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

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Males:

Oral (OECD 422), rat: NOAEL fertility ≥ 500 mg/kg bw/day

Females:

Oral (OECD 422), rat: BMDL10 (dystocia): 4.37 mg/kg bw/day

Link to relevant study records

Reference

Reference 1

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

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: 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

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

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.

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

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: 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.

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

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

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

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

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

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

Critical effects observed:

no

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

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

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.

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

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 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 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 or test system 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; 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: 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 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.

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 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

Details on study schedule:

not applicable for an OECD 422 study

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.

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 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

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

Oestrous cyclicity (parental animals):

- 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.

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:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 4 pups/sex/litter; excess pups were killed and discarded. When litters contained less than four pups of one sex, pups of the opposite sex were added to make a total of 8 pups.

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 gain (PND 0, 4 and 13), nipple retention (PND 12), thyroid homone analysis from at least two pups per litter on PND 4 and 13 (if number of pups is sufficient), anogenital distance on PND 4 (AGD)

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: Males of main group were sacrificed on Day 51.
- Maternal animals: Females of main group were sacrificed on Day 14 post-partum. Non-pregnant females were sacrificed on Gestation Day 26.
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 preservation. The testes and epididymis 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, testis, epididymis, prostate, ovaries, uterus, submandibular lymph node, mesenteric lymph node, bone marrow (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, epididymides, ovaries, uterus

Histopathological examinations were conducted as follows:
· Six males and six females from the control, low, mid and high groups (especially, focused on spermatogenesis and interstitial testicular cell structure)
· All tissues from animals found dead during the study.
· All gross, macroscopic lesions

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at postnatal Day 4 after external examination

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).

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
- 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

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.

Dermal irritation (if dermal study):

not examined

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. 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

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 by the study report authors 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:

effects observed, non-treatment-related

Description (incidence and severity):

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.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

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.

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

In the control and 1, 5 and 20 mg/kg bw/day dose groups, the mating periods were 1.8, 2.0, 3.3 and 1.8 days, the mating index was 100%, the gestation periods were 22.3, 22.1, 22.4 and 22.3 days, and the fertility indices of animals of both sexes were 100.0, 91.7, 100.0 and 100.0%, respectively. There was no statistically significant difference in the test substance dosing group.

Delivery
In the control and 1, 5 and 20 mg/kg bw/day dose groups, the post-implantation loss rates were 6.9, 2.5, 7.6 and 5.1%, the mean litter sizes were 14.2, 14.7, 14.3 and 14.7 and gestation indices were 100.0, 90.9, 100.0 and 75.0%, respectively. Numbers of abnormal delivery (dystocia) of those groups were 0, 1, 0 and 3 at 0, 1, 5
and 20 mg/kg bw/day, respectively.

Dose descriptor:

NOAEL

Remarks:

reproductive 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 on reproduction up to and including the highest dose tested

Dose descriptor:

BMDL10

Remarks:

reproductive toxicity

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive performance

Remarks on result:

other: BMDL was calculated as 1 case was seen in the low dose group, none in the mid dose group and 3 in the high dose group.

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

female reproductive system

Organ:

other: dystocia

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

In the control and 1, 5 and 20 mg/kg bw/day dose groups, live birth indices were 93.1, 97.5, 92.5 and 94.9%, the viability indices on Postnatal Day (PND) 0 were 100.0, 100.0, 100.0 and 98.4%, and the viability indices on PND Day 4 were 96.1, 98.7, 100.0 and 98.7%, respectively. In 0 (control), 1, 5 and 20 mg/kg bw/day dose groups, the sex ratios on PND 0 were 1.0, 0.9, 0.9 and 0.9, respectively.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no test substance-related changes in body weights of pups at 1, 5 and 20 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:

no effects observed

Description (incidence and severity):

There were no test substance-related changes in AGD index of pups at 1, 5 and 20 mg/kg bw/day. The nipple number was 0 in male pups at 1, 5 and 20 mg/kg bw/day on PND 12. There was no nipple retention in male pups at 1, 5 and 20 mg/kg bw/day on PND 12.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test substance-related changes in external findings of pups at 1, 5 and 20 mg/kg bw/day.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

- Thyroid hormone analysis: There were no test substance-related changes in thyroid hormone (T4) level of F1 pups at 1, 5 and 20 mg/kg/day.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

>= 20 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 offsping up to and including the highest dose tested

Critical effects observed:

no

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 in the absence of other toxic effects

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	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

Conclusions:

Based on the results of this study, the NOAEL for reproductive toxicity was considered to be 20 mg/kg bw/day for males since no effects occurred on fertility up to and including highest dose tested. Since mortality occurred in pregnant animals (dystocia) at 1 mg/kg bw/day and 20 mg/kg bw/day, but not at 5 mg/kg bw/day, a benchmark dose level (10% incidence for quantal data) was calculated. The lower 95% confidence level was 4.37 mg/kg bw/day. No adverse effects on development of offspring were observed up to and including 20 mg/kg bw/day, the highest dose tested.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

BMDL10

4.37 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, 8.7, of Regulation (EC) No 1907/2006.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Fertility

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 at least 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.

In dams, numbers of abnormal delivery were 0, 2, 0 and 1 at 0, 20, 100 and 500 mg/kg bw/day, respectively. Dystocia (death during parturition) was observed in two and one females at 20 and 500 mg/kg/day, respectively. No further effects on reproductive function (spermatogenesis) or performance (mating period, mating index, gestation period, male and female fertility indices, gestation index, pre- and post-implantation loss rates, mean litter size) were observed, compared with the control group. No test substance-related findings were observed on reproductive organs in males and females at any dose group.

Therefore, for parental fertility a LOAEL for female rats of 20 mg/kg bw/day due to death around delivery time, with a few cases of dystocia, in pregnant animals and a NOAEL for male rats of ≥ 500 mg/kg bw/day was derived.

 

OECD 422 - study 2 (2017)

Based on the 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.

In parental animals, 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.

In dams, numbers of abnormal delivery due to dystocia were 0, 1, 0 and 3 at 0, 1, 5 and 20 mg/kg bw/day, respectively. No further effects on reproductive function (spermatogenesis, estrous cycle) or performance (mating period, mating index, gestation period, male and female fertility indices, gestation index, post-implantation loss rates, mean litter size) were observed compared with the control group. No test substance-related findings were observed on reproductive organs in males and females at any dose group.

Since mortality occurred in pregnant animals (dystocia) at 1 mg/kg bw/day and 20 mg/kg bw/day, but not at 5 mg/kg bw/day, a benchmark dose level (10% incidence for quantal data) was calculated. The lower 95% confidence level was 4.37 mg/kg bw/day.

 

In conclusion, based on available studies, for parental fertility of female rats a BMDL10 of 4.37 mg/kg bw/day and for male rats a NOAEL of ≥ 500 mg/kg bw/day was derived.

The BMDL10 analysis has been conducted using study 2 (2017) where the lowest doses were used (1, 5, and 20 mg/kg bw/day). "Death" was the basis for the BMDL10 calculation with the BMDS Wizard provided by EPA. The Wizard uses different statistical models to estimate a BMDL10 (see attached BMDS Wizard Report for further details). The BMDL10 is defined as the 95% lower bound on the dose corresponding to a 10% decrease in the mean death of pregnant rats. A BMDL10 of 4.37 mg/kg bw/day was calculated with the Gamma Model as the most appropriate model out of 9 different models used in the BMDS Wizard.

Effects on developmental toxicity

Description of key information

Oral (OECD 422), rat: NOAEL developmental ≥ 500 mg/kg bw/day

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

500 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, 8.7, of Regulation (EC) No 1907/2006.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Developmental Toxicity

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 at least 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.

There were no adverse effects on pup development (including the number of dead and living pups, postnatal loss, live birth index, viability index, sex ratio and body weight) with treatment up to 500 mg/kg bw/day. Furthermore, external macroscopic examination did not reveal treatment-related findings.

Therefore, a NOAEL for developmental toxicity of ≥ 500 mg/kg bw/day was derived.

 

OECD 422 - study 2 (2017)

Based on the 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. There were no adverse effects on pup development (including the number of dead and living pups, postnatal loss, live birth index, viability index, sex ratio and body weight) with treatment up to 20 mg/kg bw/day. Furthermore, external macroscopic examination did not reveal treatment-related findings. Moreover in additional examinations, there were no test substance-related changes in the anogenital distance of pups, in the nipple retention in male pups and in the thyroid hormone (T4) level of pups. Therefore, a NOAEL for developmental toxicity of ≥ 20 mg/kg bw/day was derived.

 

In conclusion, based on available studies a NOAEL for developmental toxicity of ≥ 500 mg/kg bw/day was derived, since no adverse effects were observed in the development of F1 generation up to and including 500 mg/kg bw/day.

 

Justification for classification or non-classification

The available data on toxicity to reproduction of the test substance meet the criteria for classification as Repr. 2 (H361f) according to Regulation (EC) No 1272/2008 due to the death of dams during parturition (dystocia).