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Toxicological information

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:
July 2021 - June 2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to other study
Remarks:
dose-range finding study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2016-2017
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 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 Jul 2016
Deviations:
no
Remarks:
including 14-day recovery
GLP compliance:
yes (incl. QA statement)
Remarks:
Experimental Toxicology and Ecology, BASF SE, Ludwigshafen, Germany
Limit test:
no
Specific details on test material used for the study:
Physical state/ appearance: solid / rose
Storage conditions: room temperature
Homogeneity: given visual
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Wistar Rat, Crl:WI(Han)
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 12 - 13 weeks (males), 10 - 11 weeks (females)
- Weight at study initiation: 215 - 218 g (females) and 407 -413 g (males)
- Housing: The rats were housed individually in Makrolon type M III cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area of about 800 cm²). Pregnant animals and their litters were housed together until day of parturation (PND) 4 (end of lactation).
- Diet: ground Kliba maintenance diet mouse-rat “GLP”, meal, Provimi Kliba SA, Kaiseraugst, Switzerland, ad libitum
- Water: ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

In-life phase 27 April 2016 - 29 July 2016

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% in water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test material was applied as a suspension. To prepare this suspension, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, 0.5% Carboxymethylcellulose suspension in drinking water was filled up to the desired volume and intensely mixed with a homogenizer. During administration, the preparations was kept homogeneous with a magnetic stirrer. The test substance preparations were produced weekly, at least.
- The administration volume was 10 ml/kg bw.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration and homogeneity control of the test substance preparations was performed via total nitrogen determination (Kjeldahl).
The stability of the test substance in 0.5% Carboxymethylcellulose suspension in drinking water was demonstrated over a period of 7 days at room temperature.
Considering the low relative deviation of results within the three samples of one test substance preparation in the homogeneity analysis, it can be concluded that the test material was distributed homogeneously in 0.5% Carboxymethylcellulose suspension in drinking water.

The concentrations of the test material in 0.5% Carboxymethylcellulose suspension in drinking water were found to be in the range of 78 to 91% of the nominal concentrations. The analytical method used the solid test material as standard to assess the concentration in the frozen aqueous formulations. Spiked controls to assess the influence on test material storage and loss via absorption were not included. The determined rangeis partially below the strict specification of the test facility (90-110%), but the overall average of 84% was within generally accepted specification for concentration analysis in complex matrixes like the diet (80-120%). The authors assessed the overall accuracy of the prepared concentrations as given.
Duration of treatment / exposure:
28 days (males)
up to 63 days (females)
Frequency of treatment:
daily
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
analytically verified 840 mg/kg bw
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on a 14-day range-finding study
14-day recovery group included for high dose and control group (each 5 animals per sex and dose group)

Estrous cycle determination prior to treatment was performed in a pool of up to 60 non­ randomized female animals. Only animals with regular estrous cycle were selected for randomization before the start of the treatment period.
Females are nulliparous and non­ pregnant at the beginning of the study.

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity; a check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. Thereby, the following parameters were examined:
1. abnormal behavior during “handling”
2. fur
3. skin
4. posture
5. salivation
6. respiration
7. activity/arousal level
8. tremors
9. convulsions
10. abnormal movements
11. impairment of gait
12. lacrimation
13. palpebral closure
14. exophthalmus
15. feces (appearance/consistency)
16. urine
17. pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: before the start of the administration period in order to randomize the animals; during the administration period on study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning).
The following exceptions are notable for the female animals:
• During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter were weighed on the day of parturition (PND 0) and on PND 4.
• Females without a litter and without positive evidence of sperm in the vaginal smear were weighed weekly.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
• Food consumption was not determined during the mating period (male and female F0 animals).
• Food consumption of the F0 females with evidence of sperm was determined on GD 0, 7, 14 and 20.
• Food consumption of F0 females, which gave birth to a litter was determined for PND 4.
Food consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel) and in males after the premating period.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in the morning from the retroorbital venous plexus
- Anaesthetic used for blood collection: Yes, isoflurane (Isoba®, Essex GmbH Munich, Germany).
- Animals fasted: Yes
- How many animals: first five parental males and females per group.
- Parameters checked:
• in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany):
Parameter / Unit / Method / Reference (Operator’s Guide for Advia 120 System)
- Leukocyte count (WBC) / giga/L / cytochemistry coupled with flow cytometry
- Erythrocyte count (RBC) / tera/L / flow cytometric laserlight scattering
- Hemoglobin (HGB) / mmol/L / cyanmethemoglobin method; according to ICSH
- Hematocrit (HCT) / L/L calculation: MCV x erythrocytes
- Mean corpuscular volume (MCV) / fl / RBC/PLT method; mean of RBC volume distribution curve (histogram)
- Mean corpuscular hemoglobin (MCH) / fmol / calculation: hemoglobin, erythrocytes
- Mean corpuscular hemoglobin concentration (MCHC) / mmoL/L calculation: hemoglobin, hematocrit
- Platelet count (PLT) / giga/L / flow cytometric laserlight scattering
- Differential blood count / % and giga/L / cytochemistry coupled with flow cytometry
- Reticulocytes / % / cytochemistry coupled with flow cytometry

• Clotting tests were carried out using a ball coagulometer (AMAX destiny plus model; Trinity biotech, Lemgo, Germany).
Parameter / Unit / Method / Reference
- Prothrombin time (Hepato Quick’s test) (HQT) / seconds / citrated blood with calcium thromboplastin / Fischer, M. and Falkensammer, Ch.,
Klin. Wschr. 86, 577-583 (1974)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in the morning from the retroorbital venous plexus
- Animals fasted: Yes
- How many animals: first five parental males and females per group.
- Parameters checked:
An automatic analyzer (Hitachi 917; Roche, Mannheim, Germany) was used to examine the clinicochemical parameters.
References (ALT, AST, ALP): Recommendations of the German Society for Clinical Chemistry: "Standardization of methods for determining enzyme activities in biological liquids". J. Clin. Chem. Clin. Biochem. 8, 658-660 (1970); J. Clin. Chem. Clin. Biochem. 9, 464-465 (1971); J. Clin. Chem. Clin. Biochem. 10, 182-192 (1972); Roche working instructions

• Enzyme (systematic name and system number) / Unit / Method, wavelength and measuring temperature
- Alanine aminotransferase (ALT); EC 2.6.1.2. / μkat/L / kinetic UV test, 340 nm; 37°C /
- Aspartate aminotransferase (AST); EC 2.6.1.1. / μkat/L / kinetic UV test, 340 nm; 37°C /
- Alkaline phosphatase (ALP); EC 3.1.3.1. / μkat/L / kinetic color test, 415 nm, 37°C
- γ-Glutamyltransferase (GGT); EC 2.3.2.2. / nkat/L / kinetic color test, 415 nm, 37°C / Szasz, G. et al., J. Clin. Chem. Clin. Biochem. 12, 228 (1974); Roche working instructions

• Blood Chemistry Parameter / Unit / Method / References
- Sodium (NA) / mmol/L / ion selective electrodes (ISE) / Hitachi 917 - working instructions
- Potassium (K) / mmol/L / ion selective electrodes (ISE) / Hitachi 917 - working instructions
- Chloride (CL) / mmol/L / ion selective electrodes (ISE) / Hitachi 917 - working instructions
- Inorganic phosphate (INP) / mmol/L / molybdate reaction Henry, R.J. in: "Clinical Chemistry", Harper and Row Publishers, New York (1974);
Roche working instructions
- Calcium (CA) / mmoL/L / o-cresolphthalein complex without deproteinization / Ray Sarkar, B.C. and Chauhan, U.P.S., Anal. Biochem. 20, 155 (1967); Roche working instructions
- Urea (UREA) / mmoL/L / enzymatic determination with the urease/ glutamate dehydrogenase method / Neumann, U. and Ziegenhorn, J.: XVI,
Nordiska kongressen for klinisk kemi och klinisk fysiologi 1977, Oulu, Finland; Roche working instructions
- Creatinine (CREA) / μmoL/L / kinetic Jaffé method without deproteinization / Bartels, H. et al., Clin. Chim. Acta 37, 193 (1972); Roche working instructions
- Glucose (GLUC) / mmoL/L / hexokinase/glucose-6-phosphate dehydrogenase method / Schmidt, F.H., Klin. Wschr. 39, 1244-1247 (1961); Roche working instructions
- Total bilirubin (TBIL) / μmoL/L / DPD method Wahlefeld, A.W. et al., Scand. J. Clin. Lab. Invest. 29, Suppl. 126 (1972) Abstract 11.12; Roche working instructions
- Total protein (TPROT) / g/L / biuret method Weichselbaum, T.E., Amer. J. Clin. Path. 10, 40 (1946); Roche working instructions
- Albumin (ALB) / g/L bromocresol green method Doumas et al., Clin. Chim. Acta 31, 87 (1971); Roche working instructions
- Globulins (GLOB) / g/L / difference between total protein and albumin
- Triglycerides (TRIG) / mmoL/L / enzymatic color test with lipase esterase/ glycerokinase/ glycerol-3-phosphate oxidase/4-aminophenazone
mod. method by Wahlefeld, A.W., in "Methoden der enzymatischen Analyse" [Methods of enzymatic analysis] (Bergmeyer, H.U., ed.) Vol. II, 3rd ed.,
Verlag Chemie Weinheim, GERMANY, pp. 1878-1882 (1974); Roche working instructions
- Cholesterol (CHOL) / mmoL/L / enzymatic determination with cholesterol esterase/ cholesterol oxidase/4-amino-phenazone (CHOD-PAP method)
Siedel, J. et al., J. Clin. Chem. Clin. Biochem. 19, 838 (1981); Roche working instructions
- Magnesium (MG) / mmoL/L / xylidylblue method Mann, C.K. and Yoe, J.H., Anal. Chem. 28, 202-205 (1956); Bohnon, C., Clin. Chim. Acta 7, 811-817
(1962)

URINALYSIS: Yes
- Time schedule for collection of urine: overnight
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked:
The dry chemical reactions on test strips (Combur-10-test M, Roche, Mannheim, Germany) used to determine urine constituents semiquantitatively were evaluated with a reflection photometer (Miditron M; Roche, Mannheim, Germany).
Parameter / Method / References
- pH / methyl red and bromothymol blue / Test strip book by Roche, Mannheim, GERMANY (1977)
- Protein / tetrabromophenol-phthaleinethylester (TBPE) / Test strip book by Roche, Mannheim, GERMANY (1977)
- Glucose / GOD-POD reaction / Test strip book by Roche, Mannheim, GERMANY (1977)
- Ketones / sodium nitroprusside / Test strip book by Roche, Mannheim, GERMANY (1977)
- Urobilinogen / p-methoxyaniline-diazonium-salt / Test strip book by Roche, Mannheim, GERMANY (1977)
- Bilirubin / 2,5-dichloroaniline diazonium salt / Test strip book by Roche, Mannheim, GERMANY (1977)
- Blood / 2,5-dimethylhexane-2,5-dihydroperoxide, tetramethylbenzidine / Test strip book by Roche, Mannheim, GERMANY (1977)
- Specific gravity / refractometer / Hamilton or Atago operating instructions
- Sediment / microscopy / Hallmann, L., [Clinical Chemistry and Microscopy] 10, ed., 233-246, Georg Thieme, Stuttgart, Germany (1966)
- Color, turbidity / by visual evaluation
- Volume / graduated tubes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
- Dose groups that were examined: A functional observational battery (FOB) was performed in the first five parental males and females (with litter) per group

- Battery of functions tested:
• passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.
• Home cage observations:
1. posture
2. tremor
3. convulsions
4. abnormal movements
5. impairment of gait
6. other findings
• Open field observations:
1. behavior when removed from cage
2. fur
3. skin
4. salivation
5. nose discharge
6. lacrimation
7. eyes/pupil size
8. posture
9. palpebral closure
10. respiration
11. tremors
12. convulsions
13. abnormal movements
14. impairment of gait
15. activity/arousal level
16. feces (number of fecal pellets/appearance/consistency) within two minutes
17. urine (appearance/quantity) within two minutes
18. number of rearings within two minutes
• Sensorimotor tests/reflexes:
1. approach response
2. touch response
3. vision ("visual placing response")
4. pupillary reflex
5. pinna reflex
6. audition ("startle response")
7. coordination of movements ("righting response")
8. behavior during "handling"
9. vocalization
10. pain perception ("tail pinch")
11. grip strength of forelimbs
12. grip strength of hindlimbs
13. landing foot-splay test
14. other findings
• Motor activity assessment
- carried out in the first five parental males and females (with litter) per group at the end of the administration period.
- measured on the same day as FOB was performed.
- examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany.
The animals did not receive any food or water during the measurements.

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals were sacrificed by decapitation under Isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology.
- Weight parameters (determined on all animals):
1. Anesthetized animals
2. Epididymides
3. Testes
- The following weights were determined in 5 animals/sex and test group (females with litters, same animals as used for clinical pathology examinations):
1. Adrenal glands
2. Brain
3. Heart
4. Kidneys
5. Liver
6. Spleen
7. Thymus

- Organ / Tissue preservation
The following organs / tissues were preserved in neutral-buffered 4% formaldehyde or in modified Davidson’s solution:
1. Adrenal glands
2. All gross lesions
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulating glands
9. Colon
10. Duodenum
11. Eyes with optic nerve (modified Davidson’s solution)
12. Esophagus
13. Extraorbital lacrimal gland
14. Epididymides (modified Davidson’s solution)
15. Femur with knee joint
16. Heart
17. Ileum
18. Jejunum (with Peyer’s patches)
19. Kidneys
20. Larynx
21. Liver
22. Lungs
23. Lymph nodes (axillary and mesenteric)
24. Mammary gland (male and female)
25. Nose (nasal cavity)
26. Ovaries (modified Davidson’s solution)
27. Oviducts
28. Pancreas
29. Parathyroid glands
30. Pharynx
31. Pituitary gland
32. Prostate gland
33. Rectum
34. Salivary glands (mandibular and sublingual)
35. Sciatic nerve
36. Seminal vesicles
37. Skeletal muscle
38. Spinal cord (cervical, thoracic and lumbar cord)
39. Spleen
40. Sternum with marrow
41. Stomach (forestomach and glandular stomach)
42. Target organs
43. Testes (modified Davidson’s solution)
44. Thymus
45. Thyroid glands
46. Trachea
47. Urinary bladder
48. Uterus
49. Vagina

From the liver, each one slices of the lobus dexter medialis and the lobus sinister lateralis were fixed in Carnoy’s solution and embedded in paraplast.

HISTOPATHOLOGY: Yes
Organ samples / Methods-Scope of examinations / Test group
1. All gross lesions: Hematoxylin-eosin (H&E), all affected animals per group, all treatment groups
2. Adrenal glands: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
3. Bone marrow (femur): Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
4. Brain: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
5. Cecum: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
6. Cervix: Hematoxylin-eosin (H&E), all affected animals per group, control and high dose group
7. Coagulation glands: Hematoxylin-eosin (H&E), all affected animals per group, control and high dose group
8. Colon: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
9. Duodenum: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
10. Epididymides: Hematoxylin-eosin (H&E), all affected animals per group
11. Heart: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
12. Ileum: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
13. Jejunum: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
14. Kidneys: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
15. Liver: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group; low and mid dose group: embedded in paraplast all animals per group.
16. Lung: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
17. Lymph nodes (mesenteric and axillary lymph nodes): Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
18. Ovaries: Hematoxylin-eosin (H&E), control and high dose group, all animals per group
19. Oviducts: Hematoxylin-eosin (H&E), control and high dose group, all animals per group
20. Peyer’s patches Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
21. Prostate: Hematoxylin-eosin (H&E), control and high dose group, all animals per group
22. Rectum: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
23. Sciatic nerve: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
24. Seminal vesicles: Hematoxylin-eosin (H&E), control and high dose group, all animals per group
25. Spinal cord (cervical, thoracic and lumbar cords): Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
26. Spleen: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
27. Stomach (forestomach and glandular stomach): Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
28. Testes: Hematoxylin-eosin (H&E), control and high dose group, all animals per group
29. Thymus: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
30. Thyroid glands: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
31. Trachea: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
32. Urinary bladder: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
33. Uterus: Hematoxylin-eosin (H&E), control and high dose group, all animals per group
34. Vagina: Hematoxylin-eosin (H&E), control and high dose group, all animals per group

Other examinations:
Investigations regarding reproductive toxicity/developmental toxicity are described in the respective endpoint study record(s).
Statistics:
Food consumption, body weight and body weight change: DUNNETT-test (two-sided);
Feces, rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity; clinical pathology parameters (except for urine color and turbidity), pathology (weight parameters): Non-parametric one-way analysis using KRUSKALWALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
nominal (analytically determined: average 840 mg/kg bw)
Sex:
male/female
Basis for effect level:
other: absence of adverse effects
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022
Report date:
2022

Materials and methods

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

Test material

Constituent 1
Reference substance name:
1,3,5-Triazine-2,4,6-triamine, N2,N2’-1,6-hexanediylbis[N4,N6-dibutyl-N2,N4,N6-tris(2,2,6,6-tetramethyl-4-piperidinyl)-, N-allyl derivs., oxidized, hydrogenated
EC Number:
812-927-5
Cas Number:
1902936-62-2
Molecular formula:
not availabale
IUPAC Name:
1,3,5-Triazine-2,4,6-triamine, N2,N2’-1,6-hexanediylbis[N4,N6-dibutyl-N2,N4,N6-tris(2,2,6,6-tetramethyl-4-piperidinyl)-, N-allyl derivs., oxidized, hydrogenated
Test material form:
solid
Details on test material:
Solid, beige
Storage at room temperature
Batch identification: 0023404739

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The Sprague Dawley SD rat was the species and strain of choice because it is accepted by many regulatory authorities and there are ample experience and background data on this species and strain. The experimental laboratory has sufficient historical control data for the strain used.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: ordered from Envigo RMS srl, San Pietro al Natisone (UD), Italy and supplied by Envigo RMS B.V., Kreuzelweg 53, 5961 NM Horst, Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 27-29 days old
- Weight at study initiation: 95-121 g for males and 91-113 g for females
- Fasting period before study: no
- Housing: up to 5 of one sex in a cage (in clear polysulfone solid bottomed cages)
- Diet (e.g. ad libitum): 4 RF 21,Mucedola S.r.l., Via G. Galilei 4, 20019 SettimoMilanese (MI), Italy) was offered ad libitum
- Water (e.g. ad libitum): drinking water ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 2°C
- Humidity (%): 55% ± 15%
- Air changes (per hr): approx. 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 01-July-2021 (Arrival of animals) To: 13--Oct-2021 (Last day of necropsy)

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Oral administration is adequate since the substance is not handled in inhalable form and dermal penetration is not expected based on the data available. The oral route has been selected as it has been specifically requested by the Regulatory Authorities.
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5 % aqueous carboxymethylcellulose (0.5% CMC)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of test item was suspended in the vehicle.
The preparations were made daily/weekly (concentrations of 10, 30 and 100 mg/mL).
Concentrations were calculated and expressed in terms of test item as supplied.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis was performed in a separate study in order to validate the analytical method and the preparation procedure and to verify the stability of the preparations.

The analytical method was validated in the range from 10 to 100 mg/mL. Linearity, accuracy and precision were within the limits stated in the validation protocol (r > 0.995; accuracy 85-115%; precision CV < 10%).
A 27 hour stability at room temperature and an 8 day stabiliy at 2-8°C were verified in the range from 10 to 100 mg/mL. Suspensions are considered to be stable if concentration and homogeneity are still acceptable (85%-115% for concentration and CV < 10% for homogeneity) after the defined period of storage.
The proposed preparation procedure for the test item was checked in the range from 10 to 100 mg/mL by chemical analysis (concentration and homogeneity) to confirm that the method was suitable. Final results for all levels were within the acceptability limits for concentration (85-115%) and homogeneity (CV < 10%).
Samples of the suspensions prepared on Week 1 (Day 1) and Last week (Day 86) were analysed to check their homogeneity and concentration. Results of the analyses were within the acceptability limits for suspensions (85-115% for concentration and CV < 10% for homogeneity) and are available.
The validated software used for this activity was Chromeleon.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels have been selected based on the results obtained in a previous oral toxicity study in rats, performed according to OECD Guideline 422 - Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test. In this study, no adverse effects were noted at all dose levels (100, 300 and 1000 mg/kg bw/day) and the dose level ≥ 1000 mg/kg bw/day was considered the NOAEL in this study. Therefore, the doses were selected to include the limit dose of 1000 mg/kg bw/d and dose spacing of ca. 3 was used as standard dose spacing.

- Dose range finding studies: please refer to cross-referenced study according to OECD Guideline 422

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were checked for mortality twice per day on weekdays and once per day on weekends and public holidays. Observations for clinical signs were conducted once per day after dosing.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment and at least once per week during the study. Each animal was observed in an open arena. The test included observation and record of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, unusual respiratory pattern). Changes in fur, skin, eyes, mucous membranes, occurrences of secretions and excretions was also recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: day of allocation to treatment group, start of treatment, weekly thereafter and just prior to necropsy

FOOD CONSUMPTION:
- TIme schedule: recorded at weekly intervals starting from treatment (the group mean daily intake per rat was calculated). Measurements were performed by cage.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: all animals were examined prior to the commencement of treatment, eyes of all animals from high dose and control groups were re-examined during Week 13 of treatment

SERUM HORMONES: Yes
- Time of blood sample collection: last week of treatment
- Animals fasted: Yes
- How many animals: all
- Hormone parameters: T3, T4, TSH (determined by RadioImmuneAssay (RIA))

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters examined: Haematocrit, Haemoglobin, Red blood cell count, Reticulocyte count, Mean red blood cell volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, White blood cell count, Differential leucocyte count (Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes, Large unstained cells), Platelets, Coagulation (Prothrombin time, APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to necropsy
- Animals fasted: Yes
- How many animals: all
- Parameters examined: Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Gamma-glutamyltransferase, Urea, Blood urea nitrogen, Creatinine, Glucose, Triglycerides, Inorganic phosphorus, Total bilirubin, Total cholesterol, HDL, LDL, Total protein, Albumin, Globulin, A/G Ratio, Sodium, Potassium, Calcium, Chloride

OESTROUS CYCLE: YES
- Time schedule: at the end of the treatment and just prior to necropsy, vaginal smears were taken from all animals

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once during weeks 13 of treatment
- Dose groups that were examined: all
- Battery of functions tested: sensory activity (e.g. auditory, visual and proprioceptive stimuli) / grip strength / motor activity (at 10 minute intervals over 60 minutes)

URINALYSIS: NO

IMMUNOLOGY: NO
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
Samples of all the tissues listed below were fixed and preserved in 10% neutral buffered formalin (except eyes, optic nerves, Harderian glands, testes and epididymides which were fixed inModified Davidson’s fluid and preserved in 70% ethyl alcohol).

TISSUES PROCESSED:
see "any other information on methods including tables"
Statistics:
Standard deviations were calculated as considered appropriate. For continuous variables the significance of the differences amongst groups was assessed by analysis of variance.
Differences between each treated group and the control group were assessed by Dunnett’s test using a pooled error variance. The homogeneity of the data was verified by Bartlett’s test before Dunnett’s test. If the data were found to be inhomogeneous aModified t test (Cochran and Cox) was applied.
The mean values, standard deviations and statistical analysis were calculated from the actual values in the computer without rounding off.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related clinical signs were observed.

100 mg/kg bw/d, 1 male animal: Ocular transparent discharge and coloured staining around both eyelid in some occasions during the study
--> considered unrelated to treatment with the test item (absence of a dose-relation and other clinical signs)


No treatment-related changes were observed at the weekly clinical examination, which included an evaluation of neurotoxicity

100 mg/kg bw and 300 mg/kg bw, males and females: slight fluctuations in the number of rearing events during dosing period in a single occiasion
1000 mg/kg bw, males and females: occasional decrease in the number of rearing evens
--> not considered to be treatment-related (absence of a clear dose-relation and episodical occurence)
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
When compared to control animals, no changes were noted in mean body weights and
body weight gain in both genders during the study.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No treatment-related changes were observed in food consumption in male and female
animals, during the study.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No findings were detected in both eyes of all animals, from high dose and control groups,
following examination performed duringWeek 13 of treatment.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
100 mg/kg bw, males: higher monocytes (+78%)
--> considered to be incidental (within the range of historical data)

1000 mg/kg bw males: lower erythrocytes (-5%)
--> considered to be non-adverse (5/10 animals slightly below the historical data and slight severity of the effect; even though the relation with the test item cannot be definitively excluded)

1000 mg/kg bw, females: higher leucocytes (+41%)
--> considered to be incidental (within the range of historical data)
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes were recorded
300 and 1000 mg/kg bw, males: higher inorganic phosphorous (14% and 15%, respectively; 3/10 and 4/10 animals outside of historical date, respectively)
--> considered to be incidental (slight severity, absence of other related findings)
1000 mg/kg bw, females: decreased chloride (-2%)
--> considered to be incidental (within the range of historical data)
Endocrine findings:
no effects observed
Description (incidence and severity):
Thyroid hormone determination: no changes were recorded.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
No differences that could be considered treatment-related were observed at functional tests (sensory reaction to stimuli, landing footsplay, grip strength, motor activity) performed at the end of the treatment period.
300 and 1000 mg/kg bw, males and females: lower values in males and higher values in females of grip strength measurement (first, second trials and mean values)
--> considered to be incidental (absence of a clear dose-relation and to the inconsistency of the variations (higher or lower)

No treatment-related changes were observed at the weekly clinical examination, which included an evaluation of neurotoxicity
100 mg/kg bw and 300 mg/kg bw, males and females: slight fluctuations in the number of rearing events during dosing period in a single occiasion
1000 mg/kg bw, males and females: occasional decrease in the number of rearing events
--> not considered to be treatment-related (absence of a clear dose-relation and episodical occurence)
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
All differences between the test item-treated groups and controls were small, never reached
statistical significance and were interpreted to reflect individual variations.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
All macroscopic observations were spontaneous in nature and bore no relationship to
treatment.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
1000 mg/kg bw, males and females: aggregates of large eosinophilic macrophages in the sinuses of mesenteric lymph nodes with a score up to moderate
300 mg/kg bw, 1 female: aggregates of large eosinophilic macrophages in the sinuses of mesenteric lymph nodes with a minimal severity
--> possibly reflects accumulation of the test item; considered non adverse (due to absence of other changes in the node architecture and of associated clinical pathology changes)

1000 mg/kg bw, females: higher incidence and severity of squamous metaplasia of uterine glands
--> spontaneous squamous metaplasia is rare (indeed a higher incidence than usual is noted in controls and at intermediate dose levels in the present study); relationship to treatment cannot be completely excluded but increased incidence is likely a chance event given the high background in this study and the absence of differences in ovarian cycle distribution between control and high dose groups
Histopathological findings: neoplastic:
no effects observed

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

Table 1: Clinical Signs - Group Incidence














































































































































































































MalesFemales
Dosing Dosing 
 Dose group [mg/kg bw/d]01003001000  Dose group [mg/kg bw/d]01003001000
 Number of Animals10101010 Number of Animals10101010
 Number Examines10101010 Number Examines10101010
CategoryObservationababababCategoryObservationabababab
No significant signsNo significant signs1092.51088.51092.51092.5No significant signsNo significant signs1092.51092.51092.51092.5
APPEARANCEStaining, Both eyelids, Right, Moderate, Brown0012.00000          
Staining, Both eyelids, Right, Slight, Brown00118.00000          
Staining, Both eyelids, Right, Slight, Red0011.00000          
Staining, Eye, Slight, Brown0011.00000          
EYE - EAR - MOUTH-NOSEOcular discharge, Right, Transparent00119.00000          

a - number of animals affected


b - number of days with clinical sign/animal


 


Table 2: Body Weight - Group mean data





















































































































































































































































































































































































































































































































Males
  QuarantineDosing
Dose [mg/kg bw/d]Body Weight (g)Day 8Day 1Day 8Day 15Day 22Day 29Day 36Day 43Day 50Day 57Day 64Day 71Day 78Day 85Day 92
0(n)101010101010101010101010101010
Mean150.68186.47236.80281.32312.78339.15357.60374.00388.44396.51408.26417.40431.78435.04432.00
SD8.8279.99610.98810.76711.48613.27415.54715.20613.86412.82514.22014.13915.68115.02821.392
100(n)101010101010101010101010101010
Mean150.30186.64239.17287.20318.98345.68369.36388.32402.51410.19422.80429.83442.59445.40448.40
SD8.24812.74613.75417.39017.69321.58322.03523.35521.65422.08224.32422.78121.30922.94830.329
300(n)101010101010101010101010101010
Mean150.10184.05235.98280.84312.24338.58361.19379.70392.97403.37413.48423.71436.85439.08440.44
SD7.4496.5537.8359.18812.88017.79619.34722.13119.48424.61125.79027.48530.49827.67133.514
1000(n)101010101010101010101010101010
Mean150.52184.17234.38279.46309.39328.27350.81368.01383.58390.55404.03410.94424.02423.31422.51
SD8.7549.97611.71114.01614.06517.23018.04721.31122.23921.75722.80824.09023.69926.42828.406
Females
  QuarantineDosing
Dose group [mg/kg bw/d]Body Weight (g)Day 8Day 1Day 8Day 15Day 22Day 29Day 36Day 43Day 50Day 57Day 64Day 71Day 78Day 85Day 92
0(n)101010101010101010101010101010
Mean135.00150.48170.47191.19199.22209.94220.58226.76234.38233.67240.22241.98254.34246.63245.33
SD5.7126.9157.8337.5428.3508.6479.92410.20310.8869.71913.34712.84010.66713.6818.958
100(n)101010101010101010101010101010
Mean135.90150.82169.36187.70196.72209.38216.37222.46228.59234.15233.75237.49241.86241.94240.66
SD6.3835.3827.83110.6439.5619.56411.39613.52313.56115.11614.16017.00315.78818.13917.121
300(n)101010101010101010101010101010
Mean134.63151.08171.75185.76199.71211.50221.59227.10232.45237.24240.17243.19246.98247.21244.47
SD5.4765.5569.24811.48513.68215.30517.60017.87715.45818.12117.19717.70318.09018.83815.847
1000(n)101010101010101010101010101010
Mean133.95149.97170.25188.09199.05212.24217.10222.82230.33232.76236.21239.07244.73245.27242.53
SD6.1306.2838.50211.83410.44610.00810.73312.91311.81115.02114.08712.31615.30813.89016.520

 


Table 3: Haematology - Group mean data


































































































































































































































































































































































































































































































































































































































































































Dosing Day 92
Males
Dose group [mg/kg bw/d] RBC
(x10^6/μL)
HGB
(g/dL)
HCT
(%)
MCV
(fL)
MCH
(pg)
MCHC
(g/dL)
RET
(x10^9/L)
RETR
(%)
WBC
(x10^3/μL)
NEU
(x10^3/μL)
LYM
(x10^3/μL)
MON
(x10^3/μL)
EOS
(x10^3/μL)
BAS
(x10^3/μL)
LUC
(x10^3/μL)
NEUR
(%)
LYMR
(%)
MONR
(%)
EOSR
(%)
BASR
(%)
LUCR
(%)
PLT
(x10^3/μL)
0(n)10101010101010101010101010101010101010101010
Mean8.07914.5844.6255.2418.0532.72159.461.9735.5340.9964.2120.1440.1340.0230.02417.6976.402.602.460.430.40921.4
SD0.34140.4832.1441.4530.4450.54910.9230.094514.5950.781112.3980.07140.05910.01160.013510.66910.3241.0240.9120.1250.15692.55
100(n)8888888888888888888888
Mean8.26614.6944.6554.0817.7932.91157.091.9055.6611.2733.9440.256+D0.1310.0260.02521.3170.854.60+D2.280.460.49974.9
SD0.30730.2530.8541.6490.6940.46111.3260.156513.0500.78060.56190.08830.03360.00740.00767.3866.9771.2190.3280.0520.18950.05
300(n)10101010101010101010101010101010101010101010
Mean7.98414.5344.6355.9218.2132.56155.921.9534.8240.8963.6240.1570.1110.0200.01819.0274.523.292.360.410.40903.4
SD0.31260.3021.4801.1920.4360.57616.0100.17930.77990.11230.83020.04990.04230.00670.01143.9935.5681.0431.1120.1370.24986.77
1000(n)10101010101010101010101010101010101010101010
Mean7.683*D14.2242.9055.8218.5033.14148.731.9335.5191.0174.1590.2070.0910.0210.02319.4674.473.691.590.370.40935.7
SD0.42981.0113.0511.6190.6220.47419.9130.228214.5430.1484Feb 350.08030.03840.01100.01064.8734.1940.6790.4380.0950.09473.36
Females
Dose group [mg/kg bw/d] RBC
(x10^6/μL)
HGB
(g/dL)
HCT
(%)
MCV
(fL)
MCH
(pg)
MCHC
(g/dL)
RET
(x10^9/L)
RETR
(%)
WBC
(x10^3/μL)
NEU
(x10^3/μL)
LYM
(x10^3/μL)
MON
(x10^3/μL)
EOS
(x10^3/μL)
BAS
(x10^3/μL)
LUC
(x10^3/μL)
NEUR
(%)
LYMR
(%)
MONR
(%)
EOSR
(%)
BASR
(%)
LUCR
(%)
PLT
(x10^3/μL)
0(n)10101010101010101010101010101010101010101010
Mean6.87513.0538.1355.4618.9834.25138.652.0183.4920.5082.7780.1170.0610.0110.01915.0979.003.271.800.310.53917.5
SD0.40070.7782.4731.4520.5920.53620.8380.29850.99540.13060.86270.05460.01850.00570.00743.7244.0340.8650.4940.1290.16493.98
100(n)10101010101010101010101010101010101010101010
Mean6.70112.8137.6956.2619.1133.96151.882.2704.1380.7003.2140.1350.0550.0110.02116.5478.153.181.35*C0.290.49907.9
SD0.26010.5491.4480.8440.4890.45518.5820.304112.9850.42930.93940.05800.02550.00880.01295.3295.7240.9530.3500.1200.20855.16
300(n)10101010101010101010101010101010101010101010
Mean6.70212.7337.2555.6019.0434.23140.632.1024.1940.6243.3050.1470.0800.0150.02415.3178.323.561.920.300.60916.1
SD0.25280.4001.3631.3490.4250.30619.6890.30590.82390.11980.77700.04670.03060.00530.00974.1564.5760.8920.7180.0820.19473.22
1000(n)10101010101010101010101010101010101010101010
Mean6.81313.1238.1155.9519.2434.40153.142.2554.931*D0.7543.8730.1860.0650.0170.03415.4778.483.661.34*C0.360.68905.3
SD0.30960.4731.5581.1660.5100.40332.9090.517510.5230.22490.86820.08750.01780.00670.01964.0994.7061.2650.2950.1070.336101.97

HCT: HAEMATOCRIT [%]


RBC: RED BLOOD CELL COUNT [10^6/μL]


HGB: HAEMOGLOBIN [g/dL]


MCV: MEAN RED BLOOD CELL VOLUME [fL]


MCH: MEAN CORPUSCULAR HAEMOGLOBIN [pg]


MCHC: MEAN CORPUSCULAR HAEMOGLOBIN CONCENTRATION [g/dL]


PLT: PLATELETS [10^3/uL]


WBC: WHITE BLOOD CELL COUNT [10^3/uL]


NEUR/NEU: NEUTROPHILS [% or 10^3/μL]


LYMR/LYM. LYMPHOCYTES [% or 10^3/μL]


MONR/MON: MONOCYTES [% or 10^3/μL]


EOSR/EOS: EOSINOPHILS [% or 10^3/μL]


BASR/BAS: BASOPHILS [% or 10^3/μL]


LUCR/LUC: LARGE UNSTAINED CELLS [% or 10^3/μL]


RETR/RET: RETICULOCYTES [% or 10^3/μL]


*D Dunnett LSD Test Significant at the 0.05 level


+D Dunnett LSD Test Significant at the 0.01 level


*C Cochran and Cox Test Significant at the 0.05 level


 


Table 4: Clinical Chemistry - Group mean data
































































































































Dosing Day 92
MalesFemales
Dose group [mg/kg bw/d] Cl [nmol/L]IP [mg/dL]Group Cl [nmol/L]IP [mg/dL]
0(n)1010Control(n)1010
Mean103.175.823Mean104.806.112
SD0.8010.7791SD1.2090.8488
100(n)10102(n)1010
Mean102.876.028Mean105.386.255
SD0.7470.6315SD1.9360.7440
300(n)10103(n)1010
Mean103.646.637*DMean104.706.687
SD0.6700.5913SD1.0220.7779
 (n)10104(n)1010
1000Mean103.156.671*DMean103.06*D6.447
 SD1.0840.6880SD1.6320.6767

Cl: Chloride


IP: inorganic phosphorus


*D Dunnett LSD Test Significant at the 0.05 evel


 


Table 5: Clinical Chemistry - Historical Control Data




















































































































Historical Contral Data
MalesFemales
 Age: 9-16 weeksAge: > 16 weeks Age: 9-16 weeksAge: > 16 weeks
 Cl [nmol/L]IP [mg/dL]Cl [nmol/L]IP [mg/dL] Cl [nmol/L]IP [mg/dL]Cl [nmol/L]IP [mg/dL]
no25686369199no22475385214
Mean102.77.53103.45.81Mean103.76.10103.75.14
SD2.20.662.61.30SD2.51.292.50.99
Min obs93.65.2796.14.15Min obs94.64.0497.32.76
Max obs109.28.92111.017.14Max obs111.111.67110.88.16
Range min (perc. 5%)99.66.4099.44.61Range min (perc. 5%)100.04.4899.73.67
Range max (perc. 95%)105.78.54107.96.93Range max (perc. 95%)107.27.83107.96.89

 


Table 6: Thyroid hormones determination - Group mean data
























































































































































Dosing Day 88
MalesFemales
Dose group [mg/kg bw/d] T3 [nmol/L]T4 [nmol/L]TSH [ng/mL]Dose group [mg/kg bw/d] T3 [nmol/L]T4 [nmol/L]TSH [ng/mL]
0(n)1010100(n)101010
Mean0.95347.112.68Mean1.27139.910.31
SD0.15943.905.648SD0.14536.574.047
100(n)101010100(n)101010
Mean0.96748.911.47Mean1.22439.47.16
SD0.17614.864.181SD0.15015.681.260
300(n)101010300(n)101010
Mean1.03152.39.97Mean1.27138.78.68
SD0.12636.733.345SD0.14978.151.734
1000(n)1010101000(n)101010
Mean1.04252.311.69Mean1.34642.58.71
SD0.223606.574.888SD0.15313.272.822

 


Table 7: Estrous Cycle 



























































































































GroupStageDays SeenGroupStageDays SeenGroupStageDays SeenGroupStageDays Seen
ControlProestrus92100 mg/kg bw/dOestrus92300 mg/kg bw/dDioestrus921000 mg/kg bw/dProestrous92
Oestrus92Oestrus92Oestrus92Oestrus92
Oestrus92Oestrus92Dioestrus92Oestrus92
Oestrus92Oestrus92Oestrus92Oestrus92
Oestrus92Oestrus92Dioestrus92Proestrous92
Dioestrus93Oestrus93Oestrus93Oestrus93
Dioestrus93Metaoestrus93Oestrus93Oestrus93
Oestrus93Oestrus93Oestrus93Oestrus93
Oestrus93Oestrus93Oestrus93Oestrus93
Oestrus93Oestrus93Oestrus93Oestrus93

 


Table 8: Open Field Observation of Rearing - Group Mean Data



























































































































































































































































































































































































































































































Males
  AllocationDosing
Dose group [mg/kg bw/d] Day 2Day 3Day 10Day 17Day 24Day 31Day 38Day 45Day 52Day 59Day 66Day 73Day 80Day 89
0(n)1010101010101010101010101010
Mean10.46.78.212.912.88.79.16.97.06.97.811.25.86.1
SD0.701.251.622.181.401.161.663.981.254.251.231.553.851.37
100(n)1010101010101010101010101010
Mean10.67.28.513.88.4+D8.08.64.98.96.27.811.53.96.4
SD0.521.321.051.552.121.701.311.522.334.831.402.221.791.43
300(n)1010101010101010101010101010
Mean11.78.28.114.47.6+D8.38.77.18.95.7.769.24.66.1
SD28.1261.691.521.261.071.421.063.382.424.992.071.231.901.60
1000(n)1010101010101010101010101010
Mean12.7+C6.87.613.08.4+D8.38.46.47.85.15.87.3+D5.37.7
SD44.7141.320.841.491.171.571.512.411.621.912.972.832.581.77
Females
  AllocationDosing
Dose group [mg/kg bw/d] Day 5Day 4Day 11Day 18Day 25Day 32Day 39Day 46Day 53Day 60Day 67Day 74Day 81Day 90
0(n)1010101010101010101010101010
Mean15.116.517.213.914.214.913.912.212.912.515.517.612.112.0
SD24.1081.581.231.521.815.973.413.362.691.961.081.172.382.45
100(n)1010101010101010101010101010
Mean15.516.417.514.412.916.112.413.113.013.115.916.1*D11.611.8
SD16.1321.171.960.842.778.053.603.932.982.281.200.742.222.04
300(n)1010101010101010101010101010
Mean13.616.717.214.613.314.614.412.813.513.215.616.612.212.3
SD27.7912.001.621.172.985.582.722.572.272.101.071.432.101.49
1000(n)1010101010101010101010101010
Mean16.115.614.6+D14.513.714.611.213.512.613.816.015.5+D12.512.2
SD23.4081.841.511.182.835.254.292.802.272.572.110.971.652.30

*D Dunnett LSD Test Significant at the 0.05 level


+C Cochran and Cox Test Significant at the 0.01 level


+D Dunnett LSD Test Significant at the 0.01 level


 


Table 9: Sensory Reactivity / Grip Strength - Group Mean Data
























































































































































Dosing Day 85
MalesFemales
Dose group [mg/kg bw/d] GS1 (N)GS2 (N)GSMEDose group [mg/kg bw/d] GS1 (N)GS2 (N)GSME
0(n)1010100(n)101010
Mean17.2317.7517.490Mean46.63113.3949.315
SD2.2222.69322.704SD1.1431.3420.8377
100(n)101010100(n)101010
Mean18.3817.2017.790Mean22.92028.00410.190*D
SD2.4072.71020.556SD1.5581.0810.3658
300(n)101010300(n)101010
Mean15.18*C14.52+C14.850+CMean11.46+D11.43+D11.445+D
SD0.9851.34710.376SD1.0781.4610.7798
1000(n)1010101000(n)101010
Mean15.51*C15.43*C15.470*CMean11.63+D10.95*D11.290+D
SD0.8250.5250.4911SD1.2371.3130.5962

GS1 (N): Grip strength 1 (N) - First reading of the animal grips on horizontal bar with forelimbs.


GS2 (N): Grip strength 2 (N) - Second reading of the animal grips on horizontal bar with forelimbs.


GSME (N): Grip strength mean (N) - Averaged reading of the animal grips on horizontal bar with forelimbs.


*C Cochran and Cox Test Significant at the 0.05 level


+C Cochran and Cox Test Significant at the 0.01 level


*D Dunnett LSD Test Significant at the 0.05 level


+D Dunnett LSD Test Significant at the 0.01 level


 


Table 10: Organ Weights (absolute) - Group Mean Data





















































































































































































































































































































































































































































Males
Dose group [mg/kg bw/d] Terminal Body Weight
(g)
Adrenal glands
(g)
Brain
(g)
Epididymides
(g)
Heart
(g)
Kidneys
(g)
Liver
(g)
Pituitary gland
(g)
Seminal vesicales
(g)
Spleen
(g)
Testes
(g)
Thymus
(g)
Thyroid gland
(g)
0(n)10101010101010101010101010
Mean418.760.05721.8851.48841.4592.84211.3450.010229.3670.85163.98800.38780.0250
SD15.1480.010700.10620.115890.09940.22560.93400.001480.255470.123090.202270.088630.00279
100(n)10101010101010101010101010
Mean430.830.05471.8201.42041.4652.95211.1600.010129.1490.81683.86980.38230.0254
SD24.4560.006990.11940.167000.10520.21850.69290.002180.452060.126340.266080.049320.00334
300(n)10101010101010101010101010
Mean423.030.05501.8431.45011.4362.90011.1660.010129.5470.82984.07720.37490.0250
SD27.2230.006000.11240.158320.10170.141911.3510.001660.317160.089520.194360.058750.00397
1000(n)10101010101010101010101010
Mean404.630.05501.7941.41481.3982.77210.7910.010028.7220.78343.93530.40100.0245
SD28.8620.008840.12490.137080.13390.29060.89770.002160.382760.090600.245110.085680.00375
Females
Dose group [mg/kg bw/d] Terminal Body Weight
(g)
Adrenal glands
(g)
Brain
(g)
Heart
(g)
Kidneys
(g)
Liver
(g)
Ovaries
(g)
Pituitary gland
(g)
Spleen
(g)
Thymus
(g)
Thyroid gland
(g)
Uterus
(g)
-
0(n)101010101010101010101010 
Mean235.090.06211.6240.8871.5975.7960.10240.01230.52860.26710.02170.9126 
SD11.2520.005880.08840.08550.12370.50740.019350.002750.042180.021620.004600.36216 
100(n)101010101010101010101010 
Mean227.460.06171.6060.8861.4615.4700.10050.01200.50950.24090.021111.748 
SD17.2370.011850.12800.07140.11950.48640.017570.002110.077150.054430.004910.75968 
300(n)101010101010101010101010 
Mean233.980.06571.6520.8881.5695.7380.11430.01330.54510.23110.02260.9112 
SD17.0120.009500.09650.04720.13230.59010.020860.002450.043200.031270.005170.47996 
1000(n)101010101010101010101010 
Mean229.270.06341.6280.9331.5945.7590.10030.01320.54610.25750.02551.0257 
SD12.8130.009960.10030.09700.16930.48180.018240.004290.083100.048520.003570.84612 

 


Table 11: Microscopic observations


































































































































































Incidence and severity of selected microscopic findings (End of Treatment)



Gender



Males



Females



Group



1



2



3



4



1



2



3



4



Dose (mg/kg bw/d)



0



100



300



1000



0



100



300



1000



Mesenteric lymph node


 

Aggregates, macrophages


 

   Examined



10



10



10



10



10



10



10



10



   Absent



10



10



10



2



10



10



9



1



   Minimal



0



0



0



1



0



0



1



6



   Slight



0



0



0



4



0



0



0



3



   Moderate



0



0



0



3



0



0



0



0



Uterus


 

Squamous metaplasia


 

Examined



NA



NA



NA



NA



10



10



10



10



   Absent



-



-



-



-



7



6



7



3



   Minimal



-



-



-



-



1



2



2



2



   Slight



-



-



-



-



1



1



1



2



   Moderate


    

1



1



0



3



NA: not applicable

Applicant's summary and conclusion

Conclusions:
On the basis of the above results and under the conditions tested, no adverse effects were observed at all dose levels investigated following treatment with the test substance when administered by oral gavage for 13 consecutive weeks at the dosages of 100, 300 and 1000 mg/kg/day, when compared to controls. Therefore, it can be concluded that the No Observed Adverse Effect Level (NOAEL) for this study is 1000 mg/kg/day.
Executive summary:

The test substance was tested in a GLP-conform OECD TG 408 study:


The toxicity of the test substance was investigated in Sprague Dawley SD rats after daily oral administration for 13 weeks.


Three groups, each of 10 male and 10 female rats, received the test item by gavage at dosages of 100, 300 and 1000 mg/kg/day for 13 consecutive weeks. A fourth similarly constituted group received the vehicle alone (0.5% aqueous carboxymethylcellulose) and acted as a control. All doses were administered at a constant volume of 10 mL/kg body weight.


 


The following investigations were performed: mortality and morbidity check, daily clinical signs, weekly detailed clinical signs (removal from cage and open field observations), evaluation of sensory reactivity to stimuli and motor activity, body weight, food consumption, oestrus cycle evaluation, ophthalmoscopy, clinical pathology investigations (including haematology and coagulation, clinical chemistry and thyroid hormones determination), terminal body weight, organ weights, macroscopic observations and microscopic examination, which included the identification of the stages of the spermatogenic cycle and the evaluation of the oestrus cycle.


 


No mortality occurred and no treatment-related clinical signs were observed during the study.


No relevant changes in detailed clinical observation, that can be considered related to treatment, were observed in animals at removal from cage and in an open arena. No treatment-related changes were observed in treated animals at functional tests (sensory reaction to stumuli, landing foot splay, grip strength). No treatment-related differences were noted in the motor activity measurements.


Body weight, food consumption and oestrus cycle were not affected by treatment.


No treatment-related findings in opthalmoscopy were recorded.


No changes in haematology and coagulation which were considered to be adverse were observed. The statistically significant differences of monocytes in males and leucocytes in females were of slight severity and incidence, therefore they were considered to be incidental. The decrease of erythrocytes recorded in males dosed at 1000 mg/kg/day was considered to be non-adverse, even though the relation with the test item cannot be definitively excluded.


No treatment-related changes were recorded in coagulation and clinical chemistry.


No treatment-related changes were recorded in thyroid hormone determination.


No changes in organ weights or macroscopic effects were noted.


In microscopic observations, aggregates of large macrophages were observed in both sexes in mesenteric lymph nodes at the dose of 1000 mg/kg/day, and in a single female at the dose of 300 mg/kg/day. This change was considered non-adverse. A high incidence of squamous metaplasia of the uterus was observed at 1000 mg/kg/day, in a context of an unusually high prevalence of this finding in the study, including in control animals. It was therefore considered a chance event, unrelated to the test item.


On the basis of the above results and under the conditions tested, no adverse effects were observed at all dose levels investigated following treatment with the test substance when administered by oral gavage for 13 consecutive weeks at the dosages of 100, 300 and 1000 mg/kg/day, when compared to controls. Therefore, it can be concluded that the No Observed Adverse Effect Level (NOAEL) for this study is 1000 mg/kg/day.