Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Crl:CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc. (Hino Breeding Center)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Males and females 7 weeks old on arrival and acclimated for 19 days in total.
- Weight at study initiation: Males 357.7 to 413.1 g and females 220.9 to 269.2 g
- Fasting period before study: No
- Housing: For males and for females except the gestation and lactation periods stainless hanging-type bracket cages were used. For females during the gestation and lactation periods polymethylpentene cages were used
- Diet (e.g. ad libitum): Pellet diet for experimental animals provided ad libitum. The diet was routinely analysed.
- Water (e.g. ad libitum): Well water provided ad libitum. The water was routinely analysed.
- Acclimation period: 19 days in total
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.6 to 24.9°C
- Humidity (%): 41.7 to 74.8%
- Air changes (per hr): 10 to 20 times per hour
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

The dosing formulations were prepared once every 7 days. The vehicle (corn oil) was used as the dosing formulation for the control group. The required amount of the vehicle was divided into clear glass vials for each dosing day. The appropriate amount of the test substance was accurately weighed, the vehicle added and stirred with a magnetic stirrer to mix. The substance preparation was transferred to a measuring cylinder and made up to volume with the vehicle to prepare dosing formulations. These were divided into brown glass vials for each dosing day.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Standard vehicle
- Concentration in vehicle: 0, 1, 3 or 10 mg/mL
- Amount of vehicle (if gavage): 5 mL/Kg
Details on mating procedure:
- M/F ratio per cage: 1 male 1 female
- Length of cohabitation: Until successful evidence of copulation
- Proof of pregnancy: The vaginal smears were collected in the morning from the day after the initiation of mating, and copulation was confirmed by the presence of the vaginal plug or sperm in the smear sample. The day of successful copulation was regarded as GD 0.
- After successful mating each pregnant female was caged (how): Individually
- Any other deviations from standard protocol: None
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The determination of the test substance concentration in dosing formulations and the confirmation of homogeniety were analytically verified using GC-FID. The method had been previously established and validated.
Duration of treatment / exposure:
Males: From 14 days before mating (Days 1 to 15) until the day before necropsy through the mating period (35 days in total).
Females: From 14 days before mating (Days 1 to 15) until Day 12 of lactation (day of delivery was Day 0 of lactation) through the mating and gestation periods and delivery. Non-delivery females were maintained until the day before necropsy.
Frequency of treatment:
Daily via oral gavage
Details on study schedule:
Not applicable
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
Control
Dose / conc.:
5 mg/kg bw/day
Remarks:
Low dose
Dose / conc.:
15 mg/kg bw/day
Remarks:
Middle dose
Dose / conc.:
50 mg/kg bw/day
Remarks:
High dose
No. of animals per sex per dose:
12 males and 12 females per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on the results of a preceeding dose range finding study
- Rationale for animal assignment (if not random): On the day before the start of administration for both sexes, 7 females not showing regular 4-day estrous cycles were excluded from the group assignment on the basis of the results of estrous cycle examination. The other animals were assigned to each group by the stratified randomization on the basis of the body weights. The animals weighing within ± 20% of the mean body weights (calculated for each sex) were used for this study.
- Fasting period before blood sampling for clinical biochemistry: Not applicable
Positive control:
Not a requirement of the test guideline

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed twice a day (before and after dosing) during the administration period, and once a day in other periods.

DETAILED CLINICAL OBSERVATIONS: NO

BODY WEIGHT: Yes
- Time schedule for examinations: Males were weighed on Days 1, 8, 15, 22, 29, and 36 (necropsy day). Females were weighed on Days 1, 8, and 15 during the pre-mating period, GDs 0, 7, 14, and 20 during the gestation period, and LDs 0, 4, 7, and 13 during the lactation period.

FOOD CONSUMPTION:
- Food consumption: Males were weighed on Days 1, 7, 14, and 35. Females were weighed on Days 1, 7 and 14 during the pre-mating period, GDs 0, 6, 13, and 19 during the gestation period, and LDs 0, 3, 6, and 12 during the lactation period. Feeders containing diet were weighed and set in the animal cages in the morning. On the following morning, the feeders were weighed to calculate the food consumption for each day. The day of measurement of the set diet was designated as the day of measurement of food consumption.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
Oestrous cyclicity (parental animals):
Vaginal smears were collected with a swab from all females in the morning (approximately same time every day) from the day of the start of dosing to the day of
confirmed copulation. The obtained smears were collected on a plate for each animal, and stained with Giemsa. The estrous cycle was classified into diestrus (D), proestrus (P), estrus (E), and metestrus (M). The mean estrous cycle (number of days from the estrous period to the next estrous period) and the number of the estrous period during the test period were calculated.
Sperm parameters (parental animals):
Not examined
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes
- On PND 4, the litter size was standardized to 8 (4/sex/litter) by random removal of F1 offspring. Even when the number of either males or females per litter was less than 4, the litter size was adjusted to 8 regardless of the sex ratio. The offspring culled at the litter size adjustment (offspring excluded from blood collection) were euthanized by overdose with sodium pentobarbital (0.05 to 0.1 mL/body with undiluted solution) via an intraperitoneal injection and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD and presence of nipples/areolae in male pups.

GROSS EXAMINATION OF DEAD PUPS:
Stillborn on PND 0 and offspring at the time of death were also observed for external anomalies.

HORMONE CONCENTRATION ANALYSIS: Yes for total T4 analysis. Only males were subjected to the parental animal measurement. The offspring were subjected to the measurement on PND 13 only. No measurements were conducted on PND 4 in any offspring.

Postmortem examinations (parental animals):
SACRIFICE
- Male animals: Necropsy was conducted on the day following the final dosing (Day 36 for males).
- Maternal animals: Necropsy was conducted on the day following the final dosing (LD 13 for females).

GROSS NECROPSY
- Vaginal smears were collected from females in the morning on LD 13 (necropsy day), and the stage of the estrous cycle was examined with a microscope. The animals of both sexes were euthanized by exsanguination from the abdominal aorta after blood sampling, and the thoracoabdominal organs/tissues were examined macroscopically. The non-deliver females (Nos. 964, 968, 984, and 989) were necropsied on GD 24. Those animals were anesthetized by intraperitoneal injection of sodium pentobarbital (30 mg/kg) and euthanized by exsanguination from the abdominal aorta. Then the thoracoabdominal organs/tissues were examined macroscopically.

HISTOPATHOLOGY / ORGAN WEIGHTS
Organ weights: Thyroid (including parathyroid), testis, epididymis, seminal vesicle (including dorsolateral and coagulating gland), prostate (ventral), levator anibulbocavernosus muscle (LABC), cowper's gland, glans penis, ovary and the uterus.
Histopathology: Testis, epididmyis, ovary stomach and teeth (maxillary incisor). The stomach and tooth were additionally collected and examined because macroscopic abnormalities were observed in these organs in the high dose group at necropsy and effects attributable to the test substance treatment were suspected.
Postmortem examinations (offspring):
SACRIFICE
- The necropsy of F1 animals was performed on PND 13.

GROSS NECROPSY
- The offspring subjected to blood sampling were necropsied after blood sampling by decapitation after anesthetizing by inhalation of isoflurane. The other animals were euthanized by exsanguination from the lateral iliac artery under anesthesia with sodium pentobarbital (0.1 to 0.5 mL/body with undiluted solution,) via the intraperitoneal injection and necropsied. The thoracoabdominal organs/tissues were examined macroscopically. Furthermore, the thyroid of 1 male and 1 female (animals subjected to blood sampling for hormone level measurement) per litter were collected, fixed, and preserved in 10 vol% phosphate buffered formalin.

HISTOPATHOLOGY / ORGAN WEIGTHS
No offspring organs were weighed and no histopathology undertaken.
Statistics:
Statistical analysis was performed for the following data, except for the sex ratio, using a computer system. SAS 9.4 was used for the sex ratio. In any case, two-tailed test was used, and levels of p<0.01 and p<0.05 were judged significant. Statistical analysis of hormone concentration (total T4) was performed at the test site. The data of offspring were analyzed on the basis of litter mean values. Moreover, the body weight and food consumption after confirmation of copulation of non-pregnant females were excluded from the evaluation.

Multiple comparison test: The mean and standard deviation were calculated and homogeneity of variance was tested by Bartlett’s method (p<0.05). When the groups were accepted as homogeneous, Dunnett’s multiple comparison test was used for comparison of the groups of data. When the groups of data were found to be heterogeneous by Bartlett’s test, Steel’s multiple comparison test was conducted. Items: Body weights, food consumption, absolute and relative organ weights, estrous cycle, number of estrus, days until copulation, gestation length, number of implantations, number of delivered offspring, number of live offspring, body weight of offspring (both sexes), and AGD.

Chi-square test: For non-parametric data such as incidences, chi-square test was performed between the control and FEC-treated groups. Items: Copulation index, fertility index, gestation index, and sex ratio

Wilcoxon's rank sum test: For non-parametric data such FEC-treated groups, Wilcoxon’s rank sum test was performed between the control and FEC-treated groups. Items: Stillborn index, external anomaly index, external anomaly typing index, delivery index, birth index, viability index on Day 4, viability index on Day 13, and nipple development anomaly index.
Reproductive indices:
- Number of days requied for successful copulation
- Copulation index (%)
- Fertility index (%)
- Gestation length
- Gestation index (%)
- Delivery index (%)
Offspring viability indices:
- Birth index (%)
- Stillborn index (%)
- Viability index on day 4 (%)
- Viability index on day 13 (%)
- Sex ratio
- External anomaly index (%)
- External anomaly typing index (%)
- External anomaly index (%)
- External anomaly typing index (%)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No abnormal clinical signs were observed in either sex of any group throughout the administration period, including the gestation and lactation periods of pregnant females.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No death occurred during the examination period.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
In males, no statistically significant difference was observed between the control group and any treated group. In females, although there was no statistical significant difference, body weight was decreased from LDs 0 to 7 (-1.3% to the value on LD 0) at 50 mg/kg.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In males, food consumption was significantly low at 50 mg/kg compared with the control group on Day 35. In females, food consumption was significantly low at 50 mg/kg compared with the control group on LDs 3 to 12. Food consumption was significantly high at 5 and 50 mg/kg compared with the control group on Day 14 and significantly low at 15 mg/kg compared with the control group on LD 3; however, these changes were not judged to be treatment-related because it was transient and no changes were observed in body weight.
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, treatment-related
Description (incidence and severity):
Animals surviving to scheduled necropsy: In males, irregular arrangement of ameloblast in the maxillary incisor was observed in 1 and 10 animals at 15 and 50 mg/kg, respectively. In the maxillary incisor, in addition, degeneration of ameloblast and irregular arrangement of odontoblast were observed in 1 to 2 animals at 50 mg/kg. In the forestomach, hyperplasia of squamous cell was observed in 3 and 12 animals at 15 and 50 mg/kg, respectively, and ulcer and focal infiltrate of inflammatory cell were observed in 3 to 7 animals at 50 mg/kg. Degeneration/atrophy of seminiferous tubule in the testis and cell debris in the lumen of the epididymis were observed in 2 and 1 animals of the control group, respectively.

In females, in the maxillary incisor, degeneration of ameloblast, irregular arrangement of ameloblast, and irregular arrangement of odontoblast were observed in 1, 11, and 1 animals at 50 mg/kg, respectively. In the forestomach, ulcer, hyperplasia of squamous cell, and focal infiltrate of inflammatory cell were observed in 1, 11, and 2 animals at 50 mg/kg, respectively.

Non-pregnant and females that failed to deliver: One female at 50 mg/kg with non-pregnant showed hyperplasia of squamous cell in the forestomach.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Hormone (T4) analysis: The plasma total T4 concentration of treated groups (Groups low-, middle-, and high-dose) was equivalent to that of the control group in parental animals P0, male. There was no notable change in the plasma total T4 concentration attributable to the test substance.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
No treatment-related changes were observed in the count of estrus or estrous cycle in any treated group.
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
Fertlity: No statistically significant differences were observed in the days until copulation, copulation index, or fertility index between the control group and any treated groups.

One female (No. 968) at 5 mg/kg, 1 female (No. 984) at 15 mg/kg, and 1 female (No. 989) at 50 mg/kg did not become pregnant. Accordingly, the copulation indices
were 100% for the control and all treated groups, and the fertility indices were 100%, 91.7%, 91.7%, and 91.7% for the control, 5, 15, and 50 mg/kg groups,
respectively.

Observations during fertility and lactation: No statistically significant difference was observed in the gestation length, number of implantation sites, delivery index, or gestation index between the control group and any treated group. No abnormalities were observed in the delivery or nursing conditions of the dams.

Details on results (P0)

Repeated dose aspects:

No death occurred during the examination period.

A decrease in body weights was observed in females at 50 mg/kg and a decrease in food consumption was observed in both sexes at 50 mg/kg. These changes were considered due to mucosal irritation in the forestomach of FEC described below. In the pathological examination, changes attributable to the test substance were observed in the maxillary incisor and forestomach in males at 15 mg/kg and both sexes at 50 mg/kg. The necropsy results revealed whitish change in the maxillary incisor and the forestomach mucosa in both sexes at 50 mg/kg. For the maxillary incisor, there were histopathologically irregular arrangement of ameloblast in males at 15 mg/kg and both sexes at 50 mg/kg and degeneration of ameloblast and irregular arrangement of odontoblast in both sexes at 50 mg/kg. The fluorine compounds have toxic effects on the tooth enamel blast and odontoblast. Since FEC is the compound containing fluorine, it was considered that the influence by fluorine occurred on enamel blast cells and odontoblast cells.

For the forestomach, changes were found to be histological hyperplasia of squamous cell in males at 15 mg/kg and both sexes at 50 mg/kg, and ulcer and focal infiltrate of inflammatory cell in both sexes at 50 mg/kg, suggesting mucosal irritation of FEC.

No effects of FEC were observed in clinical signs, organ weights, or plasma total T4 concentration in any group.

Reproductive/developmental toxicity:
A decrease in body weights of live offspring was observed at 50 mg/kg. No treatment-related effects were observed on the estrous cycle, copulation index,
fertility index, gestation index, gestation length, number of implantation sites, delivery index, delivery or maternal behavior, sex ratio, birth index, viability index on PND 4 or 13, external finding, AGD, nipple development anomaly index, or plasma T4 concentrations on PND 13 in any group.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
5 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
histopathology: non-neoplastic

Target system / organ toxicity (P0)

open allclose all
Critical effects observed:
yes
Lowest effective dose / conc.:
15 mg/kg bw/day (nominal)
System:
gastrointestinal tract
Organ:
stomach
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Critical effects observed:
yes
Lowest effective dose / conc.:
15 mg/kg bw/day (nominal)
System:
musculoskeletal system
Organ:
tooth
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
not specified
Description (incidence and severity):
No external abnormalities were noted or treatment related effects to the viability index
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
A low number of pub mortality and cannibalisation occured across dose groups and was not considered related to treatment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In male and female offspring, body weight was significantly low at 50 mg/kg compared with the control group on PND 13.
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
Anogenital distance (AGD):
effects observed, non-treatment-related
Description (incidence and severity):
In males, no statistically significant difference was observed in the AGD between the control group and any treated group.

In females, relative AGD value was significantly high at 50 mg/kg compared with the control group; however, this was not judged to be treatment-related because the change was within mean ± 2 S.D. of the historical control data at the test facility and no related changes were noted in the external examination or necropsy.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
No statistically significant difference was observed in nipple development anomaly index between the control group and any treated group.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No external anomaly was observed in any offspring.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Hormone (T4) analysis: The plasma total T4 concentration of treated groups (Groups low-, middle-, and high-dose) was equivalent to that of the control group in offspring [F1, PND 13]. There was no notable change in the plasma total T4 concentration attributable to the test substance.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

A decrease in body weights of live offspring was observed at 50 mg/kg. As evidence of maternal toxicity was observed at this dose (decrease in bodyweights, food consumption, mucosal irritation in the stomach and fluorosis in the teeth) these effects are deemed to be secondary to parental toxicity.


No treatment-related effects were observed on the estrous cycle, copulation index, fertility index, gestation index, gestation length, number of implantation sites, delivery index, delivery or maternal behavior, sex ratio, birth index, viability index on PND 4 or 13, external finding, AGD, nipple development anomaly index, or plasma T4 concentrations on PND 13 in any group.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
15 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
Effects on parental animals included decreases in body weights and food consumption observed at 50 mg/kg, and histopathological abnormalities were observed in the maxillary incisor and forestomach at 15 and 50 mg/kg. It was therefore concluded that NOAEL of FEC is 5 mg/kg/day for parental animals.

There were no effects on the reproductive function of parental animals observed in any group. Effects on offspring included a decrease in body weights observed at 50 mg/kg. As evidence of maternal toxicity was observed at this dose (decrease in bodyweights, food consumption, mucosal irritation in the stomach and fluorosis in the teeth) these effects are deemed to be secondary to parental toxicity.

It was therefore concluded that NOAEL of FEC is 50 mg/kg/day for reproductive function of parent animals and 15 mg/kg/day for offspring.