4,4a,5,9b-tetrahydroindeno[1,2-d]-1,3-dioxin

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

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

Cross-reference

Reason / purpose for cross-reference:

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:

28 Apr - 17 Aug 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

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 or test system 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)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

adopted 22 Mar 1996

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4,4a,5,9b-tetrahydroindeno[1,2-d]-1,3-dioxin

EC Number:

241-997-4

EC Name:

4,4a,5,9b-tetrahydroindeno[1,2-d]-1,3-dioxin

Cas Number:

18096-62-3

Molecular formula:

C11H12O2

IUPAC Name:

2H,4H,4aH,5H,9bH-indeno[1,2-d][1,3]dioxine

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD), SPF

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 9 weeks (males), 8 weeks (females)
- Weight at study initiation: 244.8 - 281.6 g (males), 160.2 - 197.7 g (females)
- Housing: Acclimation period: 2 animals per cage; 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 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

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 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

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as Day 0 of gestation
- After successful mating each pregnant female was caged: individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses of the dosing formulations were conducted using gas chromatography and samples were taken three times from the middle of each dosing formulation prior to dosing, 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

Details on study schedule:

not applicable for an OECD 422 study

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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.

Examinations

Parental animals: Observations and examinations:

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

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

Oestrous cyclicity (parental animals):

not examined

Sperm parameters (parental animals):

Parameters examined in P male parental generations:
testis weight, epididymis weight, histopathology of testis and epididymis with focus on spermatogenesis and interstitial testicular cell structure

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
litter size, number and sex of pups, stillbirth, live birth, postnatal mortality, presence of gross anomalies, body weight

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals of main group were sacrificed 2 weeks after mating.
- Maternal animals: All surviving animals of main group were sacrificed at Day 6 post-partum. Non-pregnant females were sacrificed on Day 27 after the last day of mating.
All animals of the recovery group were sacrified 2 weeks after final dosing.

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

HISTOPATHOLOGY / ORGAN WEIGHTS
Organ Weights
Paired organs were weighed together. Animals were fasted overnight prior to necropsy and body weights were recorded on the day of necropsy. Organs were weighed and organ-to-body weight ratios were calculated. The testes and epididymides of all adult males were weighed. 6 males and 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
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 group (especially, focused on spermatogenesis and interstitial testicular cell structure)
- All tissues from animals found dead or killed in a moribund condition
- All gross, macroscopic lesions
- Target organs noted at the high dose were examined for the recovery group

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at postnatal Day 4

GROSS NECROPSY
- Gross necropsy consisted of external examinations.

HISTOPATHOLOGY / ORGAN WEIGTHS
not performed

Statistics:

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

Reproductive indices:

- Mating index (%) = (Number of females with confirmed mating / number of females placed with males) × 100
- Mating period = Day of mating confirmed – Day of initial mating (based on dosing day)
- Gestation period = Postpartum Day 0 – Gestation Day 0 (based on dosing day)
- Male fertility index (%) = (Number of males impregnating a female / number of males with confirmed mating) ×100
- Female fertility index (%) = (Number of pregnant females / number of females with confirmed mating) × 100
- Gestation index (%) = (Number of females with live pups / number of pregnant females) × 100
- Pre-implantation loss (%) = (No. of corpora lutea– No. of implantations) / No. of corpora lutea × 100
- Post-implantation loss (%) = (No. of implantations – No. of live pups) / No. of implantations × 100

Offspring viability indices:

- Live birth index (%) = (No. of live pups on postnatal Day 0 / No. of implantations) × 100
- Viability index on postnatal Day 0 (%) = (No. of pups born alive on postnatal Day 0 / total No. of pups born) × 100.
- Viability index on postnatal Day 4 (%) = (No. of pups surviving on postnatal Day 4 / No. of pups born alive on postnatal Day 0) × 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 the 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.

Dermal irritation (if dermal study):

not examined

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 (dystocia) 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 (please refer to Table 1). 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. 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 necropsy). 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.

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. 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. 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 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/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

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. Centrilobular necrosis of hepatocytes in the liver was observed at minimal to marked severity in females treated at 20 (1/3), 100 (2/2), and 500 (4/5) mg/kg bw/day. 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. 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.
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. 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

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Mating indices of 0 (control), 20, 100 and 500 mg/kg bw/day groups were 100.0, 100.0, 100.0 and 100.0%, respectively. Male and female fertility indices of those groups were 100.0, 100.0, 100.0 and 91.7%, respectively. Mating period of those groups were 2.4, 3.3, 3.6 and 1.9. There were statistically longer at 20 mg/kg bw/day compared to controls; however, this value was within in historical reference value. Gestation periods were 22.3, 22.1, 22.5 and 22.4, respectively. There were no significant differences in any dose group.

Delivery
Pre-implantation loss rates of 0 (control), 20, 100 and 500 mg/kg bw/day groups were 6.8, 6.2, 10.2 and 11.1% and post-implantation loss rates of those groups were 10.6, 11.2, 15.7 and 12.4%, respectively. In the same groups, mean litter sizes were 14.8, 13.2, 13.8 and 11.2. Gestation indices were 100.0, 100.0, 100.0 and 90.9%, respectively. There were no treatment-related changes at 20, 100 and 500 mg/kg bw/day. Numbers of abnormal delivery of those groups were 0, 2, 0 and 1, respectively. Dystocia (death during parturition) was observed for two and one dams at 20 and 500 mg/kg bw/day, respectively.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

>= 500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

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

2

Dose descriptor:

LOAEL

Remarks:

reproductive toxicity

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

mortality

reproductive performance

Target system / organ toxicity (P0)

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

female reproductive system

Organ:

other: death around delivery time, few cases of dystocia

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

Live birth indices of 0 (control), 20, 100 and 500 mg/kg bw/day groups were 89.4, 88.8, 84.4 and 87.6%, respectively. While viability indices on postnatal day (PND) 0 were 96.4, 100.0, 91.2 and 94.6%, the viability indices on PND 4 was 90.7, 97.9, 84.8 and 98.5%, respectively. In 0 (control), 20, 100 and 500 mg/kg bw/day dose groups, sex ratios were 1.4, 1.1, 0.9 and 1.1, respectively.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no treatment-related changes in body weight changes of pups at 20, 100 and 500 mg/kg bw/day.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test substance-related changes in external findings of pups at 20, 100 and 500 mg/kg bw/day.

Histopathological findings:

not examined

Other effects:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

>= 500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

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

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to other toxic effects:

reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

Any other information on results incl. tables

Table 1. Number of 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

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, the NOAEL for reproductive toxicity was considered to be 500 mg/kg bw/day for males since no effects occurred on fertility up to and including highest dose tested. For females, LOAEL was considered to be 20 mg/kg bw/day, since mortality in pregnant animals occurred around delivery time (dystocia in some dams) at 20 mg/kg bw/day. No adverse effects on development of offspring were observed up to and including 500 mg/kg bw/day, the highest dose tested.