Reaction mass of rel-(2S,4R,6S)-2,4,6-trimethyl-4-phenyl-1,3-dioxane and rel-(2S,4S,6S)-2,4,6-trimethyl-4-phenyl-1,3-dioxane

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 May - 21 Sep 2015

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:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 May - 21 Sep 2015

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:

1996

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: 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: 9 weeks (male), 8 weeks (female)
- Weight at study initiation: 312.7-358.4 g (males), 189.3-228.5 g (females)
- Housing: Acclimation period and pre-mating: 1 animal per cage; Mating: 1:1; Lactation: neonates were kept with the dam; animals were kept in stainless wire mesh cages (260W x 350D x 210H mm) and in polycarbonate cages (260W x 420L x 180H mm)
- Diet: Teklad Certified Irradiated Global 18% Protein Rodent Diet 2918C (Harlan Laboratories, Inc., USA), ad libitum
- Water: public tap water filtered and irradiated by ultraviolet light, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.5 - 23.8
- Humidity (%): 46.3 - 65.6
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was mixed with a small amount of vehicle to dissolve using a magnetic stirrer and then, the vehicle was gradually added to yield the desired concentration. The dosing formulations were stored in a refrigerator (4.4–5.5 °C). These dosing formulations were 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 dissolved in it.
- Amount of vehicle: 5 mL/kg bw

Details on mating procedure:

- M/F ratio per cage: 1:1
- Proof of pregnancy: All females were examined for the presence of a vaginal plug or sperm in the vaginal smear twice a day for confirmation of mating. Positive confirmation was defined as Day 0 of Gestation.

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 analyses were determined to be 97.13 – 99.87%. These results were within the acceptable limits (± 15% of nominal values). As a result of homogeneity and stability analyses conducted the 0.2 and 200 mg/mL dosing solutions were confirmed to be homogenous and stable for 4 h at room temperature and for 7 days under refrigeration.

Duration of treatment / exposure:

Main groups:
males: for 6 weeks, starting 2 weeks before mating, during mating and 2 weeks after mating
females: for 2 weeks prior to mating, throughout gestation and for at least 4 days after delivery up to the day before the scheduled terminal necropsy

Recovery groups:
Males and females of recovery groups were dosed once daily for 6 weeks. Animals were not mated and were assigned to 2 weeks of recovery period after the completion of administration.

Frequency of treatment:

once daily, 7 days/week

Details on study schedule:

not applicable for an OECD 422 study

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

300 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, two males and five females were found moribund or dead at 600 mg/kg bw/day on Day 2. There were no test substance-related effects in animals at 200 mg/kg bw/day. Therefore, the high dose level was selected at 300 mg/kg bw/day. Then, the mid and low dose levels were selected at 100 and 30 mg/kg bw/day, respectively.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily mortality/viability
- Cage side observations included: All animals were observed for general condition and clinical signs at least once daily throughout the study. Females were also observed for signs of abortion and pre-mature birth.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed physical examinations for signs and symptoms of adverse effects, including central and autonomic nervous system effects, motor activity and behavior, were conducted on all animals once before the test and once a week throughout the dosing and recovery periods.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights of males of the main group and animals of each sex of the recovery group were recorded just prior to dosing on Day 1 (the first day of dosing), once a week throughout the dosing and recovery periods, the day before necropsy and on the day of necropsy (fasted body weights). Body weights of females of the main group were recorded just prior to dosing on Day 1, once a week throughout the dosing and recovery periods, on Days 0, 7, 14 and 20 of gestation, on Days 0 and 4 postpartum and on the day of necropsy (fasted body weights). Fasted body weights recorded on the day of necropsy were presented, but were not included in statistical analysis.

FOOD CONSUMPTION: Yes
- Food consumptions of males of the main group and animals of each sex of the recovery group were recorded just prior to dosing on Day 1, once a week during the dosing and recovery periods and the day before necropsy. Food consumptions of females of the main group were recorded just prior to dosing on Day 0, once a week throughout the dosing and recovery periods, on Days 0, 6, 13 and 19 of gestation, on Days 0 and 3 post-partum. Food consumption was not recorded during mating.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OTHER: Haematology, clinical chemistry, urinalysis, neurobehavioural examination (for details refer to IUCLID section 7.5.1 Repeated dose toxicity: oral)

Oestrous cyclicity (parental animals):

not examined

Sperm parameters (parental animals):

Histopathological examination was conducted with focus on spermatogenesis.

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in offspring:
number and sex of pups, body weight, presence of gross abnormalities, postnatal mortality

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All animals of the main group were sacrificed 2 weeks after mating.
- Maternal animals: All animals of the main group were sacrified on Day 6 postpartum. Non-pregnant females were sacrificed on Day 27 after the last day of dosing.
All animals of the recovery group 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. The testes and epididymides of all adult males were weighed. 6 males and females were randomly selected from the main study animals in addition to all recovery animals for necropsy. Following organs were weighed: brain, heart, liver, thymus, spleen, kidneys, adrenals, ovaries, uterus

Histopathology
Tissue preservation and slide preservation
6 males and females were randomly selected from the main groups in addition to all recovery animals for tissue preparation. The testes and epididymides were fixed in Bouin's solution. The eyes with optic nerves 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, ovaries, uterus, submandibular lymph node, mesenteric lymph node, bone marrow (femur and sternum), spinal cord, sciatic nerve, eye and optic nerve, urinary bladder

Besides, from all animals except for six females and males in the main group, the following organs and tissues were harvested and preserved: brain, pituitary, heart, thymus, liver, spleen, kidneys, adrenals, prostate, testes, epididymides, ovaries, uterus

Histopathological examinations were conducted as follows:
- 6 males and females from the control, low, mid and high dose group (especially, focused on spermatogenesis and interstitial testicular cell structure)
- All tissues from animals found dead or killed in a moribund condition
- All gross, macroscopic lesions
- Target organs noted at the high dose were examined for the recovery group

Postmortem examinations (offspring):

GROSS NECROPSY
- Gross necropsy consisted of external examinations.

HISTOPATHOLOGY / ORGAN WEIGTHS
not performed

Statistics:

The statistical analysis of this study was conducted using the SAS program (SAS 9.3). For the data including body weights, food consumption, urine volume and specific gravity, hematology and blood biochemistry parameters, organ weights, mating result, birth and survival rates, sensory reactivity and motor activity, the Bartlett test was conducted to test for homogeneity of variance (significance level: 0.05). One-way analysis of variance (ANOVA) test was employed on homogeneity, if significant (significance level: 0.05), followed by Dunnett’s t-test for multiple comparisons (significance levels: 0.05 and 0.01, two-tailed). Kruskal-Wallis test was employed on heterogeneity, if significant (significance level: 0.05), followed by Steel’s test for multiple comparisons (significance levels: 0.05 and 0.01, two-tailed). Mating index, fertility index and other data associated with gestation were analyzed utilizing Fisher’s exact test (significance levels: 0.05 and 0.01). For the data of the recovery group, Folded-F test was employed to test homogeneity of variance (significance level: 0.05, two-tailed). Student t-test was employed on homogeneity, if overruled, Aspin-Welch t-test was applied (significance levels: 0.05 and 0.01, two-tailed).

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
Pre-implantation loss (%) = [(number of corpus lutea - number of implantations) / number of corpus lutea] 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

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

temporary loss of locomotor activity was observed in females at 300 mg/kg bw/day

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Clinical biochemistry findings:

effects observed, non-treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

effects observed, non-treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

reversible hepatocellular hypertrophy in the centrilobular zone of males at 100 mg/kg bw/day and in both sexes at 300 mg/kg bw/day

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY
In the main and recovery groups, all animals survived the duration of the study. There was no effect on the mortality.
In the main group, loss of fur was observed in one male at 30 mg/kg bw/day on Days 41 and 42. A decrease in locomotor activity, dirty nose and/or soiled perineal region were observed in one female after parturition (from Day 1 to 3 post partum). Wound (right shoulder) was observed in one male at 100 mg/kg bw/day from Day 40 to Day 42. Temporary loss of locomotor activity was observed frequently in females at 300 mg/kg bw/day from GD 7 to the end of dosing. In the recovery group, temporary loss of locomotor activity was observed frequently in females at 300 mg/kg bw/day from Day 25 to Day 37. Salivation was shown in two males at 300 mg/kg bw/day from Day 39 to Day 42.
In the recovery group, temporary loss of locomotor activity was observed frequently in females at 300 mg/kg/day from Day 25 to Day 37. Salivation was shown in one male at 300 mg/kg bw/day on Days 39 and 42. During the recovery period, abnormal clinical signs were not observed in both sexes at 300 mg/kg bw/day.
The symptoms such as wound and salivation were considered to occur due to individual difference because they were observed temporarily.
Temporary loss of locomotor activity observed in females at 300 mg/kg/day in the main and recovery groups was considered to be a test substance-related effect.

No clinical abnormalities in detailed examinations were observed in males and females of the main and recovery groups.

BODY WEIGHT AND WEIGHT GAIN
No statistically significant differences in body weight changes were noted in males and females of the main and recovery groups compared to the control group.

FOOD CONSUMPTION
A statistically significantly increase in food consumption was noted in males at 300 mg/kg bw/day in the main group on Day 14. A statistically significantly increase in food consumption was noted in females at 300 mg/kg bw/day in the recovery group on Day 49. These statistical significances were not considered to be test substance-related changes since these were differences of small magnitude and they were not related to body weight changes.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Mating indices at 0, 30, 100 and 300 mg/kg bw/day were 100.0, 100.0, 100.0 and 100.0%, respectively. Male and female fertility indexes of these groups were 100.0, 100.0, 91.7 and 100.0, respectively. While the mating periods of the groups were 3.3, 3.1, 4.3 and 2.5, the gestation periods were 22.1, 22.1, 22.0 and 22.2, respectively.
Pre-implantation rates at 0, 30, 100 and 300 mg/kg bw/day were 10.3, 11.6, 10.4 and 5.7% and post-implantation rates of these groups were 4.7, 12.5, 9.8 and 11.5%, respectively. In the same groups, mean litter sizes were 14.4, 14.3, 13.4 and 14.3.
Gestation indices at 0, 30, 100 and 300 mg/kg bw/day were 100.0, 100.0, 100.0 and 100.0%, respectively. Also, normal delivery was observed in all females of all groups.
There were no significant differences in any dose group.

ORGAN WEIGHTS
In the main group, increases in the absolute (+36%) and relative (+25%) organ weights of the liver were noted in males at 300 mg/kg bw/day. Relative liver weights were also increased in females (+25%) at 300 mg/kg bw/day. While the increase in the liver organ weight was not considered to be a test substance-related adverse effect since it was not accompanied by increases of ALT, AST and ALP and it was reversible in organ weights in the recovery group, the absolute liver weight increase was considered to exceed a level that should be cosidered adaptive.
Other statistical significances in the absolute and/or relative organ weights were not considered to be test substance-related effects since the differences were small in magnitude and they were within the historical range limit.

GROSS PATHOLOGY
Macroscopic examination at necropsy did not reveal any treatment-related changes. All other macroscopic findings observed in this study were considered to be incidental, and not related to the test substance.

HISTOPATHOLOGY
Following the dosing period, test substance-related microscopic findings were present in the liver. Hepatocellular hypertrophy was observed in males at 100 mg/kg bw/day and in both sexes at 300 mg/kg bw/day after six weeks of treatment. The hepatocellular hypertrophy was characterized by increased cytoplasmic volume, which was within centrilobular zone. At the end of the 2-week recovery period, this finding disappeared at 100 and 300 mg/kg bw/day. The hepatocellular hypertrophy was not considered to be adverse, since the hepatocellular hypertrophy in centrilobular zone is generally considered to be an adaptive response. Furthermore, the effect was reversible within the recovery period of the study.
All other microscopic findings seen in various organs and tissues were considered to be incidental and of no toxicological significance.

OTHER:
For results on haematology, clinical chemistry, urinalysis, neurobehavioural examination refer to IUCLID section 7.5.1. Repeated dose toxicity: oral

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

>= 300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects on reproduction up to and including the highest dose tested

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

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:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

only external

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

VIABILITY (OFFSPRING)
Viability indices at 0, 30, 100 and 300 mg/kg bw/day were 99.5, 98.1, 98.9 and 96.5% on Day 0 postnatal and 91.7, 93.9, 100.0 and 97.9% on Day 4 postnatal, respectively.
Live birth indices at 0, 30, 100 and 300 mg/kg bw/day were 95.3, 87.5, 90.2 and 88.6%, respectively.
In the same groups, the sex ratio was 0.8, 0.8, 1.1 and 0.9, respectively.

BODY WEIGHT (OFFSPRING)
There were no treatment-related changes in body weights of pups at 30, 100 and 300 mg/kg bw/day.

GROSS PATHOLOGY (OFFSPRING)
Small body was observed in one pup (0.58%) at 300 mg/kg bw/day on Day 4 postnatal. However, this change was considered to be incidental and not related to the test substance.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects on development of offspring up to and including the highest dose tested

Reproductive effects observed:

no

Conclusions:

Based on the results of this study, the NOAEL for reproductive toxicity was set at 300 mg/kg bw/day, the highest dose tested. No adverse effects on development of offspring were observed up to and including 300 mg/kg bw/day, the highest dose tested.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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:

1996

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

reaction mass of rel-(2S,4R,6S)-2,4,6-trimethyl-4-phenyl-1,3-dioxane and rel-(2S,4S,6S)-2,4,6-trimethyl-4-phenyl-1,3-dioxane

EC Number:

945-924-3

Molecular formula:

C13H18O2

IUPAC Name:

reaction mass of rel-(2S,4R,6S)-2,4,6-trimethyl-4-phenyl-1,3-dioxane and rel-(2S,4S,6S)-2,4,6-trimethyl-4-phenyl-1,3-dioxane

Test animals

Species:

rat

Strain:

other: 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: 9 weeks (male), 8 weeks (female)
- Weight at study initiation: 312.7-358.4 g (males), 189.3-228.5 g (females)
- Housing: Acclimation period and pre-mating: 1 animal per cage; Mating: 1:1; Lactation: neonates were kept with the dam; animals were kept in stainless wire mesh cages (260W x 350D x 210H mm) and in polycarbonate cages (260W x 420L x 180H mm)
- Diet: Teklad Certified Irradiated Global 18% Protein Rodent Diet 2918C (Harlan Laboratories, Inc., USA), ad libitum
- Water: public tap water filtered and irradiated by ultraviolet light, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.5 - 23.8
- Humidity (%): 46.3 - 65.6
- 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 oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was mixed with a small amount of vehicle to dissolve using a magnetic stirrer and then, the vehicle was gradually added to yield the desired concentration. The dosing formulations were stored in a refrigerator (4.4–5.5 °C). These dosing formulations were 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 dissolved in it.
- Amount of vehicle: 5 mL/kg bw

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 analyses were determined to be 97.13 – 99.87%. These results were within the acceptable limits (± 15% of nominal values). As a result of homogeneity and stability analyses conducted the 0.2 and 200 mg/mL dosing solutions were confirmed to be homogenous and stable for 4 h at room temperature and for 7 days under refrigeration.

Duration of treatment / exposure:

Main groups:
males: for 6 weeks, starting 2 weeks before mating, during mating and 2 weeks after mating
females: for 2 weeks prior to mating, throughout gestation and for at least 4 days after delivery up to the day before the scheduled terminal necropsy

Recovery groups:
Males and females of recovery groups were dosed once daily for 6 weeks. Animals were not mated and were assigned to 2 weeks of recovery period after the completion of administration.

Frequency of treatment:

once daily, 7 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

300 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, two males and five females were found moribund or dead at 600 mg/kg bw/day on Day 2. There were no test substance-related effects in animals at 200 mg/kg bw/day. Therefore, the high dose level was selected at 300 mg/kg bw/day. Then, the mid and low dose levels were selected at 100 and 30 mg/kg bw/day, respectively.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily mortality/viability
- Cage side observations included: All animals were observed for general condition and clinical signs at least once daily throughout the study. Females were also observed for signs of abortion and pre-mature birth.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed physical examinations for signs and symptoms of adverse effects, including central and autonomic nervous system effects, motor activity and behavior, were conducted on all animals once before the test and once a week throughout the dosing and recovery periods.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights of males of the main group and animals of each sex of the recovery group were recorded just prior to dosing on Day 1 (the first day of dosing), once a week throughout the dosing and recovery periods, the day before necropsy and on the day of necropsy (fasted body weights). Body weights of females of the main group were recorded just prior to dosing on Day 1, once a week throughout the dosing and recovery periods, on Days 0, 7, 14 and 20 of gestation, on Days 0 and 4 post partum and on the day of necropsy (fasted body weights). Fasted body weights recorded on the day of necropsy were presented, but were not included in statistical analysis.

FOOD CONSUMPTION: Yes
- Food consumptions of males of the main group and animals of each sex of the recovery group were recorded just prior to dosing on Day 1, once a week during the dosing and recovery periods and the day before necropsy. Food consumptions of females of the main group were recorded just prior to dosing on Day 0, once a week throughout the dosing and recovery periods, on Days 0, 6, 13 and 19 of gestation, on Days 0 and 3 post partum. Food consumption was not recorded during mating.

FOOD EFFICIENCY: No

WATER CONSUMPTION: 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
- How many animals: 6 males and females were randomly selected from the main study groups in addition to all animals from the recovery groups.
- 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
- How many animals: 6 males and females were randomly selected from the main study groups in addition to all animals from the recovery groups.
- 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), 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: The 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

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All males of the main group were sacrificed 2 weeks after mating and females of the main group were sacrificed on Day 6 post-partum. All animals of the recovery group were sacrificed 2 weeks after final dosing. Non-pregnant females were sacrificed on Day 27 after the last day of mating. Complete gross post-mortem examinations were conducted on all animals including the external and internal surfaces. 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. The testes and epididymides of all adult males were weighed. 6 males and 6 females were randomly selected from the main study animals in addition to all recovery animals for necropsy. Following organs were weighed: brain, heart, liver, thymus, spleen, kidneys, adrenals, ovaries, uterus

HISTOPATHOLOGY: Yes
Tissue preservation and slide preservation
6 males and 6 fmales were randomly selected from the main groups in addition to all recovery animals for tissue preparation. The testes and epididymides were fixed in Bouin's solution. The eyes with optic nerves 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, ovaries, uterus, submandibular lymph node, mesenteric lymph node, bone marrow (femur and sternum), spinal cord, sciatic nerve, eye and optic nerve, urinary bladder

Besides, from all animals except for six females and males in the main group, the following organs and tissues were harvested and preserved: brain, pituitary, heart, thymus, liver, spleen, kidneys, adrenals, prostate, testes, epididymides, ovaries, uterus

Histopathological examinations were conducted as follows:
- 6 males and females from the control, low, mid and high dose group (especially, focused on spermatogenesis and interstitial testicular cell structure)
- All tissues from animals found dead or killed in a moribund condition
- All gross, macroscopic lesions
- Target organs noted at the high dose were examined for the recovery group (liver)

Statistics:

The statistical analysis of this study was conducted using the SAS program (SAS 9.3). For the data including body weights, food consumption, urine volume and specific gravity, hematology and blood biochemistry parameters, organ weights, mating result, birth and survival rates, sensory reactivity and motor activity, the Bartlett test was conducted to test for homogeneity of variance (significance level: 0.05). One-way analysis of variance (ANOVA) test was employed on homogeneity, if significant (significance level: 0.05), followed by Dunnett’s t-test for multiple comparisons (significance levels: 0.05 and 0.01, two-tailed). Kruskal-Wallis test was employed on heterogeneity, if significant (significance level: 0.05), followed by Steel’s test for multiple comparisons (significance levels: 0.05 and 0.01, two-tailed). Mating index, fertility index and other data associated with gestation were analyzed utilizing Fisher’s exact test (significance levels: 0.05 and 0.01). For the data of the recovery group, Folded-F test was employed to test homogeneity of variance (significance level: 0.05, two-tailed). Student t-test was employed on homogeneity, if overruled, Aspin-Welch t-test was applied (significance levels: 0.05 and 0.01, two-tailed).

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

temporary loss of locomotor activity was observed in females at 300 mg/kg bw/day

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Clinical biochemistry findings:

effects observed, non-treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

effects observed, non-treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

increase of liver weights at 300 mg/kg bw/day in both sexes

Gross pathological findings:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

reversible hepatocellular hypertrophy in the centrilobular zone of males at 100 mg/kg bw/day and in both sexes at 300 mg/kg bw/day

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
In the main and recovery groups, all animals survived the duration of the study. There was no effect on the mortality.
In the main group, loss of fur was observed in one male at 30 mg/kg bw/day on Days 41 and 42. A decrease in locomotor activity, dirty nose and/or soiled perineal region were observed in one female after parturition (from Day 1 to 3 post partum). Wound (right shoulder) was observed in one male at 100 mg/kg bw/day from Day 40 to Day 42. Temporary loss of locomotor activity was observed frequently in females at 300 mg/kg bw/day from GD 7 to the end of dosing. In the recovery group, temporary loss of locomotor activity was observed frequently in females at 300 mg/kg bw/day from Day 25 to Day 37. Salivation was shown in two males at 300 mg/kg bw/day from Day 39 to Day 42.
In the recovery group, temporary loss of locomotor activity was observed frequently in females at 300 mg/kg/day from Day 25 to Day 37. Salivation was shown in one male at 300 mg/kg bw/day on Days 39 and 42. During the recovery period, abnormal clinical signs were not observed in both sexes at 300 mg/kg bw/day.
The symptoms such as wound and salivation were considered to occur due to individual difference because they were observed temporarily.
Temporary loss of locomotor activity observed in females at 300 mg/kg/day in the main and recovery groups was considered to be a test substance-related effect.

No clinical abnormalities in detailed examinations were observed in males and females of the main and recovery groups.

BODY WEIGHT AND WEIGHT GAIN
No statistically significant differences in body weight changes were noted in males and females of the main and recovery groups compared to the control group.

FOOD CONSUMPTION
A statistically significantly increase in food consumption was noted in males at 300 mg/kg bw/day in the main group on Day 14. A statistically significantly increase in food consumption was noted in females at 300 mg/kg bw/day in the recovery group on Day 49. These statistical significances were not considered to be test substance-related changes since these were differences of small magnitude and they were not related to body weight changes.

HAEMATOLOGY
In the main and recovery groups, no effects were noted in any animal in any dosing group.
Other statistical significances were not considered to be test substance-related changes since these were differences of small magnitude and they were within the range of historical reference data.

CLINICAL CHEMISTRY
In the main and recovery groups, no effects were noted in any animal in any dosing group.
Other statistical significances were not considered to be test substance-related changes since these were differences of small magnitude and/or they were within the range of historical reference data.

URINALYSIS
In the main and recovery groups, no effects were noted in any dosing group.

NEUROBEHAVIOUR
In the main and recovery groups, there were no test substance-related effects on auditory reflex, pinna reflex, pupillary reflex and corneal reflex test in both sexes in the test substance dosing groups when compared to the control group.
In the main and recovery groups, there were no test substance-related effects on rotarod test and spontaneous motor activity in both sexes in the test substance dosing groups when compared to the control group.
In the main group, statistically significantly decreased forelimb grip strength was noted in males at 30 mg/kg bw/day when compared to control group. However, it was not considered to have meaning of toxicological significance since it was difference of small magnitude and without dose-dependency.

ORGAN WEIGHTS
In the main group, increases in the absolute (+36%) and relative (+25%) organ weights of the liver were noted in males at 300 mg/kg bw/day. Relative liver weights were also increased in females (+25%) at 300 mg/kg bw/day. While the increase in the liver organ weight was not considered to be a test substance-related adverse effect since it was not accompanied by increases of ALT, AST and ALP and it was reversible in organ weights in the recovery group, the absolute liver weight increase was considered to exceed a level that should be cosidered adaptive.
Other statistical significances in the absolute and/or relative organ weights were not considered to be test substance-related effects since the differences were small in magnitude and they were within the historical range limit.

GROSS PATHOLOGY
Macroscopic examination at necropsy did not reveal any treatment-related changes. All other macroscopic findings observed in this study were considered to be incidental, and not related to the test substance.

HISTOPATHOLOGY
Following the dosing period, test substance-related microscopic findings were present in the liver. Hepatocellular hypertrophy was observed in males at 100 mg/kg bw/day and in both sexes at 300 mg/kg bw/day after six weeks of treatment. The hepatocellular hypertrophy was characterized by increased cytoplasmic volume, which was within centrilobular zone. At the end of the 2-week recovery period, this finding disappeared at 100 and 300 mg/kg bw/day. The hepatocellular hypertrophy was not considered to be adverse, since the hepatocellular hypertrophy in centrilobular zone is generally considered to be an adaptive response. Furthermore, the effect was reversible within the recovery period of the study.
All other microscopic findings seen in various organs and tissues were considered to be incidental and of no toxicological significance.

Effect levels

1

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

organ weights and organ / body weight ratios

2

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at 100 mg/kg bw/day

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Any other information on results incl. tables

Table 1. Absolute and relative liver weights (n=6)

	Dose level (mg/kg bw/day)
	0		30		100		300
	Male	Female	Male	Female	Male	Female	Male	Female
Liver weight
Absolute (g)	12.93 ± 1.64	11.13 ± 1.67	13.07 ± 1.67	11.58 ± 1.11	14.56 ± 2.05	12.69 ± 1.18	17.61 ± 1.68**	12.59 ± 0.84
Relative (%)	2.75 ± 0.20	3.79 ± 0.30	2.75 ± 0.22	3.85 ± 0.26	3.01 ± 0.13	4.11 ± 0.20	3.43 ± 0.17**	4.72 ± 0.30**

Significantly different from control group by Dunnett's test: *p<0.05, **p<0.01

 

Table 2. Incidence of treatment-related microscopic findings

	Dose level (mg/kg bw/day)
	0		30		100		300
	Male	Female	Male	Female	Male	Female	Male	Female
Liver
Hypertrophy, hepatocellular, centrilobular	0/6	0/6	0/6	0/6	1/6 (minimal)	0/6	5/6 (minimal)	5/6 (1x mild, 4x minimal)

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, the NOAEL for systemic toxicity was set at 100 mg/kg bw/day for males and females due to effects on liver weight.