Registration Dossier

Administrative data

Description of key information

Females:

Oral (OECD 422), rat: NOAEL systemic: 20 mg/kg bw/day, based on liver hepatocyte necrosis

Oral (OECD 422), rat: LOAEL neurobehavioral parameter: 1 mg/kg bw/day, based on decreased spontaneous motor activity

Males:

Oral (OECD 422), rat: LOAEL systemic: 20 mg/kg bw/day for male rat-specific alpha-2 -microglobulin-associated nephropathy; excluding this effect NOAEL was 100 mg/kg bw/day

Oral (OECD 422), rat: LOAEL neurobehavioral parameter: 20 mg/kg bw/day (based on no effect at highest dose tested)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

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:
28 Apr - 17 Aug 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted 22 Mar 1996
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: 9 weeks (males), 8 weeks (females)
- Weight at study initiation: 307.0 - 355.3 g (males), 191.1 - 229.6 g (females)
- Housing: Acclimation period: 2 animals per cage; Dosing period/ pre-mating: 1 animal per cage; Mating period: 1 male and 1 female; Lactation period: 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 420D x 180H mm)
- Diet: Teklad Certified Irradiated Global 18% Protein Rodent Diet 2918C (Envigo RMS, Inc., USA), ad libitum
- Water: public tap water filtered and irradiated by ultraviolet light, ad libitum
- Acclimation period: 4 days

DETAILS OF FOOD AND WATER QUALITY:
The results of feed and water analysis were confirmed to meet the allowable standard of the testing laboratory.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1 - 23.7
- Humidity (%): 46.4 - 59.7
- 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 and placed in a mortar. A small amount of vehicle, corn oil, was added and suspended. The vehicle was gradually added to yield the desired concentrations. The dosing formulations were stored in a refrigerator (4.1 – 7.1 °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 well suspended in it.
- Amount of vehicle: 5 mL/kg bw
- Lot/batch no.: MKBQ9948V, MKBL8756V, MKBS6944V
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, at Weeks 7 and 8 and analysed for verification of dose level concentration. The results of dose concentration analyses were determined to be 95.98 - 103.70% prior to dosing, 98.20 - 100.70% at Week 7 and 91.00% at Week 8. These results were within the acceptable limits (±15% of nominal values). As a result of homogeneity and stability analyses conducted in the study, the 2 and 200 mg/mL dosing solutions were confirmed to be homogenous and stable for 4 hours at room temperature and for 7 days under refrigeration.
Duration of treatment / exposure:
Main groups:
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 5 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
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
500 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 previously conducted 2-week repeated oral dose range finding study, a decrease or decreased tendency of body weight gain was noted at 100, 300 and 1000 mg/kg bw/day. Therefore, the high dose level was selected at 500 mg/kg bw/day. Then, the mid and low dose levels were selected at 100 and 20 mg/kg bw/day, respectively.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed for moribundity and mortality twice daily and for general condition and clinical signs once daily throughout the study. Females were also observed for signs of abortion and pre-mature birth.
- Cage side observations included: mortality/viability, clinical signs and general condition

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 period 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. Individual food consumption was calculated by subtracting the amount of residual feed from the amount presented.

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, for approx. 18 h
- How many animals: 6 males and 6 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, for approx. 18 h
- How many animals: 6 males and 6 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, total cholesterol (T-Chol), triglyceride (TG), glucose (Glu), calcium (Ca), potassium (K), sodium (Na), chloride (Cl)

URINALYSIS: Yes
- Time schedule for collection of urine: 6 males and 6 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 analysed.
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Animals were fasted during the fresh urine collection, but were allowed free access to drinking water.
- Parameters examined: in fresh urine samples: pH, protein, glucose, bilirubin, occult blood, color and turbidity, sediment; in 24-hour urine samples: urine volume, specific gravity

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Selected animals were examined a few days before necropsy.
- Dose groups that were examined: 6 males and 6 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 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 tissues 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

Besides, from all animals except for 6 males and 6 females 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 6 females from the control, low, mid and high dose groups (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
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).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Before females of the main group were found dead, clinical signs such as chromaturia, salivation, decrease in locomotor activity, decreased respiration, staining around mouth, nasal hemorrhage and/or irregular respiration were observed.
In surviving animals of the main group, chromaturia was observed in all males and females at 500 mg/kg bw/day from Day 2 to the end of dosing (Day 42), and hematuria was temporally observed in two males at 500 mg/kg bw/day on Day 3 and 4. Salivation was observed in five males from Days 1, 22–24 and two females on Day 1 and from GD7. Loss of fur was observed in one female of the control group from GD21.
In the recovery group, chromaturia was observed in all animals at 500 mg/kg bw/day from Day 2 until Day 45 and salivation was observed sporadically or frequently in three males and four females at 500 mg/kg bw/day during dosing period. Lacrimation was observed in two females at 500 mg/kg bw/day on Day 42. Chromaturia, hematuria and salivation were considered to be test substance-related effects, but these were recovered in animals of both sexes during a recovery period so these were considered to be reversible signs.

For detailed clinical signs, at Week 3, one male at 500 mg/kg bw/day showed mild salivation. At Week 5, one male at 500 mg/kg bw/day showed mild salivation. At Week 6, two females at 500 mg/kg bw/day showed mild lacrimation. At Week 6, one female at 100 mg/kg bw/day showed body posture (body position support impossible), grooming (no), color of skin (moderate/severe pale), color of oral mucus membrane (moderate/severe pale), respiration rate (decrease), spontaneous activity (stagger), abnormal gait (gait impossible), alertness (no activity), passivity (unresponsiveness if touch the forelimb), touch response (no reaction), pain response (no bite, vocalization), pupil size (severe/moderate ↓) and pupil reflex (no). This animal was found dead on the next day of the examination. During the recovery period, no clinical signs were observed in all animals of the control and high groups in the detailed clinical signs.
Mortality:
mortality observed, treatment-related
Description (incidence):
Two pregnant females at 20 mg/kg bw/day and one pregnant female at 500 mg/kg bw/day were found dead during parturition (gestation day (GD) 22 or 23). One female at 20 mg/kg bw/day was found dead on postpartum day (PPD) 3 and two females at 100 mg/kg bw/day were found dead on PPD1 or PPD3. One pregnant female at 500 mg/kg bw/day was found dead before parturition (GD23) and three females were found dead on PPD1, PPD2 or PPD3 (please refer to Table 1). All males of the main group and all animals of recovery group survived the duration of the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In males of the main group, body weights at 500 mg/kg bw/day were significantly lower than controls from Day 8 to the end of dosing (Day 42; about 8% at necrposy). In males of the recovery group, body weights at 500 mg/kg bw/day were significantly lower than controls from Day 8 to Day 56. There was no trend to recovery of body weights in males at 500 mg/kg bw/day. These changes were considered to be treatment related effects. In females of main group, there was a trend towards lower body weights at all dose levels between pre-mating period day 14 until GD20 which was statistically significant from controls at 100 and 500 mg/kg bw/day. There were no effects on body weight changes in females of recovery group. (please refer to Table 2, 3 and 4)
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In females of the main group, decreases in food consumptions at 20, 100 and 500 mg/kg bw/day were noted from Day 14 and GD1 (please refer to Table 5). Decreases in food consumption in females at 500 mg/kg bw/day of the main group during the dosing period were considered to be test substance-related effects since they were related to the lower value of body weights. There were no significant changes in food consumptions in males of all groups and females of the recovery group.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
In the main group, activated partial thromboplastin time (APTT) was significantly increased (p<0.01) in males at 500 mg/kg bw/day compared to the control group (please refer to Table 6). In the recovery group, no effects were noted in any of the animals at 500 mg/kg bw/day. The other statistical significances were not considered to be test substance-related changes since there were differences of small magnitude and they were within the range of historical reference data.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
In the main and recovery groups, no test substance-related effects were noted in blood chemistry examination in males or females of any dose group. The other statistical significances were not considered to be test substance-related changes since there were differences of small magnitude and they were within the range of historical reference data.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
In the main group, an increase in urine volume was noted in males at 500 mg/kg bw/day compared to the control group. No effects were noted in the other dosing groups. In the recovery group, no effects were noted in any of the animals at 500 mg/kg bw/day.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
In the main and recovery groups, there were no differences in pinna reflex, auditory reflex, corneal reflex and pupillary reflex test in both sexes at 20, 100 and 500 mg/kg bw/day when compared to the control group. In the main and recovery groups, there were no test substance-related effects in rotarod test and spontaneous motor activity test in both sexes at 20, 100 and 500 mg/kg bw/day when compared to the control group. In the main group, statistically significantly decreased hindlimb grip strength was noted in males at 500 mg/kg bw/day and in females at 20 and 500 mg/kg bw/day when compared to the control group. No effects were observed for forelimb grip strength in either sex at any dose level. No effects on hindlimb or forelimb grip strength were noted in the recovery animals in either sex.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
In the main group, relative organ weights of brain, liver, kidneys and testes in males and liver in females were significantly increased at 500 mg/kg bw/day compared to the control group. However, these changes were related to low body weights at 500 mg/kg bw/day compared to controls. Therefore, these changes were considered to have little toxicological significance. In the recovery group, relative organ weight of testes in males was significantly increased at 500 mg/kg bw/day compare to the control group. However, this change was within historical control ranges.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Unschedules death: Small thymus was observed in one female at 500 mg/kg bw/day. The other macroscopic findings were considered to be incidental.
Scheduled death: Test substance-related macroscopic changes were not observed in this study. All macroscopic findings were considered to be incidental and not related to the test substance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Unscheduled death:
Test substance-related changes were observed in the kidneys, liver, lung, thymus, spleen and submandibular lymph node. Tubular degeneration in the kidneys was observed at mild to marked severity in females treated at 20 (1/3) and 500 (1/5) mg/kg bw/day. An accumulation of hyaline droplets in the kidneys was observed in cortical tubules at mild severity in one female treated at 20 mg/kg bw/day (please refer to Table 7). Centrilobular necrosis of hepatocytes in the liver was observed at minimal severity in females treated at 20 (1/3) and from mild to marked severity in females treated at 100 (2/2) and 500 (4/5) mg/kg bw/day. Centrilobular vacuolation of hepatocytes in the liver was observed at minimal to mild severity in females treated at 100 (1/2) and 500 (4/5) mg/kg bw/day (please refer to Table 8). The vacuoles were mainly observed on the periphery of necrosis lesion. Inflammatory cell infiltration of alveolar septum in the lungs was observed at minimal to moderate severity in females treated at 20 (1/3), 100 (2/2), and 500 (5/5) mg/kg bw/day. This lesion was characterized by increased cellularity of inflammatory cells in alveolar septum without cellular or structural injury. Lymphoid atrophy in the thymus, spleen and submandibular lymph node (LN) was observed at minimal to marked severity in females treated at 20 (thymus: 3/3, spleen: 1/3, LN: 1/3), 100 (thymus: 2/2, spleen: 2/2, LN: 0/2) and/or 500 (thymus: 5/5, spleen: 4/5, LN: 3/5) mg/kg bw/day. It was not observed in surviving animals and considered to be a secondary effect to stress.
According to the study authores, the cause of death was not firmly established based on the absence of a common lesion in dead animals, but it is reasonable to assume that the test substance contributed significantly to the death considering the histopathological alterations.
All other microscopic findings seen in various organs and tissues were considered to be incidental or spontaneous and of no toxicological significance.

Scheduled death:
Test substance-related changes were observed in the kidneys and liver and these were considered to be target organs in this study.
An accumulation of hyaline droplets in the kidneys was evident in cortical tubules at minimal to moderate severity in males at 20, 100, and 500 mg/kg bw/day. Tubular degeneration was noted at moderate severity in one female treated at 100 mg/kg bw/day. At the end of the 2-week recovery period, these lesions were not observed in rats at 500 mg/kg bw/day (please refer to Table 7).
Centrilobular vacuolation of hepatocytes in the liver was noted at minimal to moderate severity in both sexes at 500 mg/kg bw/day. At the end of the 2-week recovery period, this lesion was not observed in animals of both sexes at 500 mg/kg bw/day (please refer to Table 8). All other microscopic findings seen in various organs and tissues were considered to be incidental or spontaneous and of no toxicological significance.
Histopathological findings: neoplastic:
no effects observed
Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no effect level for liver hepatocyte necrosis
Dose descriptor:
LOAEL
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Table 1. Number of unscheduled death during study

Sex

Male

Female

Dose (mg/kg bw/day)

0

20

100

500

0

20

100

500

Dead animals

0

0

0

0

0

3

2

5

Day of death

 

 

 

 

 

GD22,

GD22,

PPD3

PPD1,

PPD3

PPD1,

PPD2,

PPD3,

GD23,

PPD3,

GD23

GD: gestation day, PPD: postpartum day

Table 2. Male body weights of main group (g)

Males

 

Day of dosing

Dose (mg/kg bw/day)

1

8

14

21

28

35

42

0

 

326.0 ± 8.1

 

362.9 ± 13.3

 

390.6 ± 16.9

 

415.3 ± 16.7

 

438.9 ± 18.8

 

460.0 ± 20.8

 

472.8 ± 21.5

500

 

323.8 ± 7.6

 

342.2* ± 12.9

 

363.2* ± 20.2

 

382.3** ± 26.3

 

403.4* ± 29.2

 

424.6* ± 32.1

 

438.2* ± 32.1

Significantly different from control by Dunnett's t-test : * p<0.05

Table 3. Female body weights of main group (g)

 

Pre-mating period

Gestation period

Post-partum period

Dose (mg/kg bw/day)

1

8

14

0

7

14

20

0

4

0

212.0 ± 11.3

230.0 ± 12.1

249.5 ± 17.6

258.3 ± 13.4

300.0 ± 15.8

345.7 ± 19.2

437.1 ± 24.7

311.7 ± 30.0

329.5 ± 26.3

20

  206.7 ± 8.0

223.0 ± 7.0

235.7* ± 8.6

248.4 ± 10.3

286.9 ± 13.7

332.9 ± 19.1

417.4 ± 30.4

300.2 ± 31.3

325 ± 24.4

100

208.0 ± 8.2

221.4 ± 10.3

234.7* ± 12.7

245.6 ± 17.9

283.2* ± 21.6

324.4* ± 25.7

407.2 ± 36.0

293.6 ± 31.7

319.9 ± 27.7

500

213.2 ± 9.3

222.6 ± 8.6

236.6* ± 9.3

241.9## ± 7.0

275.6** ± 12.0

311.1** ± 12.0

380.5** ± 23.7

284.1 ± 21.4

303.3 ± 12.8

Significantly different from control by Dunnett's t-test : * p<0.05, ** p<0.01

Significantly different from control by Steel's t-test : ## p<0.01

 

Table 4. Body weights of recovery group (males and females; g)

 

Day of dosing

Day of recovery

 

1

8

14

21

28

35

42

49

56

Dose (mg/kg bw/day

 

Males

0

327.8 ± 14.0

365.7 ± 20.3

396.7 ± 23.1

424.6 ± 25.1

450.7 ± 30.2

475.4 ± 34.7

495.7 ± 37.7

513.1 ± 42.0

530.0 ± 43.5

500

328.9 ± 7.3

343.9* ± 9.9

367.7* ± 11.3

389.8* ± 14.0

412.2* ± 16.5

432.6* ± 16.5

447.2* ± 15.4

468.3# ± 13.8

480.8# ± 9.9

 

Females

0

200.9 ± 2.9

218.0 ± 5.8

226.7 ± 9.6

250.1 ± 12.1

257.2 ± 13.1

265.3 ± 14.3

275.2 ± 18.0

294.0 ± 25.3

299.6 ± 24.5

500

207.0* ± 5.9

217.8 ± 8.3

228.2 ± 10.6

238.8 ± 14.2

252.4 ± 15.8

262.8 ± 16.0

264.8 ± 15.5

282.8 ± 10.7

293.9 ± 10.3

Significantly different from control by Student t-test : * p<0.05

Significantly different from control by Aspin-Welch t-test : # p<0.05.

Table 5. Female food consumption of main groups

 

Pre-mating period

Gestation period

Post-partum period

Dose (mg/kg bw/day)

1

8

14

0

7

14

20

0

4

0

22.0 ± 4.4

21.4 ± 4.2

25.0 ± 3.2

26.5 ± 3.4

30.1 ± 4.8

30.9 ± 3.6

29.8 ± 4.6

13.7 ± 10.8

43.0 ± 9.4

20

18.4 ± 3.4

19.7 ± 2-4

19.9** ± 2.4

22.0** ± 1.9

28.0 ± 3.9

29.9 ± 3.1

29.6 ± 3.7

13.3 ± 6.7

45.0 ± 5.9

100

19.4 ± 4.4

19.3 ± 2.5

21.5* ± 3.9

21.2** ± 3.5

28.1 ± 4.8

29.4 ± 4.5

31.1 ± 4.9

17.2 ± 11.1

34.5 ± 7.7

500

22.5 ± 2.8

18.4 ± 2.5

21.6 ± 4.1

21.1** ± 2.1

25.0 ± 4.4

27.9 ± 3.5

30.4 ± 6.3

15.5 ± 9.7

35.1 ± 8.5

Significantly different from the control by Dunnett's t-test : * p<0.05, ** p<0.01.

Table 6. Haematological findings (main group)

Sex

Male

Female

Dose (mg/kg bw/day)

0

20

100

500

0

20

100

500

APTT (sec)

17.0 ± 1.4

16.7 ± 1.7

17.7 ± 2.2

23.8** ± 3.8

15.8 ± 2.7

14.2 ± 1.0

13.8 ± 2.8

16.6 ± 0.4

Significantly different from control by Dunnett's t-test : * p<0.05.

Table 7. Incidence and severity of remarkable microscopic findings in the kidney

Males

 

 

 

 

Dose (mg/kg bw/day)

0

20

100

500

Number examined

6

6

6

6

Accumulation, hyaline droplets, tubular, cortex

 

 

 

 

Minimal

0

3

0

1

Mild

0

0

3

1

Moderate

0

0

1

4

Total Number of affected

0

3

4

6

Females

 

 

 

 

Number examined

6

6

6

6

Tubular degeneration, cortex

 

 

 

 

Moderate

0

0

1

0

Total number of affected

0

0

1

0

Table 8. Incidence and severity of remarkable microscopic findings in the liver

Males

Dose (mg/kg bw/day)

0

20

100

500

Number examined

6

6

6

6

Centrilobular vacuolation, hepatocytes

 

 

 

 

 

Minimal

0

0

0

1

Total Number of affected

0

0

0

1

Females

Number examined

6

6

6

6

Tubular degeneration, cortex

 

 

 

 

Minimal

0

0

0

1

Mild

0

0

0

1

Moderate

0

0

0

1

Total number of affected

0

0

0

3

 

 

Conclusions:
Based on the results of this study, the LOAEL for systemic toxicity in males was considered to be 20 mg/kg bw/day (excluding male rat-specific, alpha-2-microglobulin-associated nephropathy the NOAEL was 100 mg/kg bw/day) and the NOAEL in female s was considered to be 20 mg/kg bw/day (for liver hepatocyte necrosis at higher doses)
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:
9 Aug - 6 Dec 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted 28 Jul 2015
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: 11 weeks (males), 12 weeks (females)
- Weight at study initiation: 381.5 - 452.5 g (males), 212.1 - 276.5 g (females)
- Housing: Acclimation and dosing period/ pre-mating: 1 animal per cage; Mating period: 1 male and 1 female per cage; Lactation period: 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 (Envigo RMS, Inc., USA), ad libitum
- Water: public tap water filtered and irradiated by ultraviolet light, ad libitum
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY:
The results of feed and water analysis met the allowable standard of the testing laboratory.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.6 - 23.2
- Humidity (%): 47.7- 66.8
- 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:
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 magnetic stirrer, and then vehicle was gradually added to yield the desired concentrations. The dosing formulations were stored in a refrigerator (3.9 – 5.3 °C). These dosing formulations were used within 7 days.
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 analysed for verification of dose level concentration. The results of dose concentration analyses were determined to be 91.20 - 94.20%. These results were within the acceptable limits (±15 % of nominal values). As a result of homogeneity and stability analyses by gas chromatography, the 0.02 and 4 mg/mL dosing solutions were confirmed to be homogenous and stable for 4 hours at room temperature and for 7 days under refrigeration.
Duration of treatment / exposure:
Main groups:
males: for 50 days, starting 2 weeks before mating, 2 weeks during mating and 22 days after mating
females: for 2 weeks prior to mating, throughout gestation and for 13 days after delivery. Females showing no evidence of parturition signs were dosed until Gestation Day 25.

Recovery groups:
Males and females of recovery groups were dosed once daily for 50 days. 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
Dose / conc.:
1 mg/kg bw/day (actual dose received)
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Dose / conc.:
20 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 selction rationale: In previously combined repeated dose toxicity study with the reproduction/developmental toxicity screening test, three females were found dead at 20 mg/kg bw/day during or after parturition. Therefore, the high dose level was selected at 20 mg/kg bw/day. Then, the mid and low dose levels were selected at 5 and 1 mg/kg bw/day, respectively.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed for moribundity and mortality twice daily and 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.
- Cage side observations included: mortality/viability, clinical signs and general condition

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed physical examinations for signs and symptoms of adverse effects, including:
· Skin, fur, eyes, mucous membranes, occurrence of secretion and excretion
· Autonomic activity (lacrimation, piloerection, pupil size, unusual respiratory pattern, etc.)
· Changes in gait, posture and response to handling, the presence of clonic or tonic movements
· Stereotypes (excessive grooming, repetitive circling, etc.) or bizarre behavior (self-mutilation, walking backward etc.)
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 both sexes of the recovery groups 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 prior 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 period, on Days 0, 7, 14 and 20 of gestation, on Days 0, 4 and 13 post-partum, the day prior to necropsy and on the day of necropsy. 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 both sexes of the recovery group were recorded just prior to dosing on Day 0 (one day before first dosing), once a week during the dosing and recovery periods (except during mating) and the day 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 period, on Days 0, 6, 13 and 19 of gestation, on Days 0, 3 and 12 post-partum and the day before necropsy. 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: 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, for approx. 18 h
- How many animals: 6 males and 6 females from the main study groups and 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, for approx. 18 h
- How many animals: 6 males and 6 females from the main study groups and 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, total cholesterol (T-Chol), triglyceride (TG), glucose (Glu), calcium (Ca), potassium (K), sodium (Na), chloride (Cl)

URINALYSIS: Yes
- Time schedule for collection of urine: 6 males were randomly selected from the main groups in addition to all male recovery animals for urinalysis two days before necropsy. Fresh, 3-hour and 24-hour urine samples were collected from the selected animals and analysed.
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Animals were fasted during the fresh urine collection, but were allowed free access to drinking water.
- Parameters examined: in fresh urine samples: pH, protein, glucose, bilirubin, occult blood, color and turbidity, sediment; in 24-hour urine samples: urine volume, specific gravity

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Selected animals were examined a few days before necropsy.
- Dose groups that were examined: 6 males and 6 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, motor activity

OTHER:
- Observation of estrous cycle: The estrous cycle for females of the main group was conducted from dosing initiation day to confirmed copulation day and necropsy day. Smears of vaginal mucosa were prepared in the morning daily. Prepared smears of 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.
- Thyroid hormone analysis: Fasted blood samples were taken from all adult males and dams of the main group at termination and analysed for T4 and TSH levels by immunoassays.
- Zinc measurement: From all pregnant females of the main group blood samples of jugular vein were collected on Gestation Day 20 and were analysed for zinc by ICP-MS or ICR-AES.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All males of the main group were sacrificed on Day 51 and females of the main group were sacrificed on Day 14 post-partum. All animals of the recovery group were sacrificed 2 weeks after final dosing. Non-pregnant females were sacrificed on Day 26 of gestation. Complete gross post-mortem examinations were conducted on all animals including the external 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. 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, thyroid, kidneys, adrenals, ovaries, uterus

HISTOPATHOLOGY: Yes
Tissue preservation and slide preservation
6 males and 6 females were randomly selected from the main groups in addition to all recovery animals for tissue preservation. The testes, epididymis and 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 tissues 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, testis, epididymis, prostate, seminal vesicle with coagulating gland, ovaries, uterus and cervix, submandibular lymph node, mesenteric lymph node, femur and sternum, spinal cord, sciatic nerve, eye and optic nerve, urinary bladder, gross lesions

Besides, from all animals except for 6 males and 6 females in the main group, the following organs and tissues were harvested and preserved: brain, pituitary, heart, thymus, liver, spleen, kidneys, adrenals, prostate, testis, seminal vesicle with coagulating gland, epididymis, ovaries, uterus and cervix, vagina

Histopathological examinations were conducted as follows:
- 6 males and 6 females from the control, low, mid and high dose groups (especially, focused on spermatogenesis and interstitial testicular cell structure)
- All tissues from animals found dead during the study
- All gross, macroscopic lesions
Statistics:
The statistical analysis of this study was conducted using the SAS program (SAS 9.3). 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, zinc value, thyroid hormone value, urine volume, hematology and clinical chemistry parameters, organ weights, 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 homogeneous data, if significant (significance level: 0.05), followed by Dunnett’s test for multiple comparisons (significance levels: 0.05 and 0.01, two-tailed). Kruskal-Wallis test was employed on heterogeneous data, 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 for homogeneity, 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):
In the main group, slight salivation was observed in two males at 20 mg/kg bw/day from Day 24 to the end of dosing. Also, slight salivation was observed in four females at 20 mg/kg bw/day from GD 5. Mass in the right chest was observed in one female on Postpartum Day 11. In the recovery group, slight salivation was observed in one female at 20 mg/kg bw/day from Day 22 to Day 35. Slight salivation was considered to be a test substance-related effect. Mass in the chest was considered to be incidental and spontaneous.
In moribund females, dystocia was observed. Slight salivation was observed in one female at 20 mg/kg bw/day since GD 8.

Detailed clinical signs:
No clinical signs were observed in males and females of the main and recovery groups in the detailed examinations once a week throughout the dosing and recovery periods.
Mortality:
mortality observed, treatment-related
Description (incidence):
One pregnant female at 1 mg/kg/day and three females at 20 mg/kg bw/day died during parturition (please refer to Table 1). All males of the main group and all animals of recovery group survived the duration of the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No test substance-related effects on the body weight changes were noted in both sexes at 1, 5 and 20 mg/kg bw/day in the main and recovery groups.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
A statistically significant decrease in food consumption was noted in females at 1 mg/kg bw/day in the main group on GD 14. A statistically significant increase in food consumption was noted in females at 1 and 5 mg/kg bw/day in the main group on postpartum day 4. A statistically significant decrease in food consumption was observed in males at 20 mg/kg bw/day in the recovery group on Day 64. However, these statistical significances were not considered to be test substance-related changes since the differences were of small magnitude and they were not related to the body weight changes.
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 recovery groups. Other statistical significances were considered by the study report authors not to be test substance-related changes because of small magnitude and the values were within the range of historical reference data.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No adverse effects were observed in any animal in the main and recovery groups. Other statistical significances were considered by the study report authors not to be test substance-related changes because of small magnitude and the values were within the range of historical reference data.
Urinalysis findings:
no effects observed
Description (incidence and severity):
In males of the main and recovery groups, no effects were noted in the test substance dosing groups.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
No test substance-related effects on auditory reflex, pinna reflex, pupillary reflex or corneal reflex were observed in animals of both sexes in the main and recovery groups when compared to the control group. In the main groups, in spontaneous motor activity, a statistically significant decrease in vertical count was noted in females at 1, 5 and 20 mg/kg bw/day when compared to the control group (please refer to Table 2). In the recovery groups, there were no test substance-related effects in the grip strength test and spontaneous motor activity of both sexes when compared to the control group. Other statistical significances were considered by the study report authors not to be test substance-related changes because of small magnitude and values.
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 relative organ weights of the thyroid were significantly increased in males at 20 mg/kg bw/day (control: 0.0277 g ± 0.0021 g; 20 mg/kg bw/day: 0.0344 g ± 0.0046 g)
Other statistical significances in the absolute and/or relative organ weights were considered by the study report authors 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):
Unscheduled death:
A total of 4 animals [1/12 females at 1 mg/kg bw/day, and 3/12 females at 20 mg/kg bw/day] were found dead during parturition. All animals commonly showed retained fetuses in the uterus at necropsy. And the most of dead animals showed poor condition/stress-related gross observations such as adrenal enlargement, black spot/focus of the stomach and/or hydrothorax of the thoracic cavity. Therefore, the study report authors considered those deaths to be attributable to dystocia.

Scheduled death:
Macroscopic examination at necropsy did not reveal treatment-related changes. The other macroscopic findings were considered by the study report authors to be incidental and not related to the test substance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Unscheduled death:
Slight to moderate uterine hemorrhage was commonly noted in all dead animals. In addition, minimal to slight increase of hematopoiesis in the bone marrow of the femur/sternum was noted in three dead animals. Those findings were closely related to the parturition process.
Minor microscopic findings observed in some dead animals were the followings:
- Thymus, stomach: slight to moderate thymic cortical lymphoid necrosis and/or minimal to slight thymic atrophy, slight mucosal necrosis of the glandular stomach (These non-specific findings were frequently noted in animals of poor condition/stress.)
- Adrenal, liver, lung: moderate adrenal necrosis, slight hepatic centrilobular necrosis, minimal pulmonary congestion (These findings were frequently noted in animals of poor condition/circulatory dysfunction.)
All microscopic findings seen in other organs and tissues were considered to be incidental and of no toxicological significance.

Scheduled death:
Treatment-related changes were not observed in this study. No test substance-related histopathological findings were noted in the reproductive organs of either sex. All microscopic findings seen in other organs and tissues were considered by the study report authors to be incidental and of no toxicological significance.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Estrous cycle:
The estrous cycle lengths (day) of females in the 0 (control), 1, 5 and 20 mg/kg bw/day dosing groups were 4.0, 4.6, 4.2 and 4.0 days, respectively. There was no statistically significant difference in any dosing group. The estrous cycle of females on the day of necropsy was all diestrus.

Zinc analysis:
Plasma zinc level was decreased at 1 mg/kg bw/day in pregnant females on GD 20. However, a decrease in plasma zinc level has little toxicological meanings since there was no dose-dependent response. Also, it was supposed that plasma zinc level had no relation with dystocia since dam with low zinc level showed normal parturition and zinc levels of dams with dystocia were within control values.

Thyroid hormone analysis:
In the main group, total thyroxine (T4) was statistically increased in F0 males at 5 and 20 mg/kg bw/day and in F0 females at 1 mg/kg bw/day. Thyroid stimulating hormone (TSH) was statistically increased in females at 5 mg/kg bw/day. However, these changes had little toxicological meanings since the values were within the range of historical control data.
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: no adverse effects observed at 20 mg/kg bw/day
Dose descriptor:
LOAEL
Remarks:
neurobehavioral toxicity
Effect level:
1 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
behaviour (functional findings)
Critical effects observed:
no

Table 1. Number of unscheduled death during study

Sex

Male

Female

Dose (mg/kg bw/day)

0

1

5

20

0

1

5

20

Dead animals

0

0

0

0

0

1

0

3

Day of death

 

 

 

 

 

GD23

 

GD23, GD22, GD23

GD: gestation day

Table 2. Spontaneous motoractivity (vertical count) in females of main groups

 

Vertical counts (minutes interval)

Dose (mg/kg bw/day)

0-10

10-20

20-30

30-40

40-50

50-60

Total

0

97 ± 29

47 ± 20

22 ± 12

29 ± 27

30 ± 18

46 ± 30

271 ± 120

1

65* ± 20

31 ± 13

25 ± 11

16 ± 10

8* ± 9

4# ± 4

149# ± 26

5

52 ** ± 15

24 ± 7

13 ± 7

8 ± 9

10* ± 8

7# ± 11

114# ± 34

20

30** ± 20

19* ± 22

15 ± 19

14 ± 14

8* ± 11

8# ± 7

94# ± 44

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

Significantly different from control by Steel test: # p<0.05.

Conclusions:
Based on the results of this study, the NOAEL for systemic toxicity was considered to be 20 mg/kg bw/day for males since no adverse effcts were observed up to the highest dose tested. The LOAEL for systemic toxicity in females was of 1 mg/kg bw/day based on decreased spontaneous motor activity.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
20 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available information comprises adequate and reliable studies (Klimisch score 1), and are thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.6, of Regulation (EC) No 1907/2006.
System:
hepatobiliary
Organ:
liver

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

OECD 422 - study 1 (2015)

The test substance was tested in a combined repeated dose oral toxicity study with the reproduction/developmental toxicity screening study according to OECD Guideline 422 and in compliance with GLP (2015). Twelve Sprague Dawley rats per sex and dose were treated via gavage with the test substance at concentrations of 20, 100 and 500 mg/kg bw/day, respectively. The control group received the vehicle corn oil. Additionally, a recovery group of 6 rats per sex was allocated to the control and high dose group. Males were treated for 6 weeks, starting 2 weeks before the mating period, during mating and 2 weeks after mating. Females were treated for 2 weeks prior to mating, throughout gestation and for 5 days after delivery. The doses were selected on the basis of a 2-week repeated oral dose range-finding toxicity study in which a decrease or decreased tendency of body weight gain was noted at 100, 300 and 1000 mg/kg bw/day.

All males of the main group and all animals of recovery group survived the duration of the study. Three females at 20 mg/kg bw/day, two females at 100 mg/kg bw/day, and five females at 500 mg/kg bw/day were dead in the main groups during the dosing period. These females died prior to, during or after parturition. Chromaturia, salivation and hematuria were observed at 500 mg/kg bw/day. However, these signs were reversible in the recovery groups during recovery period. In the detailed clinical signs, mild salivation or lacrimation was observed in some males or females at 500 mg/kg bw/day, respectively. Lower values in body weights were noted in males at 500 mg/kg bw/day of main and recovery groups and in females of the main groups. Decreases in food consumptions were noted in females of the main group at 500 mg/kg bw/day. Decreases in hindlimb grip strength were noted in both sexes at 500 mg/kg bw/day. The relatively low value in females at 20 mg/kg bw/day was not considered biologically relevant, since no significant hindlimb strength decrease was observed at a 5 -times higher dose (100 mg/kg bw/day) and no similar effect was seen in males (while comparable effects were found in both sexes at 500 mg/kg bw/day). Also the comparatively, likely incidental small standard deviation for the female 20 mg/kg bw/day dose group has probably contributed to a mathematical significance. An increase in activated partial thromboplastin time (APTT) was noted in males at 500 mg/kg bw/day. There were no test substance-related changes in blood chemistry and organ weights.

In dead females, small thymus was observed in one female at 500 mg/kg bw/day. In dead females, tubular degeneration or an accumulation of hyaline droplet in the kidneys was observed at 20 and 500 mg/kg bw/day. Centrilobular necrosis and/or centrilobular vacuolation of hepatocytes in the liver were observed at a mild to marked degree (with dose-related increase in incidence) at 100 and 500 mg/kg bw/day in females with unscheduled death. In females with scheduled necropsy, on one female of the low dose group, but none of the females of the mid or high dose group showed signs of liver damage. Inflammatory cell infiltration in the lungs and lymphoid atrophy in the thymus, spleen and submandibular lymph node were observed at 20, 100 and 500 mg/kg bw/day. In surviving animals, an accumulation of hyaline droplets in the kidneys was evident in cortical tubules in males at 20, 100 and 500 mg/kg bw/day. Tubular degeneration in the kidney cortex was noted in one female at 100 mg/kg bw/day. Centrilobular vacuolation of hepatocytes in the liver was noted in both sexes at 500 mg/kg bw/day. These lesions were not observed in the recovery groups. Therefore, for systemic toxicity the LOAEL in males and the NOAEL in females was considered to be 20 mg/kg bw/day.

 

OECD 422 - study 2 (2017)

Based on results of the first study, a second combined repeated dose oral toxicity study with the reproduction/developmental toxicity screening study according to OECD Guideline 422 and in compliance with GLP (2017) was conducted under same experimental conditions but with lower test substance concentrations of 1, 5 and 20 mg/kg bw/day. Males were treated for 50 days, starting 2 weeks before the mating period, 2 weeks during mating and 22 days after mating. Females were treated for 2 weeks prior to mating, until Postpartum Day 13. Additionally, males and females of the recovery groups were dosed once daily for 50 days.

All males of the main group and all animals of the recovery groups survived the duration of the study. One pregnant female at 1 mg/kg bw/day and 3 pregnant females at 20 mg/kg bw/day were dead in the main groups during parturition. During the dosing period, slight salivation was observed in some females at 20 mg/kg bw/day. In spontaneous motor activity, vertical count decreased dose-dependently in females at 1, 5 and 20 mg/kg bw/day in the main group. In the main group, the absolute and relative organ weights of the thyroid were increased in males at 20 mg/kg bw/day. However, there were no histopathological changes in the thyroid in both sexes in the test substance groups. In the dead females, adrenal enlargement, black spot/focus of the stomach and/or hydrothorax were observed. Microscopically, hemorrhage in the uterus, thymic cortical lymphoid necrosis, thymic atrophy, adrenal necrosis, hepatic centrilobular necrosis and/or pulmonary congestion were noted in dead females. These macroscopic and microscopic findings were frequently observed in animals of poor condition/stress. No test substance-related adverse effects were noted in the results of detailed clinical signs, body weights, food consumption, zinc level, total thyroxine (T4) and thyroid stimulating hormone (TSH), sensory function, hematology and clinical chemistry in adult animals of both sexes in the test substance-dosed groups. No test substance-related adverse effects were noted in the results of the estrous cycle.

Therefore, the NOAEL for systemic toxicity was considered to be ≥ 20 mg/kg bw/day for males and females. The NOAEL for neurobehavioral parameter was considered to be ≥ 20 mg/kg bw/day for males and for females a LOAEL of 1 mg/kg bw/day was determined based on decreased spontaneous motor activity. The finding of decreased spontaneous activity in females was considered not to have biological relevance because the effect was not observed in males, no other parameter of the functional observation battery showed any effect, no histological correlate was found, and in the 2015 OECD 422 study no changes in motor activity was observed at up much higher doses up to 500 mg/kg bw/day. In this study a decreased hindlimb strength was observed in both sexes only at 500 mg/kg bw/day.

 

In conclusion, based on available studies, the NOAEL for systemic toxicity was considered to be 20 mg/kg bw/day for females. For males a LOAEL of 20 mg/kg bw/day for systemic effects was established based on the kidney effects in the first study, although in the second study no longer adverse effects on the kidney were observed at 20 mg/kg bw/day. Since the kidney effects were caused by a male rat-specific alpha-2 -microglobulin-associated nephropathy, this effect was not considered relevant for human health risk assessment. Excluding this effect, the NOAEL for male rats was 100 mg/kg bw/day.

 

Justification for classification or non-classification

The available data on repeated oral dose toxicity of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.The adverse effects in kidney and liver histology were reversible in the recovery group and not accompanied by changes in clinical parameters or organ dysfunction. Moreover, the accumulation of hyaline droplets in males of all dose groups in the first study (2015) is most likely associated with alpha-2 -microglobulin, since no further effects were noted in the kidney of males and no accumulation of hyline droplets was noted in females selected for histopathology at the end of the study. Alpha-2 -microglobulin nephropathy is not relevant for humans.

Thus no STOT RE classification was considered.