Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 Jun - 24 Oct 2017
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:
27 Jun - 24 Oct 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted: 29 Jul 2016
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Remarks:
(Crl:CD(SD)), SPF
Details on species / strain selection:
Sprague-Dawley rats are commonly used in both the general systemic toxicity and reproductive and developmental toxicity studies with a large historical control data base. In addition, the rat is a required species in the regulatory guidelines.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: males: 10 weeks; females: 12 weeks
- Weight at study initiation: males: 327.7 - 393.2 g; females: 212.2 - 309.6 g
- Housing: individually during dosing period; one male and one female during mating period; dams were kept with their neonates during lactation; in stainless wire mesh cages (260W×350D×210H mm) or polycarbonate cages (260W×420D×180H mm)
- Diet: Teklad Certified Irradiated Global 18% Protein Rodent Diet 2918C, ad libitum
- Water: tap water filtered and irradiated by ultraviolet light; ad libitum
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY: The certificate of feed analysis was provided by the manufacturer. The results of feed analysis met the allowable standard of this facility. Samples of drinking water are analyzed for specified microorganisms once a month and all environmental contaminants once a year. The results of water analysis met the allowable standard of this facility.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.2 - 23.7
- Humidity (%): 46.8 - 59.5
- Air changes (per hr): 10 -15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
The required amount of the test substance was weighed using an electronic balance and placed in a container. The test substance was mixed with a small amount of vehicle to dissolve using a vortex mixer, and then the vehicle was gradually added to yield the desired concentration. The dosing formulations were stored in a refrigerator (3.9 - 4.9°C) and used within 7 days.

- VEHICLE
- Justification for use and choice of vehicle: Through the preliminary solubility test to determine the solubility and dispersion characteristics of the test substance, corn oil was selected as the vehicle because the test substance was well dissolved in it.
- Amount of vehicle: 2 mL/kg
- Lot/batch no.: MKBZ9899V, MKBW9504V, MKCC0462
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses of the dosing formulations were conducted using gas chromatography and samples were taken three times from the middle of each dosing formulation prior to dosing and analyzed for verification of dose level concentration. The results of dose concentration analysis were determined to be in the range of 104.17 - 113.77%. These results were within the acceptable limits (± 15% of nominal values).
As a result of homogeneity and stability analyses the 0.5 and 500 mg/mL dosing solutions were confirmed to be homogenous and stable for 4 hours at room temperature and for 7 days under refrigeration.
Duration of treatment / exposure:
Main groups:
49 days (males: 2 weeks prior to mating, during 2 weeks of mating and 21 days of post-mating; females: 2 weeks prior to mating until postpartum day 13)

Recovery groups:
males and females: once daily for 6 weeks; animals were not mated and assigned to 2 weeks of recovery period after the completion of administration
Frequency of treatment:
once daily / 7 days a week
Dose / conc.:
60 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
600 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12 (main groups)
6 (recovery groups; for control and high dose groups)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In a previously conducted 2-week repeated oral dose range-finding study, a decrease of body weight was noted in females at 1,000 mg/kg bw/day. Salivation was observed in males and females at 300 and 1,000 mg/kg bw/day. Soft stool was also observed in one male and two females at 1,000 mg/kg bw/day. Increases of the absolute and relative liver weights were noted in animals of both sexes at 1,000 mg/kg bw/day. Therefore, the high dose level was selected at 600 mg/kg bw/day. Then, the mid and low dose levels were selected at 200 and 60 mg/kg bw/day, respectively. The control group was dosed with the vehicle only with the same dose volume as the test substance- treated groups.

- Post-exposure recovery period in satellite groups: 2 weeks
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Cage side observations included: general condition and clinical signs, mortality/viability, abortion and pre-mature birth

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to dosing and once weekly thereafter
- Observations included: Skin, fur, eyes, mucous membranes, occurrence of secretion and excretion; autonomic activity (lacrimation, piloerection, pupil size, unusual respiratory pattern, etc.); changes in gait, posture and response to handling, and the presence of clonic or tonic movements; stereotypes (excessive grooming, repetitive circling, etc.) or bizarre behavior (self-mutilation, walking backward, etc.)

BODY WEIGHT: Yes
- Time schedule for examinations: males: prior to dosing on Day 1 (the first day of dosing), once a week throughout the dosing and recovery periods, the day prior to necropsy and on the day of necropsy (body weights after fasting); females: prior to dosing on Day 1 (the first day of dosing), once a week throughout the dosing period, on gestation days 0, 7, 14 and 20, on postpartum days 0, 4 and 13, the day prior to necropsy and on the day of necropsy.

FOOD CONSUMPTION:
- Food consumption of males of the main group and animals of both sexes of the recovery group was recorded just prior to dosing on Day 0 (one day before first dosing), once a week during the dosing and recovery periods (except during mating) and the day prior to necropsy. Food consumption of females of the main group was recorded just prior to dosing on Day 0 (one day before the first dosing), once a week throughout the dosing period (except during mating), on gestation Days 0, 6, 13 and 19, on postpartum Days 0, 3 and 12 and the day prior to necropsy. Residual feed was recorded on the next day. Individual food consumption was calculated by subtracting the amount of residual feed from the amount presented.

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (18 h)
- How many animals: all animals
- Parameters examined: erythrocyte count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelets (PLT), leukocyte count (WBC), neutrophils (NEU), lymphocytes (LYM), monocytes (MONO), eosinophils (EOS), basophils (BASO), reticulocytes (Reti), prothrombin time (PT), activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Animals fasted: Yes (18 h)
- How many animals: all animals
- Parameters examined: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), blood urea nitrogen (BUN), creatinine (Crea), total bilirubin (T-Bili), total protein (TP), albumin (Alb), globulin (Glo), A/G ratio, glucose (Glu), total cholesterol (T-Chol), triglyceride (TG), glucose (Glu), sodium (Na), potassium (K), chloride (Cl), calcium (Ca)

URINALYSIS: Yes
- Time schedule for collection of urine: 6 males and females were randomly selected from the main groups in addition to all recovery animals for urinalysis two days before necropsy. Fresh, 3-hour and 24-hour urine samples were collected from the selected animals and analyzed.
- Metabolism cages used for collection of urine: No data
- Animals fasted: Animals were fasted during the fresh urine collection, but were allowed free access to drinking water.
- Parameters examined: in fresh urine samples: pH, protein, glucose, bilirubin, occult blood, color and turbidity, sediment; in 24-hour urine samples: urine volume, specific gravity

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Selected animals were examined a few days before necropsy.
- Dose groups that were examined: 6 males and females were randomly selected from the main study groups in addition to all recovery animals
- Battery of functions tested: pinna reflex, auditory (sound) reflex, corneal reflex, pupillary reflex, grip strength test, rotarod test, spontaneous motor activity test

IMMUNOLOGY: No

OTHER:
Thyroid hormone analysis:
- Time schedule for collection of blood: on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- How many animals: all males
- Parameters examined: total thyroxine (Total T4) and thyroid stimulating hormone (TSH)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Males of the main groups were sacrificed on Day 50, and females of the main groups were sacrificed on Postpartum Day 14. All animals of the recovery groups were sacrificed two weeks after the final dosing. Non-pregnant females were sacrificed on Gestation Day 26. Dams, whose pups were all dead, were sacrificed as soon as possible. Complete gross postmortem examinations were conducted on all animals including the external surface and internal organs. All grossly visible abnormalities were recorded.

ORGAN WEIGHTS: Yes
Paired organs were weighed together. Animals were fasted overnight prior to necropsy and body weights were recorded on the day of necropsy. Organs were weighed and organ-to-body weight ratios were calculated. Following organs were weighed: brain, heart, liver, thymus, spleen, thyroid, kidneys, adrenals, ovaries, uterus, testis, epididymis, prostate + seminal vesicle with coagulating gland

HISTOPATHOLOGY: Yes
Tissue preservation and slide preservation
All males and females were randomly selected from the main groups in addition to all recovery animals for tissue preparation. The testes and epididymides and eyes with optic nerve were fixed in Davidson’s fixative. All other tissues were preserved in 10% neutral buffered formalin.

For the histopathological examination, the preparation of specimens of organs and tissues was carried out and the remaining organs and tissues preserved in 10% neutral buffered formalin: brain, pituitary, thymus, lung with bronchi, trachea, thyroid, esophagus, heart, liver, spleen, kidneys, adrenals, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, testes, epididymides, prostate, seminal vesicle with coagulating gland, ovaries, uterus + cervix, submandibular lymph node, mesenteric lymph node, femur and sternum, spinal cord, sciatic nerve, eye and optic nerve, urinary bladder, organs with gross lesions

Histopathological examinations were conducted as follows:
- At least six males and six females from the control, low, mid and high groups (especially focused on spermatogenesis and interstitial testicular cell structure)
- All tissues from animals found dead during the study
- All gross, macroscopic lesions
- Target organs noted at the high dose group were examined for at least the recovery group: liver
Statistics:
The statistical analysis of this study was conducted using the SAS program (SAS® 9.3, SAS Institute Inc., U.S.A.). For the data including body weights, food consumption, estrous cycle, mating period, gestation period, post-implantation loss, body weights and birth and survival rates of pups, litter size, AGD index, nipple number, thyroid hormone value, urine volume, hematology and clinical chemistry parameters, organ weights, grip strength, motor activity, the Bartlett’s test was conducted to analyze for homogeneity of variance (significance level: 0.05). One-way analysis of variance (ANOVA) test was applied on homogeneous data, then if significant (significance level: 0.05), Dunnett’s t-test was applied for multiple comparisons (significance levels: 0.05 and 0.01, two-tailed). Kruskal-Wallis test was applied on heterogeneous data, then if significant (significance level: 0.05), Steel’s test was applied for multiple comparisons (significance levels: 0.05 and 0.01, two-tailed).
The data of sensory function, mating index, fertility index and other data associated with gestation was analyzed utilizing Fisher’s exact test (significance levels: 0.05 and 0.01). For the data of the recovery group, Folded-F test was applied to analyze homogeneity of variance (significance level: 0.05, two-tailed). Student t-test was applied for homogeneous data, but if overruled, Aspin-Welch t-test was applied (significance levels: 0.05 and 0.01, two-tailed).
The organ weight data of dead animals, non-delivery females and dams whose pups were all dead were excluded from statistical analysis.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
In the main group, salivation and/or mucous stool were observed in males and females at 200 and 600 mg/kg bw/day.
Salivation was sporadically observed in 5 males and 3 females and mucous stool was temporarily observed in 2 males and 2 females at 200 mg/kg bw/day. Salivation was often observed in 8 males and all 12 females and mucous stool was sporadically observed in 9 males and 7 females at 600 mg/kg bw/day. Also, soft stool was temporarily observed in one female at 600 mg/kg bw/day. Dirty nose and soiled perineal region were observed in one female at 60 mg/kg bw/day, whose pups were all dead.
In the recovery group, salivation and mucous stool were observed in males and females at 600 mg/kg bw/day. Mucous stool was mainly observed in males and females at 600 mg/kg bw/day and it was considered to be a test substance-related effect. However, mucous stool observed in males and females at 200 mg/kg bw/day was considered to have little toxicological significance since it was observed temporarily and/or sporadically (low incidence and frequency).
However, salivation was considered to have little toxicological significance since it was caused by physicochemical characteristics. Soft stool was considered to be incidental since it was observed once in one animal.

No detailed clinical signs were observed in males and females of the main and recovery groups in the detailed examinations conducted once a week.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One pregnant female was found dead on Gestation Day 23 (GD 23) without any parturition signs at 60 mg/kg bw/day. It was considered to be incidental and had no toxicological meaning since there were no dose-dependency and no test substance-related adverse effects in other parameters at 60 mg/kg bw/day.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
In the main group, no significant differences in body weight changes were noted in males and females compared to the control group.
In the recovery group, statistically significant decreases in body weights were noted in males at 600 mg/kg bw/day from Day 35 to the end of dosing (Day 63). However, these statistical significances in body weights had little toxicological meaning since there were no statistical differences at 600 mg/kg bw/day in the main group.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No significant difference in food consumption was noted in males and females of the main and recovery groups compared to the control group.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No effects were observed in any adult animal in the main and recovery groups.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No adverse effects were observed in any adult animal in the main and recovery groups. Other statistical significant results were considered not to be test substance-related changes because of small difference and/or the values were within the range of historical reference data.
Urinalysis findings:
no effects observed
Description (incidence and severity):
In the main and recovery groups, no effects were noted in any dosing group.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related effects on the auditory reflex, pinna reflex, pupillary reflex and corneal reflex tests were observed in animals of both sexes in the main and recovery groups when compared to the control group.
In the main and recovery groups, there were no test substance-related effects in the grip strength test when compared to the control group. In the main group, there was a statistically significant temporary decrease in ambulatory count of spontaneous motor activity at 200 mg/kg bw/day. Also, there were statistically significant temporary decreases in vertical count of the spontaneous motor activity at 200 and 600 mg/kg bw/day. However, the statistical significance had little toxicological meaning because it was a small difference or temporary change and showed no dose dependency.
In the recovery group, there were no test substance-related effects on the spontaneous motor activity when compared to the control group.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
In the main group, the absolute and/or relative liver weights were significantly increased in adult males and females at 600 mg/kg bw/day (see table 1).
In males at 600 mg/kg bw/day of the recovery group, there were statistical differences in the absolute or relative weights of some organs. However, these changes were considered to have no toxicological meaning since they were related to the low body weights of males.
Other statistically significant changes in absolute and/or relative organ weights were considered not to be test substance-related effects because of small magnitude and/or the values were within the range of historical reference data.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Dead animals:
In the female which was found dead at 60 mg/kg bw/day on GD 23, no macroscopic changes were observed. The death was unrelated to the test substance based on a lack of dose-response relationship.

Surving animals:
Macroscopic examination at necropsy did not reveal test substance-related changes. At the end of the recovery period, macroscopic examination at necropsy did not reveal test substance-related changes. In addition, among four dams whose pups were all dead, only one dam showed unilateral discoloration of the kidney and focus of the stomach. The others showed no gross findings. Also, two non-delivered females were revealed as non-pregnant animals, thus there were no findings noted. The other macroscopic findings were considered to be incidental and not related to the test substance.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Dead animals:
There were no findings related to the test substance or causative of death.
Slight lymphoid atrophy of the spleen and thymus and cortical lymphoid apoptosis of the thymus were noted in one female at 60 mg/kg bw/day, and they are frequently noted in animals in poor condition. However, the severity was not severe enough to cause animal’s deaths. While all those changes probably contributed to the animal’s deaths, a clear cause of death could not be determined. Therefore, it was not considered to be related to the test substance since dose-dependent response was not clear, and these non-specific findings were frequently noted in poor condition/stress. It was difficult to perform histopathological examination of some tissues (stomach, duodenum, jejunum, ileum, cecum, colon, rectum, eye & optic nerve, and kidney) because of postmortem autolysis.

Surviving animals:
Test substance-related changes were observed in the liver (see table 2):
Centrilobular hepatocellular hypertrophy was observed at minimal to moderate severity in males and females at 600 mg/kg bw/day. At the end of the recovery period, those findings, which were observed in the main group, were not observed in all males and females of the high dose satellite group. It was considered to have little toxicological significance since hepatocellular hypertrophy in the centrilobular zone is generally considered to be an adaptive response in nature in the absence of associated inflammation or necrosis, and it was completely recovered after the recovery period in this study.

Slight to moderate thymic lymphoid atrophy and/or erosion of the stomach were noted in three dams, whose pups were all dead. It was not considered to be related to the test substance since dose-dependent response was not clear, and these non-specific findings were frequently noted in poor condition/stress.
No test substance-related histopathological findings were noted in the reproductive organs of either sex.
The microscopic findings seen in other organs and tissues were incidental and of no toxicological significance.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Malignant granular cell tumor of the abdominal cavity was noted in one male at 200 mg/kg bw/day. A high ALP value was also noted in the same animal (ALP: 1853.7 U/L). The elevated ALP level might be caused by the primary effect on the tumor itself or as secondary effect due to activation of various organs such as liver/bone due to the tumor. A clear cause of these findings could not be determined, and it was probably not related to the administration of the test substance. Therefore, there was no toxicological significance.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test substance-related adverse effects in total thyroxine (total T4) evel in adult males of the main and recovery groups at 60, 200 and 600 mg/kg bw/day.
In adult males of the main group, statistically significant increases in thyroid stimulating hormone (TSH) were noted at 200 and 600 mg/kg bw/day. However, there were no changes in T4 or no histopathological changes in the thyroid. Therefore, it was considered to have little toxicological significance.
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse systemic effects were obseved up to the highest dose tested.
Key result
Critical effects observed:
no

Table 1. Absolut and relative liver weights of main and recovery groups

Group/ Dose (mg/kg bw/day)

 

Liver (g)

Liver (g/kg bw)

males

control

Mean

13.91

2.67

 

S.D.

2.54

0.20

 

N

12

12

60

Mean

14.01

2.71

 

S.D.

2.50

0.24

 

N

12

12

200

Mean

15.22

2.97**

 

S.D.

2.09

0.17

 

N

12

12

600

Mean

15.90

3.16**

 

S.D.

2.23

0.28

 

N

12

12

Recovery groups

 

 

 

control

Mean

14.9

2.61

 

S.D.

2.32

0.18

 

N

6

6

600

Mean

12.80

2.62

 

S.D.

2.52

0.2

 

N

6

6

females

control

Mean

10.60

3.25

 

S.D.

1.19

0.18

 

N

11

11

60

Mean

10.59

3.32

 

S.D.

1.11

0.21

 

N

8

8

200

Mean

11.36

3.60**

 

S.D.

0.93

0.22

 

N

11

11

600

Mean

12.15**

4.01**

 

S.D.

0.13

0.19

 

N

11

11

Recovery groups

 

 

 

control

Mean

7.97

2.58

 

S.D.

1.03

0.12

 

N

6

6

600

Mean

8.23

2.77

 

S.D.

1.32

0.23

 

N

6

6

Significantly different from control by Dunnett's t-test: ** p<0.01.

Table 2: Incidence and severity of remarkable microscopic findings in the liver

  Main groups Recovery groups
Group/ dose (mg/kg/day) 0 60 200 600 0 600
Number examined 6 6 7 6 6 6
Liver  
Hypertrophy, hepatocellular, centrilobular
Minimal 0 0 0 3 0 0
Slight 0 0 0 1 0 0
Total Number of affected animals 0 0 0 4 0 0
  Main groups Recovery groups
Group/ dose (mg/kg/day) 0 60 200 600 0 600
Number examined 6 6 6 6 6 6
Liver            
Hypertrophy, hepatocellular, centrilobular  
Minimal 0 0 0 2 0 0
Slight 0 0 0 1 0 0
Moderate 0 0 0 2 0 0
Total Number of affected animals 0 0 0 5 0 0

 

Conclusions:
Based on the results of this study, the NOAEL for systemic toxicity was set at 600 mg/kg bw/day in males and females since no adverse effects were observed up to the highest dose tested. Increased relative organ weights of the liver and hepatocellular hypertrophy in both sexes at 600 mg/kg bw/day were considered to have only little toxicological significance since hepatocellular hypertrophy in the centrilobular zone is generally considered to be an adaptive response on the test substance.
Executive summary:

The test substance was tested in a combined repeated dose oral toxicity study with the reproduction/developmental toxicity screening study according to OECD Guideline 422 and in compliance with GLP (2017). Twelve Sprague Dawley rats per sex and dose were treated via gavage with the test substance at dose levels of 60, 200 and 600 mg/kg bw/day, respectively. The control group received the vehicle corn oil. Additionally, a non-mated recovery group of 6 rats per sex was allocated to the control and high dose group. Males of the main group were dosed once daily for a total of 49 days (for 2 weeks prior to mating, during 2 weeks of mating and 21 days of post-mating), and females of the main group were dosed once daily for 2 weeks prior to mating, throughout gestation and for 13 days after delivery. Also, males and females of the recovery groups were dosed for 49 days. Subsequently,the recovery groups were assigned to a two week treatment free period. The dose levels were selected based on the results of a repeated dose 2-week dose range-finding study in which doses of 100, 300 and 1000 mg/kg bw/day had been tested. In that study, a decrease of body weight was noted in females at 1000 mg/kg bw/day. Salivation was observed in males and females at 300 and 1000 mg/kg bw/day. Soft stool was also observed in one male and two females at 1000 mg/kg bw/day. Increases of the absolute and relative liver weights were noted in animals of both sexes at 1000 mg/kg bw/day.

In the main study, general systemic observations including mortality, general clinical signs, body weights, body weight gain, food consumption, functional behavior examination, motor activity examination, macroscopic findings, hematology, coagulation, clinical chemistry, organ weights and microscopic findings were measured and conducted. Thyroid hormone (T4 and TSH) levels in blood were also analyzed for adult males at sacrifice.

All males of the main group and all animals of both sexes in the recovery group survived the duration of the study. One pregnant female of the main group was found dead at 60 mg/kg bw/day. However, this death was considered to be incidental and had no toxicological meaning since there was no dose-dependency and no adverse effects in other parameters at 60 mg/kg bw/day.

Mucous stool was mainly observed in males and females at 600 mg/kg bw/day. Salivation was observed in males and females at 200 and 600 mg/kg bw/day, however, it was considered to have little toxicological significance since it was caused by physicochemical characteristics. In adult males of the main group, a significant increase in thyroid-stimulating hormone (TSH) was noted at 200 and 600 mg/kg bw/day. However, there were no changes in T4 and no histopathological change in the thyroid. Therefore, it was considered to have little toxicological significance.

In the main group, an increase in the relative organ weight of the liver was noted in both sexes at 600 mg/kg bw/day. Hepatocellular hypertrophy was observed in animals of both sexes at 600 mg/kg bw/day. It was considered to have little toxicological significance since hepatocellular hypertrophy in the centrilobular zone is generally considered to be an adaptive response in nature. No test substance-related adverse effects were noted in the results of body weights, food consumption, sensory function, motor activity, urinalysis, hematology and clinical chemistry in adult animals of both sexes in the test substance-dosed groups.

Therefore, the NOAEL for systemic toxicity was set at 600 mg/kg bw/day in both sexes. The effects observed at 600 mg/kg bw/day in both sexes such as mucous stool, increase in the relative organ weight of the liver and hepatocellular hypertrophy were considered to be an adaptive response to the test substance administration.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

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: 29 Jul 2016
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of 1-[(1R*,6S*)-2,2,6-trimethylcyclohexyl]hexan-3-ol and 1-[(1S*,6S*)-2,2,6-trimethylcyclohexyl]hexan-3-ol
EC Number:
947-716-8
Molecular formula:
C15H30O
IUPAC Name:
Reaction mass of 1-[(1R*,6S*)-2,2,6-trimethylcyclohexyl]hexan-3-ol and 1-[(1S*,6S*)-2,2,6-trimethylcyclohexyl]hexan-3-ol

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
(Crl:CD(SD)), SPF
Details on species / strain selection:
Sprague-Dawley rats are commonly used in both the general systemic toxicity and reproductive and developmental toxicity studies with a large historical control data base. In addition, the rat is a required species in the regulatory guidelines.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: males: 10 weeks; females: 12 weeks
- Weight at study initiation: males: 327.7 - 393.2 g; females: 212.2 - 309.6 g
- Housing: individually during dosing period; one male and one female during mating period; dams were kept with their neonates during lactation; in stainless wire mesh cages (260W×350D×210H mm) or polycarbonate cages (260W×420D×180H mm)
- Diet: Teklad Certified Irradiated Global 18% Protein Rodent Diet 2918C, ad libitum
- Water: tap water filtered and irradiated by ultraviolet light; ad libitum
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY:
The certificate of feed analysis was provided by the manufacturer. The results of feed analysis met the allowable standard of this facility. Samples of drinking water are analyzed for specified microorganisms once a month and all environmental contaminants once a year. The results of water analysis met the allowable standard of this facility.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.2 - 23.7
- Humidity (%): 46.8 - 59.5
- Air changes (per hr): 10 -15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
The required amount of the test substance was weighed using an electronic balance and placed in a container. The test substance was mixed with a small amount of vehicle to dissolve using a vortex mixer, and then the vehicle was gradually added to yield the desired concentration. The dosing formulations were stored in a refrigerator (3.9 - 4.9°C) and used within 7 days.

- VEHICLE
- Justification for use and choice of vehicle: Through the preliminary solubility test to determine the solubility and dispersion characteristics of the test substance, corn oil was selected as the vehicle because the test substance was well dissolved in it.
- Amount of vehicle: 2 mL/kg
- Lot/batch no.: MKBZ9899V, MKBW9504V, MKCC0462
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: two weeks
- Proof of pregnancy: vaginal plug and sperm in vaginal smear referred to as Day 0 of pregnancy
- After successful mating each pregnant female was caged: individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses of the dosing formulations were conducted using gas chromatography and samples were taken three times from the middle of each dosing formulation prior to dosing and analyzed for verification of dose level concentration. The results of dose concentration analysis were determined to be in the range of 104.17 - 113.77%. These results were within the acceptable limits (± 15% of nominal values).
As a result of homogeneity and stability analyses the 0.5 and 500 mg/mL dosing solutions were confirmed to be homogenous and stable for 4 hours at room temperature and for 7 days under refrigeration.
Duration of treatment / exposure:
Main groups:
49 days (males: 2 weeks prior to mating, during 2 weeks of mating and 21 days of post-mating; females: 2 weeks prior to mating until postpartum day 13)

Recovery groups:
males and females: once daily for 6 weeks; animals were not mated and assigned to 2 weeks of recovery period after the completion of administration
Frequency of treatment:
once daily/7 days a week
Doses / concentrationsopen allclose all
Dose / conc.:
60 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
600 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12 (main groups)
6 (recovery groups; for control and high dose groups)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In a previously conducted 2-week repeated oral dose range-finding study, a decrease of body weight was noted in females at 1,000 mg/kg bw/day. Salivation was observed in males and females at 300 and 1,000 mg/kg bw/day. Soft stool was also observed in one male and two females at 1,000 mg/kg bw/day. Increases of the absolute and relative liver weights were noted in animals of both sexes at 1,000 mg/kg bw/day. Therefore, the high dose level was selected at 600 mg/kg bw/day. Then, the mid and low dose levels were selected at 200 and 60 mg/kg bw/day, respectively. The control group was dosed with the vehicle only with the same dose volume as the test substance- treated groups.

- Post-exposure recovery period in satellite groups: 2 weeks

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Cage side observations included: general condition and clinical signs, mortality/viability, abortion and pre-mature birth

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
prior to dosing and once weekly thereafter
- Observations included: Skin, fur, eyes, mucous membranes, occurrence of secretion and excretion; autonomic activity (lacrimation, piloerection, pupil size, unusual respiratory pattern, etc.); changes in gait, posture and response to handling, and the presence of clonic or tonic movements; stereotypes (excessive grooming, repetitive circling, etc.) or bizarre behavior (self-mutilation, walking backward, etc.)

BODY WEIGHT: Yes
- Time schedule for examinations: males: prior to dosing on Day 1 (the first day of dosing), once a week throughout the dosing and recovery periods, the day prior to necropsy and on the day of necropsy (body weights after fasting); females: prior to dosing on Day 1 (the first day of dosing), once a week throughout the dosing period, on gestation days 0, 7, 14 and 20, on postpartum days 0, 4 and 13, the day prior to necropsy and on the day of necropsy.

FOOD CONSUMPTION:
- Food consumption of males of the main group and animals of both sexes of the recovery group was recorded just prior to dosing on Day 0 (one day before first dosing), once a week during the dosing and recovery periods (except during mating) and the day prior to necropsy. Food consumption of females of the main group was recorded just prior to dosing on Day 0 (one day before the first dosing), once a week throughout the dosing period (except during mating), on gestation Days 0, 6, 13 and 19, on postpartum Days 0, 3 and 12 and the day prior to necropsy. Residual feed was recorded on the next day. Individual food consumption was calculated by subtracting the amount of residual feed from the amount presented.


FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OTHER:
- Time schedule for collection of blood: on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- How many animals: all males
- Parameters examined: total thyroxine (Total T4) and thyroid stimulating hormone (TSH)

Haematology, clinical chemistry, urinalysis, neurobehavioural examination (for details refer to IUCLID section 7.5.1)
Oestrous cyclicity (parental animals):
The estrous cycle examination for females of the main group was conducted from the dosing initiation day to the confirmed copulation day and necropsy day. Smears of vaginal mucosa were prepared daily in the morning and were stained with Diff Quick stain (Hemacolor® for microscopy, Merck, Germany). Stained vaginal mucosa smears were examined using light microscopy. The estrous cycle is divided into four stages; proestrus, estrus, metestrus and diestrus. One estrous cycle was defined as the period between the day of estrus and the day prior to next estrus. The estrous cycle was calculated from dosing initiation day to the day before mating initiation. Vaginal smears were examined on the day of necropsy to determine the stage of the estrous cycle and allowed correlation with histopathology of the female reproductive organs.
Sperm parameters (parental animals):
Parameters examined in P0 male parental generation:
testis weight, epididymis weight, histopathology with special focus on spermatogenesis and interstitial testicular cell structure
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum
- Maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed 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), nipple retention in males

GROSS EXAMINATION OF DEAD PUPS:
yes, for external gross abnormalities (with special focus on external reproductive genitals which should be examined for signs of altered development)
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All animals of the main groups were sacrificed 21 days post-mating (Day 50).
- Maternal animals: All animals of the main groups were sacrified on Day 14 postpartum. Non-pregnant females were sacrificed on gestation day 26. Dams, whose pups were all dead, were sacrificed as soon as possible. All animals of the recovery groups were sacrified 2 weeks after final dosing.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations. All grossly visible abnormalities were recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
Organ weights:
Paired organs were weighed together. Animals were fasted overnight prior to necropsy and body weights were recorded on the day of necropsy. Organs were weighed and organ-to-body weight ratios were calculated. Following organs were weighed: brain, heart, liver, thymus, spleen, thyroid, kidneys, adrenals, ovaries, uterus, testis, epididymis, prostate + seminal vesicle with coagulating gland

Histopathology:
Tissue preservation and slide preservation
All males and females were randomly selected from the main groups in addition to all recovery animals for tissue preparation. The testes and epididymides and eyes with optic nerve were fixed in Davidson’s fixative. All other tissues were preserved in 10% neutral buffered formalin.

For the histopathological examination, the preparation of specimens of organs and tissues was carried out and the remaining organs and tissues preserved in 10% neutral buffered formalin: brain, pituitary, thymus, lung with bronchi, trachea, thyroid, esophagus, heart, liver, spleen, kidneys, adrenals, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, testes, epididymides, prostate, seminal vesicle with coagulating gland, ovaries, uterus + cervix, submandibular lymph node, mesenteric lymph node, femur and sternum, spinal cord, sciatic nerve, eye and optic nerve, urinary bladder, organs with gross lesions

Histopathological examinations were conducted as follows:
- At least six males and six females from the control, low, mid and high groups (especially focused on spermatogenesis and interstitial testicular cell structure)
- All tissues from animals found dead during the study
- All gross, macroscopic lesions
- Target organs noted at the high dose group were examined for at least the recovery group: liver
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed on PND 13

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations

HISTOPATHOLOGY / ORGAN WEIGTHS
not performed

OTHER:
Thyroid homone analysis:
- Time schedule for collection of blood: on the day of scheduled necropsy (PND 13)
- Anaesthetic used for blood collection: No
- How many animals: at least two pups (preferably one male and one female) per litter
- Parameters examined: total thyroxine (Total T4) and thyroid stimulating hormone (TSH)
Statistics:
The statistical analysis of this study was conducted using the SAS program (SAS® 9.3, SAS Institute Inc., U.S.A.). For the data including body weights, food consumption, estrous cycle, mating period, gestation period, post-implantation loss, body weights and birth and survival rates of pups, litter size, AGD index, nipple number, thyroid hormone value, urine volume, hematology and clinical chemistry parameters, organ weights, grip strength, motor activity, the Bartlett’s test was conducted to analyze for homogeneity of variance (significance level: 0.05). One-way analysis of variance (ANOVA) test was applied on homogeneous data, then if significant (significance level: 0.05), Dunnett’s t-test was applied for multiple comparisons (significance levels: 0.05 and 0.01, two-tailed). Kruskal-Wallis test was applied on heterogeneous data, then if significant (significance level: 0.05), Steel’s test was applied for multiple comparisons (significance levels: 0.05 and 0.01, two-tailed).
The data of sensory function, mating index, fertility index and other data associated with gestation was analyzed utilizing Fisher’s exact test (significance levels: 0.05 and 0.01). For the data of the recovery group, Folded-F test was applied to analyze homogeneity of variance (significance level: 0.05, two-tailed). Student t-test was applied for homogeneous data, but if overruled, Aspin-Welch t-test was applied (significance levels: 0.05 and 0.01, two-tailed).
The organ weight data of dead animals, non-delivery females and dams whose pups were all dead were excluded from statistical analysis.
Reproductive indices:
Mating index (%) = (number of females with confirmed mating / number of females placed with males) x 100
Mating period = Day of mating confirmed - Day of initial mating (based on dosing day)
Gestation period = Day 0 post partum - Day 0 of gestation (based on dosing day)
Male fertility index (%) = (number of males impregnating a female / number of males with confirmed mating) x 100
Female fertility index (%) = (number of pregnant females / number of females with confirmed mating) x 100
Gestation index (%) = (number of females with live pups / number of pregnant females) x 100
Post-implantation loss (%) = [(number of implantations - number of live pups) / number of implantations] x 100
Offspring viability indices:
Live birth index (%) = (number of live pups on postnatal Day 0 / number of implantations) x 100
Viability index on postnatal Day 0 (%) = (number of pups born alive on postnatal Day 0 / total number of pups born) x 100
Viability index on postnatal Day 4 (%) = (number of pups surviving on postnatal Day 4 / number of pups born alive on postnatal Day 0) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
In the main group, salivation and/or mucous stool were observed in males and females at 200 and 600 mg/kg bw/day.
Salivation was sporadically observed in 5 males and 3 females and mucous stool was temporarily observed in 2 males and 2 females at 200 mg/kg bw/day. Salivation was often observed in 8 males and all 12 females and mucous stool was sporadically observed in 9 males and 7 females at 600 mg/kg bw/day. Also, soft stool was temporarily observed in one female at 600 mg/kg bw/day. Dirty nose and soiled perineal region were observed in one female at 60 mg/kg bw/day, whose pups were all dead.
In the recovery group, salivation and mucous stool were observed in males and females at 600 mg/kg bw/day. Mucous stool was mainly observed in males and females at 600 mg/kg bw/day and it was considered to be a test substance-related effect. However, mucous stool observed in males and females at 200 mg/kg bw/day was considered to have little toxicological significance since it was observed temporarily and/or sporadically (low incidence and frequency).
However, salivation was considered to have little toxicological significance since it was caused by physicochemical characteristics. Soft stool was considered to be incidental since it was observed once in one animal.

No detailed clinical signs were observed in males and females of the main and recovery groups in the detailed examinations conducted once a week.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One pregnant female was found dead on Gestation Day 23 (GD 23) without any parturition signs at 60 mg/kg bw/day. It was considered to be incidental and had no toxicological meaning since there were no dose-dependency and no test substance-related adverse effects in other parameters at 60 mg/kg bw/day.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
In the main group, no significant differences in body weight changes were noted in males and females compared to the control group.
In the recovery group, statistically significant decreases in body weights were noted in males at 600 mg/kg bw/day from Day 35 to the end of dosing (Day 63). However, these statistical significances in body weights had little toxicological meaning since there were no statistical differences at 600 mg/kg bw/day in the main group.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No significant difference in food consumption was noted in males and females of the main and recovery groups compared to the control group.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No adverse effects were observed in any adult animal in the main and recovery groups. Other statistical significant results were considered not to be test substance-related changes because of small difference and/or the values were within the range of historical reference data.
Urinalysis findings:
no effects observed
Description (incidence and severity):
In the main and recovery groups, no effects were noted in any dosing group.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related effects on the auditory reflex, pinna reflex, pupillary reflex and corneal reflex tests were observed in animals of both sexes in the main and recovery groups when compared to the control group.
In the main and recovery groups, there were no test substance-related effects in the grip strength test when compared to the control group. In the main group, there was a statistically significant temporary decrease in ambulatory count of spontaneous motor activity at 200 mg/kg bw/day. Also, there were statistically significant temporary decreases in vertical count of the spontaneous motor activity at 200 and 600 mg/kg bw/day. However, the statistical significance had little toxicological meaning because it was a small difference or temporary change and showed no dose dependency.
In the recovery group, there were no test substance-related effects on the spontaneous motor activity when compared to the control group.
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):
Dead animals:
There were no findings related to the test substance or causative of death.
Slight lymphoid atrophy of the spleen and thymus and cortical lymphoid apoptosis of the thymus were noted in one female at 60 mg/kg bw/day, and they are frequently noted in animals in poor condition. However, the severity was not severe enough to cause animal’s deaths. While all those changes probably contributed to the animal’s deaths, a clear cause of death could not be determined. Therefore, it was not considered to be related to the test substance since dose-dependent response was not clear, and these non-specific findings were frequently noted in poor condition/stress. It was difficult to perform histopathological examination of some tissues (stomach, duodenum, jejunum, ileum, cecum, colon, rectum, eye & optic nerve, and kidney) because of postmortem autolysis.

Surviving animals:
Test substance-related changes were observed in the liver:
Centrilobular hepatocellular hypertrophy was observed at minimal to moderate severity in males and females at 600 mg/kg bw/day. At the end of the recovery period, those findings, which were observed in the main group, were not observed in all males and females of the high dose group. It was considered to have little toxicological significance since hepatocellular hypertrophy in the centrilobular zone is generally considered to be an adaptive response in nature) in the absence of associated inflammation or necrosis, and it was completely recovered after the recovery period in this study.

Slight to moderate thymic lymphoid atrophy and/or erosion of the stomach were noted in three dams, whose pups were all dead. It was not considered to be related to the test substance since dose-dependent response was not clear, and these non-specific findings were frequently noted in poor condition/stress.
No test substance-related histopathological findings were noted in the reproductive organs of either sex.
The microscopic findings seen in other organs and tissues were incidental and of no toxicological significance.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Malignant granular cell tumor of the abdominal cavity was noted in one male at 200 mg/kg bw/day. A high ALP value was also noted in the same animal (ALP: 1853.7 U/L). The elevated ALP level might be caused by the primary effect on the tumor itself or as secondary effect due to activation of various organs such as liver/bone due to the tumor. A clear cause of these findings could not be determined, and it was probably not related to the administration of the test substance. Therefore, there was no toxicological significance.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test substance-related adverse effects in total thyroxine (total T4) level in adult males of the main and recovery groups at 60, 200 and 600 mg/kg bw/day.
In adult males of the main group, statistically significant increases in thyroid stimulating hormone (TSH) were noted at 200 and 600 mg/kg bw/day. However, there were no changes in T4 or no histopathological changes in the thyroid. Therefore, it was considered to have little toxicological significance.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The estrous cycle lengths of females in the 0 (control), 60, 200 and 600 mg/kg bw/day dosing groups were 4.4, 4.0, 4.4 and 4.0 days, respectively. There were no statistically significant differences in any dosing group.
The estrous cycle of females on the day of necropsy was diestrus, except for one non-copulated female and one non-pregnant female, whose estrous cycles were estrous and metestrus, respectively.
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
In the control group and 60, 200 and 600 mg/kg bw/day dosing groups, the mating periods were 2.1, 2.8, 3.1 and 2.3 days, the mating index was all 100.0%, the gestation periods were 22.3, 22.3, 21.9 and 22.3 days, the gestation indices were 100.0, 91.7, 100.0 and 100.0%, the post-implantation loss rates were 10.5, 12.1, 8.8 and 11.5% and the fertility indices of animals of both sexes were 91.7, 100.0, 91.7 and 100.0%, respectively. There were no statistically significant differences in any dosing group.
In the 600 mg/kg bw/day dose group, a statistically significantly low value of total litter size was noted, and it was considered to be an adverse effect (see table 1). Reduced (not statistically significant) number of implantations might be responsible for this effect. Since dams at 600 mg/kg bw/day showed clinical signs of mucous stool and hepatocellular hypertrophy, reduced litter size can presumably be seen as a secondary effect of adaptive systemic effects of the test substance administration.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No adverse effects on fertility observed up to and including highest dose tested.
Key result
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No adverse effects on fertility observed at 200 mg/kg bw/day.
Key result
Dose descriptor:
LOAEL
Remarks:
fertility
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
reproductive performance
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse systemic effects observed at 600 mg/kg bw/day. Adaptive systemic effects observed: increased relative organ weights of the liver and hepatocellular hypertrophy at 600 mg/kg bw/day

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
In the control group and 60, 200 and 600 mg/kg bw/day dose groups, the live birth indices were 89.5, 87.9, 91.3 and 88.5%, the viability indices on Postnatal Day (PND) 0 were 98.4, 88.5, 96.6 and 98.4 , and the viability indices on PND 4 were 98.2, 77.5, 94.9 and 91.0%, respectively.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no test substance-related changes in body weights of pups at 60, 200 and 600 mg/kg bw/day on PNDs 0, 4 and 13.
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:
no effects observed
Description (incidence and severity):
There were no test substance-related changes in anogenital distance index of pups at 60, 200 and 600 mg/kg bw/day on PND 4. The nipple number was 0 in male pups at 0, 60, 200 and 600 mg/kg bw/day on PND 12 and there was no nipple retention in male pups at any dose on PND 12.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test substance-related effects in external findings in pups at 60, 200 and 600 mg/kg bw/day.
Histopathological findings:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
In F1 male pups of the main group, statistically significant increases in thyroid-stimulating hormone (TSH) were noted at 200 and 600 mg/kg bw/day. However, it was considered to have no toxicological significance since they were small differences and the values were within the range of historical reference data.

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:
600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects on development up to and including the highest dose tested.

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
600 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

Table 1. Number of pups born dead and alive and total litter size

Group/ dose
(mg/kg bw/day)
  No. of pups born (PND 0)  No. of implantations   
  Live Dead Total Left   Right
Control M ean 14.4 0.3 14.6 7.6 8.5 
  SD 1.9 0.6 2.2  1.6  2.3
  N 11 11 11  11  11
60 M ean 12.5 1.7 14.3  6.4  8.1
  SD 4.4 4.2 2.1  1.8  1.8
  N 11 11 11  12  12
200 M ean 13.5 0.5 14.1  6.7  8.8
  SD 3.1 0.8 3.4  2.0  2.9
  N 11 11 11  11  11
600 M ean 11.3 0.2 11.5 *  5.4  7.4
  SD 2.9 0.4 2.9  2.2  2.0
  N 12 12 12  12  12

PND : postnatal day

Significant difference from control by Dunnett's test : * p<0.05

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, the NOAEL for fertility was set at 600 mg/kg bw/day in males and at 200 mg/kg bw/day in females, based on reduced number of implantations and decreased litter size at 600 mg/kg bw/day. Reduced litter size can presumably be seen as a secondary effect of adaptive systemic effects (mucous stool and hepatocellular hypertrophy) in dams at the same dose level. The NOAEL for developmental toxicity in offspring was set at 600 mg/kg bw/day, since no adverse effects on development were observed up to and including highest dose tested.

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