Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 Nov 2017 - 14 Aug 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 Nov 2017 - 14 Aug 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted in 2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3550, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Version / remarks:
adopted in 2000
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Crl: WI(Han)
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. The laboratory has general and reproduction/developmental historical data in this species from the same strain and source.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: males approx. 10 weeks, females approx. 13 weeks
- Weight at study initiation: males 266-299 g, females 191-242 g
- Fasting period before study: no
- Housing: On arrival and until mating animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type, height 18 cm). During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm). During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. During pregancy and lactation females were individually housed in Macrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams.
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: municipal tap water via water bottles, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22
- Humidity (%): 43-54
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES (main study): From: 23 Jan 2018 To: 20 Mar 2018
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements.The dosing formulations were prepared weekly as a solution, filled out in daily portions and stored in the refrigerator protected from light. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing.

VEHICLE
- Concentration in vehicle: 1.67, 5 and 16.7 mg/mL
- Amount of vehicle: 3 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples were collected at one occasion during the study (week 1). Analyses were performed using a validated UPLC analytical procedure. Concentration analysis was performed for all groups and homogeneity analysis was performed for the low and high dose group.
Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤ 10%.

Stability analyses performed previously in conjunction with the method development and validation study demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.

The concentrations analysed in the formulations of all test item groups were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). No test item was detected in the vehicle control formulation. The formulations of the low and high dose groups were homogeneous (i.e. coefficient of variation ≤ 10%).
Duration of treatment / exposure:
Males were treated for 29 days up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period.
Females that delivered were treated for 50 to 56 days, i.e. 14 days prior to mating, the variable time to conception, the duration of pregnancy and at least 14 days after delivery, up to and including the day before scheduled necropsy.
One mid dose female which failed to deliver was treated for 41 days.
Frequency of treatment:
daily, 7 days/week
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Dose / conc.:
50 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 were selected based on the results of a 10-day dose range finder with oral administration of the test item in rats, and in an attempt to produce graded responses to the test item. In this range finding study, dose levels of 75 and 150 mg/kg bw/day were tested to three female rats/dose. At 150 mg/kg bw/day, all females were sacrificed in extremis on Day 3. At 75 mg/kg bw/day, clinical signs were noted consisting of hunched posture and piloerection and more incidentally abnormal gait, lethargy and/or shallow respiration, one female showed body weight loss (4%) on Day 5 and displayed gray-white foci on the heart; and liver and kidney weight were considered increased.
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily; during the dosing period, observations were performed at 0-15 min and 1 hour (±15 min) after dosing (based on the peak effect of occurrence of clinical signs observed in the dose range finder).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once weekly at 1 hour (±15 min) after dosing

FUNCTIONAL TESTS: Yes
- Time schedule: males during week 4; females during the last week of lactation
- How many animals: 5 selected males and femles/group
- Parameters examined: hearing ability, pupillary reflex, static righting reflex, fore- and hind-limb grip strength, locomotor activity

BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of treatment and weekly thereafter; mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

WATER CONSUMPTION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: males: yes, overnight with a maximum of 24 h, females: no
- How many animals: 5 males and 5 females/group
- Parameters examined: white blood cells (WBC), neutrophils (absolute), lymphocytes (absolute), monocytes (absolute), eosinophils (absolute), basophils (absolute), red blood cells (RBC), reticulocytes (absolute), red blood cell distribution width (RDW), haemoglobin, haematocrit, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelets, prothrombin time (PT), activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to scheduled necropsy
- Animals fasted: males: yes, overnight with a maximum of 24 h, females: no
- How many animals: 5 males and 5 females/group
- Parameters examined: alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), alkaline phosphatase (ALP), total protein, albumin, total bilirubin, bile acids, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate (Inorg. Phos)

THYROID HORMONES: Yes
- Time schedule for collection of blood: prior to scheduled necropsy
- How many animals: all parental males
- Parameters examined: total thyroxine (T4)
Sacrifice and pathology:
SACRIFICE
- Male animals: All animals following the mating period (a minimum of 28 days of administration)
- Maternal animals which delivered: PND 14-16
- Maternal animals which did not deliver: post-coitum day 25

GROSS NECROPSY
- All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs; the numbers of former implantation sites were recorded for all paired females.

ORGAN WEIGHTS
- Organ weights recorded for 5 males and 5 females/group: brain, cervix, epididymis, adrenal gland, coagulation gland together with seminal vesicle, prostate, parathyroid weighed together with thyroid, prostate, heart, kidney, liver, ovaries, spleen, testes, thymus, uterus
- Organ weights recorded for all remaining animals: epididymis, coagulation gland together with seminal vesicle, prostate, parathyroid weighed together with thyroid, testes

HISTOPATHOLOGY
- for 5 males and 5 females/group: bone marrow, bone (femur, sternum), brain (seven levels), cervix, epididymides, eye, adrenal, coagulation gland, mammary gland, parathyroid, pituitary, prostate, seminal vesicle, thyroid, gross lesions/masses, gut-associated lymphoid tissue, heart, kidney, large intestine (cecum, colon, rectum), liver, lung, lymph node (mandibular and mesenteric site), skeletal muscle, sciatic nerve, ovaries, small intestine (duodenum, ileum, jejunum), spinal cord, spleen, stomach, testes, thymus, trachea, urinary bladder, uterus, vagina
- for all remaining animals: cervix, epididymides, coagulation gland, mammary gland, parathyroid, pituitary, prostate, seminal vesicle, thyroid, gross lesions/masses, ovaries, testes, uterus, vagina

SPERM PARAMETERS
- for 5 parental males in the vehicle control and high dose group and one mid dose male that failed to sire:
detailed qualitative testes examination, taking into account the tubular stages of the spermatogenic cycle
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels. Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, butexcluded semi-quantitative data, and any group with less than 3 observations.

The following pairwise comparisons were made:
Low dose vs. vehicle control
Mid dose vs. vehicle control
High dose vs. vehicle control

Parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Non-Parametric: Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test).
Incidence: An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Blue discolouration of the urine was recorded for most mid and high dose females during the last week of their scheduled treatment period. This finding occurred in the absence of adverse renal pathology. It is conceivable that the bluish discolouration had occurred due to presence of the test item and/or its metabolite(-s) in the urine resulting in a specific light diffraction.

One high dose female showed restless behaviour on the day preceding scheduled necropsy. This restless behaviour was considered not test item-related since none of the other animals at this dose showed similar signs.

No treatment-related clinical signs were noted during weekly arena observations.

Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Females: In the high dose, reduced mean weight gain or slight weight loss (up to 4% of the body weight on Day 1 of treatment) was recorded for females during the premating and mating period, being statistically significant only on Day 1 of the mating period. A slightly lower mean weight gain was also recorded for high dose females over Days 4-7 and 7-13 of lactation (4% vs. 8% in the control group over Days 1-7 of lactation, and 7% vs. 12% in the control group over Days 1-13 of lactation; not statistically significant). Mean body weights and weight gain during the postcoitum phase were considered not affected by treatment.
One high dose female showed a lower weight gain during the post-coitum phase. This same female had a lower litter size (5 pups). Since none of the other animals at this dose showed a similar change in body weight, this was considered not related to treatment with the test item.

These body weight variations were not accompanied by treatment-related changes in food intake, and clinically rats appeared in a healthy state. These variations were therefore considered not to be adverse, but the slightly lower body weights at the end of lactation may have resulted in lower pup body weights.

The statistically significantly lower mean body weight of low dose females on Day 8 of the premating period occurred in the absence of a dose related trend, and was therefore considered not to be related to treatment.

Males: Body weights and body weight gain of males remained in the same range as controls over the treatment period.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
One high dose female showed a reduced food intake before and after correction for body weight during lactation. Since none of the other animals at this dose showed a similar change in food intake, this was considered unrelated to treatment with the test item.
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):
At the high dose, higher platelet counts were recorded for females (19% higher than control mean). However, these were not supported by other findings and the mean remained within the historical control data range. This finding was therefore considered not to be an adverse change.
Other haematological and coagulation parameters of treated rats were considered not to have been affected by treatment.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At the high dose, a higher aspartate aminotransferase activity (ASAT) was recorded for females (factor 3 higher than control mean). The mean exceeded the historical control data range. No morphological correlate was noted and therefore, the finding was considered an adaptive rather than an adverse finding.
Other clinical biochemistry parameters of treated rats were considered not to have been affected by treatment.

Thyroid hormone analyses:
Serum levels of T4 in F0 males were not affected by treatment.
The statistically significantly lower T4 of low dose males was considered not to be related to treatment as this occurred in the absence of a dose-related trend.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
In the high dose group, a lower mean motor activity (both total movements and ambulations) was recorded for males and females (mean total movements 26% and 46% lower than control means for males and females, respectively; not statistically significant, for details please refer to Table 1). Means remained within the historical control data range. For high dose females, the mean was predominantly affected by one low value for one animal. When this value is not included in the mean, the mean is 2851 counts (i.e. 37% lower than the control mean).
This lower motor activity was not supported by concurrent changes in grip strength measurements or clinical observations, and means remained within the historical control data range for rats of this age and strain. Histopathologically, there were no supportive morphological correlates in examined neuronal tissues. An increase in incidence and severity of myofiber degeneration and mononuclear cell infiltrate (up to slight degree) of the skeletal muscle was observed in females of the high dose group. However, there was no apparent correlation between this skeletal muscle degeneration and the measured lower motor activity on an individual animal basis. Overall, there were no indications that these findings represented an adverse effect on neurobehaviour or were detrimental to motion function or vitality of the animals. As such, these findings were considered not to be adverse.

All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was similar between control and high dose animals.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test item-related higher liver weights (relative to body weights) were noted in high dose males and females (for details please fefer to Table 2). These higher weights were not supported by histopathological or clinical biochemistry changes and means remained within the historical control data range. As such, this higher liver weight may represent an adaptive change to treatment and was considered not to represent an adverse effect.

Any other differences in organ weights were considered not to be test item-related due to the direction of the change, lack of dose-related pattern, and/or general overlap and variability in individual values.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related gross observations. All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings were noted in the kidneys of males and skeletal muscles of females of the high dose group.

A subtle exacerbation of the incidence and severity of the background finding hyaline droplet accumulation of the kidneys (up to moderate) was observed in high dose males (for details refer to Table 3). These hyaline droplets are considered to represent alpha2μglobulin, a normal protein in male rats and in absence of any other test item related signs of kidney pathology considered as non-adverse.

An increased incidence and severity of myofiber degeneration and mononuclear cell infiltrate (up to slight degree) of the skeletal muscle was observed in high dose females (for details please refer to Table 4). These changes of the skeletal muscle were multifocal (scattered) present in most of the high dose females, while in general the skeletal muscles were focally affected in the few female rats of the other dose groups.

The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Reproductive performance: There was 1/10 couples of the mid dose group with a non-pregnant female. No abnormalities were seen in the reproductive organs, which could account for their lack of fertility.
There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
No effects were noted on sperm measures.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects noted up to and including the high dose of 50 mg/kg bw/day
Critical effects observed:
no

Table 1. Motor activity test summary

Dose level
(mg/kg bw/day)

0

5

15

50

 

Males

Total movements

 

 

 

 

               Mean

3580

3596

3672

2661

               St. Dev.

1212

304

948

892

               N

5

5

5

5

Abulations

 

 

 

 

               Mean

696

791

756

550

               St. Dev.

378

121

224

210

               N

5

5

5

5

 

Females

Total movements

 

 

 

 

               Mean

4499

3569

4216

2440

               St. Dev.

1397

1004

1122

1008

               N

5

5

5

5

Abulations

 

 

 

 

               Mean

1072

851

974

512

               St. Dev.

327

211

209

258

               N

5

5

5

5

 

Table 2. Mean percent liver weights differences from control groups

 

 

Males

 

 

Females

 

Dose level (mg/kg/day):

5

15

50

5

15

50

LIVER

 

 

 

 

 

 

Absolute

5

4

8

0

5

11

Relative to body weight

2

1

8*

-2

0

11*

* P < 0.05

Table 3. Summary Test Item-Related Microscopic Findings Kidneys

 

Dose level (mg/kg/day):KIDNEYSa

Hyaline droplet accumulation

Minimal Slight Moderate

 

 

Males

 

0

5

15

50

5

5

5

5

2

-

1

-

-

-

1

2

-

-

-

1

a  = Number of tissues examined from each group

Table 4. Summary Test Item-Related Microscopic Findings Skeletal Muscle

 

Females

Dose level
(mg/kg bw/day)

0

5

15

50

Skeletal musclea

5

5

5

5

Myofiber degeneration

 

 

 

 

Minimal

-

2f

1f

1mf

Slight

-

-

-

3mf

Mononuclear cell infiltrate

 

 

 

 

Minimal

2f

1f

1f

1mf

Slight

-

-

-

3 mf

a=Number of tissues examined from each group

f=focal, mf=multifocal

Conclusions:
In the present study, the NOAEL with respect to general systemic toxicity were set at 50 mg/kg bw/day, the highest dose level tested. It should be noted that this high dose was selected based on severe toxicity observed at 75 and 150 mg/kg bw/day in the 10-day range finding study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted in 2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3550, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Version / remarks:
adopted in 2000
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl: WI(Han)
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. The laboratory has general and reproduction/developmental historical data in this species from the same strain and source.
This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: males approx. 10 weeks, females approx. 13 weeks
- Weight at study initiation: males 266-299 g, females 191-242 g
- Fasting period before study: no
- Housing: On arrival and until mating animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type, height 18 cm). During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm). During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. During pregancy and lactation females were individually housed in Macrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes.
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: municipal tap water via water bottles, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22
- Humidity (%): 43-54
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES (main study): From: 23 Jan 2018 To: 20 Mar 2018

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements.The dosing formulations were prepared weekly as a solution, filled out in daily portions and stored in the refrigerator protected from light. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing.

VEHICLE
- Concentration in vehicle: 1.67, 5 and 16.7 mg/mL
- Amount of vehicle: 3 mL/kg bw
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: all females were mated after 4 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear; referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually in Macrolon plastic cages (MIII type, height 18 cm)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples were collected at one occasion during the study (week 1). Analyses were performed using a validated UPLC analytical procedure. Concentration analysis was performed for all groups and homogeneity analysis was performed for low and high dose groups.
Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤ 10%.

Stability analyses performed previously in conjunction with the method development and validation study demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.

The concentrations analysed in the formulations of all test item groups were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). No test item was detected in the vehicle control 1 formulation. The formulations of the low and high dose group were homogeneous (i.e. coefficient of variation ≤ 10%).
Duration of treatment / exposure:
Males were treated for 29 days up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period.
Females that delivered were treated for 50 to 56 days, i.e. 14 days prior to mating, the variable time to conception, the duration of pregnancy and at least 14 days after delivery, up to and including the day before scheduled necropsy.
One mid dose female which failed to deliver was treated for 41 days.
Frequency of treatment:
daily, 7 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Dose / conc.:
50 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 were selected based on the results of a 10-day dose range finder with oral administration of the test item in rats, and in an attempt to produce graded responses to the test item. In this range finding study, dose levels of 75 and 150 mg/kg bw/day were tested to three female rats/dose. At 150 mg/kg bw/day, all females were sacrificed in extremis on Day 3. At 75 mg/kg bw/day, clinical signs were noted consisting of hunched posture and piloerection and more incidentally abnormal gait, lethargy and/or shallow respiration, one female showed body weight loss (4%) on Day 5 and displayed gray-white foci on the heart; and liver and kidney weight were considered increased.
Positive control:
no

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily; during the dosing period, observations were performed at 0-15 min and 1 hour (±15 min) after dosing (based on the peak effect of occurrence of clinical signs observed in the dose range finder).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once weekly at 1 hour (±15 min) after dosing

FUNCTIONAL TESTS: Yes
- Time schedule: males during week 4; females during the last week of lactation
- How many animals: 5 selected males and femles/group
- Parameters examined: hearing ability, pupillary reflex, static righting reflex, fore- and hind-limb grip strength, locomotor activity

BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of treatment and weekly thereafter; mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

WATER CONSUMPTION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: males: yes, overnight with a maximum of 24 h, females: no
- How many animals: 5 males and 5 females/group
- Parameters examined: white blood cells (WBC), neutrophils (absolute), lymphocytes (absolute), monocytes (absolute), eosinophils (absolute), basophils (absolute), red blood cells (RBC), reticulocytes (absolute), red blood cell distribution width (RDW), haemoglobin, haematocrit, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelets, prothrombin time (PT), activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to scheduled necropsy
- Animals fasted: males: yes, overnight with a maximum of 24 h, females: no
- How many animals: 5 males and 5 females/group
- Parameters examined: alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), alkaline phosphatase (ALP), total protein, albumin, total bilirubin, bile acids, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate (Inorg. Phos)

THYROID HORMONES: Yes
- Time schedule for collection of blood: prior to scheduled necropsy
- How many animals: all parental males
- Parameters examined: total thyroxine (T4)
Oestrous cyclicity (parental animals):
Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period.
On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrous. This was done for all females.
Sperm parameters (parental animals):
Parameters examined in all parental males:
testis weight, epididymis weight
Parameters examined in 5 selected parental males in the vehicle control and high dose group and one mid dose male that failed to sire:
detailed qualitative testes examination, taking into account the tubular stages of the spermatogenic cycle
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed after collection of blood and discarded

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals following the mating period (a minimum of 28 days of administration)
- Maternal animals which delivered: PND 14-16
- Maternal animals which did not deliver: post-coitum day 25

GROSS NECROPSY
- All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs; the numbers of former implantation sites were recorded for all paired females.

ORGAN WEIGHTS
- Organ weights recorded for 5 males and 5 females/group: brain, cervix, epididymis, adrenal gland, coagulation gland together with seminal vesicle, prostate, parathyroid weighed together with thyroid, prostate, heart, kidney, liver, ovaries, spleen, testes, thymus, uterus
- Organ weights recorded for all remaining animals: epididymis, coagulation gland together with seminal vesicle, prostate, parathyroid weighed together with thyroid, testes

HISTOPATHOLOGY
- for 5 males and 5 females/group: bone marrow, bone (femur, sternum), brain (seven levels), cervix, epididymides, eye, adrenal, coagulation gland, mammary gland, parathyroid, pituitary, prostate, seminal vesicle, thyroid, gross lesions/masses, gut-associated lymphoid tissue, heart, kidney, large intestine (cecum, colon, rectum), liver, lung, lymph node (mandibular and mesenteric site), skeletal muscle, sciatic nerve, ovaries, small intestine (duodenum, ileum, jejunum), spinal cord, spleen, stomach, testes, thymus, trachea, urinary bladder, uterus, vagina
- for all remaining animals: cervix, epididymides, coagulation gland, mammary gland, parathyroid, pituitary, prostate, seminal vesicle, thyroid, gross lesions/masses, ovaries, testes, uterus, vagina
Postmortem examinations (offspring):
SACRIFICE
- On PND 4, the surplus pups (> 8 pups per litter) were euthanized by decapitation. From two surplus pups per litter, blood was collected, if possible.
- All remaining pups were euthanized on PND 14-16.

GROSS NECROPSY
- Gross necropsy consisted of sex determination both externally and internally, recoding of external abnormalities; particular attention was paid to the external reproductive genitals to examine signs of altered development. External abnormalities were collected and fixed in 10% buffered formalin at the discretion of the Study Director.
In addition, blood was collected from two pups per litter and the thyroid from two pups per litter (if possible one male and one female pup). The pups selected for blood sampling were the same pups as selected for thyroid preservation.

HISTOPATHOLOGY / ORGAN WEIGTHS: no
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels. Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, butexcluded semi-quantitative data, and any group with less than 3 observations.

The following pairwise comparisons were made:
Low dose vs. vehicle control
Mid dose vs. vehicle control
High dose vs. vehicle control

Parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Non-Parametric: Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test).
Incidence: An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Reproductive indices:
Mating index: no. females mated/no. females paired x 100
Fertility index: no. females pregnant/no. females mated x 100
Gestation index: no. females with living pups on day 1/no. pregnant females x 100
Post-implantation survival index: total number of offspring born/total number of uterine implantation sites x 100
Offspring viability indices:
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 culling/number live offspring on Day 1 after littering x 100
Lactation index: number of live offspring on Day 13 after littering/number live offspring on Day 4 (after culling)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Blue discolouration of the urine was recorded for most mid and high dose females during the last week of their scheduled treatment period. This finding occurred in the absence of adverse renal pathology. It is conceivable that the bluish discolouration had occurred due to presence of the test item and/or its metabolite(-s) in the urine resulting in a specific light diffraction.

One high dose female showed restless behaviour on the day preceding scheduled necropsy. This restless behaviour was considered not test item-related since none of the other animals at this dose showed similar signs.

No treatment-related clinical signs were noted during weekly arena observations.

Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No mortality occurred in the course of the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Females: In the high dose, reduced mean weight gain or slight weight loss (up to 4% of the body weight on Day 1 of treatment) was recorded for females during the premating and mating period, being statistically significant only on Day 1 of the mating period. A slightly lower mean weight gain was also recorded for high dose females over Days 4-7 and 7-13 of lactation (4% vs. 8% in the control group over Days 1-7 of lactation, and 7% vs. 12% in the control group over Days 1-13 of lactation; not statistically significant). Mean body weights and weight gain during the postcoitum phase were considered not affected by treatment.
One high dose female showed a lower weight gain during the post-coitum phase. This same female had a lower litter size (5 pups). Since none of the other animals at this dose showed a similar change in body weight, this was considered not related to treatment with the test item.

These body weight variations were not accompanied by treatment-related changes in food intake, and clinically rats appeared in a healthy state. These variations were therefore considered not to be adverse, but the slightly lower body weights at the end of lactation may have resulted in lower pup body weights.

The statistically significantly lower mean body weight of low dose females on Day 8 of the premating period occurred in the absence of a dose related trend, and was therefore considered not to be related to treatment.

Males: Body weights and body weight gain of males remained in the same range as controls over the treatment period.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
One high dose female showed a reduced food intake before and after correction for body weight during lactation. Since none of the other animals at this dose showed a similar change in food intake, this was considered unrelated to treatment with the test item.
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):
At the high dose, higher platelet counts were recorded for females (19% higher than control mean). However, these were not supported by other findings and the mean remained within the historical control data range. This finding was therefore considered not to be an adverse change.
Other haematological and coagulation parameters of treated rats were considered not to have been affected by treatment.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At the high dose, a higher aspartate aminotransferase activity (ASAT) was recorded for females (factor 3 higher than control mean). The mean exceeded the historical control data range. No morphological correlate was noted and therefore, the finding was considered an adaptive rather than an adverse finding.
Other clinical biochemistry parameters of treated rats were considered not to have been affected by treatment.

Thyroid hormone analyses:
Serum levels of T4 in F0 males were not affected by treatment.
The statistically significantly lower T4 of low dose males was considered not to be related to treatment as this occurred in the absence of a dose-related trend.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
In the high dose group, a lower mean motor activity (both total movements and ambulations) was recorded for males and females (mean total movements 26% and 46% lower than control means for males and females, respectively; not statistically significant, (for details please refer to Table 1). Means remained within the historical control data range. For high dose females, the mean was predominantly affected by one low value for one animal. When this value is not included in the mean, the mean is 2851 counts (i.e. 37% lower than the control mean).
This lower motor activity was not supported by concurrent changes in grip strength measurements or clinical observations, and means remained within the historical control data range for rats of this age and strain. Histopathologically, there were no supportive morphological correlates in examined neuronal tissues. An increase in incidence and severity of myofiber degeneration and mononuclear cell infiltrate (up to slight degree) of the skeletal muscle was observed in females of the high dose group. However, there was no apparent correlation between this skeletal muscle degeneration and the measured lower motor activity on an individual animal basis. Overall, there were no indications that these findings represented an adverse effect on neurobehaviour or were detrimental to motion function or vitality of the animals. As such, these findings were considered not to be adverse.

All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was similar between control and high dose animals.
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):
Test item-related microscopic findings were noted in the kidneys of males and skeletal muscles of females of the high dose group.

A subtle exacerbation of the incidence and severity of the background finding hyaline droplet accumulation of the kidneys (up to moderate) was observed in high dose males (for details please refer to Table 3). These hyaline droplets are considered to represent alpha2μglobulin, a normal protein in male rats and in absence of any other test item related signs of kidney pathology considered as non-adverse.

An increased incidence and severity of myofiber degeneration and mononuclear cell infiltrate (up to slight degree) of the skeletal muscle was observed in high dose females (for details please refer to Table 4). These changes of the skeletal muscle were multifocal (scattered) present in most of the high dose females, while in general the skeletal muscles were focally affected in the few female rats of the other dose groups.

The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Reproductive performance: There was 1/10 couples of the mid dose group with a non-pregnant female. No abnormalities were seen in the reproductive organs, which could account for their lack of fertility.
There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis.
Histopathological findings: neoplastic:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
Mating index, precoital time, number of implantation sites, fertility index, gestation index and duration as well as parturition/maternal care, litter size and post-implantation survival index were not affected by treatment with the test item.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
>= 50 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects noted up to and including the high dose of 50 mg/kg bw/day
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
>= 50 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects noted up to and including the high dose of 50 mg/kg bw/day

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs occurred among pups that were considered to be related to treatment. The nature and incidence of clinical signs remained within the range considered normal for pups of this age, and were therefore considered not to be toxicologically relevant.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was not affected by treatment. The live birth indices were 99% for the control group and 100% for the 5, 15 and 50 mg/kg bw/day groups.
One pup of the control group was found dead at first litter check. No toxicological relevance was attributed to this single dead pup since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was considered not to be affected by treatment. Viability indices were 100, 99, 96 and 98% for the control, 5, 15 and 50 mg/kg bw/day groups, respectively.

Lactation index: The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was not affected by treatment. The lactation indices were 100, 99, 100 and 100% for the control, 5, 15 and 50 mg/kg bw/day groups, respectively.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
At the high dose, statistically significantly lower mean body weights for male and female pups were recorded on Day 13 lactation (10% lower than the control mean for males and females combined). As this was accompanied by a slightly lower weight gain of the dams in the same period (not statistically significant), it could not be excluded that these lower pup body weights had occurred secondary to lower maternal body weights. Since mean body weight remained within the range considered normal for pups of this age and strain (historical control data) and since there were no other reproductive or developmental changes noted in this study, this lower pup body weight was considered not to represent an adverse effect on pup development.

Pup body weights at low and mid dose were considered not to be affected by 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:
no effects observed
Description (incidence and severity):
Serum T4 levels in male and female pups necropsied on PND 14-16 were not affected by treatment.
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment. The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age, and were therefore considered not to be related to treatment.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment.

Treatment up to 50 mg/kg bw/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 50 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects noted up to and including the high dose of 50 mg/kg bw/day

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Table 1. Motor activity test summary

Dose level
(mg/kg bw/day)

0

5

15

50

 

Males

Total movements

 

 

 

 

               Mean

3580

3596

3672

2661

               St. Dev.

1212

304

948

892

               N

5

5

5

5

Abulations

 

 

 

 

               Mean

696

791

756

550

               St. Dev.

378

121

224

210

               N

5

5

5

5

 

Females

Total movements

 

 

 

 

               Mean

4499

3569

4216

2440

               St. Dev.

1397

1004

1122

1008

               N

5

5

5

5

Abulations

 

 

 

 

               Mean

1072

851

974

512

               St. Dev.

327

211

209

258

               N

5

5

5

5

 

Table 2. Mean percent liver weights differences from control groups

 

 

Males

 

 

Females

 

Dose level (mg/kg/day):

5

15

50

5

15

50

LIVER

 

 

 

 

 

 

Absolute

5

4

8

0

5

11

Relative to body weight

2

1

8*

-2

0

11*

* P < 0.05

Table 3. Summary Test Item-Related Microscopic Findings Kidneys

 

Dose level (mg/kg/day): KIDNEYSa

Hyaline droplet accumulation

Minimal Slight Moderate

 

 

Males

 

0

5

15

50

5

5

5

5

2

-

1

-

-

-

1

2

-

-

-

1

a  = Number of tissues examined from each group

Table 4. Summary Test Item-Related Microscopic Findings Skeletal Muscle

 

Females

Dose level
(mg/kg bw/day)

0

5

15

50

Skeletal musclea

5

5

5

5

Myofiber degeneration

 

 

 

 

Minimal

-

2f

1f

1mf

Slight

-

-

-

3mf

Mononuclear cell infiltrate

 

 

 

 

Minimal

2f

1f

1f

1mf

Slight

-

-

-

3 mf

a = Number of tissues examined from each group

f = focal, mf = multifocal

Table 5. Reproduction data summary

Dose level
(mg/kg bw/day)

0

5

15

50

 

 

 

 

 

Females paired

10

10

10

10

Females mated

10

10

10

10

Pregnant females

10

10

9

10

Females with living pups on Day 1

10

10

9

10

 

 

 

 

 

Mating index (%)

100

100

100

100

Fertility index (%)

100

100

90

100

Gestation index (%)

100

100

100

100

Table 6. Developmental data

Dose level
(mg/kg bw/day)

0

5

15

50

 

 

 

 

 

Litters total

10

10

9

10

 

 

 

 

 

Mean duration of gestation (days)

21.4

21.2

21.2

21.4

 

 

 

 

 

Dead pups at first litter check

 

 

 

 

          Litters affected (#)

1

0

0

0

  Total

1

0

0

0

 

 

 

 

 

Living pups at first litter check

 

 

 

 

        % of males/females

51/49

45/55

53/47

52/48

         Total

21

110

102

118

          Mean

12.1

11.0

11.3

11.8

 

 

 

 

 

Culled pups

41

31

27

39

 

 

 

 

 

Living pups on day 4 p.p.

 

 

 

 

        Total

80

78

71

77

        Mean

8.0

7.8

7.9

7.7

 

 

 

 

 

Breeding loss day 5-13 p.p.

 

 

 

 

    % of living pups day 4 p.p.

0.0

1.3

0.0

0.0

        Total

0

1

0

0

        Mean

0.0

0.1

0.0

0.0

 

 

 

 

 

Living pups day 13 p.p.

 

 

 

 

        % of males/females

50/50

48/52

49/51

49/51

          Total

80

77

71

77

          Mean

8.0

7.7

7.9

7.7

Applicant's summary and conclusion

Conclusions:
In the present study, the NOAELs with respect to general, reproductive and develpomental toxicity were set at 50 mg/kg bw/day, the highest dose level tested. It should be noted that this high dose was selected based on severe toxicity observed at 75 and 150 mg/kg bw/day in the 10-day range finding study.