4,4'-isopropylidenedi-o-cresol

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

OECD guideline, GLP compliant screening test is available. Some treatment effects were noted in males and females at the highest tested dose (1000 mg/kg/day) and hence NOAEL was set at 250 mg/kg. Due to the limitations of the OECD 421 screening test, it is not possible to conclude the rationale for the decrease in the fertility index to 75% at the highest tested dose (1000 mg/kg) although the authors of the study report suggest the effect is not likely due to any issues with spermatogenesis (no effects were seen when testis and epididymis were examined) or with the estrous cycle. No effects were observed in offspring (F1) throughout the duration of the study.

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

Version dated 29 July 2016

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CD (SD)

Details on species / strain selection:

This strain is widely used in reproduction/developmental toxicity studies using rodents, there is abundant historical data, and a large number of animals are available.

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: (P) 7 wks
- Weight at study initiation: (P) Males: 216.5 - 240.9g; Females: 154.8 - 185.7g
- Fasting period before study: no
- Diet (e.g. ad libitum): ad libitum. Pellet diet for experimental animals (CRF-1, Oriental Yeast Co., Ltd., radiation sterilized)
- Water (e.g. ad libitum): ad libitum. Well water admixed with sodium hypochlorite (free residual chlorine concentration: about 0.2 ppm)
- Acclimation period: The quarantine and acclimation periods (from animal receipt to the day before the start of administration) were 20 days in total, including the quarantine period for 6 days after receipt.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): Actual range: 22.5°C to 24.5°C (permissible range: 20.0°C to 26.0°C)
- Humidity (%): Actual range: 43.0% to 73.2% (permissible range: 35.0% to 75.0%)
- Air changes (per hr): 10-20 times per hour
- Photoperiod (hrs dark / hrs light): 12 hour dark; 12 hours light per day (7:00 to 19:00)

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5w/v% sodium CMC

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

Dosing formulations- Preparation method and frequency
The dosing formulations were prepared once from 8 to 11 days (permissible range: 12 days). The vehicle (0.5%CMC-Na) was used as the dosing formulation for the control group.

(Control group dosing formulations)
The required amount of the vehicle was divided into clear glass vials for each dosing day.

(BPC-treated group dosing formulation)
(1) A prescribed amount of the test substance was weighed and ground in an agate mortar.
(2) The vehicle was gradually added and mixed with a pestle. The suspension in the mortar was transferred to a measuring cylinder.
(3) The mortar and pestle were washed with the vehicle, and the washing was transferred to the measuring cylinder.
(4) The final volume was adjusted by adding a proper quantity of the vehicle to required concentrations of 6.25, 25, and 100 mg/mL.
The dosing formulations after preparation were mixed several times by end-over-end rotation and divided into brown glass vials for each dosing day.

Storage conditions and location
Refrigeration (actual temperature: 3.4°C to 6.2°C, permissible range: 1°C to 15°C), in a medical refrigerator in Dosing Formulation Storage Room A032.

Confirmation of stability and homogeneity
The test substance formulations in the brown glass vial at 1 and 100 mg/mL were confirmed to be stable and homogenous at 12 days under refrigeration, followed by the storage period of 24 hours at room temperature.

Handling of remaining dosing formulations
The remaining dosing formulations were discarded on each dosing day.

Details on mating procedure:

- M/F ratio per cage: 1 male/1 female per cage
- Length of cohabitation: The males and females of each dose level were cohabited at one-on-one basis for 24 hours from Day 15 up to the day of confirmed copulation.
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as gestation day 0 (GD0)
- After successful mating each pregnant female was caged (how): 1 per cage

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

At the first preparation, 15 mL each of the analytical samples was taken from one point of each layer (n=1 in each layer, upper, middle, and lower layers) of the whole dosing formulation at each concentration (except for the dosing formulation for the control group).

Criteria: relative standard deviation (RSD) of analytical values (concentration) should not be more than 10%, and measured concentration (mean) should be within 100 ± 10% as the ratio to the nominal concentration.

Reference standard
Test substance was used as the reference standard.
Reagents
- Acetonitrile: for HPLC Kanto Chemical Co., Inc.
- Water: It was prepared with Ultrapure Water System (Elix, ASM and Milli-Q, Merck Ltd.).

Preparation of reagent solutions
Mobile phase A: Water
Water was used as mobile phase A. Mobile phase A was degassed by sonication under reduced pressure.
Mobile phase B and needle wash solvent: Acetonitrile
Acetonitrile was used as mobile phase B and needle wash solvent. Mobile phase B and needle wash solvent were degassed by sonication under reduced pressure.

Preparation of standard solutions and processed samples
Preparation of stock standard solutions
See Table 1 below

Preapartion of standard solutions
See Table 2 below

Preparation of processed samples
See Table 3 below

High Performance Liquid Chromatography (HPLC) conditions
HPLC: Prominence UFLC (Shimadzu Corporation)
Data processing: LabSolutions (Shimadzu Corporation)
Column: Inertsil ODS-3 (3 μm, 4.6 mm I.D. × 50 mm, GL Sciences
Inc.)
Column temperature: A constant temperature of about 40°C (set at 40°C)
Mobile phase: Mobile phase A; water
Mobile phase B; acetonitrile
Ratio of mobile phases; Mobile phase A/ Mobile phase B
(50:50)
Flow rate: 1 mL/min
Analysis time: 6 minutes
Wavelength for detection: UV 240 nm
Injection volume: 10 μL
Auto-sampler set temperature: Room temperature (Not setting)
Needle wash solvent: Acetonitrile


Calculation method of the concentration
The test substance concentration in each dosing formulation was calculated by the
following equation using the peak area of BPC. The test substance concentrations of
the dosing formulations were expressed as the mean values of the concentrations of the
upper, middle, and lower layers.
Test substance concentrations in the dosing formulation (mg/mL) = (AT - b) / a × D / 1000
AT: Peak area of BPC in the processed sample
a: Slope of calibration curve
b: Intercept of calibration curve
D: Dilution factor
The regression equation for the calibration curve: Y = aX + b
X: The nominal concentration of the standard solution (μg/mL)
Y: Peak area of BPC
a: Slope
b: Intercept

Result are show in Table 4 below

Duration of treatment / exposure:

Administration period
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:

Once daily between 8:04 and 11:49 (permissible range: 8:00 and 15:00)

Dose / conc.:

62.5 mg/kg bw/day (nominal)

Remarks:

Low dose group

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

Mid-dose group

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High dose group

No. of animals per sex per dose:

12 male, 12 female per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were set based upon the results of the study entitled "A 28-Day Repeated Dose Oral Toxicity Study of Bisphenol C in Rats (Study No. 8K259, dose levels; 0, 40, 200, and 1000 mg/kg)". In this study, decreased body weight and food consumption in males were observed at 1000 mg/kg. Therefore, the high dose level was set at 1000 mg/kg which was expected to develop toxicity. The middle and low dose levels were set at 250 and 62.5 mg/kg, respectively, at a geometric ratio of 4.
- Rationale for animal assignment (if not random): On the day before the start of administration for both sexes, 10 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 (computer system; tsPharma LabSite). The animals weighing within ± 20% of the mean body weights (calculated for each sex) were used for this study.

Parental animals: Observations and examinations:

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

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, 15, and/or 22 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. All live offspring were weighed individually on PNDs 0, 4, 7, and 13.

FOOD CONSUMPTION: Yes
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.

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):

Parameters examined in P males
testis weight, epididymis weight, histopathology of testis and epididymis

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, 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, presence of nipples/areolae in male pups. Particular attention should be paid to the external reproductive genitals which should be examined for signs of altered development; gross evaluation of external genitalia.

GROSS EXAMINATION OF DEAD PUPS:
no

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals. Necropsy was conducted on the day following the final dosing (Day 36 for males)
- Maternal animals: All surviving animals. Necropsy was conducted on the day following the final dosing (LD13 for females)

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 5 were prepared for microscopic examination and weighed, respectively.

Hormone concentration (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 (offspring):

SACRIFICE
- The necropsy was performed on PND 13.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:
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.

GROSS NECROPSY
- 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.

Statistics:

Statistical analysis was performed for the following data, except for the sex ratio, using a computer system (tsPharma LabSite, Fujitsu Limited). SAS 9.4 (SAS Institute Japan[CAC Croit Corporation]) 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, count 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 BPC-treated groups.
Items: Copulation index, fertility index, gestation index, and sex ratio

Wilcoxon's rank sum test
For non-parametric data such BPC-treated groups, Wilcoxon's rank sum test was performed between the control and BPC-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:

Gestation index (%): (Number of pregnant animals delivered with live offspring / number of pregnant animals) × 100

Offspring viability indices:

Birth index (%): (Number of live offspring at birth / number of implantations) × 100
Stillborn index (%): (Number of stillborns / total number of delivered offspring) × 100
Viability index on Day 4 (%): (Number of live offspring on PND 4 / number of live offspring at birth) × 100
Viability index on Day 13 (%): (Number of live offspring on PND 13 / number of live offspring after culling) × 100
Sex ratio: (Number of male live offspring / number of male and female live offspring)
External anomaly index (%): (Number of offspring with external anomaly / number of observed offspring) × 100
External anomaly typing index (%): (Number of offspring with external anomaly by each type / number of observed offspring) × 100
Nipple development anomaly index (%): (Number of offspring with nipple development anomaly / number of observed offspring) × 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In males, decrease in locomotor activity was observed on Days 15 and 16 and soiled fur was observed from Days 14 to 23 in 1 animal (No. 640) at 1000 mg/kg. In addition,
loss of fur (chest or buttock) was observed in 2 animals (Nos. 639 and 642) at 1000 mg/kg on Day 32 or later.
In females, no abnormal clinical signs were observed in any group throughout the administration period, including the gestation and lactation periods of pregnant females.
(See Table 6 and 7 for details)

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, treatment-related

Description (incidence and severity):

In males, mean body weight was significantly low at 1000 mg/kg compared with the control group from Days 8 to 36. Individually at 1000 mg/kg, apparent decrease in
body weight was observed in 2 animals (No. 638, -19.3 g; No. 639, -56.3 g) from Days 1 to 8 and in 1 animal (No. 640, -94.0 g) from Days 8 to 15.
In females, individually, apparent decrease in body weight was observed in 2 animals (No. 687, -21.4 g; No. 689, -10.3 g) at 1000 mg/kg from Days 1 to 8. Other than the above, mean body weight was significantly low at 250 mg/kg compared with the control group on GDs 7 and 20 and LD 4; however, it was not considered to be treatment-related because there was no dose-dependency.
(See tables 8 and 9 for details)

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In males, mean food consumption was significantly low from Days 1 to 2 and successively tended to be low up to Day 15 at 1000 mg/kg compared with the control group. Individually at 1000 mg/kg, apparent decrease in food consumption was
observed in 1 animal (No. 639, 4.5 g) from Days 7 to 8 and in 1 animal (No. 640, 9.7g) from Days 14 to 15.
Other than the above, mean food consumption was significantly low at 250 mg/kg compared with the control group on Days 1 to 2; however, this change was judged to be toxicologically insignificant because it was transient and had no effect on body weight.
In females, mean food consumption was significantly low at 250 and 1000 mg/kg compared with the control group from Days 1 to 2; however, these changes were judged to be toxicologically insignificant because they were transient and had no effect on body
weight.

See Table 10 and 11 for more details)

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, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In males, in the kidney, granular cast, regeneration of the tubular epithelium, dilatation of the tubule, and infiltration of inflammatory cells in the interstitium were observed in 5 animals at 1000 mg/kg.

In the skin with macroscopic loss of fur, crust and ulcer were observed in 1 animal at 1000 mg/kg. Other than the above, there were focal atrophy of the seminiferous tubules in the testis in 1 animal each at control and 1000 mg/kg, respectively, lymphocytes infiltration into the interstitium in the epididymis in 1 and 2 animals at control and 1000 mg/kg, respectively, and sperm granuloma in the epididymis in 1 animal at 1000 mg/kg; however, these were judged to be naturally occurring changes because they were the changes spontaneously observed in normal rats or observed also in the control group with similar degree and frequency in this study.
In females, no abnormalities were observed in any female.
(See tables 14 and 15 for details)

[Non-pregnant females] No abnormalities were observed in any female.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

The plasma total T4 concentration of BPC-treated groups (Groups low-, middle-, and high-dose) was equivalent to that of the control group in parental animals [F0, male] and
offspring [F1, PND13]. There was no notable change in the plasma total T4 concentration attributable to the test substance.
All values in the quality control sample were within the range of 100 ± 25% of the nominal value, and variations in duplicate cpm of the standard solutions were within the
range of acceptability. There were no abnormalities in the procedures for the determination or in the values of the test samples. The results were therefore considered acceptable

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes were observed in the count of estrus or estrous cycle in any BPC-treated group.
In 1 female (No. 680) at 250 mg/kg, irregular estrous cycle (3.33-day cycle) was observed; however, no statistically significant differences were observed in the mean estrous cycle or count of estrus between the control group and any BPC-treated group.

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

No statistically significant differences were observed in the days until copulation, copulation index, or fertility index between the control group and any BPC-treated group.
Although there was no statistical significance, fertility index at 1000 mg/kg (75%) was lower than that in the control group (91.7%). This value was outside the range of historical data at the test facility.

Historical data (2013 to 2017, 8 studies, 12 animals/study)
Fertility index 91.7% to 100%

One female (No. 656) at control group, 1 female (No. 676) at 250 mg/kg, and 3 females (Nos. 690, 691, and 696) at 1000 mg/kg group did not become pregnant. Accordingly, the copulation indices were 100% for the control and all BPC-treated groups, and the fertility indices were 91.7%, 100%, 91.7%, and 75% for the control, 62.5, 250, and 1000 mg/kg groups, respectively.

The number of implantation sites was significantly low at 250 and 1000 mg/kg (250 mg/kg, 13.5; 1000 mg/kg, 13.6) compared with the control group; however, this was not judged to be treatment-related because the change was almost same values as historical control data at the test facility.

Historical data (2013 to 2017, 8 studies, 10 to 12 dams/study)
Number of implantation sites 13.58 to 16.00

No statistically significant differences were observed in the gestation length, delivery index, or gestation index between the control group and any BPC-treated group. No abnormalities were observed in the delivery or nursing conditions of the dams.

No death occurred during the examination period.
In the clinical signs, decrease in locomotor activity and soiled fur were observed in 1 male from Days 15 to 16 and from Days 14 to 23, respectively, at 1000 mg/kg. These changes were considered to reflect worsening of the whole body condition because
remarkable decreases in body weight and food consumption were observed in this animal around the same time. In addition, loss of fur was observed in the other 2 males at 1000 mg/kg from Day 32 or later, and crust and ulcer were observed histopathologically
in 1 male at 1000 mg/kg. The both macroscopic loss of fur and microscopic crust and ulcer formation were considered attributable to scratching behavior rather than the direct test-article effect on the skin because no abnormality was noted in the skin areas without
loss of fur in clinical observation.
Decreased body weight and food consumption were observed in males at 1000 mg/kg. Although these changes were relatively severe by Day 15, they tended to be lightened thereafter. Therefore these changes were not judged to be toxicologically serious
changes. Some females at 1000 mg/kg also showed decreases in body weight and food consumption by Day 8 and Days 1 to 2, respectively; however, these were judged to be toxicologically insignificant because they were transient and showed favorable recovery
thereafter.
In the pathological examination, changes attributable to the test substance were observed in the kidney in males at 1000 mg/kg. The necropsy results revealed enlargement of the kidney in males, and histopathological results revealed granular cast, regeneration of the tubular epithelium, dilatation of the tubule, and infiltration of inflammatory cells in the interstitium. Regenerative changes in renal tubular epithelial cells are changes following necrosis and loss of tubular epithelial cells. Meanwhile, tubular dilatation, granular cast and stromal inflammatory cell infiltration are known to occur associated
with injuries such as necrosis and degeneration . These changes observed in this study were also considered to indicate damage of tubular epithelial cells caused by test substance administration. In the cecum, necropsy results revealed dilatation of the lumen in males at 250 and 1000 mg/kg or in females at 1000 mg/kg. Administration of antibiotics, processed starches, polyols, fiber components, lactose and osmotic solvents to rodents is known to induce cecal dilatation. Although the pathogenesis is not clear, loose stool, diarrhea and cecal mucosal hypertrophy and hyperplasia is reported to occur. The cecal dilatation observed in this study did not show histological changes, and no loose stool or diarrhea was observed in the observation of general condition. Accordingly, this change was considered to be toxicologically insignificant. In the thyroid gland, enlargement of the thyroid gland was observed at 1000 mg/kg; however, since this change was observed in only one dam and had no effect on histopathological examination, it was not judged to be treatment-related.
No effects of BPC were observed in organ weights, or plasma total T4 concentration in any group.

A trend toward a decrease in fertility index was observed at 1000 mg/kg. This change was likely to be associated with failure of conception or implantation because there were no effects on estrous cycle for females or spermatogenesis for males.

No treatment-related effects were observed on the estrous cycle, count of estrus, copulation index, gestation index, gestation length, number of implantation sites, delivery index, delivery or maternal behavior,

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

histopathology: non-neoplastic

reproductive performance

Clinical signs:

no effects observed

Description (incidence and severity):

No external anomaly was observed in any offspring.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No statistically significant differences were observed between the control group and any BPC-treated 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:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

In both sexes, no statistically significant differences were observed in the AGD between the control group and any BPC-treated group.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No statistically significant differences were observed in nipple development anomaly index between the control group and any BPC-treated group.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No abnormalities were observed in any offspring.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

No statistically significant differences were observed in the number of litter, number of live newborns, birth index, sex ratio, stillborn index, or viability index on Day 4 or 13 between the control group and any BPC-treated group.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

No treatment-related effects were observed on sex ratio, birth index, viability index on PND 4 or 13, body weight, external finding, AGD, nipple development anomaly index, necropsy, or plasma total T4 concentrations on PND 13 in any group.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

No effects at highest dose

Reproductive effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Table 6 Clinical signs (Male)

Test article	Control	BPC	BPC	BPC
Dose (mg/kg)	0	62.5	250	1000
No. of animals	12	12	12	12
Treatment period - Before dosing and necropsy day
No. of animals	12	12	12	12
Normal	12	12	12	9
Decrease in locomotor activity	0	0	0	1
Loss of fur, Chest	0	0	0	1
Loss of fur, Buttock	0	0	0	1
Soiled fur, Face	0	0	0	1
Soiled fur, Abdomen	0	0	0	1
Soiled fur, Forelimb	0	0	0	1
Soiled fur, Anogenital region	0	0	0	1
Treatment period – after dosing
No. of animals	12	12	12	12
Normal	12	12	12	10
Decrease in locomotor activity	0	0	0	1
Loss of fur, Chest	0	0	0	1
Loss of fur, Buttock	0	0	0	1
Soiled fur, Face	0	0	0	1
Soiled fur, Abdomen	0	0	0	1
Soiled fur, Forelimb	0	0	0	1
Soiled fur, Anogenital region	0	0	0	1

Table 7 Clinical signs (Female)

Test article	Control	BPC	BPC	BPC
Dose (mg/kg)	0	62.5	250	1000
No. of animals	12	12	12	12
Treatment – Before dosing and necropsy day
Treatment period
No. of animals	12	12	12	12
Normal	12	12	12	12
Mating period
No. of animals	9	7	9	7
Normal	9	7	9	7
Gestation period
No. of animals	12	12	12	12
Normal	12	12	12	12
Lactation period
No. of animals	11	12	11	9
Normal	11	12	11	9
Post-treatment
Lactation period
No. of animals	11	12	11	9
Normal	11	12	11	9
Treatment – After dosing
Treatment period
No. of animals	12	12	12	12
Normal	12	12	12	12
Mating period
No. of animals	9	7	9	7
Normal	9	7	9	7
Gestation period
No. of animals	12	12	12	12
Normal	12	12	12	12

Table 8 Body weights (male) (g) (Mean ± S.D.)

Test article		Control	BPC	BPC	BPC
Dose (mg/kg)		0	62.5	250	1000
No. of animals		12	12	12	12
Treatment period	1	395.57± 19.11	394.15 ± 16.57	93.24 ± 17.68	392.45 ± 17.31
	8	427.93 ± 21.58	419.31 ± 22.42	411.10 ± 20.19	395.13 ± 30.00 ##
	15	455.11 ± 24.09	438.29 ± 28.66	434.33 ± 25.08	404.62 ± 34.14 ##
	22	476.62 ± 25.20	458.97 ± 33.54	451.40 ± 23.99	425.43 ± 29.08 ##
	29	501.61 ± 29.92	480.36 ± 38.92	417.45 ± 27.93	446.82 ± 32.64 ##
	36	522.73 ± 33.64	499.17 ± 40.41	489.40 ± 27.28	464.75 ± 33.17 ##

## significantly different from control group; p <0.01 (Dunnett test)

Table 9 Body weights (Female) (g) (Mean ± S.D.)

Test article		Control		BPC		BPC		BPC
Dose (mg/kg)		0		62.5		250		1000
No. of animals		12		12		12		12
Treatment
Treatment period	1	249.41 ± 15.28		246.90 ± 17.77		246.28 ± 16.45		246.07 ± 15.14
	8	258.44 ± 19.12		256.68 ± 15.73		251.52 ± 12.98		248.36 ± 16.52
	15	268.01 ± 18.50		265.05 ± 18.56		258.68 ± 15.01		262.15 ± 14.76
Gestation period	0	279.79 ± 25.90	(11)	270.68 ± 16.86		262.99 ± 13.01	(11)	269.40 ± 16.93	(9)
	7	316.74 ± 23.61	(11)	304.41 ± 17.30		293.03 ± 15.28 #	(11)	307.21 ± 18.21	(9)
	14	350.84 ± 26.63	(11)	338.15 ± 21.52		326.33 ± 22.96	(11)	346.14 ± 22.78	(9)
	20	433.44 ± 29.82	(11)	420.23 ± 27.28		402.53 ± 32.00 #	(11)	413.47 ± 27.80	(9)
Lactation period	0	328.31 ± 27.61	(11)	322.63 ± 19.83		308.13 ± 19.35	(11)	325.43 ± 24.46	(9)
	4	350.62 ± 244.46	(11)	341.53 ± 17.97		325.75 ± 26.12 #	(11)	341.62 ± 26.95	(9)
	7	357.41 ± 24.21	(11)	346.90 ± 19.62		335.86 ± 18.98	(11)	351.10 ± 24.80	(9)
Post-treatment
Lactation period	13	366.13 ± 26.10	(11)	358.21 ± 19.10		350.09 ± 22.24	(11)	373.17 ± 23.97	(9)

# Significantly different from the controls ; p<0.05 (Dunnett test)

Number in parentheses indicates the number of dams

Table 10: Food consumption (Male) (g) (Mean ± SD)

Test article Dose (mg/kg) No. of animals		Control 0 12	BPC 62.5 12	BPC 250 12	BPC 1000 12
Treatment period	1	28.22 ± 3.17	25.31 ± 3.04	19.86 ± 3.18 ##	18.98 ± 5.04 ##
	7	28.14 ± 2.49	27.73 ± 2.53	26.38 ± 3.08	23.37 ± 7.50
	14	27.35 ± 2.10	26.32 ± 2.87	26.73 ± 2.77	25.24 ± 5.34
	35	29.65 ± 2.91	28.24 ± 2.46	26.23 ± 5.55	30.90 ± 3.41

Significantly different from the control group; ## p <0.01 (Dunnett test)

Table 11: Food consumption (Female) (g) (Mean ± SD)

Test article Dose (mg/kg) No. of animals		Control 0 12		BPC 62.5 12		BPC 250 12		BPC 1000 12
Treatment period	1	20.36 ± 3.19		18.21 ± 2.74		14.98 ± 2.34 ##		16.36 ± 2.04 ##
	7	19.76 ± 3.23		18.85 ± 1.97		19.08 ± 2.26		17.28 ± 3.64
	14	19.63 ± 2.67		19.14 ± 3.28		17.78 ± 1.97		19.79 ± 2.92
Gestation period	0	14.23 ± 1.97	(11)	13.38 ± 3.81		12.58 ± 3.05	(11)	12.80 ± 3.04	(9)
	6	23.20 ± 2.64	(11)	22.34 ± 2.15		22.64 ± 2.71	(11)	24.74 ± 4.13	(9)
	13	23.42 ± 2.29	(11)	22.67 ± 3.05		22.14 ± 4.03	(11)	25.39 ± 3.92	(9)
	19	25.17 ± 2.40	(11)	26.71 ± 2.33		27.20 ± 3.47	(11)	28.41 ± 3.69	(9)
Lactation period	0	24.36 ± 6.71	(11)	22.02 ± 6.54		24.41 ± 5.84	(11)	27.16 ± 4.97	(9)
	3	44.79 ± 5.69	(11)	43.49 ± 3.92		45.85 ± 7.56	(11)	48.16 ± 8.75	(9)
	6	47.30 ± 3.81	(11)	45.49 ± 8.12		50.87 ± 5.49	(11)	51.06 ± 7.53	(9)
	12	60.63 ± 3.73	(11)	58.85 ± 4.85		62.27 ± 6.23	(11)	64.77 ± 5.51	(9)

Significantly different from the control group; ## p <0.01 (Dunnett test)

Number in parentheses indicates the number of dams

Table 12 Necropsy findings (male)

Test article Dose (mg/kg) No. of animals	Control 0 12	BPC 62.5 12	BPC 250 12	BPC 1000 12
Not remarkable	12	11	8	2
ALIMENTARY SYSTEM
Cecum Dilatation, Lumen	0	0	4	10
URINARY SYSTEM
Kidney Enlargement, Bilateral	0	0	0	5
GENITAL SYSTEM
Testis Small, Unilateral	0	1	0	0
Epididymis Yellow patch, Bilateral	0	0	1	0
INTEGUMENTARY SYSTEM
Skin Loss of fur	0	0	0	2

Table 13: Necropsy findings (Female)

Test article Dose (mg/kg) No. of animals	Control 0 11	BPC 62.5 12	BPC 250 11	BPC 1000 9
Not remarkable	11	11	8	5
ALIMENTARY SYSTEM
Cecum Dilatation, Lumen	0	0	0	4
GENITAL SYSTEM
Uterus Cyst, Cervix	0	0	0	1
ENDOCRINE SYSTEM
Thyroid Enlargement, Bilateral	0	0	0	1

Table 14: Histopathological findings (Male)

Test article Dose (mg/kg) No. of animals		Control 0 12		BPC 62.5 12		BPC 250 12		BPC 1000 12
ALIMENTARY SYSTEM
Cecum	Number examined	-		-		4		10
URINARY SYSTEM
Kidney	Number examined	-		-		-		5
Granular cast		-		-		-		5
minimal			-		-		-		1
mild			-		-		-		4
Regeneration, Tubular epithelium		-		-		-		5
minimal			-		-		-		1
mild			-		-		-		4
Dilation, tubule		-		-		-		5
minimal			-		-		-		1
mild			-		-		-		4
Infiltrate, inflammatory cell, Interstitium		-		-		-		5
minimal			-		-		-		1
mild			-		-		-		4
GENITAL SYSTEM
Testis	Number examined	12		-		-		12
Atrophy, seminiferous tubule, Focal		1		-		-			1
minimal			1		-		-		1
Epididymis	Number examined	12		-		-		12
Infiltrate, Lympocyte, Interstitium		1		-		-		2
minimal			1		-		-		2
Sperm granuloma		0		-		-		1
minimal			0		-		-		1
INTEGUMENTARY SYSTEM
Skin	Number examined	-		-		-		2
Crust		-		-		-		1
minimal			-		-		-		1
Ulcer		-		-		-		1
minimal			-		-		-		1

Table 15: Histopathological findings (female)

Test article Dose (mg/kg) No. of animals		Control 0 11		BPC 62.5 12		BPC 250 11		BPC 1000 9
ALIMENTARY SYSTEM
Cecum	Number examined	-		-		-		4
GENITAL SYSTEM
Ovary	Number examined	11		-		-		9
ENDOCRINE SYSTEM
Thyroid gland	Number examined	-		-		-		1

Conclusions:

Effects on parental animals: As effects of BPC on parent animals, clinical signs and histopathological abnormalities or decreases in body weights and food consumption were observed at 1000 mg/kg. It was therefore concluded that NOAEL of BPC was 250 mg/kg/day for parental animals.
Reproductive/Developmental toxicity: As effects of BPC on reproductive function of parent animals, trend toward a decreased fertility index was observed at 1000 mg/kg. As effects of BPC on offspring, no effects of BPC were observed in any group. It was therefore concluded that NOAEL of BPC was 250 mg/kg/day for reproductive performance of parent animals and 1000 mg/kg/day for offspring.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

250 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Justification for classification or non-classification

There is not enough data available to conclude on the classification and labelling under Regulation (EC) 1272/2008 for this endpoint.