Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

1.      Toxicity to Reproduction

Investigation of the toxicity to reproduction of the registered substance 4,4'-Isopropylidenediphenol, propoxylated (thereafter referred to as BPA PO) was performed in several steps.

 

1.1  Screening for toxicity to reproduction

Screening for toxicity to reproduction was performed as part of GLP-compliant studies conducted in accordance with the OECD Testing Guideline 422 on BPA 2PO and Grade 5 of the registered substance.

 

1.1.1 Screening for toxicity to reproduction conducted on BPA 2PO

The test substance was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 125, 250 and 500 mg/kg bw/day. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 43-54 days). Formulation analysis showed that the formulations were prepared accurately, were homogenous, and were stable for at least 6 hours at room temperature.

The mating, fertility and conception indices were lower for females at 500 mg/kg bw/d, along with a lower number of corpora lutea. The high mortality and limited number of litters available for evaluation likely contributed to this, though when also taking into account the evidence of impaired spermatogenesis, a treatment related effect cannot be excluded. There were no toxicologically significant changes noted in any of the remaining reproductive parameters investigated in this study (i.e. gestation index, precoital time, and number of implantation sites).

No developmental toxicity was observed up to 500 mg/kg bw/d. Due to mortality there was a lower number of litters available for assessment at 500 mg/kg bw/d, and a higher incidence of pup mortality, a lower number of living pups, and a higher number of dead pups was seen at this dose level. However, all pup mortality was attributable to a single dam and was secondary to maternal toxicity.

No treatment-related changes were noted in any of the remaining developmental parameters investigated in this study (i.e. duration of gestation, parturition, maternal care and clinical signs, body weights and macroscopy of pups).

In conclusion, treatment with BPA 2PO by oral gavage in male and female Wistar Han rats at dose levels of 125, 250 and 500 mg/kg bw/day revealed parental toxicity at 250 and 500 mg/kg bw/day, and reproduction toxicity at 500 mg/kg bw/day.

 

1.1.2 Screening for toxicity to reproduction conducted on Grade 5 of BPA PO

The test substance was administered by daily oral gavage to male and female Sprague-Dawley rats at dose levels of 30, 120 and 500 mg/kg bw/day. Males were exposed for 42 days. The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 42-53 days).

Incidence of oestrous cycle disorder increased at 500 mg/kg bw/d. No abnormal findings in pairing days until copulation, copulation index, gestation index, gestation length, number of corpora lutea, number of implantation sites, implantation index, fertility index, delivery index and delivery/lactation behaviour.

Decrease in body weight of pups was observed at 500 mg/kg bw/d. There were no changes in number of pups born, number of pups alive, sex ratio, live birth index, external findings and viability index caused by treatment with test substance.

In conclusion, treatment with Grade 5 of BPA PO resulted in high rate of oestrus cycle disorder and decrease in body weight of pups observed at 500 mg/kg bw/d.

 

1.2  Extended-One Generation Toxicity to Reproduction Study

The screening study on reproductive toxicity for Grade 5 of BPA PO indicates a possible reproductive toxicity effect of Grade 5 of BPA PO. However, the findings did not fulfil the criteria for reproductive toxicity classification according to the evaluating Competent Authority (eCA). The results concerning indications of an increased rate of oestrus cycle disorder in females (Grade 5 of BPA PO) in the screening study cause a concern for endocrine disruption in vivo. Further evaluation by QSAR model predictions on oestrogen receptor performed by the eCA indicates a potential concern that BPA PO may cause reproduction toxicity and endocrine disrupting activity through binding of constituents of BPA PO to the oestrogen receptor.

To clarify the indications of concern on reproductive toxicity, the eCA requested an Extended-One Generation Toxicity to Reproduction Study (EOGRTS) in accordance with the OECD Testing Guideline 443. This Guideline includes parameters for adverse effects on reproduction and a number of endocrine sensitive parameters which may be used to inform about endocrine disrupting modes of action.

Based on the results obtained during the screening for toxicity to reproduction, it was concluded that Grade 5 of BPA PO represented a greater concern than BPA 2PO, considering the high rate of oestrus cycle disorder induced by Grade 5 of BPA PO. However, Grade 5 of BPA PO can be regarded as a polymer under REACH, so it was ruled out as a test substance for the purpose of the EOGRTS. Since the outcome of the OECD 422 suggested that potential endocrine-disrupting properties could be related to higher degrees of propoxylation and therefore constituents with higher chain-length, Grade 4 of BPA PO was deemed as the most relevant test substance for this study by the eCA.

The following specifications were decided in regards to the study design for the EOGRTS before conducing it:

Inclusion of the extension of Cohort 1B: As there was concern on the consumer and professional exposure and a potential oestrogenic mode of action, it was considered necessary to include the extension of Cohort 1B to generate a F2 generation. This is because more severe effects on reproduction can be observed in the F1 generation.

Inclusion of Cohort 2A/2B (developmental neurotoxicity): As the screening for toxicity to reproduction conducted on Grade 5 of BPA PO showed a tendency towards increased activity and signs of impaired habituation in males as well as a potential endocrine mode of action, it was considered necessary to include Cohorts 2A and 2B to investigate the developmental neurotoxicity of the registered substance.

Inclusion of Cohort 3 (developmental immunotoxicity): No concern for developmental immunotoxicity was identified during the screening for toxicity to reproduction; however, these studies were not designed for investigation of developmental immunotoxicity and should therefore not be considered as sufficient to conclude on the potential for the registered substance to induce developmental immunotoxicity. As oestrogenic endocrine disruptors may modulate the immune system, it was considered necessary to include Cohort 3 to investigate the developmental immunotoxicity of the registered substance.

 

The test item, 4,4’Isopropylidenediphenol, propoxylated, Grade 4PO, was administered daily by oral gavage, at dose levels of 60, 180 or 540/360 mg/kg bw/d, to sexually-mature male and female rats (parental (P) generation) starting 2 weeks before mating and continuously through mating, gestation and weaning of their pups (F1 generation). At weaning, F1 generation was also exposed to graduated doses of the test item while being assigned to Cohorts of animals for reproductive/developmental toxicity, developmental neurotoxicity or developmental immunotoxicity testing.

 

 Systemic toxicity evaluation:

The No Observed Adverse Effect Levels (NOAELs) for systemic toxicity (excluded reproductive and developmental toxicity endpoints) were considered to be:

- 60 mg/kg bw/d in males based on adverse pathology findings (single cell necrosis) from 180 mg/kg bw/d in Cohort 1A males;

- 180 mg/kg bw/d in females based on adverse pathology findings (single cell necrosis) at 540/360 mg/kg bw/d in Cohort 1A females.

 

Reproductive/developmental toxicity testing:

- in P generation animals, there was an increased number of females in diestrus at 540/360 mg/kg bw/d, resulting in a prolonged duration of mean oestrous cycle associated with a lower fertility index at this dose level,

- in Cohorts 1A or 1B animals there were no adverse effects on reproductive function.

Therefore the No Observed Adverse Effect Level (NOAEL) for reproductive/developmental toxicity was considered to be 180 mg/kg bw/d.

 

Developmental neurotoxicity testing:

There were no adverse effects at neurobehavioral testing but ambiguous evidence of developmental neurotoxicity in high dose males from cohort 2A (corpus callosum increased thickness) and high dose females from cohort 2B (striatum decreased width).

Therefore the No Observed Adverse Effect Level (NOAEL) for developmental neurotoxicity was considered to be 360 mg/kg bw/d.

 

Developmental immunotoxicity testing:

in Cohort 1A:

-          moderate alterations in spleen immunophenotyping were observed differently in males (decrease of relative cytotoxic T cells count) and females (decrease of relative NK cells count and increase of T cell relative count) at 360 mg/kg bw/d,

in Cohort 3:

-          after KLH immunization and for all groups (including controls), there were no statistically significant differences.

Therefore the No Observed Adverse Effect Level (NOAEL) for developmental immunotoxicity was considered to be 360 mg/kg bw/d.

 

2.      Developmental toxicity / teratogenicity

Investigation of the developmental toxicity / teratogenicity of the registered substance was required for the purpose of the REACH Registration of the substance.

Based on the results obtained during the screening for toxicity to reproduction, it was concluded that Grade 5 of BPA PO represented a greater concern than BPA 2PO, considering developmental findings with a decrease of pup body weights observed at 500 mg/kg bw/d with Grade 5 of BPA PO. The potential endocrine modulating effect of BPA PO also strengthened the concern for developmental effects that needed to be investigated in the prenatal developmental toxicity study, thus justifying the study to be conducted on a substance with a high degree of propoxylation and containing constituents with high chain-length as previously discussed.

However Grade 5 of BPA PO can be regarded as a polymer under REACH, so it was a ruled out as a test substance for the purpose of the developmental toxicity / teratogenicity testing. Therefore Grade 4 of BPA PO was deemed as the most relevant test substance for this study by the eCA.

 

Developmental toxicity / teratogenicity of the registered substance was investigated during a GLP-compliant studies were conducted in accordance with the OECD Testing Guideline 414 on Grade 4 of BPA PO. Rats was selected as the test species.

Three groups of 24 time-mated female SpragueDawley rats received the test item, at 180, 360 or 720 mg/kg bw/d by oral route (gavage) once daily from Days 6 to 20 p.c. A constant dosage-volume of 5 mL/kg/day was used. Another group of 24 rats received the vehicle alone (olive oil) under the same experimental conditions, and acted as a control group. Formulation concentrations were checked in the start and end of the treatment period. The animals were checked daily for mortality and clinical signs. Body weight and food consumption were recorded every 2 to 3 days. On Day 21 p.c., females were sacrificed and submitted to a macroscopic postmortem examination. Hysterectomy was performed and the numbers of corpora lutea, uterine scars, implantations, early and late resorptions, and live and dead foetuses were recorded. The live foetuses were sexed, weighed and examined for external, soft tissues and skeletal (cartilages + bones) abnormalities.

In parent animals from the 720 mg/kg bw/d group, there were eight unscheduled deaths. Taking into account the similarity of the findings associated to these deaths (clinical signs, effects on body weight/food consumption and macroscopic examination at necropsy), this mortality was considered to be related to the test item treatment. In surviving animals from the highest-dose group, most of clinical signs (soiled urogenital area, lateral decubitus, piloerection, emaciated appearance, round back, cold to the touch, hypoactivity, abdominal breathing and/or eyes half-closed) were similar to those recorded in prematurely sacrificed and/or found dead animals. They were considered to be test item treatment-related and adverse. Statistically significant changes in body weight, body weight gain and food consumption were observed from 360 mg/kg bw/d, which were considered as treatment-related and adverse.

In the highest-dose there was a lower percentage of live foetuses per animal as a consequence of increased resorptions (late, early and scars) and dead foetuses which resulted in an increased incidence of mean percent of postimplantation loss. Foetuses (males and females) had a lower mean body weight, and malformations such as local oedema, cleft lip, anasarca, visceral oedema, fluid-filled thoracic cavity, fluid-filled abdomen, foetal variations (unossified bones or bones with incomplete ossification), and split supraoccipital. All these findings were considered to be treatment-related and adverse in a context of excessive maternal toxicity.

On the basis of the results obtained in this study:

- the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 180 mg/kg bw/d, based on adverse effects on body weight, body weight change and food consumption from 360 mg/kg bw/d and mortality at 720 mg/kg bw/d,

- the No Observed Adverse Effect Level (NOAEL) for embryo-foetal development was considered to be 360 mg/kg bw/d, based on embryo/foetal toxicity associated with malformations in a context of excessive maternal toxicity at 720 mg/kg bw/d.

 

 

3.      Endocrine Disrupting Properties

The screening for toxicity to reproduction performed on Grade 5 of BPA PO showed a concern for endocrine disruption in vivo. Further evaluation by QSAR model predictions on oestrogen receptor performed by the eCA indicates a potential concern that BPA PO may cause reproduction toxicity and endocrine-disrupting activity through binding of constituents of BPA PO to the oestrogen receptor.

Further toxicity to reproduction testing was therefore performed on Grade 4 of BPA PO. This test substance was selected as the potential endocrine disruption properties were considered related to higher degrees of propoxylation and Grade 5 of BPA PO is regarded as a polymer under REACH. The EOGRTS was designed to take into account the potential endocrine mode of action of the test substance.

In addition experimental testing for ER properties was requested by the eCA.

 

3.1 Stably Transfected Transactivation In Vitro Assays to Detect Estrogen Receptor Agonists and Antagonists

An assumption was made that an endocrine mode of action could be related to the binging of some constituents of Grade 5 of BPA PO to oestrogen receptor (ER). Therefore experimental in vitro testing for ER properties was performed. Stably Transfected Transactivation In Vitro Assays to Detect Oestrogen Receptor Agonists and Antagonists were conducted on various BPA PO grades and compounds – BPA 2PO and Grades 3, 4 and 5 of BPA PO – in accordance with the OECD Testing Guideline 455. The purpose was to identify if the test substances were ER agonist or antagonist and to confirm if ER properties – if any – increased with the degree of propoxylation of BPA PO compounds. These studies are reported in Section 7.12 of the Dossier.

The ability of the four BPA PO compounds to establish signal activation or blocking of the estrogenic receptor was investigated in a GLP compliant studies in accordance with the OECD Testing Guideline 455. Each of the four studies found the respective grade of BPA PO to be negative in both the ER agonist assay and ER antagonist assay when tested up to a concentration of 30 µM. All four tests concluded that the test substances do not show any estrogenic or anti-estrogenic activity when tested in stably transfected human Estrogen-α Transactivation Assay.

 

3.2 Review of endocrine disruption properties

An independent scientific review was commissioned to identify evidences of endocrine disrupting properties (as defined by the World Health Organization) for BPA PO, based on the studies performed on BPA 2PO and grades 3, 4 and 5 of the registered substance. The report is attached in Section 13 of the Dossier.

In this review, relevant endocrine endpoint activity that reflected alterations in the oestrogen (E), androgen (A), thyroid (T), and or steroidogenesis (S) pathways was extracted from identified in vivo mammalian and in vitro guideline studies. A hypothesis-driven weight-of-evidence assessment was conducted over eight defined hypotheses for endocrine activity across these pathways. This involved weighting endpoints by rank-ordered categories (Rank 1, 2, or 3) based on their relevance to a specific hypothesis. The studies reviewed were considered Level 2 to Level 5 studies within the OECD Conceptual Framework for Assessing Endocrine Disruptors (ED). Also evaluated were lines of evidence (LoE) that may suggest interference in vitamin D signalling pathways. However, at the present, there are no guidance criteria for this pathway, so endpoints that might suggest an alteration were identified but no conclusions can be made at this time.

In summary, based on the available data, BPA PO showed some changes in endpoints across LoE for both the E and T pathways. In the case of the E pathway, consistent findings across studies were not identified, with no agonist or antagonist bioactivity as measured in an in vitro ER transactivation assay. Although changes were identified across key events in the T pathway, there were no changes reported in developmental neurobehavioral measures even though several endpoints suggestive of neurotoxicity (see table below) were identified in the EOGRTS.

 

Endocrine Pathway

BPA-PO (CAS 37353-75-6)

Estrogen (E)

No ovarian weight change, with minimal interstitial gland hyperplasia; no in vitro ER transactivation. 

Androgen (A)

No significant changes of concern

Thyroid (T)

Changes in thyroid hormones that correlate with thyroid weight and histopathology and may suggest an alteration in T modality; however, no adverse effects on neurobehaviour, with ambiguous evidence of developmental neurotoxicity in high-dose rats; the only changes noted were increased thickness in corpus callosum in males, and decreased width of striatum in females.

Steroidogenesis (S)

LEI

Vitamin D signalling

Sternum: increased bone mass in trabeculae noted in adult cohorts (OECD 443), as well as increased bone (sternum) mass and osteoblast numbers in 13-week study (OECD 408).

LEI: limited endpoint information for evaluation

Link to relevant study records
Reference
Endpoint:
extended one-generation reproductive toxicity - with both developmental neuro- and immunotoxicity (Cohorts 1A, 1B without extension, 2A, 2B, and 3)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 17 January 2017 to 24 July 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Deviations:
yes
Remarks:
Deviations were considered not to have compromised the validity or integrity of the study
GLP compliance:
yes (incl. certificate)
Limit test:
no
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:

- Premating exposure duration for parental (P0) animals: Considering that the extension of Cohort 1B was included in the study design, and that this extension involved a 10-week premating exposure (covering the complete spermatogenesis and folliculogenesis before mating), a 2-week premating exposure for the P generation was deemed adequate.
- Basis for dose level selection: Doses were selected based on previous repeated-dose toxicity and toxicity to reproduction studies performed on the same grade of the registered substance. 540 mg/kg/day was selected as the high dose level. The low-dose and mid dose were selected using a ratio representing approximately a 3-fold interval (i.e. 60 and 180 mg/kg/day). After 6 days of treatment, 3 males from the high dose-group (540 mg/kg/day) had severe clinical signs and body weight losses. Therefore it was decided to lower the high-dose level to 360 mg/kg/day (2-fold interval between mid- and high-dose groups).
- Inclusion/exclusion of extension of Cohort 1B: It was considered necessary to include the extension of Cohort 1B as it allows to evaluate the potential effects on reproductive performance of the F1 animals which are exposed already during critical life stages in utero and early postnatal periods.
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B: Some observations performed during a previous toxicity to reproduction study performed on Grade 5 of the registered substance suggested potential neurodevelopmental toxicity, which supported further elucidating testing. Therefore Cohorts 2A and 2B were included.
- Inclusion/exclusion of developmental immunotoxicity Cohort 3: Findings from previous toxicity to reproduction study performed on Grade 5 suggested a potential mode of action for the registered substance that could induce developmental immunotoxicity. Therefore Cohort 3 was included.
- Route of administration: Oral was considered to be the relevant route of exposure.
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
RjHan: SD
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) males: approximately 13 weeks old. (P) females: approximately 12 weeks old.
- Weight at study initiation: (P) males: 459 g to 565 g. (P) females: 251 g to 329 g.
- Fasting period before study: No
- Housing: The P and F1 adult animals were group-housed (except 2 weeks before mating and during mating, gestation and lactation) in polycarbonate cages with stainless steel lids (in Tecniplast 2000P cages: up to 4/sex per cage (2065 cm2) for Cohort 1A animals and up to 5/sex per cage for Cohort 2A and Cohort 3 animals; in Tecniplast 2154 cages (954 cm2): up to 2/sex per cage before mating for Cohort 1B animals) containing autoclaved sawdust (SICSA, Alfortville, France) (see § Study plan adherence). Toward the end of gestation and during lactation, autoclaved wood shavings (SICSA, Alfortville, France) were provided to females and their litter as nesting material. Each cage contained two objects (rat hut and nylabone) for the environmental enrichment of the animals. The cages were placed in numerical order on the racks.
- Diet: SSNIFF R/M-H pelleted maintenance diet, batch Nos 68211320 and 89416605 (SSNIFF Spezialdiäten GmbH, Soest, Germany) ad libitum.
- Water: Tap water (filtered with a 0.22 µm filter) ad libitum.
- Acclimation period: Males were acclimated to the study conditions for a period of 17 days before treatment. Females were acclimated to the study conditions for a period of 5 days before the beginning of estrous cycle monitoring during the pre-treatment period.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%):50 ± 20%,
- Air changes (per hr): about 8 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12h/12h

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Preparation procedure (homogeneity and stability testing) for a range of concentrations covering the lowest and highest used in this study was investigated in a separate study.

VEHICLE
- Justification for use and choice of vehicle: The test substance is poorly soluble in water (<0.1 g/L) but proved to be soluble in olive oil. This vehicle was previously used during the screening for toxicity to reproduction and was therefore preferred for consistency’s sake.
- Concentration in vehicle: 0, 12, 36, 72 and 108 mg/mL
- Amount of vehicle: A constant dosage-volume of 5 mL/kg/day was used.
- Lot/batch no. BCBS0084V and BCBT7822
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation:
- Proof of pregnancy: vaginal plug or for sperm in a vaginal lavage referred to as Day 0 p.c.
- Each female was placed with the same male until mating occurred or 14 days had elapsed. When mating had not occurred after 2 weeks, the animals were separated without further opportunity for mating.
- After successful mating each pregnant female was caged: Individual housing was chosen since it is preferable for pregnant animals, littering and lactating females in order to not jeopardize gestation, littering and lactation phases, and to avoid aggressive behaviour around mating in males.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical technique: High Performance Liquid Chromatography with UV detection (HPLC/UV)

Principle and validation of the method: Analytical method developed and validated during a dedicated study prior to dose formulation analysis

Determination of test item concentrations in dose formulations:
- Eight dosages: once in each P-premating, P-mating, P gestation, P-lactation, F1-postweaning, F1-premating, F1 gestation and F1-lactation periods
- A sample was taken from control and test item dose formulations and analyzed using the validated method
- Acceptance criterion: measured concentration = nominal concentration ± 15%
Duration of treatment / exposure:
P Generation
- in the males (at least 10 weeks of treatment): 2 weeks before mating, during the mating period (up to 2 weeks), until euthanasia (after weaning of the pups),
- in the females (at least 8 to 10 weeks of treatment): 2 weeks before mating, during the mating period (up to 2 weeks), during gestation, during lactation until Day 21 p.p. (or Day 22 p.p. sacrifice after hematology, blood chemistry and urinalysis on Day 23 p.c. for the selected females), until euthanasia for females with no evidence of mating or no delivery (26 days after the last day of the mating period).

Cohort 1A
- in the males and females: from weaning (Day 22 p.p.), until euthanasia (from Days 98 to 101 p.p.).

Cohort 1B
- in the males: from weaning (Day 22 p.p.) for at least 10 weeks before mating, during the mating period (up to 2 weeks), after sacrifice of F2 pups (on Day 4 p.p.),
- in the females: from weaning (Day 22 p.p.) for at least 10 weeks before mating, during the mating period (up to 2 weeks), during gestation, during lactation until Day 4 p.p. inclusive, until euthanasia for females with no evidence of mating or no delivery (24-26 days after the last day of the mating period).

Cohort 2A
- in the males and females: from weaning (Day 22 p.p.), until euthanasia (after completion of behavioral testing: Days 75 to 78 p.p.).

Cohort 2B
There was no direct dosing in Cohort 2B animals (sacrificed on Day 22 p.p.).

Cohort 3
- in the males and females: from weaning (Day 22 p.p.), until 5 days after KLH injection on Day 56 (± 3) p.p. (sacrifice on Day 61 (± 3) p.p.).
Frequency of treatment:
Daily
Details on study schedule:
- F1 parental animals not mated until 10 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 22 days of age.
- Age at mating of the mated animals in the study: 13 or 14 weeks
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
60 mg/kg bw/day (nominal)
Dose / conc.:
180 mg/kg bw/day (nominal)
Dose / conc.:
360 mg/kg bw/day (nominal)
Remarks:
In the first instance 540 mg/kg bw/day was selected as the high dose level. After 6 days of treatment, 3 males from the high dose-group had severe clinical signs and body weight losses. Therefore it was decided to:
- lower the high-dose level to 360 mg/kg bw/day (2-fold interval between mid- and high-dose groups),
- not treat the males on Day 7,
- treat the females at 540 mg/kg/day on Day 7,
- treat all high-dose group animals at 360 mg/kg bw/day from Day 8.
No. of animals per sex per dose:
P: 24 animals/sex/dose
Cohort 1A: 20 animals/sex/dose
Cohort 1B: 20 animals/sex/dose
Cohort 2A: 10 animals/sex/dose
Cohort 2B: 10 animals/sex/dose
Cohort 3: 10 animals/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on previous repeated-dose toxicity and toxicity to reproduction studies performed on the test substance via the oral route.
Positive control:
Not included
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: From arrival, each animal was observed at least once a day as part as routine examinations. From the start of the treatment period each animal was observed at least twice a day (before and after treatment) at approximately the same time of day, for the recording of clinical signs.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical examinations were performed on all animals once a week until the end of the study. Observations included (but were not limited to) changes in the skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behavior (e.g. self mutilation, walking backwards) were also recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each male was recorded on the first day of treatment (Study Day 1), then at least once a week until euthanasia.
The body weight of each female was recorded on the first day of treatment (Study Day 1), then once a week until mated (or until euthanasia for females with no evidence of mating), on Days 0, 4, 7, 10, 14, 17 and 20 p.c. and, on Days 1, 4, 7, 14 and 21 p.p.
In addition, to the scheduled period of body weight measurements, males from high-dose group (540/360 mg/kg/day) were weighed on Day 7 (see § Study plan adherence).
Animals were weighed at euthanasia as normal procedure before pathology examination.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- The quantity of food consumed by each male was measured once a week from the first day of treatment until the start of the mating period and after the mating period until euthanasia.
The quantity of food consumed by each female was measured once a week from the first day of treatment until the start of the mating period, during gestation for the intervals: Days 0-4, 4-7, 7-10, 10-14, 14-17 and 17-20 p.c. and during lactation for the intervals: Days 1-4, 4-7, 7-14, and 14 21 p.p.
During the mating period, food consumption was not measured for males or females.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
Oestrous cyclicity (parental animals):
The estrous cycle stage was determined from a fresh vaginal lavage (stained with methylene blue), each morning:
- during 12 days of the pre-treatment period,
- during the 2 weeks of the premating period,
- during the mating period, until the females were mated or the mating period had ended,
- on the day of sacrifice (premature or scheduled)
Sperm parameters (parental animals):
Parameters examined in P and F1 male parental generations: testis weight, epididymis weight, sperm count in testes, sperm count in epididymides, sperm motility, sperm morphology.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter (5/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), presence of nipples/areolae in male pups.
The following parameters were examined in F2 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies.

GROSS EXAMINATION OF DEAD PUPS: Yes
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and euthanized by exsanguination after weaning of the F1 progeny.
- Maternal animals: All surviving animals were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and euthanized by exsanguination on Days Days 22-23 p.p, or on Days 26 or 27 p.c. for P females which did not deliver and 26 days after the end of the mating period when no delivery occurred.

GROSS NECROPSY
- Gross necropsy consisted of examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues. Special attention was paid to the reproductive organs. The numbers of implantation sites were recorded for females euthanized on Days 22-23 p.p. The numbers of corpora lutea and implantation sites were recorded for the female euthanized 26 days after the end of the mating period with no evidence of mating and for females euthanized on Day 26 p.c. due to no delivery.


HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively.
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
- Gross necropsy consisted of examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues. Special attention was paid to the reproductive organs. The numbers of implantation sites were recorded for females euthanized on Days 22-23 p.p. The numbers of corpora lutea and implantation sites were recorded for the female euthanized 26 days after the end of the mating period with no evidence of mating and for females euthanized on Day 26 p.c. due to no delivery.

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively.
Statistics:
Body weight, food consumption and reproductive data: Data were compared by one-way variance analysis and Dunnett's test, (mean values being considered as normally distributed, variances being considered as homogeneous) or by Fisher’s exact probability test (proportions).

Organ weights: PathData software was used to perform the statistical analysis of organ weight data (level of significance of 0.05 or 0.01).

Auditory startle reflex test, splenic lymphocyte immunophenotyping and number of primary follicles
Statistical analysis was performed using the SAS Enterprise Guide software. To compare the auditory startle reflex response (categorical data using positive or negative values) between treatment groups, a Fisher's exact test was used. If a global statistically significant difference is observed between treatment groups, multiple Fisher's exact tests was performed to compare the auditory startle reflex responses of each test item-treated group with that of the control group. The latency and amplitude of auditory startle reflex responses (by sex for each of the sound frequencies used) and the mean number per section of primordial follicles of P and F1 females were analyzed using the following decision tree. Animals for which auditory startle reflex data was not interpretable are excluded from statistical analyses.

Ano-genital distance, locomotor activity, number of nipples and areolae, time of preputial separation/vaginal opening, time to first estrous after vaginal opening/patency, seminology, hematology, blood biochemistry, urinalysis, thyroid hormones, anti KLH IgM and post-implantation loss: Citox software was used to perform the statistical analyses of data.
Reproductive indices:
Female mating index
Female fertility index
Lactation index
Offspring viability indices:
Live birth index
Viability index on Day 4 p.p.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Males: Ptyalism was observed in all groups (including controls) but at increased incidence in test item treated groups. This finding was considered to be test item treatment-related but not adverse.
At 540/360 mg/mg/kg/day, there were test item treatment-related effects which were considered to be adverse based on findings (thin appearance, hunched posture, piloerection, hypotonia , dyspnea and/or soiled anus/soft feces), incidence (up to 16 males affected vs. one in controls) and duration (from Day 6 up to 59).
At 180 mg/kg/day, piloerection was recorded. Taking into account the limited incidence (2 males vs. 1 male in controls) and duration (from Day 10 to 12), this findings was considered to be non-adverse.
At 60 mg/kg/day, there were no adverse findings.
Other findings observed at low and/or comparable incidence across groups (including controls) are commonly observed in this species/strain of animals and therefore not considered to be test item treatment-related.

Females: Ptyalism was observed in all groups (including controls) but at increased incidence in test item treated groups. This finding was considered to be test item treatment-related but not adverse.
At 540/360 mg/mg/kg/day, there were test item treatment-related effects which were considered to be adverse based on findings (hunched posture and piloerection), incidence (up to 3 females affected vs. none in controls) and duration (up to 5 days).
At 180 mg/kg/day, there were no adverse findings.
At 60 mg/kg/day, one female had an episode of hunched posture/piloerection on study Day 19. As this finding was limited to one animal, to one day and not observed at the mid-dose, a test item treatment-related effect was considered unlikely.
Other findings observed at low and/or comparable incidences across groups (including controls) are commonly observed in this species/strain of animals or in pregnant animals, and therefore not considered to be test item treatment-related.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
In males, there were no unscheduled deaths.

In the control group:
- One female was sacrificed for difficulties to deliver on Day 23 p.c.
- One female was sacrificed on Day 26 p.c. (study Day 43) for no delivery.
- One female was sacrificed on Day 6 p.c. (study Day 25) due to the presence of a mass close to a mammary gland.
In the 60 mg/kg/day group:
- One female was found dead on Day 24 p.c. (study Day 43).
In the 180 mg/kg/day group:
- One female was sacrificed on Day 26 p.c. (study Day 43) for no delivery.
- One female was sacrificed on study Day 57 for no mating.
In the 540/360 mg/kg/day group:
- One female was sacrificed for difficulties to deliver on Day 24 p.c. (study Day 44).
- One female was sacrificed for difficulties to deliver on Day 23 p.c. (study Day 41).
- One female was sacrificed on Day 26 p.c. (study Day 45) for no delivery.
- One female was sacrificed on Day 26 p.c. (study Day 46) for no delivery.

Overall, there were no tendencies toward any test item treatment-related deaths in females. The only potential concern could be the difficulty to deliver observed in two high-dose females. However, this is a common observation in animals of this species/strain (also observed in one control female) for which a test item treatment relationship was therefore excluded.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weight: when compared with controls, there were statistically significant differences at 180 mg/kg/day in males and, at 540/360 mg/kg/day in both sexes. While treatment-related, none of these differences raised a toxicological concern (less than 10% differences when compared with controls). There were no effects at 60 mg/kg/day.

Mean body weight change: in the 540/360 mg/kg/day group, there was a body weight loss in males (-28g vs. +22 g in controls) on Days 1 to 8. Mean body weight gain returned toward control values when the high dose level was lower to 360 mg/kg/day. In the 180 or 60 mg/kg/day group there were no adverse effects on mean body weight gains, despite a few statistically significant differences of low amplitudes which were considered to test item treatment-related.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
In the 540/360 mg/kg/day and when compared with controls, males had a lower mean food consumption (-30% vs. controls, p<0.001) on Days 1 to 8. Mean food consumption returned toward control values when the high dose level was lower to 360 mg/kg/day. In females, there were no dose related differences.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
When compared with control or Historical Control Data, there were no relevant differences in hematology and coagulation parameters. In females and when compared with Historical Control Data, there were incidental higher mean neutrophils counts in all groups, including controls.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In males:
- at 60 mg/kg/day when compared with controls and Historical Control Data, there were no relevant differences,
- from 180 mg/kg/day, there was an increased cholesterol concentration (+89% vs. controls, p<0.01) which exceeds the upper limit of the Historical Control Data (3.77 vs. 2.69 mmol/L),
- at 540/360 mg/kg/day, there were higher concentration of calcium (+11% vs. controls, p<0.01) which exceeds the upper limit of the Historical Control Data (2.87 vs. 2.83 mmol/L), higher urea and creatinine concentration (+15% and +12% vs. controls, p<0.05) which exceeds the upper limit of the Historical Control Data (42.62 vs. 40.22 µmol/L) and higher alanine aminotransferase activity (+38% vs. controls, p<0.01) which exceeds the upper limit of the Historical Control Data (65 vs. 52 U/L).

In females:
- at 60 and 180 mg/kg/day when compared with controls and Historical Control Data there were no relevant differences,
- at 540/360 mg/kg/day, there was an increased cholesterol concentration (+52% vs. controls, p<0.01) which marginally exceeds the upper limit of the Historical Control Data (4.10 vs. 4.08 mmol/L).

Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no effects on urinalysis parameters.

At 540/360 mg/kg/day and when compared with controls, there was a statistically significant higher urinary volume (12 vs. 7 mL, p<0.5) in males. However the value remained lower than the maximal range of Historical Control Data and this increase was therefore considered fortuitous.
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):
Lower final body weights were noted in males treated at 180 or 540/360 mg/kg/day, respectively at -5 and 7% (p<0.05 and 0.01).
Increased absolute and relative-to-body liver weights were recorded in males and females treated at 540/360 mg/kg/day (up to +31% in males in relative-to-body weights; p<0.01). These differences were related to the test item administration and correlated with the microscopic hepatocellular hypertrophy.
Increased absolute and relative-to-body adrenal gland weights were recorded in males and females treated at 540/360 mg/kg/day (up to +22% in females in absolute and relative-to-body weights; p<0.01). These differences were related to the test item administration and correlated with the microscopic increased cortical vacuolation.
Increased absolute and relative-to-body thyroid gland weights were recorded in males treated at 540/360 mg/kg/day (up to +24% in males in relative-to-body weights; p<0.01). These differences were related to the test item administration and correlated with the microscopic follicular cell hypertrophy.
The other organ weight changes were not considered to be test item related because they were of insufficient magnitude, were not dose-related and/or did not correlate to microscopic findings.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Slightly increased incidence of dilated pelvis was noted in the kidneys from males treated at 540/360 mg/kg/day. This change correlated with the microscopic dilation of pelvis.
Although poorly dose-related, slightly increased black/brown/red discolorations were noted in the stomach from males and females treated at 60, 180 or 540/360 mg/kg/day. This correlated with erosion/ulcer seen at minimally higher incidence in test item animals. A relationship to test item administration could not be excluded.

The few other isolated gross observations were considered to be consistent with spontaneous findings encountered in the rats of these strain and age.
This included the testes and epididymides that were small in 2 males treated at 540/360 mg/kg/day. In addition, small testes were noted in 1 male treated at 180 mg/kg/day. Although seen only in mid- and high-dose males, these findings were considered to be in the range of reference data and thus were considered to be of spontaneous origin.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The following test item-related findings were noted:
- kidney: minimal to moderate lesions including mineralization of tubules; mineralization of pelvis; scar in cortex; increased basophilia of tubules; increased infiltrates of mononuclear cells; dilation of tubule; increased dilation of pelvis in males treated at 540/360 mg/kg/day. The relationship to test item of tubular mineralization in 3/24 males treated at 180 mg/kg/day was considered to be equivocal in view of the low incidence and the absence of other renal test item-related findings,
- thyroid glands: minimal to slight follicular cell hypertrophy in males and females treated at 540/360 mg/kg/day,
- lungs: minimal foamy alveolar macrophages in males treated at 540/360 mg/kg/day,
- liver: increased incidence of minimal altered cell foci (clear cells) in males treated at 540/360 mg/kg/day that was considered to be adverse; minimal single cell necrosis/apoptosis located in centrilobular areas in males treated at 540/360 mg/kg/day and that was considered to be adverse; minimal to slight hepatocellular hypertrophy (centrilobular or centrilobular/midzonal distribution) in males and females treated at 180 or 540/360 mg/kg/day,
- adrenal glands: minimal increased vacuolation in the cortex from males treated at 540/360 mg/kg/day,
- jejunum: minimal to moderate ectasia of lymphatics in males and females treated at 180 or 540/360 mg/kg/day and considered to be related in part to the oily nature of the vehicle (olive oil),
- sternum: minimal increased bone mass in trabeculae in males treated at 540/360 mg/kg/day,
- bone marrow: minimal increased myeloid/erythroid ratio; minimal decreased cellularity in males treated at 540/360 mg/kg/day,
- skin: slight ulcer and minimal to slight serocellular crust in 2 males treated at 540/360 mg/kg/day with cutaneous macroscopic lesions,
- stomach: minimal to slight erosion/ulcer in 5/22 females treated at 540/360 mg/kg/day. In females treated at 60 or 180 mg/kg/day, the presence of isolated erosion/ulcer was considered to be unrelated to test item administration in view of their low incidence.

These findings were considered not to be adverse except when specified (i.e. altered cell foci and single cell necrosis/apoptosis in the liver from males treated at 540/360 mg/kg/day).

In addition, focal mineralization was noted in the urinary bladder from one male treated at 540/360 mg/kg/day, in association with reactive urothelium hyperplasia. Although isolated, this finding was considered to be probably related to test item administration in view of the other renal findings including mineralization.
The stomach erosion/ulcer in high-dose females was associated occasionally with degeneration/necrosis of glandular cells or infiltrate of giant cells around mineralization. These findings were considered to be related to the test item administration, although seen at low incidences.

The remaining microscopic findings were not considered to be associated with the test item administration because these findings were consistent with spontaneous and background findings described in the literature, the findings were distributed randomly among groups, or their appearance was similar to changes found in controls.

There were no test item-related changes in the male or female reproductive organs nor in nervous system.

There was a good correspondence between the vaginal smears and the histopathological examination of estrus cycle.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Thyroid hormones: At 540/360 and 180 mg/kg/day, when compared with controls, there were dose related decreases in T4 concentrations (26.4 and 30.5 vs. 35.3 ng/mL, respectively with p<0.01 or not statistically significant) associated with increased concentrations in TSH (5736 and 4777 vs. 1934 pg/mL, respectively with p <0.01). These findings were considered to be test item treatment-related and adverse at 540/360 mg/kg/day (TSH value exceeding the upper limit of Historical Control Data). At 60 mg/kg/day there were no effects,
Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
In the 540/360 mg/kg/day group and when compared with controls, there was an increased number of females in diestrus (7.3 days vs. 5.0 days, p<0.001), resulting in a prolongation of mean oestrous cycle duration (5.4 days vs. 3.9 days, p<0.001). This finding was considered to be test item treatment-related and adverse.

In the 180 and 60 mg/kg/day groups and when compared with controls, there were no effects on mean oestrous parameters.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
There were no test item treatment-related effects in sperm analysis data
Reproductive performance:
no effects observed
Description (incidence and severity):
There were no effects on mean mating and fertility data of the P generation.

In the 540/360 mg/kg/day group and when compared with controls or Historical Control Data, there was a lower fertility index (91.7% vs. 100% in controls) close to the lower value recorded in Historical Control Data (91.7% vs. 92.0% in HCD). Therefore a test-item treatment relationship was considered unlikely.
There was no test item-related mortality.

Clinical signs:
- ptyalism was observed in all groups (including controls) but at increased incidences in test item treated groups. This finding was considered to be test item treatment-related but not adverse,
- after 6 days of treatment, 3 out 24 males from the high dose-group (540 mg/kg/day) had severe clinical signs and body weight losses which resulted in the decision to decrease the high dose level to 360 mg/kg/day. At 540/360 mg/mg/kg/day, there were test item treatment-related effects which were considered to be adverse based on findings (thin appearance, hunched posture, piloerection, hypotonia, dyspnea and/or soiled anus/soft feces), incidence (up to 16 males affected vs. none in controls) and duration (from Day 6 up to 59),
- in females, at 540/360 mg/mg/kg/day, there were test item treatment-related effects which were considered to be adverse based on findings (hunched posture and piloerection), incidence (up to 3 females affected vs. none in controls) and duration (up to 5 days),
- there were no test item treatment-related adverse clinical signs at 180 or 60 mg/kg/day.

Mean body weight and mean body weight changes: When compared with controls, there were a few statistically significant differences at 180 and 540/360 mg/kg/day in males or at 540/360 mg/kg/day in females. While treatment-related, none of these differences raised a toxicological concern (less than 10% differences when compared with controls). There were no effects at 60 mg/kg/day.

Food consumption: In the 540/360 mg/kg/day and when compared with controls, males,had a lower mean food consumption (-30% vs. controls, p<0.001) on Days 1 to 8. Mean food consumption returned toward control values when the high dose level was lower to 360 mg/kg/day. In females, there were no dose related differences.

Estrous cycle: In the 540/360 mg/kg/day group and when compared with controls, there was an increased mean number of diestrus (7.3 days vs. 5.0 days, p<0.001), resulting in a prolonged duration of mean estrous cycle (5.4 days vs. 3.9 days, p<0.001). This finding was considered to be test item treatment related and adverse. In the 180 and 60 mg/kg/day groups and when compared with controls, there were no effects on mean estrous parameters.

Mating and fertility: There were no effects on mean mating and fertility data of the P generation.

Delivery data: There were no relevant effects on mean delivery data of the P generation females.

Increased liver and adrenal gland weights were recorded in males and females treated at 540/360 mg/kg/day and increased thyroid gland weights were recorded in males treated at 540/360 mg/kg/day.
Slightly increased incidence of dilated pelvis was grossly noted in the kidneys from males treated at 540/360 mg/kg/day.
The following test item-related microscopic findings were noted:
- kidney: mineralization of tubules, mineralization of pelvis, scar in cortex, increased basophilia of tubules, increased infiltrates of mononuclear cells, dilation of tubule and/or increased dilation of pelvis in males treated at 540/360 mg/kg/day,
- thyroid glands: follicular cell hypertrophy in males and females treated at 540/360 mg/kg/day,
- lungs: foamy alveolar macrophages in males treated at 540/360 mg/kg/day,
- liver: increased incidence of altered cell foci (clear cells) and single cell necrosis/apoptosis that were considered to be adverse, in males treated at 540/360 mg/kg/day, hepatocellular hypertrophy in males and females treated at 180 or 540/360 mg/kg/day,
- adrenal glands: increased vacuolation in the cortex from males treated at 540/360 mg/kg/day,
- jejunum: ectasia of lymphatics in males and females treated at 180 or 540/360 mg/kg/day,
- sternum: increased bone mass in trabeculae in males treated at 540/360 mg/kg/day,
- bone marrow: increased myeloid/erythroid ratio and decreased cellularity in males treated at 540/360 mg/kg/day,
- skin: ulcer and minimal to slight serocellular crust in 2 males treated at 540/360 mg/kg/day,
- stomach: erosion/ulcer in 5/22 females treated at 540/360 mg/kg/day.

Quantitative evaluation of primordial follicles on PCNA-stained slides: There were no differences between the high-dose and control groups, with respectively a mean total number of 14.50 and 11.42 primordial follicles per section.

Hematology and coagulation: When compared with control or Historical Control Data, there were no relevant differences in hematology and coagulation parameters.

Blood biochemistry: In males, at 60 mg/kg/day there were no relevant differences; from 180 mg/kg/day, there was an increased cholesterol concentration (+89% vs. controls, p<0.01) and at 540/360 mg/kg/day, there were higher concentrations of calcium (+11% vs. controls, p<0.01) and creatinine (+12% vs. controls, p<0.05) along with higher alanine aminotransferase activity (+38% vs. controls, p<0.01). In females, at 60 and 180 mg/kg/day there were no relevant differences; at 540/360 mg/kg/day, there was an increased cholesterol concentration (+52% vs. controls, p<0.01).

Urinalysis: There were no test item treatment-related findings on urinalysis parameters.

Thyroid hormones: At 540/360 and 180 mg/kg/day, when compared with controls, there were dose related decreases in T4 concentrations (26.4 and 30.5 vs. 35.3 ng/mL, respectively with p<0.01 or not statistically significant) associated with increased concentrations in TSH (5736 and 4777 vs. 1934 pg/mL, respectively with p <0.01). These findings were considered to be test item treatment-related and adverse at 540/360 mg/kg/day (TSH value exceeding the upper limit of Historical Control Data). At 60 mg/kg/day there were no effects.

Sperm analysis: There were no test item treatment-related effects in sperm analysis data.
Key result
Dose descriptor:
NOAEL
Effect level:
180 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
histopathology: non-neoplastic
reproductive function (oestrous cycle)
other: Thyroid hormones
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Ptyalism was observed in all treated groups at dose level related increased incidences. This finding was considered to be test item treatment-related but not adverse. There were no other test item treatment-related clinical signs.
Mortality / viability:
no mortality observed
Description (incidence and severity):
There were no test item treatment-related deaths.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no adverse effects.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no effects on mean food consumption.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
In males and females, despite a few statistically significant differences when compared with controls, there were no relevant findings when compared with Historical Control Data.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In males:
- at 60 mg/kg/day when compared with controls and Historical Control Data there were no relevant differences,
- at 180 and 540/360 mg/kg/day, there were increased cholesterol concentrations (+69% and +137% vs. controls, respectively with p<0.01) which exceeds the upper limit of the Historical Control Data (2.69 mmol/L).

In females:
- at 60 and 180 mg/kg/day when compared with controls and Historical Control Data there were no relevant differences,
- at 540/360 mg/kg/day, there was an increased cholesterol concentration (+100% vs. controls, p<0.01) which marginally exceeds the upper limit of the Historical Control Data (4.28 vs. 4.08 mmol/L).
Urinalysis findings:
no effects observed
Description (incidence and severity):
In males and females, despite a statistically significant difference in mean urinary volume in females when compared with controls, there were no relevant findings when compared with Historical Control Data.
Sexual maturation:
no effects observed
Description (incidence and severity):
When compared with controls, there were no statistically significant differences in the mean age of balanopreputial separation in males, vaginal opening in females or mean time to first oestrous after vaginal opening in females.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Cohort 1A: Increased absolute and relative-to-body kidney weights were recorded in males and females treated at 360 mg/kg/day (up to +14% in males in relative-to-body weights or in females in absolute weights; p<0.01). These differences were related to the test item administration and correlated with the microscopic renal lesions, mainly the pelvis dilatation.
Increased absolute and relative-to-body liver weights were recorded in males and females treated at 180 or 360 mg/kg/day (up to +34% in males in relative-to-body weights; p<0.01). These differences were related to the test item administration and correlated with the microscopic hepatocellular hypertrophy.
Increased absolute and relative-to-body adrenal gland weights were recorded in males and females treated at 360 mg/kg/day (up to +24% in males in relative-to-body weights or in females in absolute weights; p<0.01). These differences were related to the test item administration and correlated with the microscopic increased cortical vacuolation.
The other organ weight changes were not considered to be test item related because they were of insufficient magnitude, were not dose-related and/or did not correlate to microscopic findings.

Cohort 1B: Increased absolute and relative-to-body liver weights were recorded in males treated at 60 or 180 mg/kg/day, and in males and females treated at 360 mg/kg/day (up to +34% in males in relative to body weights; p<0.01). These differences were related to the test item administration and correlated with the microscopic hepatocellular hypertrophy.
Decreased absolute and relative-to-body pituitary gland weights were recorded in females treated at 360 mg/kg/day (up to -15% in relative-to-body weights; p<0.05). In the absence of microscopic examination of this Cohort, in view of the low magnitude of this observation and since there were no similar findings in Cohort 1A, these differences were considered to be probably not related to the test item administration.
Decreased absolute prostate weights were recorded in males treated at 360 mg/kg/day (-17%; p<0.01). In the absence of microscopic examination of this Cohort, in view of the low magnitude of this observation and since there were no similar findings in Cohort 1A, these differences were considered to be probably not related to the test item administration.
The other organ weight changes were not considered to be test item related because they were of insufficient magnitude and/or were not dose-related.

Cohort 2A: Increased absolute and relative-to-body liver weights were recorded in males and females treated at 360 mg/kg/day (up to +32% in males in absolute weights; p<0.01). These differences were related to the test item administration and correlated probably with the microscopic hepatocellular hypertrophy, although only partial microscopic examination was performed.
The other organ weight changes were not considered to be test item related because they were of insufficient magnitude, were not dose-related and/or did not correlate to microscopic findings.

Cohort 2B: There were no test item-related organ weight changes. The few organ weight changes were not considered to be test item related because they were of insufficient magnitude and/or were not dose-related.

Cohort 3: Increased absolute and relative-to-body liver weights were recorded in males and females treated at 360 mg/kg/day (up to +30% in males in relative-to-body weights; p<0.01). These differences were related to the test item administration.
Increased absolute and relative-to-body adrenal gland weights were recorded in females treated at 360 mg/kg/day (up to +25% in females in relative-to-body weights; p<0.01). These differences were related to the test item administration.
Increased absolute and relative-to-body ovaries weights were recorded in females treated at 360 mg/kg/day (up to +21% in relative-to-body weights; p<0.01). In the absence of similar change in the other cohorts and since the ovaries were not examined microscopically, the relationship to test item administration was considered to be equivocal.
The other organ weight changes were not considered to be test item related because they were of insufficient magnitude, were not dose-related and/or did not correlate to microscopic findings.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Cohort 1A: Dose-related test item-related dilated pelvis was noted in the kidneys from several males treated at 60 or 180 mg/kg/day, and in males and females treated at 360 mg/kg/day. This change correlated to the microscopic dilation of pelvis.

Cohort 1B: Dose-related dilated pelvis was noted in the kidneys from a few males treated at 60, 180 or 360 mg/kg/day.
Enlarged liver was recorded in one male treated at 360 mg/kg/day.
Cutaneous scabs were noted in the dorsal region from a few males treated at 60, 180 or 360 mg/kg/day and in one female treated at 180 mg/kg/day. Although seen also in one control female, a relationship to test item administration could not be excluded in males.
The few other isolated gross observations were considered to be consistent with spontaneous findings encountered in the rats of these strain and age.

Cohort 2A: Dilated pelvis was noted in the kidneys from 9/10 males treated at 360 mg/kg/day. This change correlated to the microscopic dilation of pelvis and was related to the test item administration. The dilated pelvis seen in isolated female treated at 60 mg/kg/day and males treated at 180 mg/kg/day were not related to the test item administration in view of the minimal incidence and the non-dose-relationship in females.
Enlarged liver was recorded in one male treated at 360 mg/kg/day and correlated with hepatocellular hypertrophy. The enlarged liver seen in one female treated at 60 mg/kg/day was not observed microscopically and thus a relationship to test item could not be ruled out.
Black discoloration was seen in the stomach from 2/10 females treated at 180 mg/kg/day and in 2/10 females 360 mg/kg/day. Although seen also in 2/10 control males, a relationship to test item administration could not be excluded.
The few other isolated gross observations were considered to be consistent with spontaneous findings encountered in the rats of these strain and age.

Cohort 2B: There were no test item-related gross changes. The few isolated gross observations were considered to be consistent with spontaneous findings encountered in the rats of these strain and age.

Cohort 3: Black discoloration was seen in the stomach from 1/10 control males and in 1/10 females treated at 360 mg/kg/day and brown discoloration was recorded in the stomach from 1/10 females treated at 360 mg/kg/day. Although seen at low incidences and also in control males, a relationship to test item administration of these changes could not be excluded in females.
The few other isolated gross observations were considered to be consistent with spontaneous findings encountered in the rats of these strain and age.
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
Cohort 1A: The following test item-related findings were noted:
- kidney: minimal to marked lesions including dystrophic mineralization in interstitium; interstitium fibrosis; dilation of tubules; pigment; cyst; dilation of pelvis; chronic inflammation in interstitium that was considered to be adverse in males and females treated at 360 mg/kg/day because the grade was noted up to marked; inflammation in pelvis. All these findings were considered to be related to test item administration in males and females treated at 360 mg/kg/day. In the other groups treated at 60 or 180 mg/kg/day, the incidence and severity were considered to be similar to those encountered in untreated rats. The isolated degeneration/necrosis of tubules seen in one male treated at 60 mg/kg/day and in one female treated at 360 mg/kg/day was considered not to be related to the test item administration in view of the low number of affected animals and the absence of dose-relationship in males,
- thyroid glands: minimal to slight follicular cell hypertrophy in males or females treated at 360 mg/kg/day,
- lungs: minimal foamy alveolar macrophages in males treated at 180 or 360 mg/kg/day and considered to be of low toxicological importance in view of the low incidence (3/20 or 2/20) and severity,
- liver: minimal single cell necrosis/apoptosis in males treated at 180 or 360 mg/kg/day that was considered to be adverse; minimal to slight hepatocellular hypertrophy in males treated at 180 mg/kg/day and in males and females treated at 360 mg/kg/day,
- jejunum: minimal to slight ectasia of lymphatics considered to be related in part to the oily nature of the vehicle (olive oil) in males and females treated at 180 or 360 mg/kg/day,
- sternum: minimal increased bone mass in trabeculae in males and females treated at 180 or 360 mg/kg/day. The relationship to test item of the minimal increased bone mass in trabeculae seen an isolated female treated at 60 mg/kg/day was considered to be equivocal in view of the low incidence of this change,
- stomach: minimal erosion/ulcer in 2/20 males treated at 360 mg/kg/day.
These findings were considered not to be adverse except when specified i.e. renal chronic inflammation in interstitium at 360 mg/kg/day and single cell necrosis/apoptosis in the liver from males treated at 180 or 360 mg/kg/day. No test item related findings were seen in the urinary bladder.
The remaining microscopic findings were not considered to be associated with the test item administration because these findings were consistent with spontaneous and background findings described in the literature, the findings were distributed randomly among groups, or their appearance was similar to changes found in controls.
There were no test item-related changes in the male or female reproductive organs nor in nervous system.
There was a good correspondence between the vaginal smears and the histopathological examination of estrus cycle.

Cohort 2A: The following test item-related findings were noted:
- kidney: minimal to moderate lesions including dystrophic mineralization in interstitium; interstitium fibrosis; mononuclear inflammatory cell infiltrate; dilation of tubules; cyst; dilation of pelvis; chronic inflammation in interstitium that was considered to be adverse at 360 mg/kg/day,
- liver: minimal single cell necrosis/apoptosis that was considered to be adverse; minimal hepatocellular hypertrophy,
- stomach: minimal to slight erosion/ulcer seen with higher incidence and/or severity in high-dose males and females,
- ureters: dilated lumen in high-dose males,
- skin: slight ulcer; moderate sero-cellular crust; minimal hyperkeratosis.
These findings were considered not to be adverse except when specified (i.e chronic inflammation in the kidneys).
The examination by the pathologist of the HE-stained multiple brain sections allowed examination of olfactory bulbs, cerebral cortex, hippocampus, basal ganglia, thalamus, hypothalamus, mid-brain (thecum, tegmentum, and cerebral peduncles), brain-stem and cerebellum. There were no test item-related findings in these areas.
There were no effects in the eyes (retina and optic nerve), peripheral nerve, muscle or spinal cord.


Cohort 2B: There were no test item-related microscopic findings.
The few microscopic findings were not considered to be associated with the test item administration because these findings were consistent with spontaneous and background findings.
The examination by the pathologist of the HE-stained multiple brain sections allowed examination of olfactory bulbs, cerebral cortex, hippocampus, basal ganglia, thalamus, hypothalamus, mid-brain (thecum, tegmentum, and cerebral peduncles), brain-stem and cerebellum. There were no test item-related findings in these areas.
There were no effects in the eyes (retina and optic nerve), peripheral nerve, muscle or spinal cord.

Cohort 3: Slight erosion/ulcer was noted in the stomach from 2/10 females treated at 540/360 mg/kg/day and was considered to be related to the test item administration.
The few other microscopic findings were not considered to be associated with the test item administration because these findings were consistent with spontaneous and background findings.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Thyroid hormones:
In non-selected F1 offspring: there were no effects on T4 or TSH plasma samples.
In Cohort 1A males and females: In males at 540/360 and 180 mg/kg/day, when compared with controls, there were increased concentrations in TSH (2937 and 3051 vs. 1421pg/mL, respectively with no statistical significance and p<0.05). These increases were not associated with a similar trend for T4 levels. Therefore a test-item treatment relationship was considered dubious. At 60 mg/kg/day there were no effects. In females, at 540/360 and 180 mg/kg/day, when compared with controls, there were increased concentrations in TSH (996 and 1085 vs. 544 pg/mL, respectively with p <0.01 and p<0.05) associated with increases in T4 levels (+12 and +13% vs. controls, respectively not statistically significant). These findings were considered to be test item treatment-related but not adverse (within the range of the Historical Control Data). At 60 mg/kg/day there were no effects.

Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Cohort 2A animals were tested once between Day 64 and 66 p.p.. There was no abnormal reactivity to manipulation or to different stimuli.

Detailed clinical examination
- "touch escape" or "ease of removal from the cage" (reactivity to handing) were normal,
- fur appearance (except for 1 or 2 animals/sex in the 180 or 540/360 mg/kg/day group, respectively), gait, posture, behavior and breathing were normal,
- there were no excessive grooming, defecation, urination in treated groups when compared with the control group,
- there were no salivation, no lacrimation, no piloerection and no palpebral closure,
- all pups had a normal pupil size (myosis) at examination and there were no exophthalmos,
- there were no tremors, no twitches, no clonic/tonic convulsions, no ataxia, no hypoactivity, no hyperactivity, no hypotonia and no stereotypies.

Reactivity to stimuli
- visual stimulus response and papillary reflex were normal in all groups,
- auditory startle reflex was normal in all groups,
- forelimb strength was normal in all groups.
Developmental immunotoxicity:
no effects observed
Description (incidence and severity):
Cohort 1A: At 540/360 mg/kg/day and when compared with controls, significant variations consisted in a moderate decrease in relative cytotoxic T cells count (-20% vs. controls) in males. In females, a moderate decrease in relative NK cells count (-32% vs. controls) and a moderate increase in relative T cells count (+28% vs. controls) was observed. Other observed changes in the studied parameters were considered to be either non-significant or non-related to the treatment.
At 180 or 60 mg/kg/day and when compared with controls, there were no effect.

Cohort 3: Before KLH immunization, all samples displayed quantifiable concentrations of anti-KLH IgM. This result can be explained by the fact that rat serum is expected to contain IgMs directed against some glycosylated moieties similar to those of KLH.
After KLH immunization and for all groups (including controls), a slight increase of anti KLH IgM concentration was observed when compared to levels before immunization (around two times more).
For all treated groups and when compared with controls, there were no statistically significant differences.
Cohort 1A
There was no test item-related mortality.
Clinical signs: Ptyalism was observed in all treated groups at dose level related increased incidences. This finding was considered to be test item treatment-related but not adverse. There were no other test item treatment-related clinical signs.
Body weight, body weight change and food consumption: There were no adverse effects.
Pathology: Increased kidney and adrenal gland weights were recorded in males and females treated at 360 mg/kg/day. Increased liver weights were recorded in males and females treated at 180 or 360 mg/kg/day.
Dose-related test item-related dilated pelvis was grossly noted in the kidneys from several males treated at 60 or 180 mg/kg/day, and in males and females treated at 360 mg/kg/day.
The following test item-related microscopic findings were noted:
- Kidney: chronic interstitial inflammation that was considered to be adverse in males and females treated at 360 mg/kg/day because of the up to moderate grade, interstitial dystrophic mineralization and fibrosis, dilation of tubules, pigment, cyst, dilation of pelvis and inflammation in pelvis in males and females treated at 360 mg/kg/day.
- Thyroid glands: follicular cell hypertrophy in males or females treated at 360 mg/kg/day.
- Lungs: foamy alveolar macrophages in males treated at 180 or 360 mg/kg/day.
- Liver: single cell necrosis/apoptosis in males treated at 180 or 360 mg/kg/day that was considered to be adverse; hepatocellular hypertrophy in males treated at 180 mg/kg/day and in males and females treated at 360 mg/kg/day.
- Jejunum: ectasia of lymphatics in males and females treated at 180 or 360 mg/kg/day.
- Sternum: increased bone mass in trabeculae in males and females treated at 180 or 360 mg/kg/day.
- Stomach: erosion/ulcer in 2/20 males treated at 360 mg/kg/day.
Quantitative evaluation of primordial follicles on PCNA-stained slides: There was a slight difference between the high-dose and control groups, with a total number of 9.63 and 15.57 primordial follicles per section respectively. A relationship to test item administration was considered to be unlikely in view of the low magnitude of the difference, of the lack of dose-relationship, of the high inter-individual variability and of the lack of reproduction abnormalities.
Sexual development: When compared with controls, there were no statistically significant differences in the mean age of balanopreputial separation in males, vaginal opening in females or mean time to first oestrous after vaginal opening in females.
Estrous cyles: There were no effects.
Mating, fertility and delivery data: There were no effects.
Blood chemistry: In males, at 60 mg/kg/day there were no relevant differences; from 180 mg/kg/day, there was an increased cholesterol concentration (+69% vs. controls, p<0.01). In females, at 60 and 180 mg/kg/day there were no relevant differences; at 540/360 mg/kg/day, there was an increased cholesterol concentration (+100% vs. controls, p<0.01).
Urinalysis: No test item treatment-related findings on urinalysis parameters.
Thyroid hormones: In males at 540/360 and 180 mg/kg/day, when compared with controls, there were increased concentrations in TSH (2937 and 3051 vs. 1421pg/mL, respectively with no statistical significance and p<0.05). These increases were not associated with a similar trend for T4 levels. Therefore a test-item treatment relationship was considered dubious. At 60 mg/kg/day there were no effects. In females, at 540/360 and 180 mg/kg/day, when compared with controls, there were increased concentrations in TSH (996 and 1085 vs. 544 pg/mL, respectively with p <0.01 and p<0.05) associated with increases in T4 levels (+12 and +13% vs. controls, respectively not statistically significant). These findings were considered to be test item treatment-related but not adverse (within the range of the Historical Control Data). At 60 mg/kg/day there were no effects.
Sperm analysis: There were no test item treatment-related effects in sperm analysis data.
Lymphocyte subtyping: At 540/360 mg/kg/day and when compared with controls, significant variations consisted in a moderate decrease in relative cytotoxic T cells count (-20% vs. controls) in males. In females, a moderate decrease in relative NK cells count (-32% vs. controls) and a moderate increase in relative T cells count (+28% vs. controls) was observed. Other observed changes in the studied parameters were considered to be either non-significant or non-related to the treatment. At 180 or 60 mg/kg/day and when compared with controls, there were no effect.


Cohort 1B
There was no test item-related mortality.
Clinical signs: Ptyalism was observed in all treated groups at dose level related increased incidences. This finding was considered to be test item treatment-related but not adverse. There were no other test item treatment-related clinical signs.
Body weight, body weight change and food consumption: There were no adverse effects.
Pathology: Increased liver weights were recorded in males treated at 60 or 180 mg/kg/day, and in males and females treated at 360 mg/kg/day.
Decreased absolute and relative-to-body pituitary gland weights were recorded in females treated at 360 mg/kg/day.
Dose-related dilated pelvis grossly was noted in the kidneys from a few males treated at 60, 180 or 360 mg/kg/day.
Enlarged liver was recorded in one male treated at 360 mg/kg/day.
No microscopic examination was performed.
Sexual development: When compared with controls, there were no statistically significant differences in the mean age of balanopreputial separation in males, vaginal opening in females or mean time to first oestrous after vaginal opening in females.
Estrous cyles: There were no effects.
Mating, fertility and delivery data: There were no effects.

Cohort 2A
There was no test item-related mortality.
Clinical signs: Ptyalism was observed with increased incidence in test item treated groups. This finding was considered to be test item treatment-related but not adverse.
Body weight, body weight change and food consumption: There were no adverse effects.
Pathology: Increased liver weights were recorded in males and females treated at 360 mg/kg/day.
Dilated pelvis was grossly noted in the kidneys from 9/10 males treated at 360 mg/kg/day.
Enlarged liver was recorded in one male treated at 360 mg/kg/day.
Black discoloration was seen in the stomach from 2/10 females treated at 180, and in 2/10 females treated at 360 mg/kg/day.
The following test item-related microscopic findings were noted:
- Kidney : chronic interstitial inflammation that was considered to be adverse; interstitial dystrophic mineralization and fibrosis; mononuclear inflammatory cell infiltrate; tubular dilation; cyst; pelvic dilation
- Liver: minimal single cell necrosis/apoptosis that was considered to be adverse; hepatocellular hypertrophy
- Stomach: erosion/ulcer
- Ureters: dilated lumen
- Skin: ulcer; sero-cellular crust; hyperkeratosis
Sexual development: When compared with controls, there were no statistically significant differences in the mean age of balanopreputial separation in males or vaginal opening in females.
Neurobehavioral testing: There were no findings at auditory startle testing, reactivity to manipulation or on different stimuli (Functional Observation Battery) and, motor activity (horizontal movements and rearing) testing.
Neurohistopathology: There were no noteworthy findings with the exception of higher cornu ammonis thickness in high dose males than in controls with ambiguous evidence.

Cohort 2B
There was no mortality during the course of the study.
Clinical signs: Ptyalism was observed with increased incidence in test item treated groups. This finding was considered to be test item treatment-related but not adverse.
Body weight, body weight change and food consumption: There were no adverse effects.
There were no test item-related organ weight changes.
There were no test item-related gross changes.
There were no test item-related microscopic findings.
Neurohistopathology: There were no differences between controls and high-dose groups for the measurements performed except for the striatum thickness that was lower in high dose females than in controls with ambiguous evidence.

Cohort 3
There was no test item-related mortality.
Clinical signs: Ptyalism was observed in all test item treated animals and at increased incidence. This finding was considered to be test item treatment-related but not adverse.
Body weight, body weight change and food consumption: There were no adverse effects.
Pathology: Increased liver weights were recorded in males and females treated at 360 mg/kg/day.
Increased adrenal gland weights were recorded in females treated at 360 mg/kg/day.
Black discoloration was seen in the stomach from 1/10 females treated at 360 mg/kg/day and brown discoloration in the stomach from 1/10 females treated at 360 mg/kg/day.
Slight erosion/ulcer was noted in the stomach from 2/10 females treated at 360 mg/kg/day.
Sexual development: When compared with controls, there were no statistically significant differences in the mean age of balanopreputial separation in males or vaginal opening in females.
Quantification of anti-KLM IgM response: For all treated groups and when compared with controls, there were no statistically significant differences.

Non selected pups
There was no mortality during the course of the study.
Decreased thymus weights were recorded in females treated at 360 mg/kg/day. In the absence of microscopic examination, no correlation could be performed.
There were no test item-related gross changes.
There were no test item-related findings.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
180 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Clinical signs:
no effects observed
Description (incidence and severity):
When compared to controls, there no relevant clinical signs in test item treated groups (both in terms of finding and associated severity) which could have reveal any impairment of maternal behaviour (nursing, care to pups).

All findings observed in lactating pups are commonly observed in this species/strain of animals at this age.
Mortality / viability:
no mortality observed
Description (incidence and severity):
In the test item treated groups and when compared with controls or Historical Control Data, there were no significant differences in the repartition of found dead or cannibalized pups and in viability index on Day 4 p.p.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
In test item treated group and when compared with controls, there was no statistically significant differences in mean body weight and mean body weight change.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
- There were no significant differences in the repartition of found dead or cannibalized pups,
- There were no effects on viability index on Day 4 p.p. in F2 lactating pups,
- There were no test item treatment-related clinical signs in F2 lactating pups,
- There were no test item treatment-related effects on mean F2 body weight and body weight gain.
Dose descriptor:
NOAEL
Generation:
F2 (cohort 1B)
Effect level:
360 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
180 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
NOAEL for reproductive/developmental toxicity = 180 mg/kg/day
NOAEL for developmental neurotoxicity = 360 mg/kg/day
NOAEL for developmental immunotoxicity = 360 mg/kg/day
NOAEL for systemic toxicity (male) = 60 mg/kg bw/d
NOAEL for systemic toxicity (female) = 180 mg/kg bw/d
Executive summary:

The test item, 4,4’Isopropylidenediphenol, propoxylated, Grade 4PO, was administered daily by oral gavage, at dose levels of 0, 60, 180 or 540/360 mg/kg bw/d, sexually-mature male and female rats (parental (P) generation) starting 2 weeks before mating and continuously through mating, gestation and weaning of their pups (F1 generation). At weaning, F1 generation was also exposed to graduated doses of the test item while being assigned to Cohorts of animals for reproductive/developmental toxicity, developmental neurotoxicity or developmental immunotoxicity testing.

 

Systemic toxicity evaluation:

The No Observed Adverse Effect Levels (NOAELs) for systemic toxicity (excluded reproductive and developmental toxicity endpoints) were considered to be:

- 60 mg/kg bw/d in males based on adverse pathology findings (single cell necrosis) from 180 mg/kg bw/d in Cohort 1A males;

- 180 mg/kg bw/d in females based on adverse pathology findings (single cell necrosis) at 540/360 mg/kg bw/d in Cohort 1A females.

 

Reproductive/developmental toxicity testing:

- in P generation animals, there was an increased number of females in diestrus at 540/360 mg/kg bw/d, resulting in a prolonged duration of mean estrous cycle associated with a lower fertility index at this dose level,

- in Cohorts 1A or 1B animals there were no adverse effects on reproductive function.

Therefore the No Observed Adverse Effect Level (NOAEL) for reproductive/developmental toxicity was considered to be 180 mg/kg bw/d.

 

Developmental neurotoxicity testing:

There were no adverse effects at neurobehavioral testing but ambiguous evidence of developmental neurotoxicity in high dose males from cohort 2A (corpus callosum increased thickness) and high dose females from cohort 2B (striatum decreased width).

Therefore the No Observed Adverse Effect Level (NOAEL) for developmental neurotoxicity was considered to be 360 mg/kg bw/d.

 

Developmental immunotoxicity testing:

in Cohort 1A:

-          moderate alterations in spleen immunophenotyping were observed differently in males (decrease of relative cytotoxic T cells count) and females (decrease of relative NK cells count and increase of T cell relative count) at 360 mg/kg bw/d,

in Cohort 3:

-          after KLH immunization and for all groups (including controls), there were no statistically significant differences.

Therefore the No Observed Adverse Effect Level (NOAEL) for developmental immunotoxicity was considered to be 360 mg/kg bw/d.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
180 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 September 2016 - 07 November 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
22 January 2001
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: breeder: Janvier, le Genest-Saint-Isle, France.
- Age at the beginning of the treatment period: approximately 10-11 weeks old
- Mean body weight at the beginning of the treatment period: 305 g (range: 261 g to 353 g)
- Fasting period before study: no
- Housing: the animals were individually housed in polycarbonate cages (Tecniplast 2154, 940 cm2) with stainless steel lids and containing autoclaved sawdust
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: for a period of 4 or 5 days before the beginning of the treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): about 8 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

IN-LIFE DATES: 10 October 2016 to 07 November 2016.
Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
PREPARATION OF DOSING FORMULATIONS:
- Emulsion in the vehicle
- Concentration in vehicle: 36, 72 and 144 mg/mL
- Amount of vehicle: 5 mL/kg/day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Type of method: HPLC-UV (High Performance Liquid Chromatography with UV detection).
Test item concentrations: remained within the acceptable range of variations (± 15%) when compared to the nominal values.
Homogeneity: the dose formulations containing the test item prepared at 1 and 200 mg/mL were found to be homogeneous.
Dose formulations ranging from 1 mg/mL to 200 mg/mL are therefore considered to be suitable for routine administration in GLP Toxicological studies.
Stability: diluted analytical samples prepared from 0.8 mg/mL and 240 mg/mL dose formulations in olive oil were found to be stable for 4 and 5 days, respectively.
Details on mating procedure:
The females were mated at the breeder's facility. The day of confirmed mating (detection of a vaginal plug) was designated as Day 0 p.c.
Duration of treatment / exposure:
The dose formulations were administered daily from Day 6 to Day 20 p.c., inclusive.
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
180 mg/kg bw/day (actual dose received)
Dose / conc.:
360 mg/kg bw/day (actual dose received)
Dose / conc.:
720 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
24 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for dose selection:
The dose levels were selected in agreement with the Sponsor, based on the results of:
- a previous 2-week dose-range finding study by the oral route (gavage) performed in the male and female rats species. In this study, three groups of six Sprague Dawley rats were treated with the test item at dose levels of 120, 500 or 1000 mg/kg/day. Clinical signs were observed in all animals given 1000 mg/kg/day in both sexes such as hunched posture, piloerection, thin appearance and ptyalism during the whole treatment period. One male treated at 1000 mg/kg/day was prematurely killed due to poor clinical condition. All animals given 500 mg/kg/day had ptyalism until the end of the study and 1/3 females showed hunched posture, piloerection and thin appearance during the first 10 days. Dose-related lower body weight, body weight change and food consumption were recorded at 500 mg/kg/day in males (-10% and -28% for body weight and body weight change on Day 14, respectively) and at 1000 mg/kg/day in both sexes (-22% and -14%, -53% and -72% for body weight and body weight change in males and females, respectively),
- a combined repeated dose toxicity study with the reproduction/developmental toxicity Screening Test (OECD 422, Ref 06-119), conducted with grade BPA 5PO, showing developmental findings with a decrease of pups weights observed at 500 mg/kg/day (the females showed no changes in body weight during premating, pregnancy or the lactation period and thereby no maternal toxicity was seen in any of the dose groups),
- an ongoing OECD 408 study in rats conducted at 30, 120 and 500 mg/kg/day. There are no findings in females over the 3 first weeks of the treatment period.

Therefore, 720 mg/kg/day was selected as the high-dose level. The low-dose and mid dose were selected using a ratio representing approximately a 2-fold interval (i.e. 180 and 360 mg/kg/day).

- Rationale for animal assignment: computerized stratification procedure.
Maternal examinations:
MORTALITY/MORBIDITY:
- Time schedule: animals were examined twice daily for mortality and morbidity.

CLINICAL OBSERVATIONS:
- Time schedule: from arrival, each animal was observed once a day as part of the routine examinations. From the start of the treatment period, each animal was observed once a day, at approximately the same time, for the recording of clinical signs.

BODY WEIGHT:
- Time schedule: the body weight of each female was recorded on Days 2, 4, 6, 9, 12, 15, 18 and 21 p.c., and prior to premature euthanasia.

FOOD CONSUMPTION:
- Time schedule: the quantity of food consumed by each female was recorded for the following intervals Days 2-4, 4-6, 6-9, 9-12, 12-15, 15-18 and 18-21 p.c.

POST-MORTEM MACROSCOPIC EXAMINATION:
- Sacrifice on Day 21 post-coitum.
- Examined: principal thoracic and abdominal organs.
Ovaries and uterine content:
The ovaries and uterus of the females were examined to determine:
- gravid uterus weight,
- number of corpora lutea,
- number and distribution of dead and live fetuses,
- number and distribution of early and late resorptions,
- number and distribution of uterine scars,
- number and distribution of implantation sites,
- gross evaluation of placentas.
Fetal examinations:
- External examinations: Yes: all fetuses per litter
- Soft tissue examinations: Yes: half fetuses per litter
- Skeletal examinations: Yes: half fetuses per litter
- Head examinations: Yes: half fetuses per litter
- Other: number dead and live, body weight, sex.
Statistics:
Data were compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fisher’s exact probability test (proportions).
Indices:
% Pre-implantation loss = 100 * (Number of corpora lutea - Number of implantation sites) / Number of corpora lutea
% Post-implantation loss = 100 * (Number of implantation sites - Number of live fetuses) / Number of implantation sites
Historical control data:
Cf attached document
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
See table 1.
At 720 mg/kg/day, most of clinical signs were also observed in prematurely sacrificed and/or found dead animals. They were considered to be test item treatment-related and adverse.
There were no test item treatment-related clinical signs in the 360 and 180 mg/kg/day groups.

Dacryorrhea, chromodacryorrhea and/or areas of hair loss on forelimbs were observed in only one animal in each low- and mid-dose group. They are common findings in this species and strain.
Mortality:
mortality observed, treatment-related
Description (incidence):
In the 720 mg/kg/day group, there were eight unscheduled deaths (all being pregnant females). Taking into account the similarity of the findings (clinical signs, effects on body weight/food consumption and macroscopic examination at necropsy), all these deaths were considered to be related to the test item treatment:
- one female (pregnant, 15 implantation sites) was prematurely sacrificed on Day 16 p.c. Prior to death, this animal progressively showed from Day 14 p.c., emaciated appearance, reddish vaginal discharge, soiled urogenital region and/or round back (in addition to ptyalism from Day 9 p.c.). This female lost 74 g of body weight on Days 6-15 p.c. (-23% vs. Day 6 p.c.) and had a markedly reduced food consumption on Days 12-15 p.c. (6 g/day). At necropsy, thymus reduced in size, forestomach with several thick and whitish colored areas, kidneys with irregular color, an enlarged adrenal and, vagina with thick and reddish content were observed,
- one female (pregnant, 13 implantation sites) was prematurely sacrificed on Day 19 p.c. Prior to death, this animal progressively showed from Day 13 p.c., piloerection, emaciated appearance, round back, hypoactivity, sedation, abdominal breathing, eyes half-closed and/or soiled urogenital area (in addition to ptyalism from Day 8 p.c.). This female lost 58 g of body weight on Days 6-18 (-20% vs. Day 6 p.c.) and had merely no food consumption on Days 15-18 p.c. At necropsy, thymus and spleen reduced in size were observed,
- one female (pregnant, 17 implantation sites) was prematurely sacrificed on Day 18 p.c. Prior to death, this animal progressively showed from Day 14 p.c., piloerection, emaciated appearance, round back, hypoactivity, loss of balance and/or abdominal breathing. This female had also half closed eyes and ptyalism on Days 8 to 17 p.c. This female lost 71 g of body weight on Days 6-18 (-24% vs. Day 6 p.c.) and had merely no food consumption on Days 15-18 p.c. At necropsy, forestomach with thickened mucosa, whitish colored areas and several brownish colored deposits were observed (this female had fetuses with reddish colored amniotic fluid),
- one female (pregnant, 16 implantation sites) was prematurely sacrificed on Day 20 p.c. Prior to death, this animal progressively showed from Day 14 p.c., piloerection, emaciated appearance, round back, soiled urogenital area and/or eyes half-closed (in addition to ptyalism from Day 13 p.c.). This female lost 41 g of body weight on Days 6-18 (-13% vs. Day 6 p.c.) and had merely no food consumption on Days 15-18 p.c. At necropsy, thymus and spleen reduced in size and, enlarged adrenals were observed,
- one female (pregnant, 14 implantation sites) was prematurely sacrificed on Day 20 p.c. Prior to death, this animal progressively showed from Day 14 p.c., piloerection, emaciated appearance, round back, hypoactivity, soiled urogenital area, brownish vaginal discharge and/or eyes half-closed (in addition to ptyalism from Day 7 p.c.). This female lost 34 g of body weight on Days 6-18 (-12% vs. Day 6 p.c.) and had merely no food consumption on Days 15 18 p.c. At necropsy, thymus and spleen reduced in size, enlarged adrenals and uterine horns with blackish colored content were observed,
- one female (pregnant, 14 implantation sites) was found dead on Day 18 p.c. Prior to death, this animal progressively showed from Day 14 p.c., piloerection, emaciated appearance, round back, hypoactivity, abdominal breathing, soiled urogenital area, reddish vaginal discharge and/or eyes half closed (in addition to ptyalism from Day 7 p.c.). This female lost 24 g of body weight on Days 6-15 (-8% vs. Day 6 p.c.) and had a low food consumption (9 g/day) on Days 12-15 p.c. At necropsy, thymus and spleen reduced in size, stomach with yellowish colored liquid count and enlarged adrenals were observed,
- one female (pregnant, 15 implantation sites) was found dead on Day 17 p.c. Prior to death, this animal progressively showed from Day 12 p.c., piloerection, emaciated appearance, round back, hypoactivity, chromorhinorrhea and/or eyes half-closed (in addition to ptyalism on Days 7-16 p.c.). This female lost 45 g of body weight on Days 6-15 (-14% vs. Day 6 p.c.) and had a low food consumption (5 g/day) on Days 12-15 p.c. At necropsy, thymus and spleen reduced in size, forestomach with several raised whitish colored foci, enlarged adrenals and uterine horns with a blackish colored content were observed,
- one female (pregnant, 16 implantation sites) was prematurely sacrificed on Day 19 p.c. Prior to death, this animal progressively showed from Day 12 p.c., piloerection, emaciated appearance, round back, cold to the touch, hypoactivity, lateral decubitus, dyspnea, brownish vaginal discharge and/or eyes half-closed (in addition to ptyalism from Day 8 p.c.). This female lost 65 g of body weight on Days 6-18 (-20% vs. Day 6 p.c.) and had merely no food consumption (1 or 0 g/day) on Days 12-18 p.c. At necropsy, thymus and spleen reduced in size, skin with reddish foci mainly on the tail region, caecum with thickened mucosa, forestomach and stomach with several brownish colored foci (depressed or not), ileum with blackish colored content and enlarged adrenals were observed.

There were no unscheduled deaths in the control, 180 and 360 mg/kg/day groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See table 2.
At 720 mg/kg/day, there were mean body weight losses on Days 9 to 15 p.c. (-1 to -19 g, p < 0.001). When compared with controls, these resulted in decreases in mean body weights (down to -24% on Day 21 p.c., p < 0.001). These findings were considered to be test item-related and adverse.

At 360 mg/kg/day and when compared with controls, there were a few transient statistically significant differences in mean body weight (-5% vs. controls on Day 12 p.c., p < 0.05) and/or mean body weight changes (+6 g vs. +14 g on Days 6-9 p.c., p < 0.001 and +17 g vs. +23 g on Days 9-12 p.c., p < 0.05) which were considered to be test item treatment-related and adverse.

At 180 mg/kg/day and when compared with controls, there was a statistically significant difference in mean body weight change (+5 g vs. +14 g in controls on Days 6-9 p.c., p < 0.001) which was considered to be test item treatment-related but of limited toxicological significance (transient observation with no effect on mean body weight).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
See table 3.
At 720 mg/kg/day, there were decreases in mean food consumption (down to -60% vs. controls on Days 15-18 p.c., p < 0.001). These findings were considered to be test item-related and adverse.

At 360 mg/kg/day and when compared with controls, there were transient decreases in mean food consumption (down to -18% vs. controls on Days 6-9 p.c., p < 0.001) with a return to control values from Days 12-15 p.c. These findings were considered to be test item-related and adverse but of minor toxicological concern (less than 20% difference vs. controls).

At 180 mg/kg/day and when compared with controls, there was a transient decrease in mean food consumption (-14% vs. controls, p < 0.01) on Days 6-9 p.c. with a return to control values thereafter. This finding was considered to be test item-related but of minor toxicological concern.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
See table 4.
At 720 mg/kg/day, there was a series of macroscopic findings affecting the thymus, spleen, stomach/forestomach, intestines and adrenals which were considered to be test item-related (also observed in almost all prematurely or found dead animals).

At 360 and 180 mg/kg/day, there were no test item treatment-related findings. The few findings recorded were also observed in the control group (enlarged lymph nodes) or are common observations in this species and strain (pancreas with edema and placenta with colored deposit).
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Net body weight change
See table 5.
At 720 mg/kg/day and when compared with controls, there were lower mean gravid uterus weight (-26%, p < 0.001), carcass weight (-24%, p < 0.001) and net body weight change (-28.4 g vs. +54.1 g, p < 0.001). These adverse findings were considered to be consecutive to the effects recorded on mean body weight and food consumption at this dose level and to the lower number of live fetuses.

At 360 mg/kg/day and when compared with controls, there was a decreased carcass weight (-6%, p < 0.05) resulting in a lower net body weight change (+33.4 g vs. +54.1 g, p < 0.05). These findings were considered to be test item treatment-related but of minor toxicological concern taking into account the amplitude of the differences.

At 180 mg/kg/day, there were no test item treatment-related effects on mean gravid uterus weight, carcass weight and net body weight change.
Number of abortions:
not examined
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
See table 6.
At 720 mg/kg/day and when compared with control, there was a lower number of live fetuses per animal (9.3 vs. 12.1, p < 0.05) as a consequence of increased resorptions (early and scars) and dead fetuses (5.5 vs. 0.0) which resulted in an increased incidence of mean percent of post-implantation loss (26.8% vs. 5.3%, p < 0.01). All these findings were outside the range of the HCD and, considered to be test item treatment-related and adverse.

When compared with controls, there were no test item treatment-related effects on hysterectomy data in the 360 and 180 mg/kg/day groups.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
effects observed, treatment-related
Description (incidence and severity):
See table 6.
At 720 mg/kg/day and when compared with control, there was a lower number of live fetuses per animal (9.3 vs. 12.1, p < 0.05) as a consequence of increased resorptions (early and scars) and dead fetuses (5.5 vs. 0.0) which resulted in an increased incidence of mean percent of post-implantation loss (26.8% vs. 5.3%, p < 0.01). All these findings were outside the range of the HCD and, considered to be test item treatment-related and adverse.

When compared with controls, there were no test item treatment-related effects on hysterectomy data in the 360 and 180 mg/kg/day groups.
Dead fetuses:
effects observed, treatment-related
Description (incidence and severity):
See table 6.
At 720 mg/kg/day and when compared with control, there was a lower number of live fetuses per animal (9.3 vs. 12.1, p < 0.05) as a consequence of increased resorptions (early and scars) and dead fetuses (5.5 vs. 0.0) which resulted in an increased incidence of mean percent of post-implantation loss (26.8% vs. 5.3%, p < 0.01). All these findings were outside the range of the HCD and, considered to be test item treatment-related and adverse.

When compared with controls, there were no test item treatment-related effects on hysterectomy data in the 360 and 180 mg/kg/day groups.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
effects observed, treatment-related
Description (incidence and severity):
See table 15.
All females were pregnant with the exception of two in the low-dose group.

At hysterectomy on Day 21 p.c., 24/24, 22/24, 24/24 and 14/16 surviving females were pregnant with live fetuses in the groups treated at 0, 180, 360 and 720 mg/kg/day, respectively.

In the 720 mg/kg/day group, two females had only implantation scars, two other pregnant females were found dead on Days 17 or 18 p.c., and six pregnant females were prematurely sacrificed on Day 16, 18, 19 or 20 p.c. for ethical grounds.

In the 180 mg/kg/day group, two females had no corpora lutea and no implantation sites.
Dose descriptor:
NOAEL
Effect level:
180 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
mortality
body weight and weight gain
food consumption and compound intake
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
See table 7.
At 720 mg/kg/day and when compared with control, fetuses (males and females) had a lower mean body weight (-23% vs. controls, p < 0.001). At this dose level, mean fetal body weight was below the lower limit of the HCD. This finding was considered to be test item treatment-related and adverse.
When compared with controls, there were no effects on mean fetal body weight in the 360 and 180 mg/kg/day groups.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
See table 7.
There were no effects on mean percentage of male fetuses (sex-ratio) at any dose tested.
External malformations:
effects observed, treatment-related
Description (incidence and severity):
Malformations
See table 8.
At 720 mg/kg/day, one female had nine fetuses with anasarca (generalized edema) and another female had a fetus with a cleft lip. One female had also four different fetuses with local edema (variation). While limited to two litters, these findings were observed in the high-dose group only and at incidences above the upper limit of the HCD. Therefore a test item treatment relationship cannot be excluded.
At 360 and 180 mg/kg/day, there were no malformations at external examination of the fetuses.
In the control group, one fetus had a gastroschisis, a malformation commonly observed in this species and strain.

Variations
See table 9
At 720 mg/kg/day there were fetuses with local edema (14.3% of litters vs. none in controls, p < 0.001). This observation was also recorded at higher litter and fetal incidences when compared with HCD. Therefore, this finding was considered to be test item treatment-related but not adverse (variation).
There were no external variations in fetuses from control, 180 and 360 mg/kg/day groups.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Cartilage
See table 12.
When compared with controls, at 360 and/or 720 mg/kg/day, there was a series of statistically significant increases of cartilage present (related to absence of ossification). In the 720 mg/kg/day group only and at some occasions (cartilages of caudal vertebrae, sternebrae, metacarpal bones, distal phalanx, metatarsal bones and pelvic girdle), the litters and/or fetal incidences were above the upper limit of HCD. These findings were considered to be test item treatment-related and not adverse (the bone concerned being present).

In the 180 mg/kg/day group, one fetus had absent cartilage of cervical vertebrae (associated to absent hemicentrum). This finding was isolated, not dose-related or associated with any other findings. Therefore a test item treatment-related effect was considered unlikely.

Variations
See table 13.
From 360 mg/kg/day and when compared with controls, there were dose-related increases in the litter and/or fetal incidences of unossified bones or bones with incomplete ossification . In the 720 mg/kg/day group only, the litter and/or fetal incidences were above the upper limit of HCD. These findings were considered to be test item treatment-related and not adverse (variations of ossification degree, the cartilages being present).
At 180 mg/kg/day and when compared with controls, there were no test item treatment-related findings.

Malformations
See table 14.
At 720 mg/kg/day, there were increases in the litter and fetal incidences of split supraoccipital. This finding affected four fetuses from the same litter. While limited to one litter, this finding was observed in the high-dose group only and not recorded in the HCD. Therefore a test item treatment relationship cannot be excluded.

At 360 mg/kg/day, there were no skeletal malformations.

At 180 mg/kg/day, one fetus had a cervical vertebra with absent hemicentrum. This observation was isolated, not dose-related or associated with any other findings. Therefore a test item treatment-related effect was considered unlikely.



Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
Variations
See table 10.
At 720 mg/kg/day, one female had four fetuses with visceral edema, another female had one fetus with fluid-filled thoracic cavity and, another female had four fetuses with fluid-filled thoracic cavity and abdomen. While limited to three litters, these findings were observed in the high-dose group only and at incidences above the upper limit of the HCD. Therefore a test item treatment relationship cannot be excluded.
When compared with controls, there were no test item treatment-related variations at soft tissue examination of the fetuses from the 360 and 180 mg/kg/day groups.

Malformations
See table 11.
There were no test item treatment-related malformations at soft tissue examination of the fetuses.
In the control group, one fetus had an absent kidney/ureter, a malformation commonly observed in this species and strain.
Dose descriptor:
NOAEL
Effect level:
360 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
external malformations
skeletal malformations
visceral malformations
other: embryo-fetal toxicity
Developmental effects observed:
yes
Lowest effective dose / conc.:
720 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified

Table 1: Clinical signs

Dose level (mg/kg/day)

0

      180

     360

     720

Ptyalism

 

       22

       24

       16

Soiled urogenital area

 

 

 

         4

Lateral decubitus

 

 

 

         1

Piloerection

 

 

 

       16

Emaciated appearance

 

 

 

       12

Round back

 

 

 

       14

Cold to the touch

 

 

 

         3

Hypoactivity

 

 

 

         6

Abdominal breathing

 

 

 

         3

Eyes half-closed

 

 

 

         9

Dacryorrhea

 

         1

 

 

Chromodacryorrhea

 

         1

 

           

Chromorhinorrhea

 

           

 

         1

Area of hair loss on forelimbs

 

 

         1

 

Number of affected animals

0/24

22/24

24/24

16/16

 

Table 2: Body weight

Dose level (mg/kg/day)

0

180

360

720

Body weight (g)

 

 

 

 

Day 2p.c.

281

282
(0)

281
(0)

280
(0)

Day 4p.c.

293

296
(+1)

295
(+1)

295
(+1)

Day 6p.c.

305

307
(+1)

304
(0)

303
(-1)

Day 9p.c.

320

312
(-3)

310
(-3)

310
(-3)

Day 12p.c.

343

332
(-3)

327*
(-5)

309#
(-10)

Day 15p.c.

364

352
(-3)

347
(-5)

290#
(-20)

Day 18p.c.

406

399
(-2)

391
(-4)

299#
(-26)

Day 21p.c.

459

454
(-1)

443
(-3)

347#
(-24)

Body weight change (g)

 

 

 

 

Days 2-4p.c.

+12

+14

+14

+14

Days 4-6p.c.

+13

+12

+9*

+9*

Days 6-9p.c.

+14

+5#

+6#

+6**

Days 9-12p.c.

+23

+20

+17*

-1#

Days 12-15p.c.

+21

+21

+20

-19#

Days 15-18p.c.

+43

+47

+44

+5#

Days 18-21p.c.

+53

+55

+52

+30#

Days 6-21p.c.

+154

+147

+138

+45#

( ): in brackets, percentage differencevs.controls (%).

Statistical significance: *: p < 0.05; **: p < 0.01; #: p < 0.001.

Table 3: Food consumption

Dose level (mg/kg/day)

            0

180

360

720

Days 6-9p.c.

          22

19**
(-14)

18#
(-18)

17#
(-23)

Days 9-12p.c.

          25

22
(-12)

21**
(-16)

17#
(-32)

Days 12-15p.c.

          26

26
(0)

25
(-4)

13#
(-50)

Days 15-18p.c.

          30

30
(0)

30
(0)

12#
(-60)

Days 18-21p.c.

          30

30
(0)

30
(0)

23#
(-23)

(): in brackets, percentage difference (%)vs. controls.

Statistical significance: **: p < 0.01; #: p < 0.001.

Table 4: Macroscopic examination

 

Dose level (mg/kg/day)

0

180

360

720

Lymph node(s): enlarged

1

      2

      2

      0

Pancreas: edema

0

      0

      1

      0

Thymus: reduced in size

0

      0

      0

      3

Spleen: reduced in size

0

      0

      0

      2

Stomach: colored deposit

0

      0

      0

      1

Stomach: colored focus

0

      0

      0

      2

Intestines: colored contents

0

      0

      0

      1

Intestines: colored focus

0

      0

      0

      1

Adrenal: enlarged

0

      0

      0

      3

Uterus: colored contents in uterine horn

0

      0

      0

      1

Vagina: colored contents

0

      0

      0

      1

Placenta: colored deposit

0

      0

      1

      0

Number of affected animals

1/24

2/24

3/24

4/16

 

Table 5: Net body weight change

Dose level (mg/kg/day)

0

180

360

720

Gravid uterus weight

99.9

104.6
(+5)

105.1
(+5)

73.6#
(-26)

Carcass weight

359.5

349.3
(-3)

337.6*
(-6)

273.3#
(-24)

Net body weight change (g) from Day 6p.c.

+54.1

+42.1

+33.4*

-28.4#

(): in brackets, percentage difference (%)vs. controls.

Statistical significance: *: p < 0.05; #: p < 0.001.

Table 6: Hysterectimy data

Dose level (mg/kg/day)

  0

180

360

 720

HCD

Number of pregnant females

 24

  22

  24

  16

388

Mean number ofcorpora luteaper animal

 14.5

  15.2

  15.5

  13.9

[13.8; 16.0]

Mean number of implantation sites

 12.8

  13.4

  14.0

  12.5

[12.5; 14.5]

Mean pre-implantation loss (%)

 11.6

  11.4

    9.3

  10.2

[6.2; 14.0]

Mean number of live fetuses per animal

 12.1

  12.8

  13.2

    9.3*

[11.6; 13.8]

Mean number of dead fetuses

  0.0

   0.0

    0.0

    5.5

[0.00; 0.50]

Mean number of resorptions + scars

  0.6

   0.6

    0.9

    2.4*

[0.50; 1.35]

Mean number of implantation scars

  0.0

   0.0

    0.0

    1.3*

/

Mean number of early resorptions

  0.5

   0.5

    0.8

    0.9

/

Mean number of late resorptions per animal

  0.1

   0.1

    0.0

    0.2

/

Mean post-implantation loss (%)

  5.3

   4.3

    6.2

  26.8**

[3.5; 11.1]

HCD: Historical Control Data (Sprague-Dawley rats, March 2014 - July 2016, n = 18 studies) [min.; max.].

/: not reported in HCD.

Statistical significance:*: p < 0.05; **: p < 0.01.

 

Table 7: Fetal body weights and sex ratio

Dose level (mg/kg/day)

    0

180

360

720

HCD

Mean male fetal body weight (g)

    6.02

5.94
(-1)

5.88
(-4)

4.59#
(-24)

[5.7; 6.1]

Mean female fetal body weight, (g)

    5.75

5.69
(-1)

5.55
(-4)

4.53#
(-21)

[5.4;5.7]

Mean percentage of male fetuses (%)

   48.1

46.9

48.4

53.9

[44.0; 55.4]

HCD: Historical Control Data (Sprague-Dawley rats, March 2014 - July 2016, n = 18 studies) [min.; max.].

(): in brackets, percentage difference (%)vs.controls.

Statistical significance:#: p < 0.001.

Table 8: External malformations

 

Dose level (mg/kg/day)

        0

    180

     360

      720

HCD

Number of litters

      24

      22

       24

       14

          388

Number of fetuses

    291

    282

     316

      148

         5000

Anasarca, F (L)

0 (0)

0 (0)

0 (0)

6.1# (7.1)

0 (0)(a)

Cleft lip, F (L)

0 (0)

0 (0)

0 (0)

0.7 (7.1)

0.4 (5.0)(a)

Gastroschisis, F (L)

0.3 (4.2)

0 (0)

0 (0)

0 (0)

0.4 (5.6)(a)

Litters affected, n (%)

1 (4.2)

0 (0)

0 (0)

2 (14.3)

11 (2.8)(b)

Fetus affected, n (%)

1 (0.3)

0 (0)

0 (0)

10# (6.8)

13 (0.3)(b)

n: number; F: fetal incidence; L: litter incidence.

HCD: Historical Control Data (Sprague-Dawley rats, March 2014 - July 2016, n = 18 studies).

(a): max.(b): mean.Statistical significance: #: p < 0.001.


 

Table 9: External variations

Dose level (mg/kg/day)

        0

    180

  360

         720

HCD

Number of litters

      24

      22

    24

          14

       388

Number of fetuses

    291

    282

  316

         148

     5000

Local edema, F (L)

0 (0)

0 (0)

0 (0)

6.1# (14.3)

0.8 (9.5)(a)

Litters affected, n (%)

0 (0)

0 (0)

0 (0)

2 (14.3)

6 (1.5)(b)

Fetus affected, n (%)

0 (0)

0 (0)

0 (0)

9# (6.1)

7 (0.1)(b)

n: number; F: fetal incidence; L: litter incidence.

HCD: Historical Control Data (Sprague-Dawley rats, March 2014 - July 2016, n = 18 studies).

(a): max.(b): mean.Statistical significance: #: p < 0.001.

Table 10: Soft tissue variations

Dose level (mg/kg/day)

       0

   180

    360

     720

HCD

Number of litters

     24

     22

      24

       13

       387

Number of fetuses

   140

   134

    153

       71

     2404

Dilated renal pelvis, F (L)

2.9 (12.5)

0 (0)

4.6 (20.8)

2.8 (7.7)

9.5 (28.6)(a)

Small testis, F (L)

0.7 (4.2)

0 (0)

0 (0)

0 (0)

0 (0)(a)

Absent innominate artery, F (L)

1.4 (8.3)

1.5 (9.1)

1.3 (8.3)

4.2 (7.7)

5.1 (25.0)(a)

Short innominate artery, F (L)

1.4 (4.2)

0.7 (4.5)

0 (0)

0 (0)

3.7 (22.7)(a)

Dilated ureter, F (L)

2.9 (12.5)

2.2 (13.6)

2.6 (12.5)

0 (0)

7.1 (28.0)(a)

Thymus: reddish foci, F (L)

0 (0)

1.5 (9.1)

0 (0)

0 (0)

0 (0)(a)

Edema, F (L)

0 (0)

0 (0)

0 (0)

5.6* (7.7)

0 (0)(a)

Fluid-filled thoracic cavity, F (L)

0 (0)

0 (0)

0 (0)

7.0** (15.4)

0 (0)(a)

Fluid-filled abdomen, F (L)

0 (0)

0 (0)

0 (0)

5.6* (7.7)

0.9 (5.0)(a)

Litters affected, n (%)

7 (29.2)

6 (27.3)

7 (29.2)

4 (30.8)

86 (22.2)(b)

Fetus affected, n (%)

9 (6.4)

7 (5.2)

10 (6.5)

14** (19.7)

119 (5.0)(b)

n: number; F: fetal incidence, L: litter incidence.

HCD: Historical Control Data (Sprague-Dawley rats, March 2014 - July 2016, n = 18 studies).

(a): max.(b): mean.

Statistical significance: *: p < 0.05; **: p < 0.01.

 

Table 11: Soft tissue malformations

Dose level (mg/kg/day)

       0

   180

    360

     720

HCD

Number of litters

     24

     22

      24

       13

       387

Number of fetuses

   140

   134

    153

       71

     2404

Absent kidney, F (L)

0.7 (4.2)

0 (0)

0 (0)

0 (0)

0.7 (4.5)(a)

Absent ureter, F (L)

0.7 (4.2)

0 (0)

0 (0)

0 (0)

/

Litters affected, n (%)

1 (4.2)

0 (0)

0 (0)

0 (0)

7 (1.8)(b)

Fetus affected, n (%)

1 (0.7)

0 (0)

0 (0)

0 (0)

13 (0.5)(b)

n: number; F: fetal incidence, L: litter incidence.

HCD: Historical Control Data (Sprague-Dawley rats, March 2014 - July 2016, n = 18 studies);/: not reported in HCD.

(a): max.(b): mean.

 

Table 12: Skeletal examination (cartilage)

Dose level (mg/kg/day)

           0

     180

       360

     720

HCD

Number of litters

         24

      22

         24

      14

       388

Number of fetuses

       151

     148

       163

      77

     2596

Cartilage of hyoid: present, F (L)

0 (0)

1.4 (4.5)

4.3* (16.7)

0 (0)

7.4 (30.4) (a)

Cartilage of cervical vertebra(e): present, F (L)

2.6 (12.5)

2.7 (18.2)

3.1 (8.3)

20.8# (42.9)

22.1 (58.3) (a)

Cartilage of cervical vertebra(e): absent, F (L)

0 (0)

0.7 (4.5)

0 (0)

0 (0)

0 (0) (a)

Cartilage of thoracic vertebra(e): present, F (L)

6.0 (25.0)

5.4 (22.7)

14.1* (45.8)

14.3* (50.0)

31.5 (87.5) (a)

Cartilage of lumbar vertebra(e): present, F (L)

0.7 (4.2)

0 (0)

0 (0)

0 (0)

0.7 (4.5) (a)

Cartilage of sacral vertebra(e): present, F (L)

0 (0)

0 (0)

0 (0)

3.9* (14.3)

0.7 (5.0) (a)

Cartilage of caudal vertebra(e): present, F (L)

0.7 (4.2)

0 (0)

0 (0)

22.1# (28.6)

2.2 (15.0) (a)

Cartilage of sternebra(e): present, F (L)

0 (0)

0.7 (4.5)

0.6 (4.2)

23.4# (35.7**)

3.8 (21.7) (a)

Cartilage of rib(s): present, F (L)

2.6 (16.7)

1.4 (9.1)

0 (0)

0 (0)

5.1 (26.1) (a)

Cartilage of rib(s): absent, F (L)

0.7 (4.2)

0 (0)

0 (0)

1.3 (7.1)

5.1 (21.7) (a)

Cartilage of metacarpal bone(s): present, F (L)

0.7 (4.2)

0.7 (4.5)

0.6 (4.2)

26.0# (42.9**)

5.0 (20.0) (a)

Forepaw: cartilage of proximal phalanx present, F (L)

24.5 (75.0)

18.9 (54.5)

26.4 (62.5)

45.5** (85.7)

50.0 (85.0) (a)

Forepaw: cartilage of distal phalanx present, F (L)

59.6 (79.2)

46.6 (68.2)

56.4 (83.3)

46.8 (78.6)

59.0 (87.5) (a)

Cartilage of metatarsal bone(s): present, F (L)

5.3 (25.0)

10.8 (36.4)

19.0# (66.7**)

66.2# (85.7#)

36.8 (72.7) (a)

Hindpaw: cartilage of distal phalanx: present, F (L)

41.7 (75.0)

31.1 (63.6)

35.6 (75.0)

32.5 (64.3)

43.5 (75.0) (a)

Cartilage of pelvic girdle: present, F (L)

0 (0)

0 (0)

0 (0)

2.6 (14.3)

0.7 (5.0) (a)

Litters affected, n (%)

24 (100.0)

20 (90.9)

23 (95.8)

13 (92.9)

236 (60.8) (b)

Fetus affected, n (%)

110 (72.8)

93 (62.8)

113 (69.3)

62 (80.5)

990 (38.1) (b)

n: number; F: fetal incidence, L: litter incidence.

HCD: Historical Control Data (Sprague-Dawley rats, March 2014 - July 2016, n = 18 studies).

(a): max.(b): mean.

Statistical significance: *: p < 0.05; **: p < 0.01; #: p < 0.001.

 

Table 13: Skeletal variations

Dose level (mg/kg/day)

        0

     180

       360

       720

HCD

Number of litters

      24

      22

         24

         14

       388

Number of fetuses

     151

     148

       163

         77

     2596

Parietal: incomplete ossification, F (L)

0.7 (4.2)

0.7 (4.5)

0 (0)

20.8# (28.6)

9.2 (35.0)(a)

Interparietal: incomplete ossification, F (L)

0.7 (4.2)

0.7 (4.5)

0 (0)

16.9# (28.6)

9.2 (45.0)(a)

Supraoccipital: incomplete ossification, F (L)

0.7 (4.2)

0 (0)

0 (0)

9.1** (28.6)

5.6 (25.0)(a)

Frontal: incomplete ossification, F (L)

0 (0)

0.7 (4.5)

0 (0)

18.2# (28.6*)

1.7 (11.1)(a)

Nasal: incomplete ossification, F (L)

0 (0)

0 (0)

0 (0)

14.3# (28.6*)

0.8 (5.6)(a)

Hyoid: incomplete ossification, F (L)

0 (0)

1.4 (4.5)

4.3* (16.7)

0 (0)

14.8 (55.0)(a)

Incisive bone: incomplete ossification, F (L)

0 (0)

0.7 (4.5)

0 (0)

14.3# (21.4*)

07 (5.0)(a)

Cervical vertebra(e): incomplete ossification of centrum, F (L)

2.6 (12.5)

2.0 (13.6)

2.5 (8.3)

9.1* (35.7)

18.3 (54.2)(a)

Cervical vertebra(e): unossified centrum, F (L)

0 (0)

0 (0)

0.6 (4.2)

11.7# (14.3)

4.9 (29.2)(a)

Cervical vertebra(e): incomplete ossification of arch, F (L)

0 (0)

0 (0)

0 (0)

3.9* (7.1)

0.8 (5.6)(a)

Thoracic vertebra(e): bipartite ossification of centrum, F (L)

0 (0)

1.4 (9.1)

1.8 (8.3)

6.5** (28.6*)

5.0 (16.7)(a)

Thoracic vertebra(e): dumbbell ossification of centrum, F (L)

0.7 (4.2)

0 (0)

2.5 (16.7)

5.2* (28.6)

6.5 (30.0)(a)

Sacral vertebra(e): incomplete ossification of arch, F (L)

0 (0)

0 (0)

0 (0)

3.9* (14.3)

0.7 (5.0)(a)

Caudal vertebra(e): unossified arch, F (L)

0 (0)

0 (0)

0 (0)

7.8** (21.4*)

1.5 (10.0)(a)

Caudal vertebra(e): unossified centrum, F (L)

0 (0)

0 (0)

0 (0)

18.2# (28.6*)

1.4 (5.6)(a)

Caudal vertebra(e): incomplete ossification of arch, F (L)

0.7 (4.2)

0 (0)

0 (0)

14.3# (21.4)

2.3 (15.0)(a)

Caudal vertebra(e): incomplete ossification of centrum, F (L)

0 (0)

0 (0)

0 (0)

6.5** (21.4*)

1.4 (10.0) (a)

Incomplete ossification of 1st to 4th sternebrae, F (L)

0 (0)

0.7 (4.5)

0.6 (4.2)

7.8** (21.4*)

3.1 (15.0) (a)

Incomplete ossification of 6th sternebra, F (L)

0 (0)

0 (0)

0 (0)

19.5# (28.6*)

2.9 (16.7)(a)

Unossified 5thsternebra, F (L)

0 (0)

0 (0)

0 (0)

6.5** (14.3)

1.7 (11.1)(a)

Unossified 6thsternebra, F (L)

0 (0)

0 (0)

0 (0)

3.9* (14.3)

0.7 (5.0)(a)

Incomplete ossification of metacarpal(s), F (L)

0.7 (4.2)

0 (0)

0.6 (4.2)

13.0# (28.6)

2.2 (10.0)(a)

Unossified metacarpal(s), F (L)

0 (0)

0.7 (4.5)

0 (0)

16.9# (28.6*)

2.9 (15.0)(a)

Forepaw: unossified proximal phalanx, F (L)

24.5 (75.0)

18.9 (54.5)

26.4 (62.5)

45.5** (85.7)

50.0 (85.0)(a)

Unossified 1stmetatarsal, F (L)

5.3 (25.0)

11.5 (40.9)

19.0# (66.7**)

66.2# (85.7#)

36.8 (72.7)(a)

Litters affected, n (%)

24 (100.0)

22 (100.0)

23 (95.8)

13 (92.9)

345 (88.9)(b)

Fetus affected, n (%)

115 (76.2)

104 (70.3)

117 (71.8)

62 (80.5)

1267 (48.8)(b)

n: number; F: fetal incidence, L: litter incidence.

HCD: Historical Control Data (Sprague-Dawley rats, March 2014 - July 2016, n = 18 studies).

(a): max.(b): mean.

Statistical significance: *: p < 0.05; **: p < 0.01; #: p < 0.001.

Table 14: Skeletal malformations

Dose level (mg/kg/day)

      0

     180

   360

     720

HCD

Number of litters

    24

      22

    24

      14

      388

Number of fetuses

   151

     148

   163

      77

    2596

Supraoccipital: split, F (L)

0 (0)

0 (0)

0 (0)

5.2* (7.1)

0 (0)(a)

Cervical vertebra(e): absent hemicentrum, F (L)

0 (0)

0.7 (4.5)

0 (0)

0 (0)

0 (0)(a)

Litters affected, n (%)

0 (0)

1 (4.5)

0 (0)

1 (7.1)

15 (3.9)(b)

Fetus affected, n (%)

0 (0)

1 (0.7)

0 (0)

4* (5.2)

18 (0.7)(b)

n: number; F: fetal incidence; L: litter incidence.

HCD: Historical Control Data (Sprague-Dawley rats, March 2014 - July 2016, n = 18 studies).(a): max;(b): mean.

Statistical significance: *: p < 0.05.

Table 15: Pregnancy status

Dose level (mg/kg/day)

         0

   180

        360

      720

Number of females

       24

     24

          24

        24

Females found dead

         0

      0

           0

         2

Females prematurely sacrificed

         0

      0

           0

         6

Non-pregnant females

         0

      2

           0

         0

Females with live fetuses on Day 21p.c.

       24

     22

          24

        16

 

Conclusions:
The test item was administered to time-mated female Sprague-Dawley rats, by gavage, once daily, from Days 6 to 20 p.c., at dose levels of 180, 360 or 720 mg/kg/day.

On the basis of the results obtained in this study:
- the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 180 mg/kg/day, based on adverse effects on body weight, body weight change and food consumption from 360 mg/kg/day and mortality at 720 mg/kg/day,
- the No Observed Adverse Effect Level (NOAEL) for embryo-fetal development was considered to be 360 mg/kg/day, based on embryo/fetal toxicity associated with malformations in a context of excessive maternal toxicity at 720 mg/kg/day.
Executive summary:

The objective of this GLP study was to evaluate the potentialtoxic effects of the test item, on the pregnant female and on embryonic and fetal development, following daily oral administration (gavage) to pregnant female rats from implantation to the day before scheduled hysterectomy [Day 6 to Day 20 post-coitum (p.c.) inclusive].

 

Methods

Three groups of 24 time-mated female Sprague‑Dawley rats received the test item, at 180, 360 or 720 mg/kg/day by oral route (gavage) once daily from Days 6 to 20 p.c. A constant dosage-volume of 5 mL/kg/day was used. Another group of 24 rats received the vehicle alone (olive oil) under the same experimental conditions, and acted as a control group.

 

Formulation concentrations were checked in the start and end of the treatment period.

 

The animals were checked daily for mortality and clinical signs. Body weight and food consumption were recorded every 2 to 3 days. On Day 21 p.c., females were sacrificed and submitted to a macroscopic post‑mortem examination. Hysterectomy was performed and the numbers of corpora lutea, uterine scars, implantations, early and late resorptions, and live and dead fetuses were recorded. The live fetuses were sexed, weighed and examined for external, soft tissues and skeletal (cartilages + bones) abnormalities.

 

Results

Chemical analysis of dose formulations:

. the test item concentrations (-5.2% to -1.8%) in the administered dose formulations analyzed in Weeks 1 and 4 remained within the acceptable range of variations (± 15%) when compared to the nominal values,

. no test item was observed in the control dose formulation.

 

Pregnancy status:

. at hysterectomy on Day 21 p.c., 24/24, 22/24, 24/24 and 14/16 surviving females were pregnant (with live fetuses) in the groups treated at 0, 180, 360 and 720 mg/kg/day, respectively.

 

Mortality:

. in the 720 mg/kg/day group, there were eight unscheduled deaths. Taking into account the similarity of the findings associated to these deaths (clinical signs, effects on body weight/food consumption and macroscopic examination at necropsy), this mortality was considered to be related to the test item treatment,

. there were no unscheduled deaths in the control group, 180 and 360 mg/kg/day groups.

 

Clinical signs:

. at 720 mg/kg/day in surviving females, most of clinical signs (soiled urogenital area, lateral decubitus, piloerection, emaciated appearance, round back, cold to the touch, hypoactivity, abdominal breathing and/or eyes half-closed) were similar to those recorded in prematurely sacrificed and/or found dead animals. They were considered to be test item treatment-related and adverse,

. there were no test item treatment-related clinical signs in the 360 and 180 mg/kg/day groups.


Body weight and body weight change:

. at 720 mg/kg/day, there were body weight losses on Days 9 to 15 p.c. (-1 to -19 g, p < 0.001). When compared with controls, these resulted in decreased mean body weights (down to -24% on Day 21 p.c., p < 0.001). These findings were considered to be test item-related and adverse,

. at 360 mg/kg/day and when compared with controls, there were a few transient statistically significant differences in mean body weight (-5% vs. controls on Day 12 p.c., p < 0.05) and/or mean body weight changes (+6 g vs. +14 g on Days 6-9 p.c., p < 0.001 and +17 g vs. +23 g on Days 9-12 p.c., p < 0.05) which were considered to be test item treatment-related and adverse,

. at 180 mg/kg/day and when compared with controls, there were no toxicologically significant effects on mean body weight or mean body weight changes. However, the transient statistically significant difference in mean body weight change (+5 g vs. +14 g in controls on Days 6 -9 p.c., p < 0.001) was considered to be test item treatment-related but of limited toxicological significance.

 

Food consumption:

. at 720 mg/kg/day, there were decreases in mean food consumption (down to -60% vs. controls on Days 15-18 p.c., p < 0.001). These findings were considered to be test item-related and adverse,

. at 360 mg/kg/day and when compared with controls, there were transient decreases in mean food consumption (down to -18% vs. controls on Days 6-9 p.c., p < 0.001) with a return to control values from Days 12-15 p.c. These findings were considered to be test item-related but of minor toxicological concern (less than 20% difference vs. controls),

. at 180 mg/kg/day and when compared with controls, there was a transient decrease in mean food consumption (-14% vs. controls, p < 0.01) on Days 6 -9 p.c. with a return to control values thereafter. This finding was considered to be test item-related but of minor toxicological concern.

 

Macroscopic post-mortem examination of the dams:

. at 720 mg/kg/day, there was a series of macroscopic findings affecting the thymus, spleen, stomach/forestomach, intestines and adrenals which were considered to be test item-related (also observed in almost all prematurely or found dead animals),

. at 360 and 180 mg/kg/day, there were no test item treatment-related findings.

 

Gravid uterus weight, carcass weight and net body weight change:

. at 720 mg/kg/day and when compared with controls, there were weight losses affecting the gravid uterus (-26%, p < 0.001), carcass (-24%, p < 0.001) and net body (-28.4 g vs. +54.1 g, p < 0.001). These adverse findings were considered to be consecutive to the effects recorded on mean body weight and food consumption at this dose level,

. at 360 mg/kg/day and when compared with controls, there was a decreased carcass weight (-6%, p < 0.05) resulting in a lower net body weight change (+33.4 g vs. +54.1 g, p < 0.05). These findings were considered to be test item treatment-related but of minor toxicological concern taking into account the amplitude of the differences,

. at 180 mg/kg/day, there were no test item treatment-related effects.

 

Hysterectomy data:

. at 720 mg/kg/day and when compared with control, there was a lower percentage of live fetuses per animal (73.2 vs. 94.7, p < 0.01) as a consequence of increased resorptions (late, early and scars) and dead fetuses (5.5% vs. 0.0%) which resulted in an increased incidence of mean percent of post‑implantation loss (26.8% vs. 5.3%, p < 0.01). All these findings were outside the range of the Historical Control Data and, considered to be test item treatment-related and adverse,

. there were no test item treatment-related effects on hysterectomy data in the 360 and 180 mg/kg/day groups.

Fetal examination:

Fetal weights:

. at 720 mg/kg/day and when compared with control, fetuses (males and females) had a lower mean body weight (-23% vs. controls, p < 0.001). At this dose level, mean fetal body weight was below the lower limit of the HCD. This finding was considered to be test item treatment-related and adverse,

. there were no effects on mean fetal body weight in the 360 and 180 mg/kg/day groups.

 

Sex ratio:

. there were no effects on mean percentage of male fetuses (sex-ratio) at any dose tested.

 

External examination:

. at 720 mg/kg/day there were fetuses with local edema (a variation affecting 14.3% of litters vs. none in controls, p < 0.001). One female had nine fetuses with anasarca (a term used to define a malformation corresponding to a generalized edema) and another one had a fetus with a cleft lip. These findings were observed in the high-dose group only and/or at incidences above the upper limit of the HCD. Therefore a test item treatment relationship was not excluded,

. at 360 and 180 mg/kg/day, there were no variations or malformations at external examination of the fetuses.

 

Soft tissue examination:

. at 720 mg/kg/day, three females had fetuses with visceral edema, fluid-filled thoracic cavity and/or, fluid-filled abdomen. While limited to 3 litters, these variations were observed in the high-dose group only and at incidences above the upper limit of the HCD. Therefore a test item treatment relationship cannot be excluded. There were no test item treatment-related variations at soft tissue examination of the fetuses from 360 and 180 mg/kg/day groups,

. there were no test item treatment-related malformations at soft tissue examination of the fetuses from any group.

 

Skeletal examination:

. from 360 mg/kg/day and when compared with controls, there were dose-related increases in the litter and/or fetal incidences of skeletal variations (unossified bones or bones with incomplete ossification). In the 720 mg/kg/day group only, the litter and/or fetal incidences were above the upper limit of HCD. These findings were considered to be test item treatment-related and not adverse. At 180 mg/kg/day, there were no test item treatment-related skeletal variations,

. at 720 mg/kg/day, there were increases in the litter and fetal incidences of split supraoccipital. This malformation affected four fetuses from the same litter. While limited to one litter, this finding was observed in the high-dose group only and not recorded in the HCD. Therefore a test item treatment relationship cannot be excluded,

. at 360 and 180 mg/kg/day, there were no test item treatment-related skeletal malformations.

 

Conclusion

The test item was administered to time-mated female Sprague-Dawley rats, by gavage, once daily, from Days 6 to 20 p.c., at dose levels of 180, 360 or 720 mg/kg/day. 

On the basis of the results obtained in this study:

. the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 180 mg/kg/day, based on adverse effects on body weight, body weight change and food consumption from 360 mg/kg/day and mortality at 720 mg/kg/day,

. the No Observed Adverse Effect Level (NOAEL) for embryo-fetal development was considered to be 360 mg/kg/day, based on embryo/fetal toxicity associated with malformations in a context of excessive maternal toxicity at 720 mg/kg/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
180 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

The classification was determined in accordance with Regulation (EC) N°1272/2008.

There is no available evidence suggesting that the registered substance is a human reproductive toxicant.

In the EOGRTS no significant developmental neurotoxicity or developmental immunotoxicity was observed. At the highest-dose and when compared with controls, there was an increased number of females in diestrus (7.3 days vs. 5.0 days, p<0.001) considered as test-item related, resulting in a prolongation of mean oestrous cycle duration (5.4 days vs. 3.9 days, p<0.001). A lower fertility index was observed (91.7% vs. 100% in controls) close to the lower value recorded in Historical Control Data (91.7% vs. 92.0% in HCD), therefore a test-item treatment relationship was considered unlikely. These effects were only observed in Parental animals and not in animals from the F1 generation, therefore their relevance for the purpose of classification is considered as dubious.

In the developmental toxicity / teratogenicity study performed in accordance with the OECD Testing Guideline 414, embryo/foetal toxicity associated with malformations was only observed in a context of excessive maternal toxicity and is therefore considered as a secondary effect to the systemic toxicity in the pregnant animals.

There is conflicting evidence regarding the registered substance displaying an endocrine mode of action as defined by the WHO, therefore no conclusion can be made regarding the endocrine disrupting properties of BPA PO.

Therefore it is concluded that the registered substance does not meet the criteria for classification as toxic to reproduction in accordance with Regulation (EC) N°1272/2008.