SUMILIZER GP

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 May - 29 Oct 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

adopted in 2016

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

442-450-4

EC Name:

-

Cas Number:

203255-81-6

Molecular formula:

C42H61O4P

IUPAC Name:

6-(3-(3-tert-Butyl-4-hydroxy-5-methylphenyl)propoxy)-2,4,8,1 0-tetra-tert-butyldibenz(d,f)(1,3,2)dioxaphosphepin

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc.
- Age at study initiation: 9 weeks
- Weight at study initiation: 270 - 360 g (males) and 180 - 240 g (females)
- Housing: Animals were housed in groups of 2/sex in Metal bracket-type cages (300 W x 410 D x 200 H, mm) with wire mesh floors during quarantine and acclimatisation and individually after group assignment. During the mating period, males and females were cohabitated on a 1:1 basis. Thereafter, females were housed individually during gestation and one liter/cage was housed during lactation. White flakes (Charles River Laboratories Japan, Inc.) were used as bedding.
- Diet: Pelleted diet, CRF-1 (Oriental Yeast Co., Ltd.), ad libitum. The diet of the lots used was analysed for contaminants and the results were confirmed to be within an acceptable range.
- Water: Tap water, supplied from an automatic watering system or water containers, ad libitum. Samples of drinking water were collected twice during the study and analysed for contaminants. The resutls were confirmed to be within an acceptable range.
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 (actual range: 21 - 25 °C)
- Humidity (%): 50 ± 20 (actual range: 43 - 58%)
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/ 12 (8:00 am - 8:00 pm)

IN-LIFE DATES: From: 27 May 2020 To: 05 Aug 2020

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: methyl cellulose

Remarks:

(MC), 0.5% aqueous solution

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dose formulations were prepared 9 times during the study at intervals of 6 - 7 days. The amount of the test article required for each dose was accurately weighed out into an agate mortar and pulverized with an agate pestle to mix with a small amount of the vehicle. When the suspension became 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 volume, and the mixture was stirred to make homogeneous suspension.

VEHICLE
- Justification for use and choice of vehicle: Because the test article is insoluble in water, 0.5% aqueous methylcellulose (MC) was chosen as vehicle.
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Amount of vehicle: 10 mL/kg bw

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: Mating pairs were cohabited continuously from the evening of the first day of mating until confirmation of copulation.
- Proof of pregnancy: Successful copulation was confirmed by the presence of vaginal plugs in the vagina or fallen on the cage trays, or sperm in vaginal smears. The day was designated as gestation Day 0.
- After successful mating each pregnant female was caged individually.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity and stability of the 1 and 100 mg/mL dosing suspensions were assayed in a previous study. The results showed homogeneity of the test article in the dosing suspensions and their stability for 8 days after preparation at room temperature in an airtight brown glass container. At the first preparation, the 10 and 100 mg/mL dosing suspensions were analysed for homogeneity of the test article. Samples were collected from the upper, middle and bottom part of the formulation. The results showed that the relative standard deviations (RSDs) were 2.0% and 1.1% in the 10 and 100 mg/mL dosing suspensions, respectively, which met the acceptance criteria (RSD, 5.0% or less).
Dosing suspensions of all concentrations were analysed for concentrations of the test article at the first preparation and at the final preparation for males. The results showed that the contents and RSDs of the 10, 30, and 100 mg/mL dosing suspensions ranged from 94.0 - 102.3% and from 0.0 - 2.0%, respectively, which met the acceptance criteria (content, 100 ± 10.0%; RSD, 5.0% or less).

Duration of treatment / exposure:

Males were treated for 28 days, starting 14 days prior to mating. Females were treated starting 14 days prior to mating and during the mating period until successful copulation. Females with successful copulation were mated during gestation through Day 13 after parturition (lactation Day 13). Females with no evidence of parturition until Day 25 after successful copulation (1/12 females of the control group and 1/12 females of the high dose group) were treated until gestation Day 25.

Frequency of treatment:

daily, 7 days/week

Details on study schedule:

not applicable for an OECD 421 study.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

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: Dose levels were chosed based on the results of a preliminary 90-day oral repeated dose toxicity study in rats, in which 16 or 26 each of males and females were administered dose levels of 0, 100, 300 and 1000 mg/kg bw/day.
Dose levels of 300 mg/kg bw/day an above induced an increase in the relative liver weight in females. At 1000 mg/kg bw/day, an increase in absolute and relative liver weight was also noted for males, as well as an increase in absolute kidney weight in males. Females of the 1000 mg/kg bw/day group further showed increased cholesterol levels in clinical chemistry. There were no abnormal findings in males and females of the 100 mg/kg bw/day dose group. Based on these findings, the highest dose level of the present study was chosen to be 1000 mg/kg bw/day, and 300 and 100 mg/kg bw/day were selected as the middle and low dose level.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily (before and after dosing) during the administration period, and once on the day of necropsy.
- Cage side observations included observations for mortality and clinical signs of toxicity including the external appearance, behaviour etc.. Any abnormalities found were recorded with the times of onset and duration.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight of males was recorded before dosing and on administration Days 1, 4, 7, 14, 21 and 28 and on the day of necropsy. Body weight of females was recorded before dosing, on administration Days 1, 4, 7 and 14, before dosing on gestation Days 0, 7, 14 and 20, and before dosing on lactation Days 0, 4, 7, 13 and on Day 14 after parturition at scheduled necropsy. For the 2 females with no evidence of parturition, body weight on Day 26 after successfull copulation was recorded (day of necropsy for these animals).

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/rat/day: Yes. Measurements for food consumption were made on the same days as body weight measurements, except the weeks during mating and the day of necropsy.

WATER CONSUMPTION: No

THYROID HORMONE ANALYSIS:
- Time schedule for collection of blood: Blood was collected in the morning on the day of necropsy from the abdominal aorta from all parental males.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: No
- How many animals: all parental males of all groups.
- Parameters examined: T4 serum levels

Oestrous cyclicity (parental animals):

Estrous cycles were examined for all parental females from the first day of administration to the day of successful copulation. Vaginal smears were stained with Giemsa solution and examined with a light microscope to determine the stage of estrous cycle. Females showing repetition of a 4-, or 5-day estrous cycle (proestrus, estrus, metestrus, and diestrus) were determined to be normal. During 14 days from administration Days 1 to 14, length of estrous cycle, number of estrus, and the number of animals with abnormal estrous cycle were calculated.

Sperm parameters (parental animals):

Tissues collected from all animals in the control and high dose groups were examined microscopically for histopathological changes, including the qualitative assessment of spermatogenesis and the structure of interstitital testicular cells in the testis.

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). Offspring not selected (> 8 pups per litter) were euthanized by decapitation for those for blood collection (2 pups/litter) or by an intraperitoneal injection of a 60 mg/mL pentobarbital sodium solution for the others.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, body weight gain on PND 0, 4, 7 and 13, clinical signs, anogenital distance (AGD) on PND 4, presence of nipples/areolae in male pups on Day 13

GROSS EXAMINATION OF DEAD PUPS:
Dead pups were observed for external appearance (including the oral cavity) and all organs and tissues were macroscopically examined. The thyroid of 1 male and 1 female per litter was fixed and preserved in 10% neutral buffered formalin.

Postmortem examinations (parental animals):

SACRIFICE
All animals were observed for external appearance and blood was collected from the abdominal aorta under anesthesia with isoflurane. The animals were then euthanised by exsanguination.
- Male animals: All surviving animals were sacrificed following the day after 28 days of administration.
- Maternal animals: All surviving animals were sacrificed on Day 14 after parturition. Two females with no evidence of parturition were sacrificed on Day 26 after successful copulation.

GROSS NECROPSY
All animals were subjected to a post mortem examination at which all organs and tissues were macroscopically observed. For females, the number of implantation sites was counted. From all animals of all dose groups, the organs and tissues listed 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 was punctured at both poles. For bilateral organs, both sides of them were fixed and preserved.
Thyroids, testes, epididymides, prostate, seminal vesicles (with coagulating glands), levator ani plus bulbocavernosus muscle complex, Cowper's gland, glans penis, liver and kidneys in males; thyroids, ovaries, uterus including cervix, liver and kidneys in females.

ORGAN WEIGHTS
The following organs were weighed for all male animals at necropsy: Testes, epididymides, seminal vesicles (with coagulating glands) and prostate.

HISTOPATHOLOGY
Microscopic examinations were confined to the animals of the control and high dose groups. Organs and tissues were embedded in paraffin way, thin-sectioned and stained with hematoxylin and eosin prior to examination. For males, testes and epididymides were histopathological examined. The testes were examined for spermatogenesis and the structure of testicular interstitial cells. For females, the ovaries were histopathologically examined.

Postmortem examinations (offspring):

SACRIFICE:
On PND 4, the surplus pups (> 8 per litter) were euthanized by decapitation. From two surplus pups per litter, blood was colleced, if possible. All remaining pups were sacrificed on PND 13 by intraperitoneal inection of a 60 mg/mL pentobarbital sodium solution.

THYROID HORMONE ANALYSIS:
- Time schedule for collection of blood: Blood was collected on PND 4 (from the offspring not selected by adjustment of the number of offspring) and PND 13.
- Anaesthetic used for blood collection: No.
- How many animals: F1: two surplus pups per litter (if possible) on PND 4 and two pups per litter on PND 13
- Parameters examined: T4

GROSS NECROPSY:
All pups were observed for external appearance (including the oral cavity) and all organs and tissues were macroscopically examined. The thyroid of 1 male and 1 female per litter was fixed and preserved in 10% neutral buffered formalin.

HISTOPATHOLOGY / ORGAN WEIGTHS: not performed

Statistics:

Group means and standard deviations were calculated for body weight, body weight gain, food consumption, absolute & relative organ weight, serum T4 level, estrous cycle, number of estrus, days required for copulation, numbers of implantations, offspring delivered, live and dead newborns, delivery index, gestation length, viability indices of offspring, and the incidence of offspring with external anomalies. The data were analysed 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. When a significant difference was detected (p < 0.1) by the Kruskal-Wallis test, Steel’s test was performed.
Body weight and AGD of offspring were analysed by a linear mixed model with litter as a random effect. For body weight of offspring, litter size was used as a covariate, and on PND 0, gestation length was also used as a covariate. The number of nipples was analysed by a generalized linear mixed model with litter as a random effect. For body weight, AGD, and the number of nipples of offspring, Dunnett’s test was performed for comparison with the control group.
The incidence of abnormal estrous cycles, copulation index, fertility index, gestation index, sex ratio of live offspring, and incidence of dams with offspring showing external anomalies, and the results of histopathology were analysed by Fisher’s exact probability test.
For non-pregnant animals, body weight, body weight gain, food consumption during gestation and histopathological findings were excluded from evaluation. Statistically significance level was set at 5% for comparative analysis with the control group. All statistical tests were two-tailed.

Reproductive indices:

- Copulation index [%] = (Number of animals with successful copulation/Number of animals used for mating) x 100
- Fertility index [%] = (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
- Gestation length: Number of days from gestation Day 0 to lactation Day 0.

Offspring viability indices:

Delivery index [%] = (Number of offspring delivered/Number of implantations) x 100
Sex ratio [%] = (Number of live male offspring/Number if live male and female offspring) x 100
Incidence of offspring with external anomalities [%] = (Number of live offspring with external anomalities/Number of live offspring examined) x 100
Incidence of dams with offspring showing external anomalities = Number of dams having offspring with external anomalities/Number of dams that delivered
- Viability index [%] on PND 0 = (Number of live offspring on PND 0/Number of offspring delivered) x 100
- Viability index [%] on PND 4 = (Number of live offspring on PND 4/Number of offspring delivered) x 100
- Viability index [%] on PND 13 = (Number of live offspring on PND 13/Number of offspring delivered) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

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:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

In the epididymis, spermatic granuloma was observed in 1/12 males (mild in degree) in the 1000 mg/kg bw group. The findings was not attributed to treatment with the test item due to the absence of statistical significance and because the finding was reported as spontaneous observation in animals of this age and strain.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Thyroid hormone T4 levels in males were not affected by treatment.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

1/12 females in the 300 mg/kg bw group showed diestrus continuously for 13 days, which was determined to be an abnormal estrous cycle (persistent diestrus). The finding was considered to be incidental.

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Effect levels (P0)

1

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 effects observed

2

Key result

Dose descriptor:

NOAEL

Remarks:

fertility

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 effects observed

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:

mortality observed, non-treatment-related

Description (incidence and severity):

Dead or missing (possible canibalised by maternal rats) were sporadically observed in all test groups including the control group from PND 0 - 4. These occurred at low incidence and no significant differences were noted in the viability indices on postnatal days 0 and 4 between any test article-treated group and the control group. No deaths occurred from PND 5 - 13.
The corresponding viability indices were 99.46 ± 1.78%, 98.29 ± 2.93% and 100 ± 0.00% for the control group on PND 0, 4 and 13, 95.18 ± 13.37%, 98.93 ± 2.51% and 100 ± 0.0% at 100 mg/kg bw/day on PND 0, 4 and 13, 97.93 ± 4.08%, 94.85 ± 15.99% and 100 ± 0.00% at 300 mg/kg bw/day on PND 0, 4 and 13 and 100.0 ± 0.00%, 98.87 ± 2.51% and 100.0 ± 0.00% at 1000 mg/kg bw/day on PND 0, 4 and 13.

Body weight and weight changes:

no effects observed

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

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopical abnormalities were noted in any offspring found dead on PND 0 - 13 and no abnormal findings were noted in any surviving pup sacrificed at scheduled necropsy on PND 13.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Thyroid hormone T4 levels in the pups were not affected by treatment.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

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 effects observed

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:

Administration of test item doses up to 1000 mg/kg bw/day had no effect on fertility and development. The NOAEL for fertility and for development were both derived to be 1000 mg/kg bw/day.