Dose / conc.:
400 mg/kg bw/day (actual dose received)
Remarks:
High dose
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
High dose
No. of animals per sex per dose:
50
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
CLINICAL SIGNS:
The animals were observed individually before and after dosing at each time of dosing for any signs of behavioural changes, reaction to treatment or illness, and additionally regularly throughout the working day. Cageside observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns.

NEUROLOGICAL SCREENING:
At the end of the treatment period, screening of sensory reactivity to stimuli of different types (e.g. auditory, visual and proprioceptive stimuli; based on Gad5), as well as the assessment of grip strength (Meyer6) and motor activity assessment was conducted for all animals.
In detail the test battery included testing of righting reflex, body temperature, salivation, startle response, respiration, mouth breathing, urination, convulsions, piloerection, diarrhoea, pupil size, pupil response, lacrimation, impaired gait, stereotypy, toe pinch, tail pinch, wire manoeuvre, hind leg splay, positional passivity, tremors, positive geotropism, limb rotation, auditory function.

MORTALITY:
Checks on mortality were made early in the morning and again in the afternoon of each day to look for dead or moribund animals.

BODY WEIGTH:
The weight of each rat was recorded at group allocation (test day -7), on test day 1 (before first administration), and once a week thereafter always on the same day of the week throughout the experimental period (test days 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, 85 and 90).

FOOD CONSUMPTION:
The quantity of food left by individual animals was recorded on a weekly basis throughout the experimental period. The food intake per animal (g/animal/week) was calculated using the total amount of food given to and left by each rat in each group on completion of a treatment week.

DRINKING WATER CONSUMPTION:
The drinking water consumption was recorded weekly by weighing the water bottles when filled and the residues upon removal at the end of the test week. Any residue was discarded. Weekly mean values per animal were calculated.

LABORATORY EXAMINATIONS:
Blood samples were taken from the retrobulbar venous plexus under isoflurane anaesthesia from animals fasted overnight. The blood samples were collected at the end of test week 13 (on test day 91, before necropsy) and were used for haematological investigations, coagulation tests, clinical chemistry tests, and for bile acid determination.

HAEMATOLOGICAL PARAMETERS:
Haemoglobin content (HGB), erythrocytes (RBC), leucocytes (WBC), reticulocytes (Reti), platelets (PLT), haematocrit value (HCT), absolute and relative differential blood count, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC).

COAGULATION PARAMETERS:
Thromboplastin time (TPT), activated partial thromboplastin time (aPTT).

CLINICAL CHEMISTRY PARAMETERS:
Albumin, globulin, albumin/globulin ratio, bile acids, bilirubin (total), cholesterol, creatinine, glucose, protein, triglycerides, urea (in blood), calcium, chloride, potassium, sodium, alanine amino-transferase (ALAT), alkaline phosphatase (aP), aspartate aminotransferase (ASAT), lactate dehydrogenase (LDH)

URINALYSIS:
Urine samples were collected from all animals fasted overnight on test day 91 (test week 13). Investigated parameters were volume, pH and specific gravity. The analytes of qualitative urinalyis were protein, glucose, bilirubin, urobilinogen, ketones, heamoglobin and nitrite. A microscopic examination of urine samples was carried out by centrifuging samples and spreading the resulting deposit on a microscope slide. The deposit was examined for the presence of epithelial cells, leucocytes, erythrocytes, organisms, further constituents (i.e. sperm, cassts), crystalluria.

OPHTHALMOLOGICAL AND AUDITORY EXAMINATIONS:
Examinations were performed pre-dose (test day 1, prior to first dosing) and in test week 13 (end of the treatment period, test day 90). The eyes were examined with a HEINE ophthalmoscope. Prior to examination, mydriasis was produced after instillation of STULLIN®10 eye drops into the conjunctival sacs.
The following ocular structures were examined:
• Adnexa oculi (i.e. lids, lacrimal apparatus), conjunctiva
• Cornea, anterior chamber
• Lens, vitreous body, fundus (retina, optic disc)
The auditory acuity was checked with a simple noise test.
Sacrifice and pathology:
SACRIFICE:
Starting on test day 91 (approx. 24 hours after the last administration), the animals were dissected following a randomisation scheme. Animals not dissected on test day 91, were dosed again on test day 91 and dissected on test day 92. The animals were euthanized by carbon dioxide (CO2), exsanguinated by cutting the aorta abdominalis, weighed, dissected, and inspected macroscopically.The weights of the following organs of all animals were determined before fixation: adrenal gland, kidney, spleen, uterus (incl. cervix), brain, liver, testicle, prostate and seminal vesicles (with coagulating glands as a whole), epididymis, ovary, thymus, heart, pancreas. The paired organs (adrenal glands, gonads and kidneys) were identified as left or
right and weighed individually.
The organs or parts of organs of all animals were fixed in 7% buffered formalin. Eyes were preserved in Davidson's solution and testes in Bouin's solution for optimum fixation.

HISTOPATHOLOGY:
The organs listed below of all animals of groups 1 and 4 (control and high dose) were examined histologically after preparation of paraffin sections and haematoxylin-eosin (H & E) staining:
Adrenal gland (2)
Aorta abdominalis
Bone (os femoris with joint)
Bone marrow (os femoris)
Brain (cerebrum, cerebellum, brain stem)
Epididymis (2)
Eye with optic nerve (2)
Gross lesions observed
Heart (left and right ventricle, septum)
Intestine, small (duodenum, jejunum, ileum, incl. Peyer's patches, Swiss roll method)
Intestine, large (colon, rectum)
Kidney and ureter (2)
Liver
Lungs (with mainstem bronchi and bronchioles, preserved by inflation with fixative and then immersion)
Lymph node (1, cervical)
Lymph node (1, mesenteric)
Mammary gland
Muscle (skeletal, leg)
Nerve (sciatic)
Oesophagus
Ovary and oviducts (2)
Pancreas
Pituitary
Prostate and seminal vesicles with coagulating glands
Salivary glands (mandibular, parotid, sublingual)
Skin (left flank)
Spinal cord (3 sections)
Spleen
Stomach
Testicle (1)
Thymus
Thyroid (2) (incl. parathyroids)
Tissue masses or tumours (including regional lymph nodes)
Trachea (incl. larynx)
Urinary bladder
Uterus (incl. cervix)
Vagina

In addition, frozen sections of the heart, liver and one kidney were prepared, stained with Oil Red O, and examined microscopically, and the stomach and the kidneys (2) of the animals of group 2 (low dose) and group 3 (intermediate dose) were examined microscopically following H & E) staining.
Furthermore, a detailed histomorphological examination was performed on one testicle and one epididymis of all male animals of groups 1 to 4 (control and all dose groups) following H & E and PAS staining (with special emphasis on the qualitative stages of the spermatogenesis and the histopathology of the interstitial testicular structure). Sperm count an viabilty was assessed and sperm morphology was examined for all male animals.
Statistics:
Data for toxicology and pathology were captured, as far as possible, using the departmental computerized systems (Provantis® Integrated preclinical software, version 9.4.0, Instem LSS Ltd., Stone, Staffordshire ST15 0SD, United Kingdom). Raw data not fully compatible with the computerized systems were maintained on paper according to appropriate SOPs. The test item-treated groups 2 to 4 were compared with the control group 1.
The following statistical methods were used for the data captured with the Provantis system: Multiple t-test based on DUNNETT, Exact Test of R.A. Fisher.

Homogeneity of variances and normality of distribution were tested using BARTLETT's test and the SHAPIRO-WILKS test. In case of heterogeneity and/or non-normality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), intergroup comparisons with the control group were made by DUNNETT's test (see above).

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
None of the male and female rats repeatedly treated orally with 50 or 200 mg TMT/kg b.w./day revealed any changes in behaviour, external appearance, consistency of faeces, external appearance, body posture, movement and coordination capabilities. In the high dose group (400/500 mg/kg b.w./day) slight salivation was noted in all animals. Further clinical symptoms observed at the high dose are considered to be test item-related (piloerection, swelling of the left ear, necrosis of the ears, inflammation of the tail, necrosis of the tail tip, missing tail tip, inflammation of the scrotum, reddened mucous membranes); see also gross pathology. Skin lesions and inflammation persisted in the recovery period.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Two decedent animals were reported in the high dose group on test days 33 and 60. As a consequence the high dose was reduced from 500 to 400 mg/kg.
The macroscopic inspection at necropsy revealed a reddish stained thymus, a pale pancreas, dark red stained lungs, and a haemorrhagic mucosa in the stomach for both prematurely deceased animals. The microscopic examination revealed a moderate congestion in the lungs that correlated to the macroscopic finding of dark red stained lungs, as well as slight pulmonary edema and presence of eosinophilic material in the bronchi/bronchioles. All findings reported might be visible also following gavage accidents. Hence, the cause of death remains unclear.

Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No test item-related influence was observed on the body weight and the body weight gain of the male and female animals repeatedly treated orally with 50 or 200 mg TMT/kg b.w./day compared to the control animals throughout the treatment period. No test item-related differences were noted for the body weight at autopsy between the animals treated with the low or intermediate dose and the control animals.

No test item-related influence was noted for the body weight, body weight gain, and body weight at autopsy of the female high dosed animals during and at the end of the treatment period. The body weight of the low dosed and of the high dosed animals appeared to be slightly reduced by approx. 6% (not statistically significant at p ≤ 0.05 or p ≤ 0.01) compared to the control group at the end of the treatment period. However, no noteworthy differences in body weight were noted for the animals of the intermediate dose group compared to the control group during the course of the study. As no dose-response-relationship was observed, the slight changes at the low and at the high dose are considered coincidental.

The body weight of the male animals treated with 500/400 mg TMT/kg b.w./day was slightly reduced by up to 11.2% during the treatment period (statistically significant at p ≤ 0.05 or p ≤ 0.01 from test day 15 to test day 64). Accordingly, the body weight gain was reduced by approx. 16%, and the body weight at autopsy was approx. 10% lower compared to the control.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No test item-related influence was observed on the food consumption of the male and female animals treated orally with 50, 200 or 500/400 mg TMT/kg b.w./day compared to the control animals throughout the treatment period and the recovery period.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Description (incidence and severity):
The ophthalmological examination did not reveal any test item-related changes of the eyes and the optic region in any animal after repeated oral treatment with 50, 200 or 500/400 mg TMT/kg b.w./day in test week 13 (test day 90) and test week 17 (test day 121). No test item-related pathological changes were noted on the adnexa oculi (i.e. lids, lacrimal apparatus), conjunctiva, cornea, anterior chamber, lens, vitreous body, and fundus (retina, optic disc).
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related influence was observed on the haematological parameters for the male and female animals treated orally with 50, 200 or 500/400 mg TMT/kg b.w./day compared to the control animals at the end of the treatment period (test day 91, only the main study animals) and at the end of the recovery period (test day 122).
A statistically significant decrease in platelet count was observed in males of the high-dose group on test day 91, however, the effect is supposed to be due to the relatively low value observed for the control group. Furthermore, monocytes were reduced in males of the high-dose group on test day 91, and MCHC was increased at the end of the recovery period in high-dose females. For both changes the difference to the control group is considered to have no toxicological significance. Therefore, the haematological findings are not considered to be treatment-related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related influence was observed on the biochemical parameters for the male and female animals treated orally with 50, 200 or 500/400 mg TMT/kg b.w./day compared to the control animals at the end of the treatment period (test day 91, only the main study animals) and at the end of the recovery period (test day 122).

A statistically significant decrease in triglycerides was observed in males of the high-dose group on test day 91, however, the effect is supposed to be due to the relatively low value observed for the control group. Furthermore, glucose was reduced in males of the high-dose group on test day 91, and potassium was increased at the end of the recovery period in high-dose females. For both changes the difference to the control group is considered to have no toxicological significance. Therefore, the findings in clinical biochemistry are not considered to be treatment-related.

Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
There were statistically significant changes noted in form of an increase by 1% for the specific gravity and an increase by 7% for the pH value of the urine of the male main study animals at the end of the treatment period. It is deemed that these changes are related to the copper excretion into the urine; large portions of the pigments were found within the tubules. It remains unclear, for what reason in a number of animals (females from groups 2-4; males from group 4), a high erythrocyte count was observed. However, a treatment-relationship is likely.
Behaviour (functional findings):
no effects observed
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Minor differences in organ weight changes are not deemed to be attributable to treatment with the test item.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The macroscopic inspection at necropsy did not reveal any test item-related changes in the organs and tissues of the animals treated orally with 50, 200 or 500/400 mg TMT/kg b.w./day after terminal sacrifice at the end of the treatment period (test day 91 or 92) and at the end of the recovery period (test day 122) except for necrotic skin lesions in high dose animals.
Necrosis was observed in the ears of four animals per sex of the high dose group, respectively. Furthermore, there was necrosis of the tail or missing tip of tail in one male and three females of the high dose group. In addition, there were skin lesions consisting of tail base inflammation or scrotal inflammation in one high dose male, respectively.
Neuropathological findings:
no effects observed
Description (incidence and severity):
No test item-related influence was noted on any of the parameters examined during the functional observation tests, on the fore- and hind limb grip strength, or on the spontaneous motility for any of the male and female animals after repeated oral treatment with 50, 200 or 500/400 mg TMT/kg b.w./day in test week 13 and in test week 17.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The histomorphological examination of a variety of organs and tissues was restricted to the control group 1 and the high dose group 4 treated with 500/400 mg TMT/kg b.w./day. However, the kidneys of the male and female animals of all dose groups were examined histologically. In animals of all treatment groups including recovery, significant changes were observed in the kidneys. Yellow-brown pigment accumulation was observed in both in the proximal and distal tubules of the kidneys of these animals. The majority of the pigment was located at the apical part of the tubular epithelial and within the tubular lumen without affecting the integrity of the cells.
Different special stains were applied. The pigment was negative for bile pigment (Hall’s stain) and iron (Perl’s stain). Staining by the PAS method for acid mucopolysacharides revealed a slight positivity that is deemed to be partly due to the dense accumulation of larger depositions but is considered partly also due to the adhesion or morphological relationship to acidic mucopolysacharides. In Schmorl’s stained sections, the pigment was strongly positive and stained dark blue-green. Thus, although this staining is considered to be unspecific, the pigment was initially supposed to be lipofuscin.
Lipofuscin is a fine yellow-brown pigment that forms granules composed of lipid-containing residues of lysosomal digestion. Hence, this pigment is stored in tertiary lysosomes. Tertiary lysosomes have limited dimensions and form round structures. They are located intracytoplasmically surrounding the nucleus at a random distribution. However, since the pigment was formed by polygonal structures forming clumps, the pigment morphology, but also the localization was atypical for lipofuscin.
There was no specific morphological lesion in the kidneys that could be related to the pigment storage. In order to evaluate the nature of the pigment in kidneys, the samples were evaluated by scanning electron microscopy and energy dispersive X-ray (EDX) analysis. As a result, in test item treated animals, in all evaluated spots copper (Cu) was detected at mean weight percentages of 3-4%. Since the test item is employed in the formation of metal complexes, the observed pigment deposition most probably represents a copper chelate complex.
Histopathological findings: neoplastic:
no effects observed

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
gross pathology
histopathology: non-neoplastic
Dose descriptor:
LOEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: Not regarded adverse as independent effect.

Target system / organ toxicity

open allclose all
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
400 mg/kg bw/day (actual dose received)
System:
other: skin
Organ:
skin
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Critical effects observed:
no
Lowest effective dose / conc.:
50 mg/kg bw/day (actual dose received)
System:
other: kidney

Any other information on results incl. tables

No influence was noted on the sperm count (number of ultrasound-resistant spermatids per gram testicular tissue) and on the sperm motility (determined as percentage of motile spermatozoa in the epididymal cauda of the males) for any of the test item-treated groups in comparison to the control group in test week 13 (end of treatment) and in test week 17 (end of recovery). Furthermore, the examination of the sperm morphology did not reveal any test item-related

differences between the control group and the test item-treated groups in test week 13 (end of treatment) and in test week 17 (end of recovery).

The results of the sperm viability determination and the sperm morphology examination is supported by the fact that the microscopic evaluation did not reveal any test item related effects on the male reproductive organs at the histomorphological level.

Applicant's summary and conclusion

Conclusions:
Oral treatment with 1,3,5-triazine-2,4,6-trithione trisodium salt (TMT) for 90 days did not cause any effects of biological or toxicological significance at the low and intermediate doses of 50 and 200 mg TMT/kg b.w./day.
Based on the accumulation of a yellow-brown pigment in the proximal and distal tubules of the kidneys in all treated animals, which was identified as a copper chelate complex, a NOEL could not be established; the low dose of 50 mg/kg was considered to be a LOEL instead. As cell integrity of the cells was not affected, the effect was not regarded to be adverse. However, in combination with the presence of inflammations and/or necrosis in the ears, tails and scrotum of several animals at 500/400 mg/kg, which were likely caused by a copper deficiency, and the adverse nature of these findings, the NOAEL was established at 200 mg/kg.
Executive summary:

In the 90-day study with the test item TMT in rats, animals were treated daily by gavage at doses of 50, 200, or 400 mg/kg (initial dose of 500 mg/kg was reduced). As a result, two major issues were reported: 1) clinical signs, i.e. gross pathology effects (necrosis of ears and tail (tip), inflammation of the tail and scrotum) in individual high-dose animals, and 2) accumulation of a yellow-brown pigment in the proximal and distal tubules of the kidneys in all treated animals. The majority of the pigment was located at the apical part of the tubular epithelial and within the tubular lumen. It was formed by polygonal structures forming clumps. As the test item TMT is employed in the formation of metal complexes, a special energy dispersive X-ray (EDX) technology was used on parts of the formalin-fixed kidneys to determine the chemical compositon of the deposited pigment. As a result, copper turned out to be a major component of the pigment. The complex formation with copper likely caused a copper deficiency that resulted in the described skin inflammatory lesions at the high dose group, which has to be considered adverse. Minor changes in the hematology and clinical chemistry parameters could be related to these inflammatory lesions.

There was no further specific morphological lesion in the kidneys that could be related to the pigment storage. However, there were statistically significant changes noted in form of an increase by 1% for the specific gravity and an increase by 7% for the pH value of the urine of the male main study animals at the end of the treatment period that are related to pigment excretion.

Two decedent animals were reported. The gross lesions were unremarkable and also histopathology evaluation did not reveal specific indications for systemic toxicity. All findings reported might be visible also following gavage accidents. Hence, the cause of death remains unclear.