1,3-bis(trimethylsilyl)urea

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 Feb - 18 Aug 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 Feb - 18 Aug 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

adopted in 2016

Deviations:

yes

Remarks:

prothrombin time (PT) was described as "unusually high" but no historical control data on PT were provided.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

RccHanTM

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS, UK
- Age at study initiation: 69 – 76 days (males) and 98 – 104 days (females)
- Weight at study initiation: 311 – 336 g (males) and 189 – 237 g (females)
- Housing: Prior to mating, the animals were housed in groups of 4/sex in polycarbonate cages with a stainless steel mesh lid and with softwood based bark-free fiber bedding. During mating, males and females were housed in a 1 : 1 ratio in grid bottomed cages with absorbent paper. After mating, males were housed in up to 4 animals while females were housed individually. Throughout the study, the cages were enriched with an aspen chew block (except during lactation), a plastic shelter (except during pairing and lactation) and with paper shavings from Day 20 after mating and throughout lactation.
- Diet: SDS VRF1 certified pelleted diet, ad libitum
- Water: potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum
- Acclimation period: 6 days (males) and 20 days (females)

DETAILS OF FOOD AND WATER QUALITY:
Certificates of analysis for the diet were scrutinized and approved before any batch of diet was released for use. Certificates of analysis were routinely provided by the water supplier.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 – 24
- Humidity (%): 40 - 70
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 26 Feb 2020 To: 10 May 2020

Route of administration:

oral: gavage

Vehicle:

corn oil

Remarks:

dried, deacidified

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
A correction factor of 1.03 was used when calculating quantities of test item used during dose preparation. The required amount of test material was weighed, transferred to a mortar and ground to a fine powder using a pestle. Small amounts of the pre-weighed vehicle were added and mixed to form a smooth paste. The suspension was poured into a measuring cylinder which had been wetted with the vehicle (dried, de-acidified corn oil) and the mortar was rinsed thoroughly with the vehicle and the rinsed residue was added to the measuring cylinder. The required volume was achieved by addition of the remaining vehicle and the suspension was transferred to a beaker for mixing using a high shear homogenizer until visibly homogenous. A series of formulations at the required concentrations were prepared by dilution of individual weighings of the test item. Test item dosing formulations were prepared daily and stored refrigerated (2 – 8 °C).
VEHICLE:
- Nominal concentration in vehicle: 8.34, 25 and 75 mg/mL, corresponding to a formulated concentration of 8.6, 25.8 and 77.3 mg/mL
- Amount of vehicle: 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples from each ofthe first and the last preparation were analysed for achieved concentrations of the test item after extraction with ethylacetate, using gas chromatographic analysis with flame ionisation detection. For the low dose (41.7 mg/kg bw/day) and the mid dose formulation (125 mg/kg bw/day), the mean concentrations were within 10% of the nominal concentration, confirming the accuracy of formulation. For the highest dose formulation (375 mg/kg bw), the mean concentrations were slightly outside the acceptable 10% of the nominal concentration (+12% for the first preparation and +13% for the last preparation, respectively). The difference from mean and coefficient of variation remained within 5%, confirming precise analysis. The procedural recoveries remained in the validated range confirming the continued accuracy of the analytical method, with the exception of one procedural recovery prepared during the last preparation which was excluded as an outlier.
In addition, the homogeneity and stability of the formulations during storage were confirmed as part of another study. Results indicated that the formulations were stable for 2 hours when stored at ambient temperature (15 - 25 °C) ond for 1 day when stored refrigerated (2 - 8 °C).

Duration of treatment / exposure:

Males were treated daily for 2 weeks before pairing, up to necropsy after a minimum of 5 consecutive weeks.
Females that delivered were treated daily for 2 weeks before pairing, throughout pairing, gestation and until Day 13 of lactation.
Females that failed to produce viable litter were treated daily for 2 weeks before pairing, throughout pairing until 25 days after mating.

Frequency of treatment:

daily, 7 days/week

Dose / conc.:

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

Remarks:

A correction factor of 1.03 was used when calculating quantities of test item used during dose preparation.

Dose / conc.:

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

Remarks:

A correction factor of 1.03 was used when calculating quantities of test item used during dose preparation.

Dose / conc.:

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

Remarks:

A correction factor of 1.03 was used when calculating quantities of test item used during dose preparation.

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Dose levels were selected based on the results of a preliminary 14-days dose-range finding study in rats. Groups of 3 males and 3 females were exposed to test item doses of 300, 450, 650 or 1000 mg/kg bw/day. After the first dose administration, males and females administered ≥ 650 mg/kg bw/day showed signs comprised of gasping, irregular and shallow breathing, decreased activity, unsteady gait and prostrate posture. Subsequently, these groups were prematurely killed on the grounds of animal welfare. Macroscopic examination of these animals revealed instances of esophagus distention and abnormal contents in the gastrointestinal tract. Animals receiving 450 mg/kg bw/day showed similar clinical signs on Day 1 and 2 of treatment but the extent was less severe, showed no respiratory changes and no treatment-related signs were observed from Day 4 to 15 of treatment. In males and females of the 450 mg/kg bw/day group there was a reduction in body weight gain over the study period and a slight reduction in overall group mean food consumption, when compared to the 300 mg/kg bw/day group. There were no macroscopic findings after 14 days of treatment in both sexes that received 300 or 450 mg/kg bw/day. In addition, absolute liver weight was low for males receiving 450 mg/kg bw/day, when compared to the 300 mg/kg bw/day group, while the organ weight of females was unaffected by treatment. Accordingly, 375 mg/kg bw/day was selected as a suitable high dose for the present OECD 422 screening study.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily
- Cage side observations included: signs associated with dosing, clinical signs of ill-health and mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to treatment, once per week during treatment, on Days 0, 6, 13 and 20 after mating and on Days 1, 6 and 12 of lactation.
- Detailed clinical observations included: Detailed physical examinations and arena observations.

BODY WEIGHT: Yes
- Time schedule for examinations in males: Prior to dosing on the day that treatment commenced (Day 1) and weekly thereafter.
- Time schedule for examinations in females: Prior to dosing on the day that treatment commenced (Day 1), weekly before pairing, on Days 0, 7, 14 and 20 after mating, in Day 1, 4, 7 and 13 of lactation and on the day of necropsy.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

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

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At termination, blood samples of approx. 0.5 mL were collected from the sublingual vein into tubes containing EDTA anticoagulant.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: No
- How many animals: The 5 lowest numbered surviving males and the first 5 lactating females with a surviving litter per group.
- Parameters examined: haematocrit (Hct), haemoglobin concentration (Hb), erythrocyte count (RBC), absolute reticulocyte count (Retic), mean cell haemoglobin (MCH), mean cell haemoglobin concentration (MCHC), mean cell volume (MCV), red cell distribution width (RDW), total leucocyte count (WBC), differential leucocyte count (neutrophils (N), lymphocytes (L), eosinophils (E), basophils (B), monocytes (M), large unstained cells (LUC)), platelet count (Plt), prothrombin time (PT) and activated partial thromboplastin time (APTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At termination, blood samples of approx. 0.5 mL were collected from the sublingual vein into tubes containing lithium heparin as anticoagulant.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: No
- How many animals: The 5 lowest numbered surviving males and the first 5 lactating females with a surviving litter per group.
- Parameters examined: alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (Bili), bile acids (Bi Ac), urea, creatinine (Creat), glucose (Gluc), total cholesterol (Chol), triglycerides (Trig), sodium (Na), potassium (K), chloride (Cl), calcium (Ca), inorganic phosphorus (Phos), total protein (Total Prot), albumin (Alb) and albumin/globulin ratio (A/G Ratio).

THYROID HORMONE ANALYSIS: Yes
- Time schedule for collection of blood: At termination, blood samples were collected from surviving F0 males and F0 females from the sublingual vein. On PND 4 and 13, blood was collected from F1 offspring by decapitation.
- Anaesthetic used for blood collection: In adult animals, blood was collected under isoflurane anaesthesia. For the offspring, no anaesthesia was used.
- Animals fasted: No
- How many animals:
At termination: All surviving F0 males and all surviving F0 reproductive phase females (no samples obtained from females that failed to litter).
On PND 4: Offspring from up to 2 females per litter.
On PND 13: 2 males and 2 females per litter (where possible).
- Parameters examined: thyroxine (T4) and thyroid stimulating hormone (TSH).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations and dose groups examined: Prior to dosing, on the 5 lowest numbered surviving males in each group during week 5 of treatment and on the first 5 lactating females per group at Day 7 – 9 of lactation
- Battery of functions tested: sensory activity (approach response, pinna reflex, auditory startle reflex and tail pinch response), grip strength (forelimb and hindlimp grip strength) and motor activity

IMMUNOLOGY: No

Sacrifice and pathology:

SACRIFICE:
F0 Males were sacrificed after 5 weeks of treatment by carbon dioxide inhalation.
F0 females were sacrificed on lactation Day 13 by carbon dioxide inhalation.
F0 females that failed to produce a viable litter were sacrificed 25 days after mating by carbon dioxide inhalation.
F1 offspring for thyroid hormone sampling was sacrificed on PND 4 or 13 by decapitation. F1 offspring not selected for thyroid hormone sampling was sacrificed on PND 13 by intraperitoneal injection of phenobarbitone.

GROSS PATHOLOGY:
All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any of the organs and tissues (external and cut surface) mentioned below was recorded and the required tissue samples preserved in appropriate fixative.
ORGAN WEIGHTS:
The following organ weights were collected for the first 5 F0 males and for the first 5 lactating F0 females: adrenals, brain, epididymides, heart, kidneys, liver, prostrate, seminal vesicles with coagulating glands, spleen, testes and uterus with cervix and oviducts. For bilateral organs, left and right organs were weighed together. Requisite organs were weighed for animals killed at scheduled intervals.
HISTOPATHOLOGY:
Tissue samples were dehydrated, embedded in paraffin wax and sectioned. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required. Sections were stained with hematoxylin and eosin; in addition samples of the testes were stained using a standard periodic acid/Schiff (PAS) method.
Histopathological examination of the following organs and tissues was performed for animals of the control (corn oil) and high dose group (375 mg/kg bw/day) only, for the first 5 F0 males and for the first 5 lactating F0 females per group:
Adrenals, brain (including cerebrum, cerebellum and pons), caecum, colon, duodenum, epididymides, eyes, heart (including auricular and ventricular regions), ileum, jejunum, kidneys, liver (sections from 2 lobes), lungs (section from 2 major lobes including bronchi), lymph nodes (left axillary and mesenteric), ovaries, Peyer’s patches, prostate, rectum, seminal vesicles with coagulating glands, skin with mammary glands (inguinal area), spinal cord (transverse and longitudinal sections at the cervical level), spleen, sternum (with marrow), stomach, testes, thymus, thyroids, trachea, urinary bladder and vagina. Samples from the sciatic nerve, from the skeletal muscle and from the uterus with cervix and oviducts were fixed, but not examined histopathologically.

In addition to the organs listed above, all abnormalities were investigated microscopically for all F0 males and females. Further, kidneys and livers from the 5 lowest surviving F0 males of the low (41.7 mg/kg bw/day) and mid dose groups (125 mg/kg bw/day) underwent histopathological examination.


For details on terminal procedures regarding reproductive and developmental parameters, please refer to section 7.8.

Statistics:

Please refer to the document "Statistical Analyis_OECD 422" under "Attached background material".

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 375 mg/kg bw/day, 1/10 females showed decreased activity and unsteady gait at the 1 – 2 and 4 – 5 hour observation after the first dose. The animal further showed abnormally cold to touch, piloerection, swaying and uncoordinated gait and hunched posture and was killed prematurely on animal welfare reasons. Further findings became evident at macroscopical analysis.
For the remaining animals treated at 375 mg/kg bw/day, decreased activity (2/10 males and 6/10 females) and unsteady gait (5/10 males and 4/10 females) were noted in both sexes on Days 1 and 2 of treatment. Unsteady gait was further noted in 1/10 males on Day 7 of treatment. In addition, there was a single incidence of unsteady gait on Day 2 in 1/10 males of the 125 mg/kg bw/day dose group. The clinical signs were attributed to treatment and considered to be of toxicological relevance. There were no treatment-related findings with regard to physical examination and arena observation in males or females before pairing, in females during gestation or in females during lactation. For details please refer to Table no. 1 under “Any other information on results incl. tables”.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

1/10 females of the 375 mg/kg bw/day group was sacrificed prematurely on animal welfare reasons, as severe clinical signs of toxicity were observed. There were also macroscopic lesions observed in this animal, but because no such additional signs and findings were noted in the remaining animals of this group, the mortality was not related to treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 375 mg/kg bw/day, the group mean body weight gain of males was slightly reduced from Day 1 – 36 of the treatment period (-27.5%) and attained statistical significance from Day 1 – 8 of the study (-55.5%). The group mean body weight gain was also reduced for females of this group (-35.3%) from Day 1 – 15 of the study when compared to the control group, but did not attain statistical significance. The findings were in line with a reduced food consumption in both sexes during study Week 1 and in males during study Week 2 and considered treatment-related.
There was no effect on body weight gain in males (treatment from Day 1 – 36) or females (before pairing, treatment from Day 1 – 15) at the lower dose groups. In addition, there was no effect on body weight gain in females of any dose group during Day 0 - 20 of gestation. During lactation, a reduced body weight gain was noted in females at 375 mg/kg bw/day from Day 4 – 7 only (statistically not significant), but the overall weight gain was similar to those of control animals. For details please refer to Table no. 2 under “Any other information on results incl. tables”.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 375 mg/kg bw/day, there was a slight but statistically significant reduction in group mean food intake for males (-17.7%) and females (-8.8%, statistically not significant) during the first week of administration. The overall food intake in males of the high dose group during the whole treatment period (study weeks 1 – 5) was slightly low (statistically not significant) when compared to control animals. The findings on reduced food consumption during the first study Week were consistent with a reduction in body weight and therefore considered treatment-related. There was no adverse effect on food consumption in female animals during the whole course of the study and no abnormal food intake in animals of the lower dose groups.
For details please refer to Table no. 3 under “Any other information on results incl. tables”.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In females there was a statistically significant increase in mean cell haemoglobin (MCH) levels at 125 (+6.7%) and 375 mg/kg bw/day (+3.9%) on Day 13 of lactation. Slight but statistically not significant increases in MCH were also noted in females at 41.7 mg/kg bw/day (+2.8%) and in males at 375 mg/kg bw/day (+4.3%). Females treated at 375 mg/kg bw/day, further showed a statistically significant increase (+ 4.3%) in mean cell haemoglobin concentration (MCHC). MCHC levels in females were also slightly increased at 125 mg/kg bw/day (+1.8%, statistically not significant).
In addition, prothrombin time (PT) was unusually high for females that received 375 mg/kg bw/day, however, a review of the individual data for this parameter reveals an abnormal value for one female only, therefore the finding was not attributed to treatment. A slight increase in activated partial thromboplastin time (APTT) was found in females at 125 and 375 mg/kg bw/day (+20.7% and +16.2%, statistically not significant) when compared to control animals. All findings were only marginal or occurred without a clear dose response relationship and were therefore considered non-adverse. For details please refer to Table no. 4 under “Any other information on results incl. tables”.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At 125 and 375 mg/kg bw/day, there was an increase in male bilirubin levels after 5 weeks of treatment (+100% at both, 125 and 375 mg/kg bw/day, statistically significant only for the 375 mg/kg bw/day group) when compared to control animals. In addition, males of the high dose group showed a statistically significant decrease in albumin levels at 41.7 (-5.1%), 125 (-2.6%) and 375 mg/kg bw/day (-2.6%). In addition, there was a statistically significant increase in inorganic phosphorus (+18.6%) at 125 mg/kg bw/day only. The findings occurred without a clear dose-response relationship and in the absence of any microscopical findings and were therefore considered to be non-adverse.
In females, there was a statistically significant increase in total protein levels (+8.9%) at 375 mg/kg bw/day. In addition, there was a slight but statistically significant increase in sodium levels at 375 mg/kg bw/day (+2.9%) and in chloride at 125 (+5.5%) and 375 mg/kg bw/day (+8.4%). Potassium levels were also increased in females at 375 mg/kg bw/day (+3.4%, statistically not significant). There was further an increase in triglyceride levels at 125 (+30.0%) and 375 mg/kg bw/day (+26.7%), an increase in glucose (+23.9, +12.1 and +18.1% at 41.7, 125 and 375 mg/kg bw/day) and bile acids (+60.3, +19.0 and +39.7% at 41.7, 125 and 375 mg/kg bw/day), but the findings were statistically not significant when compared to control levels and occurred without any dose-response relationship.
Statistically significant reductions in clinical chemistry parameters in females were noted for the high dose group in the albumin/globulin ratio (-12.1%). Reductions were also noted for alkaline phosphatase (+0.7, +9.6 and -30.4% at 41.7, 125 and 375 mg/kg bw/day), asparate aminotransferase (-10.9% at 375 mg/kg bw/day) and phosphorus (-24.2, -9.9 and -26.4% at 41.7, 125 and 375 mg/kg bw/day), but the findings occurred without any dose-response relationship and were statistically not significant. All observations in females were considered to be without toxicological relevance. For details please refer to Table no. 5 under “Any other information on results incl. tables”.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

At 375 mg/kg bw/day, the hindlimb grip strength in males was slightly decreased (-10.2%) when compared to control animals. In females at 375 mg/kg bw/day, the forelimb grip strength was slightly increased (+16.0%) when compared to control animals. Both findings attained statistical significance, but due to the different direction of change in males and females, the findings were attributed to biological variation.
Group mean motor activity scores for males and females showed some inter-group variation with data for males that received 375 mg/kg/day showing an isolated statistical significance for high beams at the 6-minute interval, but none of these differences were considered to be associated with treatment.
There were no sensory or motor activity findings in animals of the lower dose groups. For details please refer to Tables no. 6 and 7 under “Any other information on results incl. tables”.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In males of the 375 mg/kg bw/day group, there was a statistically significant increase in the body weight adjusted mean heart (+10.3%), liver (+14.1%) and kidney weight (+10.4%) when compared to control animals. In females, the mean body weight adjusted liver (+19.1% and +9.5%) and kidney weights (+16.1% and +11.1%) were statistically significantly increased at 125 and 375 mg/kg bw/day. The findings in liver and kidneys in males correlated with microscopical abnormalities and were therefore considered treatment-related and of toxicological significance. The findings in females did not follow a dose-response relationship and, in the absence of histopathological changes, were therefore considered to be without toxicological relevance. There were no organ weight changes in animals of the lower dose groups. For details please refer to Table no. 8 under “Any other information on results incl. tables”.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no treatment related macroscopic abnormalities at scheduled termination for males and females.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At 375 mg/kg bw/day, treatment-related microscopic abnormalities were predominantly observed in the kidneys of male rats, comprising hyaline droplets (5/5 males), granular casts (4/5 males) and tubular basophilia (3/5 males). These findings correlated with higher adjusted mean kidney weights in this group and were considered adverse.
Findings in the liver comprised minimal centrilobular hepatocyte hypertrophy and was noted in 3/5 males administered 375 mg/kg bw/day. The finding correlated with increased adjusted mean liver weights in the same sex group. This finding was however, considered non-adverse. There were no findings in histopathology at the lower dose groups. For details please refer to Table no. 9 under “Any other information on results incl. tables”.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Thyroid hormone analysis: There were no treatment-related findings, all levels in treated animals were comparable to those of control animals.

Key result

Dose descriptor:

LOAEL

Remarks:

systemic

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no adverse effects observed

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse effects observed.

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Table 1: Clinical signs of toxicity

Category	Observation	Males				Females
Dose (mg/kg bw/day)		0	41.7	125	375	0	41.7	125	375
Abnormal gait	Unsteady	0/10	0/10	1/10	2/10	0/10	0/10	0/10	6/10
Behaviour	Decreased activity	0/10	0/10	0/10	5/10	0/10	0/10	0/10	4/10
	Eating of bedding material	0/10	0/10	0/10	1/10	0/10	0/10	0/10	1/10
Body Temperature	Abnormally cold to touch	0/10	0/10	0/10	1/10	0/10	0/10	0/10	0/10
Coat	Piloerection	0/10	0/10	0/10	1/10	0/10	0/10	0/10	0/10
Posture	Prostate	0/10	0/10	0/10	1/10	0/10	0/10	0/10	0/10

Table 2: Body weight and body weight gain in males and females before pairing and in females during lactation

Days

Change

Body weight (g) and body weight change (g) group mean values for males and females before pairing

Sex

Dose group (mg/kg bw/day)

1

8

15

22

29

36

1 - 8

8 - 15

15 - 22

22 - 29

22 - 36

1 - 15

1 - 36

Male

0

324 ± 7.9

342 ± 12.6

360 ± 18.2

369 ± 23.1

384 ± 26.8

393 ± 30.7

18 ± 7.1

18 ± 6.3

9 ± 6.9

15 ± 4.5

24 ± 10.2

36 ± 12.3

69 ± 24.2

41.7

325 ± 7.2

346 ± 15.6

368 ± 23.9

379 ± 29.2

395 ± 33.7

410 ± 42.6

21 ± 9.5

22 ± 9.2

12 ± 7.7

16 ± 4.8

31 ± 13.7

43 ± 18.0

85 ± 37.3

125

322 ± 6.4

340 ± 8.4

358 ± 10.8

368 ± 12.1

381 ± 14.5

393 ± 14.7

18 ± 4.7

18 ± 5.3

10 ± 5.6

13 ± 4.6

24 ± 8.2

36 ± 8.1

70 ± 13.1

375

321 ± 5.2

329 ± 13.1*

343 ± 20.5

348 ± 22.6

362 ± 28.1

371 ± 34.0

8 ± 8.7**

14 ± 8.6

6 ± 6.8

13 ± 6.1

23 ± 12.4

21 ± 16.4*

50 ± 29.6

Female

0

211 ± 12.2

220 ± 12.6

228 ± 13.5

9 ± 5.1

8 ± 5.2

17 ± 7.4

41.7

208 ± 12.6

216 ± 13.4

224 ± 14.3

8 ± 4.9

8 ± 4.4

16 ± 7.3

125

211 ± 10.3

219 ± 117

228 ± 12.1

8 ± 3.6

8 ± 6.2

17 ± 6.4

375

210 ± 11.8

217 ± 16.5

222 ± 17.2

6 ± 7.4

5 ± 6.0

11 ± 8.3

Body weight (g) and body weight change (g) females during lactation

Sex

Dose group (mg/kg bw/day)

1

4

7

13

1 - 4

4 - 7

7 - 13

1 - 13

Female

0

260 ± 20.3

261 ± 21.0

267 ± 22.9

281 ± 23.7

2 ± 4.8

6 ± 6.6

13 ± 5.6

21 ± 10.8

41.7

252 ± 23.1

256 ± 25.2

265 ± 25.6

277 ± 18.7

4 ± 4.0

10 ± 8.6

11 ± 10.9

25 ± 10.4

125

260 ± 21.9

265 ± 21.9

272 ± 23.0

285 ± 26.7

5 ± 4.1

8 ± 5.0

12 ± 12.0

25 ± 13.5

375

248 ± 16.0

250 ± 17.1

252 ± 18.3

268 ± 21.2

1 ± 7.1

2 ± 7.4

16 ± 9.2

20 ± 12.3

 * and **: statistical significance at p < 0.05 and p < 0.01

Table 3: Food consumption in males and females before pairing

		Week				Change
Food consumption (g/animal/week) group mean values for males and females before pairing (F0)
Sex	Dose group (mg/kg bw/day)	1	2	4	5	1 - 2	1 - 5
Male	0	129 ± 0.4	130 ± 8.2	124 ± 0.2	122 ± 4.7	130 ± 4.2	126 ± 2.3
	41.7	130 ± 14.9	129 ± 16.1	131 ± 13.8	131 ± 12.3	129 ± 15.5	130 ± 14.2
	125	123 ± 11.4	122 ± 7.0	123 ± 7.2	124 ± 1.6	122 ± 9.1	123 ± 6.5
	375	101 ± 8.2*	112 ± 3.6	120 ± 4.4	118 ± 5.3	107 ± 5.8*	113 ± 5.3
Female	0	92 ± 3.2	89 ± 2.5			91 ± 2.1
	41.7	89 ± 7.4	88 ± 9.4			89 ± 8.4
	125	89 ± 7.6	92 ± 10.9			91 ± 9.2
	375	79 ± 4.2*	87 ± 2.7			83 ± 3.4

 *: statistical significance at p < 0.05

Table 4: Haematology parameters at study termination

Parameter	Males				Females
Dose (mg/kg bw/day)	0	41.7	125	375	0	41.7	125	375
MCH (pg)	18.5 ± 0.81	18.5 ± 1.02	18.5 ± 1.09	19.3 ± 1.31	17.9 ± 0.16	18.4 ± 0.43	19.1 ± 0.84*	18.6 ± 0.70*
MCHC (g/dL)	28.9 ± 0.84	28.7 ± 0.75	28.7 ± 0.97	29.7 ± 0.82	27.9 ± 0.99	28.4 ± 0.53	28.4 ± 0.52	29.1 ± 0.71*
PT (sec)	23.9 ± 3.99	22.2 ± 2.42	20.4 ± 0.97	22.3 ± 2.73	20.4 ± 0.70	21.8 ± 1.21	22.4 ± 2.18	31.9 ± 22.95
APTT (sec)	17.1 ± 3.20	15.7 ± 2.17	16.1 ± 1.85	15.5 ± 1.21	11.1 ± 2.74	11.2 ± 2.73	13.4 ± 5.32	12.9 ± 5.82
MCH: mean corpuscular haemoglobin; MCHC: mean corpuscular haemoglobin concentration, PT: prothrombine time; APTT: activated partial thromboplastine time  *: statistical significance at p < 0.05

Table 5: Clinical chemistry parameters at study termination

Parameter	Males				Females
Dose (mg/kg bw/day)	0	41.7	125	375	0	41.7	125	375
A/G	1.47 ± 0.121	1.34 ± 0.078	1.43 ± 0.047	1.46 ± 0.080	1.66 ± 0.065	1.65 ± 0.049	1.61 ± 0.062	1.46 ± 0.130**
Alb (g/L)	39 ± 0.8	37 ± 0.5*	38 ± 1.1*	38 ± 0.8*	35 ± 1.3	36 ± 1.1	36 ± 1.2	36 ± 2.2
ALP (U/L)	128 ± 23.1	154 ± 34.1	119 ± 16.0	111 ± 35.9	135 ± 50.5	136 ± 37.3	148 ± 57.4	94 ± 21.2
AST (U/L)	76 ± 17.4	65 ± 15.5	67 ± 15.9	68 ± 14.9	101 ± 16.4	86 ± 8.3	90 ± 9.4	90 ± 17.5
Bile Ac. (µmol/L)	14.8 ± 7.19	18.2 ± 6.30	5.6 ± 2.70	21.0 ± 11.25	12.6 ± 5.03	20.2 ± 11.88	15.0 ± 4.64	17.6 ± 6.69
Bili (µmol/L)	0 ± 0.4	0 ± 0.5	1 ± 0.5	1 ± 0.0*	0 ± 0.5	1 ± 0.5	0 ± 0.5	0 ± 0.5
Cl- (mmol/L)	99.2 ± 1.59	99.2 ± 0.97	99.0 ± 1.67	98.3 ± 0.98	95.7 ± 0.82	96.9 ± 1.22	101.0 ± 5.08*	103.7 ± 6.48*
Glc (mmol/L)	12.71 ± 0.938	11.72 ± 1.171	10.74 ± 1.060	11.72 ± 1.545	6.53 ± 1.636	8.09 ± 1.211	7.32 ± 1.484	7.71 ± 1.644
K+ (mmol/L)	4.02 ± 0.248	4.39 ± 0.157	4.24 ± 0.240	4.09 ± 0.265	4.94 ± 0.149	5.00 ± 0.378	5.76 ± 1.052	5.11 ± 0.589
Na+ (mmol/L)	140 ± 1.3	140 ± 1.3	140 ± 1.0	139 ± 0.8	139 ± 1.1	140 ± 0.0	141 ± 1.2	143 ± 4.0*
P (mmol/L)	1.40 ± 0.074	1.62 ± 0.122	1.66 ± 0.116*	1.37 ± 0.227	0.91 ± 0.344	0.69 ± 0.469	0.82 ± 0.212	0.67 ± 0.418
Total prot. (g/L)	66 ± 1.6	65 ± 1.7	64 ± 1.3	64 ± 1.5	56 ± 2.0	57 ± 1.5	58 ± 2.7	61 ± 4.8*
Trig (mmol/L)	1.03 ± 0.286	1.13 ± 0.399	1.68 ± 0.678	0.99 ± 0.599	0.30 ± 0.084	0.32 ± 0.089	0.39 ± 0.061	0.38 ± 0.077
A/G: albumin/globulin ratio; Alb: albumin; ALP: alkaline phosphatase; AST: aspartate aminotransferase; Bile Ac.: bile acids; bili: bilirubin; Cl-: chloride; Na+: sodium; K+: potassium; P: inorganic phosphorus; Total prot.: total protein; Trig: triglycerides; * and **: statistical significance at p < 0.05 and p < 0.01

Table 6: Grip strength measurements during week 5 of treatment (males) or during Days 7 - 9 of lactation (females)

	Males				Females
	0	41.7	125	375	0	41.7	125	375
Forelimb grip strength (kg)	1.17 ± 0.08	1.13 ± 0.16	1.19 ± 0.16	1.12 ± 0.09	1.00 ± 0.13	0.98 ± 0.11	1.06 ± 0.06	1.16 ± 0.12*
HCD mean (range)	1.13 (1.03 - 1.23)				1.01 (0.90 - 1.16)
Hindlimb grip strength (kg)	0.49 ± 0.02	0.51 ± 0.02	0.55 ± 0.07	0.44 ± 0.01*	0.52 ± 0.05	0.52 ± 0.06	0.57 ± 0.05	0.51 ± 0.08
HCD mean (range)	0.51 (0.39 - 0.66)				0.48 (0.36 - 0.62)
* statistical significance at p < 0.05; HCD: historical control data summarising 15 studies conducted in Jan 2017 - Nov 2019

Table 7: Motor activity measurements during week 5 of treatment (males) or during Days 7 - 9 of lactation (females)

Sex	Dose group (mg/kg bw/day)	High beam level
		6 min	30 min	60 min	Total
Male	0	90.4 ± 13.8	5.8 ± 11.3	9.4 ± 7.1	222.0 ± 57.2
	41.7	84.4 ± 16.7	2.4 ± 5.4	4.2 ± 9.4	156.2 ± 25.7
	125	67.2 ± 16.6	10.2 ± 10.9	19.8 ± 32.1	195.4 ± 120.8
	375	58.2 ± 21.9*	8.4 ± 10.4	10.0 ± 8.6	169.8 ± 25.4
Female	0	49.8 ± 7.3	16.2 ± 10.2	3.6 ± 8.0	162.4 ± 69.7
	41.7	75.5 ± 24.9	20.0 ± 32.5	12.0 ± 14.2	250.5 ± 132.9
	125	71.2 ± 17.3	30.0 ± 18.4	27.8 ± 15.9	285.6 ± 63.8
	375	69.4 ± 30.7	13.6 ± 9.0	8.2 ± 8.5	187.6 ± 87.8

 *: statistical significance at p < 0.05

Table 8: Organ weight analysis

Parameter	Males				Females
Dose (mg/kg bw/day)	0	41.7	125	375	0	41.7	125	375
Liver weight
absolute (g)	11.754 ± 1.350	14.848 ± 2.215	12.997 ± 1.274	13.260 ± 1.315	12.753 ± 1.571	12.283 ± 0.574	14.877 ± 0.604	12.587 ± 2.017
relative to bw (g)	12.569	13.023	13.399	13.867**	12.263	12.204	14.599*	13.433*
Kidney weight
absolute (g)	2.107 ± 0.269	2.464 ± 0.237	2.310 ± 0.249	2.446 ± 0.284	1.616 ± 0.146	1.691 ± 0.173	1.840 ± 0.090	1.619 ± 0.201
relative to bw (g)	2.209	2.237	2.360	2.521 *	1.554	1.681	1.804**	1.727**
Heart weight
absolute (g)	0.961 ± 0.088	1.114 ± 0.127	1.042 ± 0.072	1.079 ± 0.121	0.898 ± 0.066	0.870 ± 0.068	0.919 ± 0.049	0.834 ± 0.099
relative to bw (g)	1.012	1.000	1.067	1.117**	0.885	0.868	0.911	0.857
* and **: statistical significance at p < 0.05 and p < 0.01

Table 9: Microscopic findings in males after 5 weeks of treatment

Dose Level (mg/kg bw/day)	0	41.7	125	375
Kidney
Number Examined	5	5	5	5
Accumulation, Hyaline Droplets
Minimal	0	0	0	3
Slight	0	0	0	2
Cast(s), Granular
Minimal	0	0	0	3
Slight	0	0	0	1
Basophilia, Tubular
Minimal	1	0	1	2
Slight	0	0	0	1
Liver
Number Examined	5	5	5	5
Hypertrophy, Centrilobular
Minimal	0	0	0	3

Conclusions:

Under the conditions of the study, the NOAEL for systemic toxicity was 125 mg/kg bw/day in male rats, based on alpha 2u-globulin nephropathy observed at 375 mg/kg bw/day. For females, a NOAEL of 375 mg/kg bw/day was established. Alpha 2u-globulin nephropathy is specific for male rats only and not relevant for humans. Thus, a NOAEL of 375 mg/kg bw/day will be used for hazard and risk assessment in humans.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

adopted in 2016

Deviations:

yes

Remarks:

no historical control data provided for several parameters in haematology (prothrombine time, although the results were described as "unusually high"), clinical chemistry, reproductive performance, viability indices or sexual maturation of the offspring

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1,3-bis(trimethylsilyl)urea

EC Number:

242-177-9

EC Name:

1,3-bis(trimethylsilyl)urea

Cas Number:

18297-63-7

Molecular formula:

C7H20N2OSi2

IUPAC Name:

1,3-bis(trimethylsilyl)urea

Test animals

Species:

rat

Strain:

Wistar

Remarks:

RccHanTM

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS, UK
- Age at study initiation: 69 – 76 days (males) and 98 – 104 days (females)
- Weight at study initiation: 311 – 336 g (males) and 189 – 237 g (females)
- Housing: Prior to mating, the animals were housed in groups of 4/sex in polycarbonate cages with a stainless steel mesh lid and with softwood based bark-free fiber bedding. During mating, males and females were housed in a 1 : 1 ratio in grid bottomed cages with absorbent paper. After mating, males were housed in up to 4 animals while females were housed individually. Throughout the study, the cages were enriched with an aspen chew block (except during lactation), a plastic shelter (ecept during pairing and lactation) and with paper shravings from Day 20 after mating and throughout lactation.
- Diet: SDS VRF1 certified pelleted diet, ad libitum
- Water: potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum
- Acclimation period: 6 days (males) and 20 days (females)

DETAILS OF FOOD AND WATER QUALITY:
Certificates of analysis for the diet were scrutinized and approved before any batch of diet was released for use. Certificates of analysis were routinely provided by the water supplier.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 – 24
- Humidity (%): 40 - 70
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 26 Feb 2020 To: 10 May 2020

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Remarks:

dried, deacidified

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
A correction factor of 1.03 was used when calculating quantities of test item used during dose preparation. The required amount of test material was weighed, transferred to a mortar and ground to a fine powder using a pestle. Small amounts of the pre-weighed vehicle were added and mixed to form a smooth paste. The suspension was poured into a measuring cylinder which had been wetted with the vehicle (dried, de-acidified corn oil) and the mortar was rinsed thoroughly with the vehicle and the rinsed residue was added to the measuring cylinder. The required volume was achieved by addition of the remaining vehicle and the suspension was transferred to a beaker for mixing using a high shear homogenizer until visibly homogenous. A series of formulations at the required concentrations were prepared by dilution of individual weighings of the test item. Test item dosing formulations were prepared daily and stored refrigerated (2 – 8 °C).
VEHICLE:
- Nominal concentration in vehicle: 8.34, 25 and 75 mg/mL, corresponding to a formulated concentration of 8.6, 25.8 and 77.3 mg/mL
- Amount of vehicle: 5 mL/kg bw

Details on mating procedure:

- M/F ratio per cage: 1:1 from within the same treatment groups.
- Length of cohabitation: Up to two weeks.
- Proof of pregnancy: Ejected copulation plugs in cage tray or sperm in vaginal smear referred to as Day 0 of gestation.
- After successful mating each pregnant female was caged individually.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples from each ofthe first and the last preparation were analysed for achieved concentrations of the test item after extraction with ethylacetate, using gas chromatographic analysis with flame ionisation detection. For the low dose (41.7 mg/kg bw/day) and the mid dose formulation (125 mg/kg bw/day), the mean concentrations were within 10% of the nominal concentration, confirming the accuracy of formulation. For the highest dose formulation (375 mg/kg bw), the mean concentrations were slightly outside the acceptable 10% of the nominal concentration (+12% for the first preparation and +13% for the last preparation, respectively). The difference from mean and coefficient of variation remained within 5%, confirming precise analysis. The procedural recoveries remained in the validated range confirming the continued accuracy of the analytical method, with the exception of one procedural recovery prepared during the last preparation which was excluded as an outlier.
In addition, the homogeneity and stability of the formulations during storage were confirmed as part of another study. Results indicated that the formulations were stable for 2 hours when stored at ambient temperature (15 - 25 °C) ond for 1 day when stored refrigerated (2 - 8 °C).

Duration of treatment / exposure:

Males were treated daily for 2 weeks before pairing, up to necropsy after a minimum of 5 consecutive weeks.
Females were treated daily for 2 weeks before pairing, throughout pairing, gestation and until Day 13 of lactation.

Frequency of treatment:

daily, 7 days/week

Details on study schedule:

not applicable for an OECD 422 study

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

A correction factor of 1.03 was used when calculating quantities of test item used during dose preparation.

Doses / concentrations 2

Dose / conc.:

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

Remarks:

A correction factor of 1.03 was used when calculating quantities of test item used during dose preparation.

Doses / concentrations 3

Dose / conc.:

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

Remarks:

A correction factor of 1.03 was used when calculating quantities of test item used during dose preparation.

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Dose levels were selected based on the results of a preliminary 14-days dose-range finding study in rats. Groups of 3 males and 3 females were exposed to test item doses of 300, 450, 650 or 1000 mg/kg bw/day. After the first dose administration, males and females administered ≥ 650 mg/kg bw/day showed signs comprised of gasping, irregular and shallow breathing, decreased activity, unsteady gait and prostrate posture. Subsequently, these groups were prematurely killed on the grounds of animal welfare. Macroscopic examination of these animals revealed instances of esophagus distention and abnormal contents in the gastrointestinal tract. Animals receiving 450 mg/kg bw/day showed similar clinical signs on Day 1 and 2 of treatment but the extent was less severe, showed no respiratory changes and no treatment-related signs were observed from Day 4 to 15 of treatment. In males and females of the 450 mg/kg bw/day group there was a reduction in body weight gain over the study period and a slight reduction in overall group mean food consumption, when compared to the 300 mg/kg bw/day group. There were no macroscopic findings after 14 days of treatment in both sexes that received 300 or 450 mg/kg bw/day. In addition, absolute liver weight was low for males receiving 450 mg/kg bw/day, when compared to the 300 mg/kg bw/day group, while the organ weight of females was unaffected by treatment. Accordingly, 375 mg/kg bw/day was selected as a suitable high dose for the present OECD 422 screening study.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily
- Cage side observations included: signs associated with dosing, clinical signs of ill-health and mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to treatment, once per week during treatment, on Days 0, 6, 13 and 20 after mating and on Days 1, 6 and 12 of lactation.
- Detailed clinical observations included: Detailed physical examinations and arena observations.

BODY WEIGHT: Yes
- Time schedule for examinations in males: Prior to dosing on the day that treatment commenced (Day 1) and weekly thereafter.
- Time schedule for examinations in females: Prior to dosing on the day that treatment commenced (Day 1), weekly before pairing, on Days 0, 7, 14 and 20 after mating, in Day 1, 4, 7 and 13 of lactation and on the day of necropsy.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

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

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At termination, blood samples of approx. 0.5 mL were collected from the sublingual vein into tubes containing EDTA anticoagulant.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: No
- How many animals: The 5 lowest numbered surviving males and the first 5 lactating females with a surviving litter per group.
- Parameters examined: haematocrit (Hct), haemoglobin concentration (Hb), erythrocyte count (RBC), absolute reticulocyte count (Retic), mean cell haemoglobin (MCH), mean cell haemoglobin concentration (MCHC), mean cell volume (MCV), red cell distribution width (RDW), total leucocyte count (WBC), differential leucocyte count (neutrophils (N), lymphocytes (L), eosinophils (E), basophils (B), monocytes (M), large unstained cells (LUC)), platelet count (Plt), prothrombin time (PT) and activated partial thromboplastin time (APTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At termination, blood samples of approx. 0.5 mL were collected from the sublingual vein into tubes containing lithium heparin as anticoagulant.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: No
- How many animals: The 5 lowest numbered surviving males and the first 5 lactating females with a surviving litter per group.
- Parameters examined: alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (Bili), bile acids (Bi Ac), urea, creatinine (Creat), glucose (Gluc), total cholesterol (Chol), triglycerides (Trig), sodium (Na), potassium (K), chloride (Cl), calcium (Ca), inorganic phosphorus (Phos), total protein (Total Prot), albumin (Alb) and albumin/globulin ratio (A/G Ratio).

THYROID HORMONE ANALYSIS: Yes
- Time schedule for collection of blood: At termination, blood samples were collected from surviving F0 males and F0 females from the sublingual vein.
- Anaesthetic used for blood collection: In adult animals, blood was collected under isoflurane anesthesia.
- Animals fasted: No
- How many animals: All surviving F0 males and all surviving F0 reproductive phase females (no samples obtained from females that failed to litter).
- Parameters examined: thyroxine (T4) and thyroid stimulating hormone (TSH).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations and dose groups examined: Prior to dosing, on the 5 lowest numbered surviving males in each group during week 5 of treatment and on the first 5 lactating females per group at Day 7 – 9 of lactation
- Battery of functions tested: sensory activity (approach response, pinna reflex, auditory startle reflex and tail pinch response), grip strength (forelimb and hindlimp grip strength) and motor activity

IMMUNOLOGY: No

Oestrous cyclicity (parental animals):

Oestrous cycles were evaluated by examination of vaginal smears. Dry smears were taken for 15 days before pairing using cotton swabs. Wet smears were taken for 14 days before treatment, after pairing until mating using pipette lavage and four days before scheduled termination.

Sperm parameters (parental animals):

A detailed qualitative examination of the testes was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen. Any cell- or stage-specificity of testicular findings was noted.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: not specified

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number of pups (daily), sex ratio (on PND 1, 4, 7 and 13), stillbirths, live births, postnatal mortality, presence of gross anomalies, body weight (on PND 1, 4, 7 and 13), physical or behavioural abnormalities, anogenital distance (AGD, on PND 1) and presence of nipples/areolae in male pups (on PND 13).

GROSS EXAMINATION OF DEAD PUPS:
Yes, where possible a macroscopic examination (external) with an assessment of stomach for milk content was performed for prematurely dead pups.

THYROID HORMONE ANALYSIS:
- Time schedule for collection of blood: On PND 4 and 13, blood was collected from F1 offspring by decapitation.
- Anaesthetic used for blood collection: No anaesthesia was used.
- Animals fasted: No
- How many animals:
On PND 4: Offspring from up to 2 females per litter.
On PND 13: 2 males and 2 females per litter (where possible).
- Parameters examined: thyroxine (T4) and thyroid stimulating hormone (TSH). T4 examination was given priority over TSH.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

Postmortem examinations (parental animals):

SACRIFICE:
F0 Males were sacrificed after 5 weeks of treatment by carbon dioxide inhalation.
F0 females were sacrificed on lactation Day 13 by carbon dioxide inhalation.
F0 females that failed to produce a viable litter were sacrificed 25 days after mating by carbon dioxide inhalation.

GROSS PATHOLOGY:
All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any of the organs and tissues (external and cut surface) mentioned below was recorded and the required tissue samples preserved in appropriate fixative.

ORGAN WEIGHTS:
The following organ weights were collected for the first 5 F0 males and for the first 5 lactating F0 females: adrenals, brain, epididymides, heart, kidneys, liver, prostrate, seminal vesicles with coagulating glands, spleen, testes and uterus with cervix and oviducts. For bilateral organs, left and right organs were weighed together. Requisite organs were weighed for animals killed at scheduled intervals.

HISTOPATHOLOGY:
Tissue samples were dehydrated, embedded in paraffin wax and sectioned. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required. Sections were stained with hematoxylin and eosin; in addition samples of the testes were stained using a standard periodic acid/Schiff (PAS) method.
Histopathological examination of the following organs and tissues was performed for animals of the control (corn oil) and high dose group (375 mg/kg bw/day) only, for the first 5 F0 males and for the first 5 lactating F0 females per group:
Adrenals, brain (including cerebrum, cerebellum and pons), caecum, colon, duodenum, epididymides, eyes, heart (including auricular and ventricular regions), ileum, jejunum, kidneys, liver (sections from 2 lobes), lungs (section from 2 major lobes including bronchi), lymph nodes (left axillary and mesenteric), ovaries, Peyer’s patches, prostate, rectum, seminal vesicles with coagulating glands, skin with mammary glands (inguinal area), spinal cord (transverse and longitudinal sections at the cervical level), spleen, sternum (with marrow), stomach, testes, thymus, thyroids, trachea, urinary bladder and vagina. Samples from the sciatic nerve, from the skeletal muscle and from the uterus with cervix and oviducts were fixed, but not examined histopathologically.

In addition to the organs listed above, all abnormalities were investigated microscopically for all F0 males and females. Further, kidneys and livers from the 5 lowest surviving F0 males of the low (41.7 mg/kg bw/day) and mid dose groups (125 mg/kg bw/day) underwent histopathological examination.

Postmortem examinations (offspring):

SACRIFICE
- Offspring selected for thyroid hormone sampling on PND 4 and 13 was sacrificed by decapitation. Offspring not selected for thyroid hormone sampling was sacrificed on PND 13 by intraperitoneal injection of sodium pentobarbitone.

GROSS NECROPSY
- Gross necropsy consisted of external examination. Particular attention was paid to external genitals.

HISTOPATHOLOGY / ORGAN WEIGTHS: not performed

Statistics:

Please refer to the document "Statistical Analyis_OECD 422" under "Attached background material".

Reproductive indices:

Percentage mating (%) = (Number of animals mating / Animals paired) x 100
Conception rate (%) = (Number of animals achieving pregnancy / Animals mated) x 100
Fertility index (%) = (Number of animals achieving pregnancy / Animals paired) x 100
Gestation index (%) = (Number of live litters born / Number of pregnant females) x 100
Duration of gestation = Time elapsing between the detection of mating and commencement of parturition
Pre-coital interval = Time between the first pairing and evidence of mating

Offspring viability indices:

Offspring viability indices
- Post-implantation survival index (%) = (Total number of offspring born / Total number of uterine implantation sites) x 100%
- Live birth index (%) = (Number of live offspring on Day 1 after littering / Total number of offspring born) x 100
- Viability index (%) = (Number of live offspring on Day 4 (before blood sampling) / Number of live offspring on Day 1 after littering) x 100
- Lactation index (%) = (Number of live offspring on Day 13 after littering / Number of live offspring on Day 4 (after blood sampling)) x 100
Offspring sex ratio:
Percentage males (%) = Number of males in litter / Total number of offspring in litter

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 375 mg/kg bw/day, 1/10 females showed decreased activity and unsteady gait at the 1 – 2 and 4 – 5 hour observation after the first dose. The animal further showed abnormally cold to touch, piloerection, swaying and uncoordinated gait and hunched posture and was killed prematurely on animal welfare reasons. Further findings became evident at macroscopical analysis.
For the remaining animals treated at 375 mg/kg bw/day, deacreased activity (2/10 males and 6/10 females) and unsteady gait (5/10 males and 4/10 females) were noted in both sexes on Days 1 and 2 of treatment. Unsteady gait was further noted in a 1/10 males on Day 7 of treatment. In addition, there was a single incidence of unsteady gait on Day 2 in 1/10 males of the 125 mg/kg bw/day dose group. The clinical signs were attributed to treatment and considered to be of toxicological relevance. There were no treatment-related findings with regard to physical examination and arena observation in males or females before pairing, in females during gestation or in females during lactation. For details please refer to Table no. 1 under “Any other information on results incl. tables”.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

1/10 females of the 375 mg/kg bw/day group was sacrificed prematurely on animal welfare reasons, as severe clinical signs of toxicity were observed. There were also macroscopic lesions observed in this animal, but because no such additional signs and findings were noted in the remaining animals of this group, the mortality was not related to treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 375 mg/kg bw/day, the group mean body weight gain of males was slightly reduced from Day 1 – 36 of the treatment period (-27.5%) and attained statistical significance from Day 1 – 8 of the study (-55.5%). The group mean body weight gain was also reduced for females of this group (-35.3%) from Day 1 – 15 of the study when compared to the control group, but did not attain statistical significance. The findings were in line with a reduced food consumption in both sexes during study Week 1 and in males during study Week 2 and considered treatment-related.
There was no effect on body weight gain in males (treatment from Day 1 – 36) or females (before pairing, treatment from Day 1 – 15) at the lower dose groups. In addition, there was no effect on body weight gain in females of any dose group during Day 0 - 20 of gestation. During lactation, a reduced body weight gain was noted in females at 375 mg/kg bw/day from Day 4 – 7 only (statistically not significant), but the overall weight gain was similar to those of control animals. For details please refer to Table no. 2 under “Any other information on results incl. tables”.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 375 mg/kg bw/day, there was a slight but statistically significant reduction in group mean food intake for males (-17.7%) and females (-8.8%, statistically not significant) during the first week of administration. The overall food intake in males of the high dose group during the whole treatment period (study weeks 1 – 5) was slightly low (statistically not significant) when compared to control animals. The findings on reduced food consumption during the first study Week were consistent with a reduction in body weight and therefore considered treatment-related. There was no adverse effect on food consumption in female animals during the whole course of the study.
For details please refer to Table no. 3 under “Any other information on results incl. tables”.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In females there was a statistically significant increase in mean cell haemoglobin (MCH) levels at 125 (+6.7%) and 375 mg/kg bw/day (+3.9%) on Day 13 of lactation. Slight but statistically not significant increases in MCH were also noted in females at 41.7 mg/kg bw/day (+2.8%) and in males at 375 mg/kg bw/day (+4.3%). Females treated at 375 mg/kg bw/day, further showed a statistically significant increase (+ 4.3%) in mean cell haemoglobin concentration (MCHC). MCHC levels in females were also slightly increased at 125 mg/kg bw/day (+1.8%, statistically not significant).
In addition, prothrombin time (PT) was unusually high for females that received 375 mg/kg bw/day, however, a review of the individual data for this parameter reveals an abnormal value for one female only, therefore the finding was not attributed to treatment. A slight increase in activated partial thromboplastin time (APTT) was found in females at 125 and 375 mg/kg bw/day (+20.7% and +16.2%, statistically not significant) when compared to control animals. All findings were only marginal or occurred without a clear dose response relationship and were therefore considered non-adverse. For details please refer to Table no. 4 under “Any other information on results incl. tables”.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At 125 and 375 mg/kg bw/day, there was an increase in male bilirubin levels after 5 weeks of treatment (+100% at both, 125 and 375 mg/kg bw/day, statistically significant only for the 375 mg/kg bw/day group) when compared to control animals. In addition, males of the high dose group showed a statistically significant decrease in albumin levels at 41.7 (-5.1%), 125 (-2.6%) and 375 mg/kg bw/day (-2.6%). In addition, there was a statistically significant increase in inorganic phosphorus (+18.6%) at 125 mg/kg bw/day only. The findings occurred without a clear dose-response relationship and in the absence of any microscopical findings and were therefore considered to be non-adverse.
In females, there was a statistically significant increase in total protein levels (+8.9%) at 375 mg/kg bw/day. In addition, there was a slight but statistically significant increase in sodium levels at 375 mg/kg bw/day (+2.9%) and in chloride at 125 (+5.5%) and 375 mg/kg bw/day (+8.4%). Potassium levels were also increased in females at 375 mg/kg bw/day (+3.4%, statistically not significant). There was further an increase in triglyceride levels at 125 (+30.0%) and 375 mg/kg bw/day (+26.7%), an increase in glucose (+23.9, +12.1 and +18.1% at 41.7, 125 and 375 mg/kg bw/day) and bile acids (+60.3, +19.0 and +39.7% at 41.7, 125 and 375 mg/kg bw/day), but the findings were statistically not significant when compared to control levels and occurred without any dose-response relationship.
Statistically significant reductions in clinical chemistry parameters in females were noted for the high dose group in the albumin/globulin ratio (-12.1%). Reductions were also noted for alkaline phosphatase (+0.7, +9.6 and -30.4% at 41.7, 125 and 375 mg/kg bw/day), asparate aminotransferase (-10.9% at 375 mg/kg bw/day) and phosphorus (-24.2, -9.9 and -26.4% at 41.7, 125 and 375 mg/kg bw/day), but the findings occurred without any dose-response relationship and were statistically not significant. All observations in females were considered to be without toxicological relevance. For details please refer to Table no. 5 under “Any other information on results incl. tables”.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

At 375 mg/kg bw/day, the hindlimb grip strength in males was slightly decreased (-10.2%) when compared to control animals. In females at 375 mg/kg bw/day, the forelimb grip strength was slightly increased (+16.0%) when compared to control animals. Both findings attained statistical significance, but due to the different direction of change in males and females, the findings were attributed to biological variation.
Group mean motor activity scores for males and females show some inter-group variation with data for males that received 375 mg/kg/day showing an isolated statistical significance for high beams at the 6-minute interval, but none of these differences were considered to be associated with treatment.
There were no sensory or motor activity findings in animals of the lower dose groups. For details please refer to Tables no. 6 and 7 under “Any other information on results incl. tables”.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At 375 mg/kg bw/day, treatment-related microscopic abnormalities were predominantly observed in the kidneys of male rats, comprising hyaline droplets (5/5 males), granular casts (4/5 males) and tubular basophilia (3/5 males). These findings correlated with higher adjusted mean kidney weights in this group and were considered adverse.
Findings in the liver comprised minimal centrilobular hepatocyte hypertrophy and was noted in 3/5 males administered 375 mg/kg bw/day. The finding correlated with increased adjusted mean liver weights in the same sex group. This finding was however, considered non-adverse. For details please refer to Table no. 9 under “Any other information on results incl. tables”.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Thyroid hormone analysis: There were no treatment-related findings, all levels in treated animals were comparable to those of control animals.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

A statistically significant shift in oestrus cycles during treatment was noted for females of the high dose group (375 mg/kg bw/day). While all 10 females of the control group had an oestrous cycle of 4 days, 6/9 surviving females at 375 mg/kg bw/day had an oestrous cycle of 4 days, 2/9 females had an oestrous cycle of 4-5 days and 1/9 females had an irregular cycle. The finding was consistent with a shift in gestation length, a reduction in copulation plug count and sperm estimate in the vaginal smear of this group and therefore considered treatment-related.

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

At 375 mg/kg bw/day, the number of animals with a higher sperm count in the vaginal smear after mating was statistically significantly less than in control animals. However, there was no correlation between sperm estimates in the vaginal smear or low copulation plugs and siring of a pregnancy and there was no effect on reproductive performance or in histopathology of male reproductive organs. Thus, the findings were concluded to be non-adverse and of uncertain relationship to treatment. For details please refer to Table no. 10 under “Any other information on results incl. tables”.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Percentage mating: At 375 mg/kg bw/day, 8/9 mated females achieved pregnancy. In the remaining groups, all animals were successfully mated.
There was a statistically significant shift in the number of copulation plugs in the animals of the high dose group. In the control group, the amount of animals with a higher copulation plug number was higher (1/10 animals with 2 copulation plus, 4/10 animals with 3 copulation plugs and 5/10 animals with 4 – 6 copulation plugs), there were less animals with a high copulation plug number observed at 375 mg/kg bw/day (1/9 animals with 1 copulation plug, 4/9 animals with 2 copulation plugs, 3/9 animals with 3 copulation plugs and 1/9 animals with 4 – 6 copulation plugs).

Conception rate: There was a very slight decrease in conception rate in females at 375 mg/kg bw/day (-11%, statistically not significant) which was a result of one female not having achieved pregnancy. Due to the single occurrence the finding was not attributed to treatment.

Fertility index: There was a very slight decrease in fertility index in females at 375 mg/kg bw/day (-11%, statistically not significant) which was a result of one female not having achieved pregnancy. Due to the single occurrence the finding was not attributed to treatment.

Gestation index: Gestation indices were considered not to be affected by treatment and were 100% for all groups.

Duration of gestation: A statistically significant shift in gestation length was observed for females at 375 mg/kg bw/day. While females of the control group had a gestation length of 22 (2/10), 22.5 (5/10) or 23 (3/10) days, females of the high dose group had a gestation length of 22.5 (2/8), 23 (5/8) or 23.5 (1/8) days.

Pre-coital interval: 1/10 males of the 375 mg/kg bw/day dose group showed a longer pre-coital interval of 5 – 8 days, however, the same finding was noted for 1/10 males at 41.7 mg/kg bw/day, therefore the pre-coital interval was considered to be unaffected by treatment.

Effect levels (P0)

1

Key result

Dose descriptor:

LOAEL

Remarks:

systemic

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no adverse effects observed

3

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse effects observed

4

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Post-implantation survival index: The total number of offspring born compared to the total number of uterine implantations was considered not to be affected by treatment. Post-implantation survival indices were 94.9 ± 6.96 % (control group), 96.2 ± 7.30 % (41.7 mg/kg bw/day), 91.2 ± 10.07 % (125 mg/kg bw/day) and 97.9 ± 5.89 % (375 mg/kg bw/day), respectively.

Litter size: The mean number of living pups at first litter check was considered not to be affected by treatment. Live litter sizes were 11.1 ± 2.77 (control group), 11.2 ± 2.04 (41.7 mg/kg bw/day), 11.8 ± 2.20 (125 mg/kg bw/day) and 11.0 ± 4.90 (375 mg/kg bw/day), respectively.

Live birth index: The number of live offspring on Day 1 after littering compared to the total number of offspring born was considered not affected by treatment. Live birth indices were 100% (control group), 100% (41.7 mg/kg bw/day), 100% (125 mg/kg bw/day) and 97.5% (375 mg/kg bw/day), respectively.

Viability index: The number of live offspring on PND 4 before blood sampling compared to the number of offspring on Day 1 after littering was considered not to be affected by treatment. Viability indices were 100% (control group), 100% (41.7 mg/kg bw/day), 100% (125 mg/kg bw/day) and 99.2% (375 mg/kg bw/day), respectively.

Lactation index: The number of live offspring on PND 13 after littering compared to the number of live offspring on PND 4 after blood sampling was considered not to be affected by treatment. The lactation indices were 99.2% (control group), 100% (41.7 mg/kg bw/day), 100% (125 mg/kg bw/day) and 100% (375 mg/kg bw/day), respectively.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

At 375 mg/kg bw/day, the mean body weight gains of male and female offspring was marginally lower than in control animals (-8.8%, statistically not significant). The effect was considered to be not related to treatment.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

effects observed, non-treatment-related

Description (incidence and severity):

At 375 mg/kg bw/day, the sex ratio of males to females was slightly low (total number of males: 39.3% at 375 mg/kg bw/day vs. 53.3% in the control group); however there was considerable inter-group variation (44.1% at 41.7 mg/kg bw/day, 54.6% at 125 mg/kg bw/day and 39.3% at 375 mg/kg bw/day) and therefore the effect was not attributed to treatment.

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

THYROID HORMONE ANALYSIS: There were no treatment-related findings.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

developmental

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Table 1: Clinical signs of toxicity in parental animals (F0)

Category	Observation	Males				Females
Dose (mg/kg bw/day)		0	41.7	125	375	0	41.7	125	375
Abnormal gait	Unsteady	0/10	0/10	1/10	2/10	0/10	0/10	0/10	6/10
Behaviour	Decreased activity	0/10	0/10	0/10	5/10	0/10	0/10	0/10	4/10
	Eating of bedding material	0/10	0/10	0/10	1/10	0/10	0/10	0/10	1/10
Body Temperature	Abnormally cold to touch	0/10	0/10	0/10	1/10	0/10	0/10	0/10	0/10
Coat	Piloerection	0/10	0/10	0/10	1/10	0/10	0/10	0/10	0/10
Posture	Prostate	0/10	0/10	0/10	1/10	0/10	0/10	0/10	0/10

Table 2: Body weight and body weight gain in males and females before pairing and in females during lactation (F0)

Days

Change

Body weight (g) and body weight change (g) group mean values for males and females before pairing

Sex

Dose group (mg/kg bw/day)

1

8

15

22

29

36

1 - 8

8 - 15

15 - 22

22 - 29

22 - 36

1 - 15

1 - 36

Male

0

324 ± 7.9

342 ± 12.6

360 ± 18.2

369 ± 23.1

384 ± 26.8

393 ± 30.7

18 ± 7.1

18 ± 6.3

9 ± 6.9

15 ± 4.5

24 ± 10.2

36 ± 12.3

69 ± 24.2

41.7

325 ± 7.2

346 ± 15.6

368 ± 23.9

379 ± 29.2

395 ± 33.7

410 ± 42.6

21 ± 9.5

22 ± 9.2

12 ± 7.7

16 ± 4.8

31 ± 13.7

43 ± 18.0

85 ± 37.3

125

322 ± 6.4

340 ± 8.4

358 ± 10.8

368 ± 12.1

381 ± 14.5

393 ± 14.7

18 ± 4.7

18 ± 5.3

10 ± 5.6

13 ± 4.6

24 ± 8.2

36 ± 8.1

70 ± 13.1

375

321 ± 5.2

329 ± 13.1*

343 ± 20.5

348 ± 22.6

362 ± 28.1

371 ± 34.0

8 ± 8.7**

14 ± 8.6

6 ± 6.8

13 ± 6.1

23 ± 12.4

21 ± 16.4*

50 ± 29.6

Female

0

211 ± 12.2

220 ± 12.6

228 ± 13.5

9 ± 5.1

8 ± 5.2

17 ± 7.4

41.7

208 ± 12.6

216 ± 13.4

224 ± 14.3

8 ± 4.9

8 ± 4.4

16 ± 7.3

125

211 ± 10.3

219 ± 117

228 ± 12.1

8 ± 3.6

8 ± 6.2

17 ± 6.4

375

210 ± 11.8

217 ± 16.5

222 ± 17.2

6 ± 7.4

5 ± 6.0

11 ± 8.3

Body weight (g) and body weight change (g) females during lactation

Sex

Dose group (mg/kg bw/day)

1

4

7

13

1 - 4

4 - 7

7 - 13

1 - 13

Female

0

260 ± 20.3

261 ± 21.0

267 ± 22.9

281 ± 23.7

2 ± 4.8

6 ± 6.6

13 ± 5.6

21 ± 10.8

41.7

252 ± 23.1

256 ± 25.2

265 ± 25.6

277 ± 18.7

4 ± 4.0

10 ± 8.6

11 ± 10.9

25 ± 10.4

125

260 ± 21.9

265 ± 21.9

272 ± 23.0

285 ± 26.7

5 ± 4.1

8 ± 5.0

12 ± 12.0

25 ± 13.5

375

248 ± 16.0

250 ± 17.1

252 ± 18.3

268 ± 21.2

1 ± 7.1

2 ± 7.4

16 ± 9.2

20 ± 12.3

 * and **: statistical significance at p < 0.05 and p < 0.01

Table 3: Food consumption in males and females before pairing (F0)

		Week				Change
Food consumption (g/animal/week) group mean values for males and females before pairing (F0)
Sex	Dose group (mg/kg bw/day)	1	2	4	5	1 - 2	1 - 5
Male	0	129 ± 0.4	130 ± 8.2	124 ± 0.2	122 ± 4.7	130 ± 4.2	126 ± 2.3
	41.7	130 ± 14.9	129 ± 16.1	131 ± 13.8	131 ± 12.3	129 ± 15.5	130 ± 14.2
	125	123 ± 11.4	122 ± 7.0	123 ± 7.2	124 ± 1.6	122 ± 9.1	123 ± 6.5
	375	101 ± 8.2*	112 ± 3.6	120 ± 4.4	118 ± 5.3	107 ± 5.8*	113 ± 5.3
Female	0	92 ± 3.2	89 ± 2.5			91 ± 2.1
	41.7	89 ± 7.4	88 ± 9.4			89 ± 8.4
	125	89 ± 7.6	92 ± 10.9			91 ± 9.2
	375	79 ± 4.2*	87 ± 2.7			83 ± 3.4

 *: statistical significance at p < 0.05

Table 4: Haematology parameters at study termination (F0 animals)

Parameter	Males				Females
Dose (mg/kg bw/day)	0	41.7	125	375	0	41.7	125	375
MCH (pg)	18.5 ± 0.81	18.5 ± 1.02	18.5 ± 1.09	19.3 ± 1.31	17.9 ± 0.16	18.4 ± 0.43	19.1 ± 0.84*	18.6 ± 0.70*
MCHC (g/dL)	28.9 ± 0.84	28.7 ± 0.75	28.7 ± 0.97	29.7 ± 0.82	27.9 ± 0.99	28.4 ± 0.53	28.4 ± 0.52	29.1 ± 0.71*
PT (sec)	23.9 ± 3.99	22.2 ± 2.42	20.4 ± 0.97	22.3 ± 2.73	20.4 ± 0.70	21.8 ± 1.21	22.4 ± 2.18	31.9 ± 22.95
APTT (sec)	17.1 ± 3.20	15.7 ± 2.17	16.1 ± 1.85	15.5 ± 1.21	11.1 ± 2.74	11.2 ± 2.73	13.4 ± 5.32	12.9 ± 5.82
MCH: mean corpuscular haemoglobin; MCHC: mean corpuscular haemoglobin concentration, PT: prothrombine time; APTT: activated partial thromboplastine time  *: statistical significance at p < 0.05

Table 5: Clinical chemistry parameters at study termination (F0 animals)

Parameter	Males				Females
Dose (mg/kg bw/day)	0	41.7	125	375	0	41.7	125	375
A/G	1.47 ± 0.121	1.34 ± 0.078	1.43 ± 0.047	1.46 ± 0.080	1.66 ± 0.065	1.65 ± 0.049	1.61 ± 0.062	1.46 ± 0.130**
Alb (g/L)	39 ± 0.8	37 ± 0.5*	38 ± 1.1*	38 ± 0.8*	35 ± 1.3	36 ± 1.1	36 ± 1.2	36 ± 2.2
ALP (U/L)	128 ± 23.1	154 ± 34.1	119 ± 16.0	111 ± 35.9	135 ± 50.5	136 ± 37.3	148 ± 57.4	94 ± 21.2
AST (U/L)	76 ± 17.4	65 ± 15.5	67 ± 15.9	68 ± 14.9	101 ± 16.4	86 ± 8.3	90 ± 9.4	90 ± 17.5
Bile Ac. (µmol/L)	14.8 ± 7.19	18.2 ± 6.30	5.6 ± 2.70	21.0 ± 11.25	12.6 ± 5.03	20.2 ± 11.88	15.0 ± 4.64	17.6 ± 6.69
Bili (µmol/L)	0 ± 0.4	0 ± 0.5	1 ± 0.5	1 ± 0.0*	0 ± 0.5	1 ± 0.5	0 ± 0.5	0 ± 0.5
Cl- (mmol/L)	99.2 ± 1.59	99.2 ± 0.97	99.0 ± 1.67	98.3 ± 0.98	95.7 ± 0.82	96.9 ± 1.22	101.0 ± 5.08*	103.7 ± 6.48*
Glc (mmol/L)	12.71 ± 0.938	11.72 ± 1.171	10.74 ± 1.060	11.72 ± 1.545	6.53 ± 1.636	8.09 ± 1.211	7.32 ± 1.484	7.71 ± 1.644
K+ (mmol/L)	4.02 ± 0.248	4.39 ± 0.157	4.24 ± 0.240	4.09 ± 0.265	4.94 ± 0.149	5.00 ± 0.378	5.76 ± 1.052	5.11 ± 0.589
Na+ (mmol/L)	140 ± 1.3	140 ± 1.3	140 ± 1.0	139 ± 0.8	139 ± 1.1	140 ± 0.0	141 ± 1.2	143 ± 4.0*
P (mmol/L)	1.40 ± 0.074	1.62 ± 0.122	1.66 ± 0.116*	1.37 ± 0.227	0.91 ± 0.344	0.69 ± 0.469	0.82 ± 0.212	0.67 ± 0.418
Total prot. (g/L)	66 ± 1.6	65 ± 1.7	64 ± 1.3	64 ± 1.5	56 ± 2.0	57 ± 1.5	58 ± 2.7	61 ± 48*
Trig (mmol/L)	1.03 ± 0.286	1.13 ± 0.399	1.68 ± 0.678	0.99 ± 0.599	0.30 ± 0.084	0.32 ± 0.089	0.39 ± 0.061	0.38 ± 0.077
A/G: albumin/globulin ratio; Alb: albumin; ALP: alkaline phosphatase; AST: aspartate aminotransferase; Bile Ac.: bile acids; bili: bilirubin; Cl-: chloride; Na+: sodium; K+: potassium; P: inorganic phosphorus; Total prot.: total protein; Trig: triglycerides; * and **: statistical significance at p < 0.05 and p < 0.01

Table 6: Grip strength measurements during week 5 of treatment (males) or during Days 7 - 9 of lactation (females)

	Males				Females
	0	41.7	125	375	0	41.7	125	375
Forelimb grip strength (kg)	1.17 ± 0.08	1.13 ± 0.16	1.19 ± 0.16	1.12 ± 0.09	1.00 ± 0.13	0.98 ± 0.11	1.06 ± 0.06	1.16 ± 0.12*
HCD mean (range)	1.13 (1.03 - 1.23)				1.01 (0.90 - 1.16)
Hindlimb grip strength (kg)	0.49 ± 0.02	0.51 ± 0.02	0.55 ± 0.07	0.44 ± 0.01*	0.52 ± 0.05	0.52 ± 0.06	0.57 ± 0.05	0.51 ± 0.08
HCD mean (range)	0.51 (0.39 - 0.66)				0.48 (0.36 - 0.62)
* statistical significance at p < 0.05; HCD: historical control data summarising 15 studies conducted in Jan 2017 - Nov 2019

Table 7: Motor activity measurements during week 5 of treatment (males) or during Days 7 - 9 of lactation (females), F0 animals

Sex	Dose group (mg/kg bw/day)	High beam level
		6 min	30 min	60 min	Total
Male	0	90.4 ± 13.8	5.8 ± 11.3	9.4 ± 7.1	222.0 ± 57.2
	41.7	84.4 ± 16.7	2.4 ± 5.4	4.2 ± 9.4	156.2 ± 25.7
	125	67.2 ± 16.6	10.2 ± 10.9	19.8 ± 32.1	195.4 ± 120.8
	375	58.2 ± 21.9*	8.4 ± 10.4	10.0 ± 8.6	169.8 ± 25.4
Female	0	49.8 ± 7.3	16.2 ± 10.2	3.6 ± 8.0	162.4 ± 69.7
	41.7	75.5 ± 24.9	20.0 ± 32.5	12.0 ± 14.2	250.5 ± 132.9
	125	71.2 ± 17.3	30.0 ± 18.4	27.8 ± 15.9	285.6 ± 63.8
	375	69.4 ± 30.7	13.6 ± 9.0	8.2 ± 8.5	187.6 ± 87.8

 *: statistical significance at p < 0.05

Table 8: Organ weight analysis in parental animals (F0)

Parameter	Males				Females
Dose (mg/kg bw/day)	0	41.7	125	375	0	41.7	125	375
Liver weight
absolute (g)	11.754 ± 1.350	14.848 ± 2.215	12.997 ± 1.274	13.260 ± 1.315	12.753 ± 1.571	12.283 ± 0.574	14.877 ± 0.604	12.587 ± 2.017
relative to bw (g)	12.569	13.023	13.399	13.867**	12.263	12.204	14.599*	13.433*
Kidney weight
absolute (g)	2.107 ± 0.269	2.464 ± 0.237	2.310 ± 0.249	2.446 ± 0.284	1.616 ± 0.146	1.691 ± 0.173	1.840 ± 0.090	1.619 ± 0.201
relative to bw (g)	2.209	2.237	2.360	2.521 *	1.554	1.681	1.804**	1.727**
Heart weight
absolute (g)	0.961 ± 0.088	1.114 ± 0.127	1.042 ± 0.072	1.079 ± 0.121	0.898 ± 0.066	0.870 ± 0.068	0.919 ± 0.049	0.834 ± 0.099
relative to bw (g)	1.012	1.000	1.067	1.117**	0.885	0.868	0.911	0.857
* and **: statistical significance at p < 0.05 and p < 0.01

Table 9: Microscopic findings in F0 males after 5 weeks of treatment

Dose Level (mg/kg bw/day)	0	41.7	125	375
Kidney
Number Examined	5	5	5	5
Accumulation, Hyaline Droplets
Minimal	0	0	0	3
Slight	0	0	0	2
Cast(s), Granular
Minimal	0	0	0	3
Slight	0	0	0	1
Basophilia, Tubular
Minimal	1	0	1	2
Slight	0	0	0	1
Liver
Number Examined	5	5	5	5
Hypertrophy, Centrilobular
Minimal	0	0	0	3

Table 10: Sperm count estimates from vaginal smears at mating in F0 animals

Dose group (mg/kg bw/day)	Sperm count category
	0	1	2	3	4
0	0/10	0/10	1/10	3/10	6/10
41.7	0/10	0/10	2/10	4/10	4/10
125	0/10	0/10	1/10	5/10	4/10
375**	1/9	1/9	3/9	3/9	1/9

Category 0: no sperm; category 1: occasional sperm; category 2: continuous few sperm; category 3: many scattered sperm; category 4: solid masses of sperm

**: statistically significant when compared to control animals

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, the NOAEL for systemic toxicity was 125 mg/kg bw/day in males, based on alpha 2u-nephropathy in male rats at 375 mg/kg bw/day. The finding is specific for male rats only and not relevant for humans. The NOAEL for systemic toxicity in females was 375 mg/kg bw/day. The NOAELs for reproduction and for development were both determined to be 375 mg/kg bw/day for male and female rats.