Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 Oct 2016 - 03 Mar 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted Jul 2016
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc.
- Age at study initiation: 11 weeks
- Weight at study initiation: Males: 377 - 454 g; Females: 222 - 286 g
- Fasting period before study:
- Housing: The animals were individually housed in metal bracket cages (300 W × 410 D × 200 H, mm) with wire mesh floors except for the mating period when 1 male and 1 female were housed together.
- Diet: Pellet feed, CRF-1 (by Oriental Yeast Co., Ltd; LOT No.: 160509, 160705, and 160804), ad libitum
- Water: Tap water, ad libitum
- Acclimation period: From the day of receipt to the day of group assignment, including the quarantine period

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 23
- Humidity (%): 40 - 58
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 18 Oct 2016 To: 03 Mar 2017

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance at the amount required for each dose was accurately weighed out into a mortar and pulverized with a pestle to mix with a small amount of the control substance (vehicle). When the suspension becomes fluidal, the suspension was transferred to a graduated glass using a syringe. The mortar was rinsed with the vehicle several times until the rinse appeared clear and the rinse was also transferred to the graduated glass. The content of the graduated glass was stirred with a stirrer to make a suspension. To the suspension, the vehicle was added to achieve the prescribed concentration, and the test substance was suspended in the vehicle.

VEHICLE
- Concentration in vehicle: 10, 30 and 100 mg test substance per mL
- Amount of vehicle: 10 mL/kg bw
- Lot/batch no.: V6B7838, V6H8492, and V6P9446
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: from the evening of the first day of mating until confirmation of copulation
- Proof of pregnancy: vaginal plugs in the vagina or fallen on the cage trays, or sperm in vaginal smears referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: Females were individually housed. For females with successful copulation, small trays with bedding for experimental animals (White flake, Charles River Laboratories Japan, Inc.) were used from gestation day 17 to lactation day 13.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dosing suspensions of all concentrations were analyzed for concentrations of the test substance at the first preparation and at the final preparation for males. The contents of the 10, 30, and 100 mg/mL dosing suspensions prepared at the first and final preparation were 93.7% to 100.3% (relative standard deviations 0.3% to 4.1%), which were within the range of acceptance criteria (content: 100% ± 10.0%, relative standard deviation, 5.0% or less).

The 10 and 100 mg/mL preparations were confirmed to be homogeneous and stable during 7 days (calculated from day 0 designated as the day of preparation) at room temperature. The stability test results were as follows: The contents at preparation were 100.0% and 99.8% (relative standard deviations 0.6% and 1.1%, respectively); and the remaining rates after storage were 98.7% and 93.7% (relative standard deviations 0.6% and 1.3%, respectively). These values were within the ranges of acceptance criteria (content and remaining rate 100% ± 10.0%, relative standard deviation 5.0% or less).
The homogeneity test results were as follows: the relative standard deviations of the 10 and 100 mg/mL preparations were 0.6% and 1.0%, respectively, which were within the range of acceptance criteria.
Duration of treatment / exposure:
(P) Males: for 35 days from 14 days before the start of mating
(P) Females: for 14 days before the start of mating and during mating period until successful copulation, thereafter during gestation period and until lactation day 13
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dosage levels were selected based on the results of an acute oral toxicity study of the same test substance in mice in which mice were dosed at 2500 and 5000 mg/kg bw/day and a 90-day oral (Dietary) toxicity study in rats with the test substance in which rats were dosed at 2500, 5000, 10000 and 30000 ppm. There was no evidence of toxicity noted at any dose level in both studies. Therefore, 1000 mg/kg bw/day was considered to be appropriate for the high dose in the current study. In addition, mid- and low-dose levels of 300 and 100 mg/kg bw/day, respectively were considered appropriate to assess the dose response of the test substance in the current study.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes, all males and females were observed for mortality, behaviour, and external appearance.
- Time schedule: Twice daily (before and after administration), and once on the day of necropsy

BODY WEIGHT: Yes
- Time schedule for examinations: For males, the body weight was recorded before administration and on administration days 1, 4, 7, 14, 21, 28, and 35, and on the day of necropsy. For females, before administration on administration days 1, 4, 7, and 14; before administration on gestation days 0, 7, 14, and 20; and before administration on lactation days 0, 4, 7, and 13; and on day 14 after parturition (day of necropsy). For the animals that were euthanized due to moribundity, body weight on the day of euthanasia was recorded.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, food consumption of all males was determined on the same days as body weight measurement, except the weeks during mating and the day of necropsy. For females, same as those for body weight measurement except cohabitation period and the day of necropsy. However, only remaining amount was measured and recorded on gestation day 20 and lactation day 13, and only amount supplied was measured and recorded on gestation day 0 and lactation day 0.
Estrous cyclicity (parental animals):
Before necropsy on the day of necropsy, vaginal smears were collected from all females and stained with Giemsa solution to determine the stage of estrous cycles under a light microscope.
Sperm parameters (parental animals):
Parameters examined in all male parental generations:
testis weight, epididymis weight, spermatogenesis and the structure of testicular interstitial cells
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, postnatal mortality, presence of gross anomalies, weight gain, physical abnormalities, anogenital distance (AGD), number of papillae in male pups
Postmortem examinations (parental animals):
SACRIFICE
Males were sacrificed on administration day 35, and maternal animals were sacrificed nn day 14 after parturition. All parental animals were observed for external appearance and blood was collected from the abdominal aorta under anesthesia with isoflurane. Animals were then euthanized by exsanguination and all organs and tissues were macroscopically observed. The organs and tissues described below were fixed and preserved in 10% neutral buffered formalin. The testes and epididymides were fixed in Bouin’s solution and preserved in 70% ethanol. For fixing the testes, the tunica albuginea were punctured at both poles. For bilateral organs, both sides of them were fixed and preserved.

GROSS NECROPSY
- Male animals: Thyroids, testes, epididymides, prostate, seminal vesicles (with coagulating glands), levator ani plus bulbocavernosus muscle complex, Cowper's gland, and glans penis.
- Female animals: Thyroids, ovaries, uterus including cervix, and macroscopic lesions (with border to normal tissue)

HISTOPATHOLOGY / ORGAN WEIGHTS
- Male animals: The organ weights of testes, epididymides, seminal vesicles (with coagulating glands), thyroids, prostate, levator ani plus bulbocavernosus muscle complex, Cowper's gland and glans penis were recorded for all males. The testes, epididymides, and thyroids of the control and high-dose males were investigated at the histopathological examination. The testes were examined for spermatogenesis and the structure of testicular interstitial cells.
- Female animals: The organ weights of thyroids, uterus including cervix and ovaries were recorded for all females. Ovaries of the control and high-dose females and organs with gross lesions found at necropsy were investigated at the histopathological examination.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at [#?] days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
All animals were observed for external appearance (including the oral cavity), and those for blood collection were euthanized by decapitation, and the others were euthanized by excessive dose of pentobarbital sodium. All organs and tissues were macroscopically observed. The thyroids of 1 male and 1 female per litter were fixed and preserved in 10% neutral buffered formalin.

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.

OTHER
Measurement of serum T4 concentration by LC/MS/MS was performed for all females and all males during blood collection at necropsy.
Statistics:
Statistical analyses were performed using the computer system (MiTOX). Group means and standard deviations were calculated for body weight, body weight gain, food consumption, absolute and relative organ weight, length of estrous cycle, number of estrus, number of days required for copulation, numbers of implantations, numbers of offspring delivered, and live newborns and dead newborns, birth index, gestation period, body weight of offspring on postnatal days 0 and 4, and viability index on postnatal day 4, and the incidence of offspring with external anomalies. These data were analyzed by the Bartlett test for homogeneity of variances, followed by the one-way analysis of variance when group variances were homogeneous (p≥0.05), or by the Kruskal-Wallis test when group variances were heterogeneous (p<0.05). When a significant difference was detected (p<0.1) by the one-way analysis of variance, Dunnett’s test was performed for comparison with the control group. When a significant difference was detected (p<0.1) by the Kruskal-Wallis test, Steel’s test was performed for comparison with the control group. For serum T4 concentration, viability index on postnatal days 0 and 13, body weight of offspring on postnatal days 7 and 13, number of papillae, and anogenital distance were separately analyzed by the same statistical procedures as described above using a statistical analysis system (Sanken System Co., Ltd.) and SAS system (SAS Institute Inc.).
The incidence of abnormal estrous cycles, copulation index, fertility index, gestation index, sex rate of live offspring, and incidence of dams with offspring showing external anomalies, and the results of histopathology were analyzed by Fisher's exact probability test using SAS system or MiTOX.
For the comparative analysis with the control group, statistical significance level was set at 5%.
Reproductive indices:
- Fertility Index female [%] = number of animals that impregnated a female or were pregnant/number of animals with successful copulation x 100
- Gestation Index [%] = number of females with normal parturition/number of pregnant females x 100
- Copulation index [%] = number of animals with successful copulation/number of animals used for mating x 100
Offspring viability indices:
- Birth index [%] = number of live offspring on postnatal day 0/number of implantations x 100
- Sex rate [%] = number of live male offspring/(number of live male offspring + number of live female offspring) x 100
- Incidence of offspring with external anomalies [%] = (number of live offspring with external anomalies/number of live offspring examined) × 100
- Incidence of dams with offspring showing external anomalies = number of dams having offspring with external anomalies / number of dams that delivered
- Viability index [%] on postnatal day 0 = (number of live offspring on postnatal day 0/number of offspring delivered x 100
- Viability index [%] on postnatal day 4 = (number of live offspring on postnatal day 4/number of live offspring on postnatal day 0 x 100
- Viability index [%] on postnatal day 13 = (number of live offspring on postnatal day 13/number of offspring selected on postnatal day 4 x 100

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Females:
In the control group, piloerection, tachypnea, hypothermia, exhaustion, and dystocia were noted in 1/12 females during parturition on gestation day 23. This animal was determined to have a poor prognosis and euthanized within the day.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Females:
1/12 females of the control group was euthanized on gestation day 23 due to a poor prognosis caused by clinical signs.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Males:
In the 1000 mg/kg bw/day group, body weight gain of males was significantly lower than that in the control group. This change was considered not to be toxicologically significant because it was slight in degree, and no changes were noted in body weight.
In the 100 mg/kg bw/day group, body weight gain was significantly low in males, which was considered unrelated to the test substance administration because no changes were noted in the 300 mg/kg bw/day group.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Males:
In the 100 mg/kg bw/day group, food consumption was significantly low in males on administration day 14, which was not dose-dependent and was considered unrelated to the test substance administration.
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Males:
In the 1000 mg/kg bw/day group, the relative weight of the levator ani plus bulbocavernosus muscle was significantly high in males; however, since no significant difference was found in the absolute weight, and no changes were noted in other reproductive organs, this was considered not to be toxicologically significant.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Females:
In the control group, necropsy revealed dark red discoloration of medulla in the kidney in 1/12 females, which was euthanized because of dystocia.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Males:
In the 1000 mg/kg bw/day group, slight ultimobranchial body in the thyroid was noted in 1/12 males, which was considered to be a spontaneous change because it was also noted in the control group. Other than this, slight degeneration/necrosis of round spermatid in stage I-VIII and atrophy of the seminiferous tubule in the testis were noted in 1/12 males each in the control group.
Females:
Histopathological examination at the gross lesions revealed moderate congestion, thrombus, and necrosis of the renal tubule in the kidney in 1/12 females, which was euthanized because of dystocia in the control group.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Serum T4 concentration:
In males or females, no significant differences were noted in any dose group.

Reproductive function / performance (P0)

Reproductive function: estrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
Normal parturition was observed in all animals except the one female with dystocia in the control group. No significant differences were noted in the number of implantations, gestation period, gestation index, number of offspring, number of live newborns, number of dead newborns, or sex rate of offspring in any dose group compared with the control group. The birth index was significantly high in the 1000 mg/kg bw/day group, which was considered unrelated to the test substance administration because what is toxicologically significant is a low value.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse and treatment-related effects observed in parental animals up to and including the highest tested dose level.
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive performance
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse and treatment-related effects on reproduction were observed in parental animals up to and including the highest tested dose level.

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
In the 1000 mg/kg bw/day group, body weight was significantly low in males and females on postnatal days 0 to 13.
In the 300 mg/kg bw/day group, body weight was significantly low in males on postnatal day 4. There were no significant differences in the body weight of offspring in the 100 mg/kg bw/day group compared with the control group.

The significantly low body weight of male offspring in the 300 mg/kg bw/day group was considered incidental because the value was within the range of historical control data according to the study director (mean: 10.5 g, range: 9.2 to 11.4 g). In the 1000 mg/kg bw/day group, the number of live offspring was larger and the gestation period was shorter than those in the control group. According to following analyses, these may be factors that caused significantly low body weight of male and female offspring on postnatal day 0 in this group. An analysis of covariance (using the number of live offspring in each litter as a covariate) revealed no significant differences in the mean body weight by sex on postnatal day 0 between the 1000 mg/kg bw/day group and the control group, in 5 litters in the control group and 11 litters in the 1000 mg/kg bw/day group with same conditions for the gestation period. Based on this result, the low body weight of offspring on postnatal day 0 in the 1000 mg/kg bw/day group was considered attributable to the large number of offspring and the short gestation period in this group, and not test substance-related.
As a result of the statistical analysis of the body weight gain calculated for postnatal days 0–4, 4–7, and 7–13 between the 1000 mg/kg group and the control group, significantly low values were detected on postnatal days 0-4 in male and female offspring in the 1000 mg/kg group; however, no significant differences were noted on postnatal days 4–7 and 7–13 (after standardization) in either male or female offspring. The body weight gain on postnatal days 0-4 in the 1000 mg/kg group (3.233 g in males, 2.898 g in females) was within the range of historical control data3) (mean 3.7 g, range 2.8 to 4.4 g in males; mean 3.5 g, range 2.9 to 4.4 g in females). These indicated that the low body weight gain on postnatal days 0–4 was associated with the large number of offspring. Accordingly, the low body weight on and after postnatal day 4 in the 1000 mg/kg group was not considered toxicologically significant because this was due to the low body weight on postnatal day 0 and large number of offspring in this group.
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
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Serum T4 concentration: No significant differences were noted in the serum T4 concentrations on postnatal day 4 or 13 between any dose group and the control group.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

No external anomalies were noted in any offspring.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse and treatment-related effects were observed in the offspring up to and including the highest tested dose level.

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
The test substance had no effect on reproductive performance.