Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 May 2017 - 09 Sep 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 May 2017 - 09 Sep 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
Jul 29, 2016
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD (SD), SPF
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: males: 10 weeks; females: 12 weeks
- Weight at study initiation: males: 355.5 - 403.1 g; females: 228.1 - 285.7 g
- Housing: acclimation period: 2 animals per cage; mating: 1:1; dosing period: 1 animal; lactation: neonates were kept with the dam; animals were kept in stainless-steel cages (260W×350L×210H mm) and in poly sulfone cages (260W×420L×180 mm)
- Diet: Teklad Certified Irradiated Global 18% Protein Rodent Diet 2918C, ad libitum
- Water: filtered, ultraviolet light-irradiated tap water, ad libitum
- Acclimation period: 7 or 9 days

DETAILS OF FOOD AND WATER QUALITY:
The certificate of feed analysis was provided by the manufacturer, Envigo RMS, Inc. The results of feed analysis met the allowable standard of this facility. Samples of drinking water were 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): 21.0 - 23.3
- Humidity (%): 44.8 - 60.2
- 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 concentrations. The dosing formulations were prepared every day before dosing.

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

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. Samples were taken three times from the middle layer 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 100.90–109.40%. These results were within the acceptable limits (±15% of nominal values).
Duration of treatment / exposure:
Main groups:
males: 49 days (for 2 weeks prior to mating, during 2 weeks of mating and 21 days of post-mating)
females: starting 2 weeks prior to mating and continued through gestation and for 13 days after delivery

Recovery groups:
males and females: 49 days
Frequency of treatment:
once daily, 7 days/week
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
400 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:
The dose level was selected based on the results of a repeated dose 2-week dose range-finding study in Sprague-Dawley rats. In this study, salivation was observed in males and females at 500 mg/kg bw/day. Increases of the absolute and/or relative liver weights were noted in males and females at 500 mg/kg bw/day. Increases of the absolute and relative uterus weights were noted in females at 500 mg/kg bw/day. Therefore, the high dose level was selected at 400 mg/kg bw/day. Then, the mid and low dose levels were selected at 100 and 25 mg/kg bw/day, respectively.
The control group animals were 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: twice daily
- Cage side observations included: mortality, moribundity, clinical signs and general condition, as well as signs of abortion or premature delivery in dams during gestation

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once per week
- Observations included: evaluation of fur, skin, eyes, mucous membranes, occurrence of secretions and excretions, autonomic nervous system activity (lacrimation, piloerection, pupil size, and unusual respiratory pattern), changes in gait, posture, response to handling, clonic and tonic movements, stereotypical and bizarre behavior

BODY WEIGHT: Yes
- Time schedule for examinations: first day of dosing and thereafter once a week throughout the dosing period and the recovery period, on Gestation Days 0, 7, 14 and 20, on Postpartum Days 0, 4 and 13, on the day prior to necropsy and on the day of necropsy (fasted body weights)

FOOD CONSUMPTION: Yes
- Food consumption for each animal was determined prior to dosing on Day 0 (one day before 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. Food consumption was not recorded during mating. 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: at necrospy
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes, for 18 h
- How many animals: all animals
- Parameters checked: 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: at necropsy
- Animals fasted: Yes, for 18 h
- How many animals: all animals
- Parameters checked: 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, total cholesterol (T-Chol), triglyceride (TG), glucose (Glu), calcium (Ca), potassium (K), sodium (Na), chloride (Cl)

URINALYSIS: Yes
- Time schedule for collection of urine: two days before sacrifice
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes
- Parameters checked: fresh (3-h) urine: pH, protein, glucose, bilirubin, occult blood, color and turbidity, sediment; 24-h urine: urine volume, specific gravity

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: a few days before sacrifice, for dams on lactation days 6-11
- Dose groups that were examined: 6 males and 6 females, selected from each group and in all animals of the recovery group
- Battery of functions tested: pinna reflex, auditory reflex, corneal reflex, pupillary reflex, grip strength test (forelimb and hindlimb grip strength), motor activity

IMMUNOLOGY: No

OTHER:
Thyroid hormone analysis:
- Time schedule for collection of blood: dams: LD 13; male adults: at termination
- Anaesthetic used: Yes, isoflurane
- Animals fasted: Yes, for at least 18 h
- How many animals: all dams and all adult males
- Parameters checked: total thyroxine (T4) and total 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 (PPD) 14. All animals of the recovery groups were sacrificed two weeks after the final dosing. Non-delivered females were sacrificed on Gestation Day (GD) 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# (weight after fixation), adrenals#, kidneys#, ovaries#, uterus, epididymis#, prostate + seminal vesicle with coagulating gland

HISTOPATHOLOGY: Yes
All males and all females from the main groups in addition to all animals of the recovery groups were selected for tissue preservation. The testis and 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 tissues of the remaining organs were 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, urinary bladder, 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 or killed in a moribund condition during the study.
· All gross, macroscopic lesions
· Target organs noted at the high dose group were examined for at least the recovery group: stomach, mesenteric lymph node, 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, AGD index, nipple number, thyroid hormone value, urine volume, hematology and clinical chemistry parameters, organ weights, grip strength and 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 recovery group, Folded-F test was applied to analyze homogeneity of variance (significance level: 0.05, two-tailed). The 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).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Moribund or dead animals:
In the main group, one female was found moribund at 400 mg/kg bw/day on Day 14. In the main and recovery groups, two males and one female were found dead at 400 mg/kg bw/day on Day 40 and Day 39, respectively. When animals were found in a moribund state or before death occured, dirty nose, soft stool, soiled perineal region, decrease in stool, abdominal distention or irregular respiration were observed in females at 400 mg/kg bw/day.Since the test substance is corrosive to the skin, irregular respiration might be related to gavage-related reflux.
Salivation was observed in three animals at 400 mg/kg bw/day.

Surviving animals:
In the main group, salivation was observed in five males and seven females at 400 mg/kg bw/day starting on Day 5. In the recovery group, salivation was observed in five males and three females at 400 mg/kg bw/day. However, salivation was considered to have little toxicological significance since it was caused by physicochemical characteristics. Soiled perineal region or dirty nose was observed in two females at 400 mg/kg bw/day on PPD 1.

Detailed clinical signs:
At Week 2, one female showed a decrease of respiration rate. This animal was found dead on the next day after the examination. No clinical signs were observed in males of the main groups and in both sexes of the recovery group in the detailed examinations once a week.
Mortality:
mortality observed, treatment-related
Description (incidence):
In the main and recovery groups, two males and one female were found dead at 400 mg/kg bw/day on Day 40 and Day 39, respectively.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
In the main groups, statistically significant decreases in body weights were temporarily noted in females at 100 and 400 mg/kg bw/day on GD 7 and PPD 0, respectively. In the recovery group, statistically significant decreases in body weights were temporarily noted in males at 400 mg/kg bw/day on Day 8 and Day 14. However, these statistical significances in body weights had little toxicological meaning since they were temporary changes and of small difference.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
In the main groups, a statistically significant decrease or increase in food consumption were sporadically and/or temporarily noted in females at 100 and 400 mg/kg bw/day. In the recovery group, a statistically significant increase in food consumption was temporarily noted in females at 400 mg/kg bw/day on Day 63.
However, these statistical significances in food consumption had little toxicological meaning since they were temporary changes, of small difference and/or not related to any body weight development.
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 adverse effects were observed in any animal in the main and the recovery groups. Other statistical significances were considered not to be test substance-related changes because of small differences and the values were found to be within the range of historical reference data.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
In the main group, increases on Creatinin were noted in males at 400 mg/kg bw/day. High values of blood urea nitrogen (BUN) and creatinine (Crea) were noted in one moribund female and one surviving female at 400 mg/kg bw/day. However, since there were no suspicious findings in the kidneys during the histopathological examination, they were considered to be incidental and of no toxicological significance. No adverse effects were observed in any animal of the recovery group. Other statistical significances were considered not to be test substance-related changes because of a small difference and/or the values were within the range of historical reference data.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
In the main groups, occult blood and/or erythrocytes were observed in the urine in one male at 100 and 400 mg/kg bw/day, respectively. However, since there were no common findings of the kidney in the histopathological examination, they were considered to be incidental and of no toxicological significance. In the recovery group, urine volume was statistically significantly decreased in males at 400 mg/kg bw/day. However, it was considered to be of no toxicological relevance since it showed the same level as the control males of the main group.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related effects on auditory reflex, pinna reflex, pupillary reflex and corneal reflex test were observed in animals of both sexes in the main groups and recovery groups when compared to the control group. In the main and the 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 increase in vertical count of the spontaneous motor activity at 100 mg/kg bw/day. However, the statistical significance had little toxicological meaning because there was a small difference or temporary change and showed no dose-dependency. In recovery groups, there were no test substance-related effects in 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 organ weights of the liver were significantly increased in males and females at 400 mg/kg bw/day. Other statistical significances in the 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:
effects observed, treatment-related
Description (incidence and severity):
Moribund or dead animals:
Two males and two females in the high dose (400 mg/kg bw/day) group were found moribund or dead during the treatment period. Thickening/focus of the forestomach was noted in 2 females at 400 mg/kg bw/day. And the dead animals showed poor condition/stress-related gross observations such as adrenal enlargement, black area/red discoloration of the glandular stomach, and small thymus or spleen. The other macroscopic findings were incidental and not related to the test substance.

Surviving animals:
Thickening of the forestomach was noted in 2/12 males at 100 mg/kg bw/day and in 8/10 males and 7/11 females at 400 mg/kg bw/day. In addition, small thymus was noted in 2/11 females at 400 mg/kg bw/day.

At the end of the recovery period, the macroscopic examination at necropsy did not reveal treatment-related changes in the animals of the high-dose recovery group. In addition, no gross findings were noted in the two non-pregnant females and in the one dam whose pups were all dead. Small thymus was commonly noted limited to the dams whose pups were all dead. The other macroscopic findings were regarded as incidental and not related to the test substance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Moribund or dead animals:
The 2 males and 2 females at 400 mg/kg bw/day which did not survive until the scheduled necropsy, were found dead between Day 14 and 40. Minimal to slight diffuse hyperkeratosis and minimal to slight diffuse squamous cell hyperplasia in the forestomach were observed in the 3 animals found dead/moribund, and these findings were also noted during scheduled necropsy in animals of the main group. Slight or moderate submucosal hemorrhage/edema and multifocal necrosis/focal erosion of the forestomach were noted in one animal at 400 mg/kg bw/day. It was considered a test substance-related change and probably related to the observed deaths. These findings were limited to the forstomach.
In addition, slight to moderate mucosal necrosis of the trachea was noted in 2 animals at 400 mg/kg bw/day. There was no clear gavage error since no irritancy-related morphological changes in lungs or bronchi were observed. The occurrence of gavage-related reflux might be facilitated by the skin corosivity of the test formulation.
Minimal to slight centrilobular hepatocellular hypertrophy of the liver was noted in 2 animals and also noted in the scheduled necropsied main group.
And nearly all of the dead animals showed poor condition/stress-related gross lesions such as cortical hypertrophy of the adrenal gland, lymphoid atrophy of the spleen, atrophy or increased lymphocytic apoptosis of the thymus, hemorrhage of the glandular stomach, and a diffuse atrophy of hepatocytes of the liver.
All microscopic findings seen in other organs and tissues were considered to be incidental and of no toxicological significance.

Surving animals:
Test substance-related changes were observed in the liver, stomach and mesenteric lymph nodes.
- Liver:
Centrilobular hepatocellular hypertrophy was observed at minimal to slight severity in 4/6 males and at minimal severity in 4/6 females at 400 mg/kg bw/day. At the end of the recovery period, those findings, which were observed in the main group, were not observed in males and females at 400 mg/kg bw/day. Centrilobular hepatocellular hypertrophy was regarded as adaptive response to the test substance and it was considered not to be harmful to the animals since it was not accompanied by inflammation, degeneration or necrosis.
- Stomach:
Minimal to moderate diffuse hyperkeratosis and slight to moderate diffuse squamous cell hyperplasia were observed limited to the forestomach in males at 100 mg/kg bw/day and males and females at 400 mg/kg bw/day. At the end of the recovery period, those findings, which were observed in the main groups, were not observed in males and females of the high dose recovery group.
- Mesenteric lymph node:
Minimal to moderate erythrophagocytosis and minimal to slight diffuse lymphoid hyperplasia were observed in males and females at 400 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 at 400 mg/kg bw/day.
- Tymus:
Moderate to severe thymic atrophy was commonly noted limited to dams, whose pups were all dead, and it correlated with macroscopic finding of small thymus.
- Kidney:
There was no suspicious morphological change in the kidneys and no clear dose relationship increased pattern for animals with high values for BUN, Crea and high occult blood/erythrocytes in the urine at 400 mg/kg bw/day. Therefore, there was no toxicological significance.

No test substance-related histopathological findings were noted in the reproductive organs of either sex. The other microscopic findings seen in other organs and tissues were incidental and of no toxicological significance.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
There were no test substance-related adverse effects in total thyroxine (total T4) and total thyroid-stimulating hormone (total TSH) levels in adult males of the main and the recovery groups at 25, 100 and 400 mg/kg bw/day.
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
100 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 100 mg/kg bw/day
Key result
Dose descriptor:
LOAEL
Remarks:
systemic
Effect level:
400 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
gross pathology
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
local
Effect level:
25 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No adverse effects observed at 25 mg/kg bw/day
Key result
Dose descriptor:
LOAEL
Remarks:
local
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical signs
mortality
gross pathology
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
local
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No local adverse effects observed at 100 mg/kg bw/day
Key result
Dose descriptor:
LOAEL
Remarks:
local
Effect level:
400 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical signs
mortality
gross pathology
histopathology: non-neoplastic
Key result
Critical effects observed:
no

 Table 1. Incidence and Severity of Remarkable Microscopic Findings in the Stomach
Sex Male          
Main group Recovery group
Tissue Group / G1 G2 G3 G4 G1 G4
Dose Control Low Mid High Control High
(mg/kg/day) 0 25 100 400 0 400
Number examined 6 6 8 9 6 6
Stomach
Hyperkeratosis, diffuse, forestomach
Minimal 0 0 1 1 0 0
Slight 0 0 1 3 0 0
Moderate 0 0 0 5 0 0
Total Number of affected animals 0 0 2 9 0 0
Squamous cell hyperplasia, diffuse, forestomach
Slight 0 0 1 2 0 0
Moderate 0 0 0 7 0 0
Total Number of affected animals 0 0 1 9 0 0
Sex Female          
Main group Recovery group
Tissue Group / G1 G2 G3 G4 G1 G4
Dose Control Low Mid High Control High
(mg/kg/day) 0 25 100 400 0 400
Number examined 8 7 6 9 6 5
Stomach
Hyperkeratosis, diffuse, forestomach
Minimal 0 0 0 2 0 0
Slight 0 0 0 5 0 0
Moderate 0 0 0 1 0 0
Total Number of affected animals 0 0 0 8 0 0
Squamous cell hyperplasia, diffuse, forestomach
Slight 0 0 0 6 0 0
Moderate 0 0 0 1 0 0
Total Number of affected animals 0 0 0 7 0 0

Table 2. Incidence and Severity of Remarkable Microscopic Findings in the Mesenteric lymph node
Sex Male
Main group Recovery group
Number examined 6 6 6 6 6 6
Tissue G1 G2 G3 G4 G1 G4
Group / Dose (mg/kg/day) Control Low Mid High Control High 
0 25 100 400 0 400
Mesenteric lymph node
Erythrophagocytosis
Minimal 0 0 0 3 0 0
Slight 0 0 0 1 0 0
Moderate 0 0 0 1 0 0
Total Number of affected animals 0 0 0 5 0 0
Hyperplasia, lymphoid, diffuse
Minimal 0 0 0 1 0 0
Slight 0 0 0 1 0 0
Total Number of affected animals 0 0 0 2 0 0
Sex Female
Main group Recovery group
Tissue G1 G2 G3 G4 G1 G4
Group / Dose (mg/kg/day) Control Low Mid High Control High 
0 25 100 400 0 400
Number examined 6 6 6 6 6 5
Mesenteric lymph node
Erythrophagocytosis
Minimal 0 0 0 1 0 0
Moderate 0 0 0 1 0 0
Total Number of affected animals 0 0 0 2 0 0
Hyperplasia, lymphoid, diffuse
Minimal 0 0 0 1 0 0
Total Number of affected animals 0 0 0 1 0 0
Conclusions:
In conclusion, the NOAEL of the test substance for local toxicity was considered to be 25 mg/kg bw/day for males and 100 mg/kg/day for females, based on thickening of the forestomach with diffuse hyperkeratosis and squamous cell hyperplasia in males at 100 mg/kg bw/day and in females at 400 mg/kg bw/day. Additionally, erythrophagocytosis and diffuse lymphoid hyperplasia of mesenteric lymph nodes in both sexes as well as thymus atrophy in females were observed at 400 mg/kg bw/day. Thus, the NOAEL and LOAEL for systemic repeated dose toxicity were found to be 100 and 400 mg/kg bw/day respectively in both sexes. Since the adverse effects can mainly be attributed to local effects caused by the corrosive properties (Skin corr. 1B) of the test substance, no STOT-RE was assigned.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
Jul 29, 2016
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD (SD), SPF
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: males: 10 weeks; females: 12 weeks
- Weight at study initiation: males: 355.5 - 403.1 g; females: 228.1 - 285.7 g
- Housing: acclimation period: 2 animals per cage; mating: 1:1; dosing period: 1 animal; lactation: neonates were kept with the dam; animals were kept in stainless-steel cages (260W×350L×210H mm) and in poly sulfone cages (260W×420L×180 mm)
- Diet: Teklad Certified Irradiated Global 18% Protein Rodent Diet 2918C, ad libitum
- Water: filtered, ultraviolet light-irradiated tap water, ad libitum
- Acclimation period: 7 or 9 days

DETAILS OF FOOD AND WATER QUALITY:
The certificate of feed analysis was provided by the manufacturer, Envigo RMS, Inc. The results of feed analysis met the allowable standard of this facility. Samples of drinking water were 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): 21.0 - 23.3
- Humidity (%): 44.8 - 60.2
- 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 concentrations. The dosing formulations were prepared every day before dosing.

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
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: two weeks
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually
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. Samples were taken three times from the middle layer 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 100.90–109.40%. These results were within the acceptable limits (±15% of nominal values).
Duration of treatment / exposure:
Main groups:
males: 49 days (for 2 weeks prior to mating, during 2 weeks of mating and 21 days of post-mating)
females: starting 2 weeks prior to mating and continued through gestation and for 13 days after delivery

Recovery groups:
males and females: 49 days
Frequency of treatment:
once daily, 7 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
400 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:
The dose level was selected based on the results of a repeated dose 2-week dose range-finding study in Sprague-Dawley rats. In this study, salivation was observed in males and females at 500 mg/kg bw/day. Increases of the absolute and/or relative liver weights were noted in males and females at 500 mg/kg bw/day. Increases of the absolute and relative uterus weights were noted in females at 500 mg/kg bw/day. Therefore, the high dose level was selected at 400 mg/kg bw/day. Then, the mid and low dose levels were selected at 100 and 25 mg/kg bw/day, respectively.
The control group animals were 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: twice daily
- Cage side observations included: mortality, moribundity, clinical signs and general condition, as well as signs of abortion or premature delivery in dams during gestation

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once per week
- Observations included: evaluation of fur, skin, eyes, mucous membranes, occurrence of secretions and excretions, autonomic nervous system activity (lacrimation, piloerection, pupil size, and unusual respiratory pattern), changes in gait, posture, response to handling, clonic and tonic movements, stereotypical and bizarre behavior

BODY WEIGHT: Yes
- Time schedule for examinations: first day of dosing and thereafter once a week throughout the dosing period and the recovery period, on Gestation Days 0, 7, 14 and 20, on Postpartum Days 0, 4 and 13, on the day prior to necropsy and on the day of necropsy (fasted body weights)

FOOD CONSUMPTION: Yes
- Food consumption for each animal was determined prior to dosing on Day 0 (one day before 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. Food consumption was not recorded during mating. 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


OTHER: Haematology, clinical chemistry, thyroid hormone analysis, urinalysis, neurobehavioural examination (for details refer to IUCLID section 7.5.1 Repeated dose toxicity: oral)
Oestrous cyclicity (parental animals):
The estrous cycle examination for females of the main groups was conducted from dosing initiation day to the confirmed copulation day and necropsy day. Smears of vaginal mucosa were prepared daily in the morning. Prepared smears of the vaginal mucosa 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.
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 F0 male parental generation:
testis weight, epididymis weight, other: histopathology especially focused on spermatogenesis and interstitial testicular cell structure
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed 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

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

OTHER
Thyroid hormone analysis:
Blood samples for thyroid hormone analysis (total thyroxine and total thyroid stimulating hormone) were taken from at least two pups (preferably one male and one female) per litter on PND 4 and on PND 13, each, respectively. When the litter size was large enough, from pups which were available above the culling target of 8 pups/litter, and from at least two pups per litter on both PNDs 4 and 13, otherwise. When only one pup was available above the culling target, only one pup was used for the determination of PND 4. When litter size was below the culling target, blood collection was not carried out on PND 4. On PND 13, blood samples of all male and female pups were taken and pooled according to sex for analysis.
Postmortem examinations (parental animals):
SACRIFICE
Males of the main groups were sacrificed on Day 50, and females of the main groups were sacrificed on Postpartum Day (PPD) 14. All animals of the recovery groups were sacrificed two weeks after the final dosing. Non-delivered females were sacrificed on Gestation Day (GD) 26. Dams, whose pups were all dead, were sacrificed as soon as possible.

GROSS NECROPSY
Complete gross postmortem examinations were conducted on all animals including the external surface and internal organs. All grossly visible abnormalities were recorded.

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# (weight after fixation), adrenals#, kidneys#, ovaries#, uterus, epididymis#, prostate + seminal vesicle with coagulating gland

HISTOPATHOLOGY
All males and all females from the main groups in addition to all animals of the recovery groups were selected for tissue preservation. The testis and 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 tissues of the remaining organs were 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, urinary bladder, 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 or killed in a moribund condition during the study.
· All gross, macroscopic lesions
· Target organs noted at the high dose group were examined for at least the recovery group: stomach, mesenteric lymph node, liver.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed on PND 13
- All animals were subjected to postmortem examinations (macroscopic examination)

GROSS NECROPSY
Pups were carefully examined externally for gross abnormalities. Particular attention was paid to the external reproductive genitals which should be examined for signs of altered development.



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, AGD index, nipple number, thyroid hormone value, urine volume, hematology and clinical chemistry parameters, organ weights, grip strength and 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 recovery group, Folded-F test was applied to analyze homogeneity of variance (significance level: 0.05, two-tailed). The 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).
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 animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Moribund or dead animals:
In the main group, one female was found moribund at 400 mg/kg bw/day on Day 14. In the main and recovery groups, two males and one female were found dead at 400 mg/kg bw/day on Day 40 and Day 39, respectively. When animals were found in a moribund state or before death occured, dirty nose, soft stool, soiled perineal region, decrease in stool, abdominal distention or irregular respiration were observed in females at 400 mg/kg bw/day. Since the test substance is corrosive to the skin, irregular respiration might be related to gavage-related reflux.
Salivation was observed in three animals at 400 mg/kg bw/day.

Surviving animals:
In the main group, salivation was observed in five males and seven females at 400 mg/kg bw/day starting on Day 5. In the recovery group, salivation was observed in five males and three females at 400 mg/kg bw/day. However, salivation was considered to have little toxicological significance since it was caused by physicochemical characteristics. Soiled perineal region or dirty nose was observed in two females at 400 mg/kg bw/day on PPD 1.

Detailed clinical signs:
At Week 2, one female showed a decrease of respiration rate. This animal was found dead on the next day after the examination. No clinical signs were observed in males of the main groups and in both sexes of the recovery group in the detailed examinations once a week.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
In the main and recovery groups, two males and one female were found dead at 400 mg/kg bw/day on Day 40 and Day 39, respectively.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
In the main groups, statistically significant decreases in body weights were temporarily noted in females at 100 and 400 mg/kg bw/day on GD 7 and PPD 0, respectively. In the recovery group, statistically significant decreases in body weights were temporarily noted in males at 400 mg/kg bw/day on Day 8 and Day 14. However, these statistical significances in body weights had little toxicological meaning since they were temporary changes and of small difference.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
In the main groups, a statistically significant decrease or increase in food consumption were sporadically and/or temporarily noted in females at 100 and 400 mg/kg bw/day. In the recovery group, a statistically significant increase in food consumption was temporarily noted in females at 400 mg/kg bw/day on Day 63.
However, these statistical significances in food consumption had little toxicological meaning since they were temporary changes, of small difference and/or not related to any body weight development.
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 adverse effects were observed in any animal in the main and the recovery groups. Other statistical significances were considered not to be test substance-related changes because of small differences and the values were found to be within the range of historical reference data.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
In the main group, increases on Creatinin were noted in males at 400 mg/kg bw/day. High values of blood urea nitrogen (BUN) and creatinine (Crea) were noted in one moribund female and one surviving female at 400 mg/kg bw/day. However, since there were no suspicious findings in the kidneys during the histopathological examination, they were considered to be incidental and of no toxicological significance. No adverse effects were observed in any animal of the recovery group. Other statistical significances were considered not to be test substance-related changes because of a small difference and/or the values were within the range of historical reference data.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
In the main groups, occult blood and/or erythrocytes were observed in the urine in one male at 100 and 400 mg/kg bw/day, respectively. However, since there were no common findings of the kidney in the histopathological examination, they were considered to be incidental and of no toxicological significance. In the recovery group, urine volume was statistically significantly decreased in males at 400 mg/kg bw/day. However, it was considered to be of no toxicological relevance since it showed the same level as the control males of the main group.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related effects on auditory reflex, pinna reflex, pupillary reflex and corneal reflex test were observed in animals of both sexes in the main groups and recovery groups when compared to the control group. In the main and the 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 increase in vertical count of the spontaneous motor activity at 100 mg/kg bw/day. However, the statistical significance had little toxicological meaning because there was a small difference or temporary change and showed no dose-dependency. In recovery groups, there were no test substance-related effects in 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 organ weights of the liver were significantly increased in males and females at 400 mg/kg bw/day. Other statistical significances in the 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:
effects observed, treatment-related
Description (incidence and severity):
Moribund or dead animals:
Two males and two females in the high dose (400 mg/kg bw/day) group were found moribund or dead during the treatment period. Thickening/focus of the forestomach was noted in 2 females at 400 mg/kg bw/day. And the dead animals showed poor condition/stress-related gross observations such as adrenal enlargement, black area/red discoloration of the glandular stomach, and small thymus or spleen. The other macroscopic findings were incidental and not related to the test substance.

Surviving animals:
Thickening of the forestomach was noted in 2/12 males at 100 mg/kg bw/day and in 8/10 males and 7/11 females at 400 mg/kg bw/day. In addition, small thymus was noted in 2/11 females at 400 mg/kg bw/day.

At the end of the recovery period, the macroscopic examination at necropsy did not reveal treatment-related changes in the animals of the high-dose recovery group. In addition, no gross findings were noted in the two non-pregnant females and in the one dam whose pups were all dead. Small thymus was commonly noted limited to the dams whose pups were all dead. The other macroscopic findings were regarded as incidental and not related to the test substance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Moribund or dead animals:
The 2 males and 2 females at 400 mg/kg bw/day which did not survive until the scheduled necropsy, were found dead between Day 14 and 40. Minimal to slight diffuse hyperkeratosis and minimal to slight diffuse squamous cell hyperplasia in the forestomach were observed in the 3 animals found dead/moribund, and these findings were also noted during scheduled necropsy in animals of the main group. Slight or moderate submucosal hemorrhage/edema and multifocal necrosis/focal erosion of the forestomach were noted in one animal at 400 mg/kg bw/day. It was considered a test substance-related change and probably related to the observed deaths. These findings were limited to the forstomach.
In addition, slight to moderate mucosal necrosis of the trachea was noted in 2 animals at 400 mg/kg bw/day. There was no clear gavage error since no irritancy-related morphological changes in lungs or bronchi were observed. The occurrence of gavage-related reflux might be facilitated by the skin corosivity of the test formulation.
Minimal to slight centrilobular hepatocellular hypertrophy of the liver was noted in 2 animals and also noted in the scheduled necropsied main group.
And nearly all of the dead animals showed poor condition/stress-related gross lesions such as cortical hypertrophy of the adrenal gland, lymphoid atrophy of the spleen, atrophy or increased lymphocytic apoptosis of the thymus, hemorrhage of the glandular stomach, and a diffuse atrophy of hepatocytes of the liver.
All microscopic findings seen in other organs and tissues were considered to be incidental and of no toxicological significance.

Surving animals:
Test substance-related changes were observed in the liver, stomach and mesenteric lymph nodes.
- Liver:
Centrilobular hepatocellular hypertrophy was observed at minimal to slight severity in 4/6 males and at minimal severity in 4/6 females at 400 mg/kg bw/day. At the end of the recovery period, those findings, which were observed in the main group, were not observed in males and females of recovery group at 400 mg/kg bw/day. Centrilobular hepatocellular hypertrophy was regarded as adaptive response to the test substance and it was considered not to be harmful to the animals since it was not accompanied by inflammation, degeneration or necrosis.
- Stomach:
Minimal to moderate diffuse hyperkeratosis and slight to moderate diffuse squamous cell hyperplasia were observed limited to the forestomach in males at 100 mg/kg bw/day and males and females at 400 mg/kg bw/day. At the end of the recovery period, those findings, which were observed in the main groups, were not observed in males and females of the high dose recovery group.
- Mesenteric lymph node:
Minimal to moderate erythrophagocytosis and minimal to slight diffuse lymphoid hyperplasia were observed in males and females at 400 mg/kg bw/day. At the end of the recovery period, those findings, which were observed in the main group, were not observed in males and females of recovery group at 400 mg/kg bw/day.
- Tymus:
Moderate to severe thymic atrophy was commonly noted limited to dams, whose pups were all dead, and it correlated with the macroscopic finding of small thymus.
- Kidney:
There was no suspicious morphological change in the kidneys and no clear dose relationship increased pattern for animals with high values for BUN, Crea and high occult blood/erythrocytes in the urine at 400 mg/kg bw/day. Therefore, there was no toxicological significance.

No test substance-related histopathological findings were noted in the reproductive organs of either sex. The other microscopic findings seen in other organs and tissues were incidental and of no toxicological significance.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
There were no test substance-related adverse effects in total thyroxine (total T4) and total thyroid-stimulating hormone (total TSH) levels in adult males of the main and the recovery groups at 25, 100 and 400 mg/kg bw/day.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The estrous cycle lengths (day) of females in the 0 (control), 25, 100 and 400 mg/kg bw/day dosing groups were 4.0, 4.0, 4.0 and 4.3 days, respectively. There were no statistically significant differences in any dosing group. The estrous cycles of females were diestrus on the day of necropsy, except for the two non-pregnant females and the moribund female. The estrous cycles of the two non-pregnant females were metestrus and proestrus on the day of necropsy, respectively.
Reproductive function: sperm measures:
not examined
Description (incidence and severity):
In the control and the 25, 100 and 400 mg/kg bw/day dose groups, the gestation index was all 100%, the post-implantation loss rates were 2.8, 6.8, 4.3 and 19.6%, and the live birth indices were 97.2, 93.2, 95.7 and 80.4%, respectively. The rate on postimplantation loss was significantly increased and live birth index was significantly decreased at 400 mg/kg bw/day.
The total litter sizes were 16.1, 14.1, 15.8 and 14.7, the viability indices on postnatal day (PND) 0 were 100.0, 100.0, 97.7 and 89.5, and the viability indices on PND 4 were 97.4, 96.1, 98.8 and 68.9%, respectively. The decrease in viability on PND 4 was not statistically significant at 400 mg/kg bw/day, however it is of toxicological relevance since the differences were substantial.
In addition, normal delivery was observed in all animals of the control and 25, 100 and 400 mg/kg bw/day dose groups, and the sex ratios on PND 0 were 1.2, 0.8, 1.0 and 1.0, respectively.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No adverse effects observed at 100 mg/kg bw/day in females
Key result
Dose descriptor:
LOAEL
Remarks:
fertility
Effect level:
400 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:
fertility
Effect level:
400 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No adverse effects observed up to the highest dose tested
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
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
Key result
Dose descriptor:
LOAEL
Remarks:
systemic
Effect level:
400 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
gross pathology
histopathology: non-neoplastic

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:
mortality observed, treatment-related
Description (incidence and severity):
Live birth index was significantly decreased at 400 mg/kg bw/day.
The viability indices on postnatal day (PND) 0 were 100.0, 100.0, 97.7 and 89.5, and the viability indices on PND 4 were 97.4, 96.1, 98.8 and 68.9%, respectively. The decrease in viability on PND 4 was not statistically significant at 400 mg/kg bw/day, however it is of toxicological relevance since the differences were substantial.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
A significant increase in body weights of pups was temporarily noted in male pups at 25 mg/kg bw/day on PND 4. However, this statistical significance in body weights of pups was not considered to be an adverse effect since it was a temporary change and there was no dose dependency.
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 AGD index of pups at 0, 25, 100 and 400 mg/kg bw/day on PND 4.
The nipple number was 0 in the male pups at 0, 25, 100 and 400 mg/kg bw/day on PND 12. There was no nipple retention in the male pups at 0, 25, 100 and 400 mg/kg bw/day 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 changes in external findings in pups at 0 (control), 25, 100 and 400 mg/kg bw/day.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Thyroid hormone analysis:
There were no test substance-related adverse effects in total thyroxine (total T4) and thyroid-stimulating hormone (TSH) levels in F1 male and female pups on PND 13 at 25, 100 and 400 mg/kg bw/day.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
development
Generation:
F1
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
Key result
Dose descriptor:
LOAEL
Remarks:
development
Generation:
F1
Effect level:
400 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Table 1. Summary of delivery and pups data

  No. of implantations Post-implantation loss rate (%) Live birth index (%) PND 0 PND 4 Viability index (%) Sex ratio (male/female) Gestation index (%) Delivery (No. of abnormality)
Group /
Dose No. of pups born No. of live pups No. of live pups
(mg/kg/day)  
    Left Right Live Dead Total Male Female Male Female PND 0 PND 4
   
G1 Mean 8.1 8.5 2.8 97.2 16.1 0 16.1 8.6 7.5 8.4 7.3 100 97.4 1.2 100 0
0 S.D. 2.9 2.5 3.3 3.3 1.6 0 1.6 2.3 2.5 2.1 2.5 0 5.5 95 / 82
N 11 11 11 11 11 11 11 11 11 11 11 11 11 11
G2 Mean 7.1 7.8 6.8 93.2 14.1 0 14.1 6.2 7.9 5.8 7.6 100 96.1 0.8 100 0
25 S.D. 3.4 1.7 9.5 9.5 3.9 0 3.9 2.7 2.5 2.4 2.9 0 11.9 74 / 95
N 12 12 12 12 12 12 12 12 12 12 12 12 12 12
G3 Mean 7.3 8.8 4.3 95.7 15.4 0.3 15.8 7.8 7.7 7.7 7.6 97.7 98.8 1 100 0
100 S.D. 1.7 1.8 7.5 7.4 1.9 0.9 1.4 1.9 2.1 1.9 2.1 5.9 2.8 93 / 92
N 12 12 12 12 12 12 12 12 12 12 12 12 12 12
G4 Mean 8.4 8 19.6 ## 80.4 ## 13.2 1.5 14.7 6.6 6.6 5.3 4.7 89.5 68.9 1 100 0
400 S.D. 1.6 1.6 20.9 20.9 3.8 3.2 1.7 2.5 2.3 4.1 3.5 22 47.7 66 / 66
N 10 10 10 10 10 10 10 10 10 10 10 10 10 10

Significant difference from control by Steel's test: ## p<0.01          

PND : Postnatal day.  

Post-imp. loss rate (%) = ((No. of implantations-No. of live pups) / No. of implantations) × 100 ×100

Live birth index (%) = (No. of live pups on postnatal day 0 / No. of implantations) × 100 

Viability index on PND 0 (%) = (No. of pups born alive on PND 0 / Total No. of pups born) × 100

Viability index on PND 4 (%) = (No. of pups surviving on PND 4 / No. of pups born alive on PND 0)

Gestation index (%) = (No. of females with live pups / No. of pregnant females) × 100

Sex ratio = Total No. of live male pups / Total No. of live female pups (on PND 0)

Applicant's summary and conclusion

Conclusions:
In conclusion, the NOAEL for parental fertility was considered to be 400 mg/kg bw/day for males and 100 mg/kg bw/day for females, based on increased post-implantation loss rate and decreased live birth index. The NOAEL for developmental toxicity was considered to be 100 mg/kg bw/day for pups, based on decreased viability on post-natal day 4. These effects can presumably be attributed to secondary effects of parental toxicity at same dose level and thus no hazard on reproduction or development was identified.