Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-01-31 to 2018-mm-dd
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
On study day 5, 3 female animals of MD group and all male and female animals of the HD group were treated with an unintended test item formulation. Thyroid/parathyroid glands from 1 pup/sex of 8 animals were not preserved.
GLP compliance:
yes (incl. certificate)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Name: 2,6-Di-tert-butyl-4-nonylphenol
Batch No.: 222375101
Purity: 93.1%
Storage Conditions: room temperature, closed bottle
Expiry Date: 02 May 2018
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Test System
Species/strain: healthy Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Sex: male and female; the female animals were non-pregnant and nulliparous.
Age at the start of the treatment period: approx. 13-14 weeks old
Body weight at the allocation of the animals to the experimental groups: males: 327 – 362 g (mean: 344.50 g, ± 20% = 275.60 – 413.40 g)
females: 209 – 236 g (mean: 222.45 g, ± 20% = 177.96 – 266.94 g)
The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to the German Act on Animal Welfare the animals were bred for experimental purposes. This study was performed in an AAALAC-accredited laboratory. According to German animal protection law, the study type has been reviewed and accepted by local authorities. Furthermore, the study has been subjected to Ethical Review Process and was authorised by the Bavarian animal welfare administration.

Housing and Feeding Conditions
- Full barrier in an air-conditioned room
- Temperature: 22 ± 3°C
- Relative humidity: 55 ± 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Animals were housed in groups of 5 animals / sex / cage in type IV polysulphone cages) during the premating period for both males and females and during postmating period for males depending on the mating status. During mating period, males and females were housed together in ratio 1:1 (male to female). After the confirmation of mating, females were kept individually during gestation/lactation period in type III H, polysulphone cages and males were returned to their original cage. In each cage Altromin saw fibre was used as bedding.
- Nesting material was provided latest on GD 18 for all mated females
- Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich
- Adequate acclimatisation period (at least 5 days) under laboratory conditions

Preparation of the Animals
Prior to the start of the treatment period, a detailed clinical observation outside the home cage was made. None of the animal showed pathological signs before the first administration.
Before initiation of dosing, all females were screened for two weeks for regular estrous cyclicity and females (10 females/ group) with regular estrous cycle (4-5 day cycle) were used in the study.
Before the first administration, all animals to be used for the study were weighed and assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females, respectively. Randomisation was performed with validated IDBS Workbook 10.1.2 software.
Each animal was marked with its identification number by individual ear tattoo marking.



Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
Manufacturer: Sigma-Aldrich; Batch No.: MKBW9504V, MKBQ9948V; Physical State: liquid; Storage Conditions: at room temperature; Expiry Date: 14 April 2017 (MKBW9504V) 28 April 2017 (MKBQ9948)
Details on exposure:
The test item was weighed into a tared plastic vial on a precision balance.
The test item was dissolved in a suitable vehicle (corn oil). The dose formulations were prepared by adding the required volume of vehicle and further vortexing it for 2-3 minutes.
The vehicle was selected following solubility check and also in consultation with sponsor. The same vehicle was used during the dose range finding study. The test item and control formulations were prepared once in 10 days before the administration procedure.
The vehicle was also be used as control item.

According to the results of a previous dose range finding study (BSL Munich Study No. 166596) and in consultation with the sponsor the following doses were selected for the 3 dose groups (LD = low dose, MD = medium dose, HD = high dose).

Control: 0 mg/kg/d
Low Dose: 100 mg/kg/d
Medium Dose: 300 mg/kg/d
High Dose: 1000 mg/kg/d


The highest dose level was chosen with the aim of inducing toxic effects, but no death or severe suffering. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dosage related response and NOAEL.
The animals in the control group were handled in an identical manner to the test group subjects and received the vehicle using the same volume as used for the high dose group.

The test item and vehicle were administered at a single dose to the animals by oral gavage. The application volume for all groups was 4 mL/kg body weight.
For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.
Inadvertently, on study day 5, 3 female animals of MD group (animal no. 68-70) and all male and female animals of the HD group (animal no. 31-40 and 71-80) were treated with an unintended test item. The unintended test item was administered at a dose of 50 mg/kg (MD) and 100 mg/kg bw (HD) with an application volume of 4 mL/kg corn oil.





Details on mating procedure:
Mating was performed using a ratio of 1:1 (male to female) (if possible). The vaginal smear of the females were checked every morning after the start of the mating period to confirm the mating. If the vaginal smear of a particular female was not found to be sperm-positive, the actual stage of the estrus cycle on that day was documented. The day of the vaginal plug and/or sperm was considered as day 0 of gestation.
The cages were arranged in such a way that possible effects due to cage placement were minimised.


Analytical verification of doses or concentrations:
yes
Remarks:
During the study samples were collected for the investigation of substance concentration. Sampling for investigation of substance concentration in study week 1 was repeated as formulations had not been stirred sufficiently.
Details on analytical verification of doses or concentrations:
During the study samples were collected for the investigation of homogeneity and substance concentration.
Samples were taken from the top, middle and bottom of prepared formulations from all dose groups and from the middle of the control group in study week 1
(pre-mating period), 3 (first week of mating), 5 (gestation) and in the last week of the study (gestation / lactation) from all groups (40 samples).
Each sample taken during the study was retained in duplicate (sample A, sample B, each of at least 5 mL). The A-samples were analysed at Eurofins Munich and until then stored under appropriate conditions based on available stability data. The procedures followed for the study
sample analysis were mentioned in a phase plan amended to the study plan. The B-samples were retained at -15 to -35 °C at BSL Munich
(test facility) and were discarded after completion of the final study report.

Duration of treatment / exposure:
The animals were treated with the test item formulation or vehicle on 7 days per week for a maximum period of 63 days, i.e. during 14 days of pre-mating and
maximum 14 days of mating in both males and females. Then in females, treatment was done during the gestation period and up to post-natal day 12.
Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.
Frequency of treatment:
daily
Details on study schedule:
Arrival of the Test Item: 13 September 2016
Study Initiation Date: 31 January 2017
Amendment to Study Plan: 20 February 2017
2nd Amendment to Study Plan: 03 March 2017
3rd Amendment to Study Plan: 23 March 2017
4th Amendment to Study Plan: 23 June 2017
Delivery of Animals: 26 January 2017
Acclimatisation Period: 26 January 2017 – 08 February 2017
Experimental Starting Date: 08 February 2017
Treatment Period: 14 February 2017
Necropsies: 1 5 March 2017, 16 March 2017
Experimental Completion Date: 16 March 2017
Completion Date of Delegated
Phase (Histopathology): xx
Completion Date of Delegated
Phase (Formulation Analysis): xx
Study Completion Date: xx



Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
100 animals (40 males and 60 females) were included in the study. All females were screened for regular estrous cycles for 14 days before the treatment initiation and only 40 females (10 females/group) showing regular estrous cycles were continued in the study.
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
Clinical Observations
General clinical observations were made at least once a day, preferably at the same time each day. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations are made once daily.
Once before the first exposure, and at least once a week thereafter, detailed clinical observations were made in all animals outside the home cage in a standard arena. Clinical observations include spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.

Functional Observations
Multiple detailed behavioural observations were made in the week before the first treatment and during the last week of the treatment in 5 randomly selected males and during the last week of lactation of the lactation period in 5 randomly selected females (only lactating females were evaluated) of each group outside the home cage using a functional observational battery of tests:
Sensory reactivity to different modalities, grip strength and motor activity assessments and other behavioural observations as well as rearing supported and not supported, urination, defecation, startle/ auditory response, equilibrium reflex, positional passivity, visual placing, fore and hind limb grip strength, tail pinch response, toe pinch reflex, extensor thrust/limb tone, hind limb reflex, righting reflex on the ground, air righting reflex, pupil response, body temperature and ophthalmoscopy (anterior chamber of the eye and fundus of eye).

Body Weight and Food Consumption
The animals were weighed once before the assignment to the experimental groups, on the first day of dosing and weekly thereafter as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum), on PND 4 and PND 13 along with the pups. All animals were weighed directly before termination. Any animals prematurely sacrificed were weighed prior to the sacrifice.
Food consumption was measured on the corresponding days of the body weight measurements after the beginning of the dose administration. Food consumption was not measured during the mating period in males and females and the post-mating period in males.

Haematology
Haematological parameters were examined in 5 randomly selected males and all females (only lactating females were evaluated) from each group at the end of the treatment prior to or as part of the sacrifice of the animals. Blood from the abdominal aorta of the animals was collected in EDTA-coated tubes. The following haematological parameters were examined: haematocrit value (Hct), haemoglobin content (Hb), red blood cell count (RBC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), reticulocytes (Re), platelet count (PLT), white blood cells (WBC), neutrophils (Neu), lymphocytes (Lym), monocytes (Mono),
eosinophils (Eos), basophils (Baso) and large unstained cells (Luc).

Blood Coagulation
Coagulation parameters from 5 randomly selected males and all females (only lactating females were evaluated) of each group were examined at the end of the treatment prior to or as part of the sacrifice of the animals. Blood from the abdominal aorta of the animals was collected in citrate tubes. The following coagulation parameters were examined: prothrombin time (PT) and activated partial thromboplastin time (aPTT).

Clinical Biochemistry
Parameters of clinical biochemistry from 5 randomly selected males and all females (only lactating females were evaluated) of each group were examined at the end of the treatment prior to or as part of the sacrifice of the animals. Blood from the abdominal aorta of the animals was collected in serum separator tubes. The following parameters of clinical biochemistry were examined: alanine aminotransferase (ALAT), aspartate-aminotransferase (ASAT), alkaline phosphatase (AP), creatinine (Crea), total protein (TP),
albumin (Alb), urea, total bile acids (TBA), total cholesterol (Chol), glucose (Gluc), sodium (Na) and potassium (K).
From 2 female (if possible) pups/litter on day 4 after birth, from all dams and 2 pups/litter (1 male and 1 female, if possible) at termination on day 13 and from all adult males at termination blood samples were collected from the defined site. All blood samples were stored under appropriate conditions. Blood samples from the day 13 pups and the adult males were assessed for serum levels for thyroid hormones (T4). Further assessment of T4 in blood samples from the main study dams and day 4 pups were not deemed necessary. Additionally, assessment of TSH in day 4 pups, adult females, day 13 pups, and adult males were not considered necessary based on the fact that no histopathological findings were observed in thyroid/parathyroid gland of selected male and female adult animals and T4 hormone levels of males and day 13 pups. The study monitor was consulted for these decisions. Pup blood was pooled by litter for thyroid hormone analysis.

Urinalysis
A urinalysis was performed with samples collected from 5 randomly selected males and females (only lactating females were evaluated) prior to or as part of the sacrifice of the animals. Additionally, urine colour/ appearance were recorded. The following parameters were measured using qualitative indicators (Henry Schein Medical GmbH, Urine Stripes): specific gravity, nitrite, ph-value (pH), protein, glucose, ketone bodies (ketones), urobilinogen (ubg), bilirubin, blood and leukocytes.












Oestrous cyclicity (parental animals):
Estrous cycles were monitored before treatment starts to select females with regular cyclicity (using vaginal smears) for the study. Further on, vaginal smears were also examined daily from the beginning of the treatment period until evidence of mating.
Litter observations:
The duration of gestation was recorded and is calculated from day 0 of the pregnancy. Each litter was examined as soon as possible after the delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities.
Live pups were counted and sexed and litters weighed within 24 hours of littering (PND 0) and on PND 4 and PND 13. Live pups were identified by tattooing. In addition to the observations of the parent animals, any abnormal behaviour of the offspring was recorded.
The anogenital distance (AGD) of each pup was measured on PND 0. Pup body weight measured on PND 0 was converted to cube root and used for the calculation of relative AGD (Relative AGD = AGD / Cube root of pup weight). The number of nipples/areolae in male pups was counted on PND 12.

Postmortem examinations (parental animals):
Pathology
Males were sacrificed any time after the completion of the mating period (after a minimum dosing period of 28 days) and females were sacrificed on the respective PND 13 by using anesthesia (ketamine/xylazine). All surviving pups were killed by cervical dislocation on PND 13.
Vaginal smears were examined on the day of necropsy to determine the stage of estrous cycle.
Non-pregnant females were sacrificed on day 26 from the day of sperm positive vaginal smear or from the last day of mating period.
All adult animals were subjected to a detailed gross necropsy which includes careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents. 
Special attention was paid to the organs of the reproductive system. The ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coagulating glands as a whole), the thyroid/parathyroid glands and all organs showing macroscopic lesions of all adult animals were preserved in 4% neutral-buffered formaldehyde, except for testes and epididymides which were preserved in modified Davidson’s Solution for 24 hours and then transferred to 70% ethanol.
The number of implantation sites and corpora lutea were recorded for each parental female at necropsy. The number of corpora lutea and implantation sites were recorded for females sacrificed 26 days after the end of the mating period with no evidence of mating and or on day 26 post-coitum due to non-delivery.


Organ Weights
The wet weight of the organs [testes (paired weight), levator ani + bulbocavernosus, muscle complex (complete weight), epididymides (paired weight), glans penis, prostate, seminal vesicles and coagulating glands (complete weight), uterus with cervix, Cowper’s gland (paired weight), ovaries (paired weight), thyroid/parathyroid glands (from 1 pup/sex/litter/group and from all adult males and females) – weighed after fixation (complete weight), thymus, liver, spleen, kidneys (paired weight), brain, adrenal (paired weight), heart and pituitary gland] of 5 randomly selected male and female animals (only lactating females were evaluated) from each group were recorded as soon as possible. Paired organs were weighed together. Organ weights of animals found dead or euthanised for animal welfare reasons were not recorded.
Reproductive organs were weighed from all animals.
Thyroid/parathyroid glands from 1 pup/sex/litter/group (sacrificed on PND 13) and from all adult males and females were preserved. Weight of thyroid/parathyroid glands were measured after fixation.
Adrenal glands, all gross lesions, aorta, brain (incl. medulla/pons, cerebellar and cerebral cortex), caecum, colon, duodenum, epididymides, eyes with optic nerve and Harderian gland, femur with knee joint, heart, ileum (including Peyer´s patches), jejunum, kidneys, liver, lungs, lymph nodes (mandibular), lymph nodes (mesenteric and axillary), mammary gland area (male and female), oesophagus, ovaries, oviducts, pancreas, parathyroid glands, pituitary, prostate and seminal vesicles with coagulating glands as a whole, rectum, salivary glands (sublingual, submandibular), sciatic nerve, skeletal muscle, skin, spinal cord (cervical, thoracic and lumbar segments),
spleen, sternum (with bone marrow), stomach, testes, thymus, thyroid/parathyroid glands, tongue, trachea, ureters, urinary bladder, uterus with cervix and vagina of the same selected animals from each group were preserved in 4% neutral-buffered formaldehyde except for testes and epididymides that were fixed in Modified Davidson’s Fixative for approximately 24 hours before they were transferred to 70% ethanol. All animals found dead and/or intercurrently euthanised for animal welfare reasons were subjected to a gross necropsy and the organs preserved for a histopathological examination.
Thyroid/parathyroid glands from 1 pup/sex/litter/group sacrificed on PND 13 and non-selected adult animals were preserved for potential histopathological examination. The thyroid gland weight was measured after fixation.


Histopathology
A full histopathology was carried out on the preserved organs and tissues of the selected animals of the control and high dose groups which were sacrificed at the end of the treatment period. Thyroid/parathyroid glands from pups and from the remaining non-selected adult animals was not deemed necessary as no test item related histopathological findings were observed in thyroid/parathyroid gland of selected animals and there was also no test item related effect observed on T4 hormone level in males and pups sacrificed on PND 13.
A full histopathology was carried out on the preserved organs and tissues of all animals which died during the study or which were euthanised due to morbidity.

Testes, epididymides, ovaries, uterus with cervix, vagina, accessory sex organs (prostate, seminal vesicle with coagulating gland) were trimmed, embedded into paraffin, cut at an approximated thickness of 2-4 µm and stained with hematoxylin and eosin and examined in control and HD animals and in non-pregnant female animals of the LD and MD animals. Testes, epididymides and accessory sex organs (prostate, seminal vesicle with coagulating gland) were also examined in the mating partners of the non-pregnant female LD and MD animals.
In addition, from 5 randomly selected males and females from the LD and MD group liver was processed and evaluated. Further, from the 5 selected males from the LD and MD group lung, axillary lymph nodes and kidney were processed and evaluated.
Any gross lesion macroscopically identified was examined microscopically in all animals. Discoloration possibly due to the test item was evaluated in the organs of all dose groups.
For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides.
The histological processing of tissues to microscope slides was performed at the GLP-certified contract laboratory AnaPath GmbH, AnaPath Services, Hammerstrasse 49, 4410 Liestal, Switzerland (test site for tissue processing). The histopathological evaluation was performed at the GLP-certified contract laboratory AnaPath GmbH, AnaPath Services, Hammerstrasse 49, 4410 Liestal, Switzerland (test site for histopathology). The study phases from test site 1 and 2 were performed in compliance with the Swiss Ordinance relating to Good Laboratory Practice adopted 18 May 2005 [SR 813.112.1] (Status as of 01 December 2012). Blocking, embedding, cutting, H&E staining and scientific slide evaluation were performed according to the corresponding SOP’s of the test sites.
The principal investigator for histopathological tissue processing sent all raw data (including blocks, slides, paper raw data, statement of compliance and quality assurance statement) to the study director.
The principal investigator for histopathological evaluation provided the histopathology results to the study director by e-mail and sent a pathology phase report to the study director upon the completion of the study. The histopathology phase report was incorporated as an appendix to the final study report.



Postmortem examinations (offspring):
Dead pups and all surviving pups on PND 13 were carefully examined externally for gross abnormalities before terminal sacrifice.
Statistics:
A statistical assessment of the results of body weight, food consumption, parameters of haematology, blood coagulation, clinical biochemistry and litter data was performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and
relative organ weights were statistically analyzed by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. These statistics were performed with GraphPad Prism V.6.01 software or Ascentos 1.1.3 software (p<0.05 was considered as statistically significant).

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
see Details on results P0
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
see Details on results P0
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
see Details on results P0
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
see Details on results P0
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
see Details on results P0
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
see Details on results P0
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
see Details on results P0
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
see Details on results P0
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see Details on results P0
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
see Details on results P0

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
see Details on results P0
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
see Details on results P0

Details on results (P0)


Clinical Observations
In terminally sacrificed males, predominant clinical sign observed during the treatment period (premating day 1 to mating day 14) was slightly to moderately
increased salivation in few animals of LD and almost all animals of MD and HD group. Isolated incidences of diarrhoea in 2 males (no. 24 and 25) of MD group was considered to be incidental.

In terminally sacrificed females, predominant clinical signs observed during the treatment period (PD 2 to PND 12) were moving the bedding, slightly to
moderately increased salivation in few animals of LD, MD and HD group.
Isolated incidences of alopecia on various body parts, moderate to severe piloerection, sunken flanks, hunched posture, anemia and bloody vagina were
observed on few occasions in very few female animals in all group including control group and considered to be incidental in nature.
Moving the bedding was observed transiently in few males and all females of the LD and almost all male and female animals of MD and HD group. The clinical signs salivation and moving the bedding were observed immediately after the dose administration and therefore were considered to be a sign of discomfort
due to a local reaction to the test item rather than a systemic adverse effect and has no toxicological relevance.
None of the females showed signs of abortion or premature delivery.
During the weekly detailed clinical observation, no relevant differences between the groups were found.

Mortality
During the treatment period of this study, few mortalities/moribund sacrifice were observed as follows:
- Female no. 42 (C) was found dead on gestation day (GD) 9. Clinical signs observed before death on GD 8 were abnormal breathing and severe salivation.
- Male no. 35 (HD) was found dead on day 27. No specific clinical signs were observed before death except moving the bedding from day 17-26 and
salivation from day 21-26.
- Male no. 38 (HD) was euthanised on day 29 in moribund condition for animal welfare reasons. Clinical signs observed in this animal were moving the
bedding from day 18-28, salivation on day 28, spontaneous activity markedly reduced, marked piloerection, nasal discharge, anaemia and slightly reduced grip strength on day 29.
- Male no. 40 (HD) was found dead on day 14. No specific clinical signs were observed before death.
- Female no. 72 (HD) was found dead on gestation day (GD) 13. Clinical signs observed before death were diarrhoea from premating day 3-4, moving the
edding from GD 1-9 and 12, moderate abnormal breathing and moderate piloerection on GD 9, slightly reduced spontaneous activity on GD 9-12, slight
piloerection on GD 11-12 and nasal discharge (red) on GD 12.
- Female no. 78 (HD) was found dead on gestation day (GD) 12. Clinical signs observed before death were moving the bedding on premating day 2, 4 and GD1-11, slight to severe salivation on GD 5 and GD 7-9 and slight piloerection on GD 11.
- Female no. 80 (HD) was euthanised in moribund condition on post natal day (PND) 0 due to animal welfare reasons. Predominant clinical signs observed
were moving the bedding on GD 0-21, slight to moderate salivation on GD 1-3 and diarrhoea on GD 13. On PND (post-natal day) 0, severely reduced
spontaneous activity, sunken flanks, severe piloerection and bloody vagina was observed.
Histopathologically, the cause of death/morbidity of animal no. 42 (C- female), 38 (HD-male) and 72 (HD- female) was not evident. The cause of morbidity of
animal no. 80 (HD- female) was considered to be incidental intra-uterine hemorrhage (hemometra) and most likely of traumatic nature. The cause of death of
animal no. 35 (HD- male) was inflammatory process involving the heart (myocardium). The cause of death of animal no. 78 (HD- female) was inflammatory
process involving the heart (myocardium and pericardium) and intra-uterine hemorrhage (hemometra) which was deemed to be incidental and most likely of
traumatic nature. Death of animal no. 40 (HD- male) was attributed to peri-renal hemorrhage.

Body Weight Development
In males, there was no statistically significant difference observed on body weight between the LD, MD and HD dose groups and the control group during the entire study period.
Group mean body weight gain in HD group was statistically significantly lower during premating day 1-7, premating day 14 to mating and post mating day 7
and premating day 1 to terminal sacrifice when compared with the control. There was also a lower body weight gain observed in HD group during premating day 1 and post mating day 14 without achieving the statistical significance when compared with the control.
In females, statistically significantly higher group mean body weight was observed during premating day 14, lactation day 4 and 13 in LD group when
compared with the controls.
There was statistically significantly lower group mean body weight gain observed during gestation day 14-20 and during overall gestation period i.e. gestationday 0-20 in HD group when compared with the controls.
This statistically and biologically significant effect on body weight gain in HD group male and female was considered as test item related and toxicologically relevant. However, due to lack of dose dependency and consistency, statistically significant effect on female group mean body weight in LD group was not considered to be toxicologically relevant.

Food Consumption
In males, in correlation to the body weight and body weight gain, the food consumption during treatment period tended to increase with the progress of the
study in the control, the LD, the MD and the HD group. However, In HD group, during premating period, food consumption was lower without achieving
statistical significance compared to the control group and this effect on food consumption in males was considered to be test item related.
In females, no statistically significant effect on food consumption was observed during premating period, gestation and lactation period in treatment groups
when compared with the controls. However, in correlation to body weight development, marginally lower food consumption was observed in MD and HD
animals during gestation day 7-14 and 14-20 when compared with the controls.

Haematology and Coagulation
In males sacrificed at the end of treatment period, no test item related adverse effects were observed for haematological parameters. However, there was a statistically significantly higher platelets (PLT) and reticulocytes (RE) count observed in HD group compared to the control group. There was also statistically
significantly higher prothrombin time (PT) observed in MD group when compared to the controls. In HD group, due to clotted samples and few invalid values,
no group mean data was available.
All mean and most of the individual values were within the historical control data range. As group mean value was within historical control data limit,
statistically significant effect on PLT and RE in HD group was considered to be incidental and not related to the treatment with test item. However, increase in
prothrombin time (PT) in MD, could be attributed to hepatocellular injury of the liver revealed after histopathology evaluation and inability of liver to synthesise
clotting factors.
In females sacrificed at the end of treatment period, higher but statistically insignificant PLT count was observed in HD group when compared with the
controls. There was also statistically significantly lower prothrombin time (PT) was observed in LD, MD and HD group when compared to the controls.
All mean and most of the individual values were within the historical control data range. As group mean value was within historical control data limit,
statistically significant effect on PLT in HD group was considered to be incidental and not related to the treatment with test item. Lover prothrombin time has noclinical significance and therefore, this effect on prothrombin time in female was not considered to be adverse.

Clinical Biochemistry
In males sacrificed at the end of treatment period, significantly lower total bile acids (TBA) was observed in MD and HD group although statistical significance was only achieved in HD group when compared with the controls. There was also marginally higher alanine aminotransferase (ALAT) and alkaline
phosphatase (AP) observed in MD and HD group without achieving statistical significance when compared with the controls.
In females sacrificed at the end of treatment period, higher aspartate aminotransferase (ASAT) ALAT and AP were observed in HD group without achieving
statistical significance when compared with the controls. There was also statistically significantly higher total protein (TP), cholesterol, Potassium in HD group and statistically significantly lower creatinine and TBA in MD and glucose in MD and HD group when compared with the controls.
This effect on clinical biochemistry parameters (TBA, ASAT, ALAT and AP) in male and female was considered to be test item related and could be attributed to hepatocellular injury of liver as correlated with histopathology evaluation.
Furthermore, there was data variation observed in individual values for sodium and potassium in few males (Control 2/5 and HD 1/5) and females (Control 3/9,LD 4/10, MD 6/10) of all groups including controls.
It is common to have data variation in electrolyte measurement without effect on animal health for either individual or group of animals which do not appear to be dose related or correlate with histopathological findings in kidney. Excess water intake occurs when excessive thirst is induced by the test item or where renal excretion does not act as a compensatory mechanism, this result in lowering of the sodium. However, no indication of excess water intake was
observed during clinical examination of the animals in this study. In the light of fact that no major test item related clinical signs (except moving the bedding andoccasional salivation in few LD and MD females), absence of relevant histopathological lesions in kidney, animals with lower sodium values in control group
and no dose dependency in the dose groups, this effect was considered to be incidental and not related to treatment with test item.
Generally, lower potassium is observed in animals with diarrhoea, renal tubular defects and thrombocytopenia. As no such clinical signs, neither relevant
kidney lesions nor effect on platelet count was observed in these animals, this effect on potassium in few male and female animals was not considered to be of toxicological relevance.
In both male and female animals of all groups including controls, there were few individual values below the 95% range for TP, ALB (males: Control 2/5, LD
1/5, HD 1/5 and females: Control 2/9, LD 3/10, MD 3/8).
See below our historical data for these parameters (Na, K, TP and ALB). The biological range corresponds to the range where 95% of the values can be
expected within our laboratory as these biological parameters follow a normal distribution. In this study the values outside of the range were distributed
throughout all the groups and in both sexes.
Although these out-of-range data could suggest altered hoemeostasis potentially resulting in an impaired health condition, as the remaining other hematologicaland clinical chemistry parameters did not indicate any such deviations, these data are considered incidental and the origin whether technical, analytical or
biological, could not be identified.

Urinanalysis
The urinalysis performed in selected male and female animals sacrificed at the end of treatment period revealed no test item treatment related effect and all
urinary parameters were in the normal range of variation. High protein levels were found in the urine of few male and females of all groups including control
group. Therefore, this effect on urine parameters was not considered to be test item related.

Functional Obsrevations
In males, no relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period.
There were no biologically relevant differences in body temperature between the groups.
In females, no relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period
except statistically significantly higher supported rearing count was observed in last week of the treatment in HD group when compared with the controls. As
this type of difference was marginal, it has no toxicological relevance and could be considered as biological variation. There were no biologically relevant
differences in body temperature between the groups.

Organ Weights
In males sacrificed at the end of treatment period, there were statistically significantly higher thyroid/parathyroid and liver weight (absolute and relative to
body weight) in MD and HD group when compared with the controls. There was also statistically significantly lower relative (to body weight) liver weights
observed in LD group when compared with the controls. In the light of absence of histopathological findings and T4 hormone levels in males, this effect on
thyroid/parathyroid weight is not considered to be adverse.
In females sacrificed at the end of treatment period, statistically significantly higher absolute and relative liver weights (relative to body weight) were
observed in LD, MD and HD group when compared with the controls.

This effect on male and female liver weight was considered as test item related as histopathologically hepatocytes vacuolization (fatty changes) was
observed in all dose groups and in both males and females. The hepatocytes vacuolization sometimes associated with hepatocytes hypertrophy which was correlated with increased liver weights in male and females.

Pathology
Few specific macroscopic changes were recorded for the male and female animals, which based on microscopic examination were not considered to be of
test item treatment relevance.
The macroscopic changes observed were fluid filled- thoracic cavity (male no. 34 of HD group, female no. 78 of HD group), oesophagus – red fluid filled
(male no. 34 of HD group), heart – hard (female no. 76 of HD group), pericardium - oily fluid filled and enlarged (female no. 78 of HD group ), lung – abnormal
surface (female no. 76 of HD group), stomach – filled with dark fluid or (male no. 38 of HD group, female no. 80 of HD group), Jejunum - mass 5-8 cm in
diameter and adhesion with right kidney and left testes (male no. 40 of HD group), payer’s patches - dark discolouration (female no. 78 of HD group), caecum - gas filled (female no. 42 of control group), caecum - filled with dark fluid (male no. 38 of HD group), liver- abnormal colour and spotted (female no. 42 of control group), kidneys - dark abnormal colour (male no. 40 of HD group), uterus - filled with blood (female no. 72 of HD group), spleen- abnormal colour – black
(male no. 40 of HD group, female no. 42 of control group), spleen - small (male no. 38 of HD group), thymus - dark abnormal colour and enlarged on both sides(male no. 35 of HD group) testes (male no. 40 of HD group) and seminal vesicles - dark abnormal colour (male no. 35 of HD group), mesenteric lymph node -
dark discolouration (female no. 78 of HD group), mammary gland- grey abnormal colour (female no. 42 of control group), skin - black abnormal colour (male no.40 of HD group), corpora lutea - abnormal colour – dark (female no. 42 of control group) and implantation sites - enlarged - Both sides (female no. 42 of
control group).
Macroscopic findings correlating with histopathological observations were observed in following animals from the HD group:
- Animal no. 35 (Male – HD group)- the enlarged and dark coloured (abnormal color, dark) thymus observed at necropsy histologically correlated with severe thymic hemorrhages.
- Animal no. 38 (Male – HD group) – the small spleen (small complete) observed at necropsy histologically correlated with splenic atrophy.
- Animal no. 40 (Male – HD group) – a black mass surrounding the kidney was observed and histologically correlated with severe peri-renal hemorrhage.
- Animal no. 78 (Female – HD group) - the enlarged pericardium histologically correlated with the mixed cell infiltration observed in the heart.
The above mentioned findings were deemed incidental. All other observed gross lesions reflected the animal physiology or, in absence of corresponding
histopathological findings, were considered to be of no toxicological relevance.

Histopathology
Under the conditions of this experiment, repeated dose exposure to 2,6-di-tert-butyl-4-nonylphenol induced microscopic changes at all doses in the liver of
treated animals.
There were 7 animals (6 from the HD group and one control animal) found dead or sacrificed in moribund condition during the treatment period.
The lymphocyte depletion observed in spleen, mesenteric lymph node, axillary lymph nodes and thymus of the decedents and moribund animals could be
induced by the test item application or, could be stress related.
In the heart there was a mixed inflammatory cell infiltrates in few decedents from the high dose group. The observed heart changes were deemed most likely incidental not treatment related. Further, the inflammatory cell infiltrates and hemorrhages observed in the pericardium of one female decedent were also
considered incidental not treatment related.
Furthermore, the hemorrhages observed in different organs (i.e. thymus, epididymis) were considered to be of traumatic nature and not test item related.
In liver, hepatocellular vacuolation was observed in both surviving and decedent animals. The hepatocellular vacuolation in all groups showed dose
dependency and was considered adverse.
Further, in liver, glycogen accumulation was observed in surviving females from the mid and high dose groups. This finding could also be related to the animal feeding schedule. However, the glycogen accumulation in hepatocytes, although test item related, was considered as non-adverse.
In kidneys, hyaline droplets were observed in the cytoplasm of tubular epithelial cells. The observed hyaline droplets represent an accumulation of secondary lysosomes containing alpha-2u globulin in the cytoplasm of tubular epithelial cells. This process is considered to be a male rat specific phenomenon not
occurring in humans and is of no toxicological relevance for humans.
In lung, accumulation of vacuolated alveolar macrophages (histiocytosis) were observed in decedent and surviving animals of all groups including controls.
This histopathologic change could be related to aspiration of small amounts of vehicle (corn oil) and, in decedents, to an ongoing inflammatory process
whereas a relationship with the administration of the 2,6-Di-tert-butyl-4-nonylphenol cannot be fully excluded. However, based on the incidence, severity and distribution the observed lung change was considered to be most likely incidental in nature
In axillary lymph nodes, accumulation of vacuolated alveolar macrophages (histiocytosis) were observed in decedent and surviving animals. There was also a minor degree of lymphocytes depletion observed. This histopathologic change was considered most likely stress related, whereas a relationship with the
administration of 2,6-Di-tert-butyl-4-nonylphenol cannot be fully excluded.
The vacuolization of adrenal gland cells from the Zona fasciculata was considered to be most likely secondary to stress from various causes and was
deemed incidental and non-adverse.
The histopathologic changes observed in the esophagus of one animal were considered related to the route of administration (gavage) of the test item and
were deemed incidental.
Microscopic evaluation of reproductive organs in surviving animals and decedent revealed no histological evidence of toxicity in the reproductive organs and tissues including testes, epididymides, prostate gland, seminal vesicles, coagulating glands, ovaries, oviducts, uterus and cervix, and vagina. In decedents
(animal no. 78 and 80), intrauterine hemorrhage was observed and considered most likely to be incidental in nature.
In testes, a detailed qualitative examination of hematoxylin-PAS (Periodic Acid Schiff) stained slides after taking into account the tubular stages of the
spermatogenic cycle (Sperm staging) revealed no treatment-related effects on the testicular histomorphology including spermatogenesis and interstitial cell
structure.
In non-pregnant females and mating partners, no treatment related histomorphological effects were observed in the reproductive organs of the non-pregnant control female (no. 42) and its pairing partner (no. 2), the non-pregnant mid dose group females 63 and 65 and their pairing partners 23 and 25 and the
non-pregnat high-dose group females 72 and 78 and their pairing partners (no. 32 and 38).
Summing up, in high dose animals the repeated dose administration of 2,6-di-tert-butyl-4-nonylphenol induced adverse changes in the liver.
In addition, in high and mid dose groups several overlapping histopathologic changes were observed, whereas in the low dose group only hepatocellular
vacuolation was present. In addition, the comparison between low dose and control animals revealed that hepatocellular vacuolation was present at slightly
higher incidence (2/5 in low dose animals vs. 1/5 animals in controls) but similar severity in low dose animals. Therefore the hepatocellular vacuolation in
animals from the low dose group was regarded as low adverse effect and a LOAEL (low adverse effect level) was established at 100 mg/kg bw/day for
repeated dose administration of 2,6-di-tert-butyl-4-nonylphenol in male and female Wistar rats.

Precoital Interval and Duration of Gestation
There were no effects on the duration of precoital interval and the duration of gestation in the dose groups when compared to the control group.

Estrous Cyclicity
Test item had no biologically significant effect on the estrous cycle analysed during 2 weeks premating period after the first administration. There were no
considerable differences in the length or sequence of cycle stages between the dose groups and the control group. Deviations from the physiological 4 or 5 day cycle in the rat were observed occasionally in 3 animals of HD treatment group. There was also statistically significant increase in mean number of
abnormal cycles observed in HD group. However, this effect was attributed to just these 3/10 animals (no. 71, 73 and 80) with 3 day cycle from HD group
and therefore this effect was considered as biological variation and not related to the treatment with the test item.

Pre- and Postnatal Data
There were no test item treatment related effects observed on the number of corpora lutea, percent preimplantation loss and post implantation loss in
treatment groups when compared with the control group. However, statistically significantly lower number of implantation sites were observed in HD group
when compared with the controls. This was also correlated with slightly low group mean values for live pups on PND 0, 4 and 13 in HD group without
achieving statistical significance when compared with the controls. This difference in number of implantation sites and number of live pups in HD group was marginal but could be attributed to treatment with test item and considered to be toxicologically relevant.

Reproductive Indices
There were no test item related effects on the reproductive indices (copulation, viability and delivery indices) in the dose groups when compared to the
control group. However, a reduced fertility index (number of females pregnant/ No. of females copulated X 100) of 80 % in the MD and HD group was
observed compared to 90 % in control group. Although there was reduction in fertility index in MD and HD group, it was within the standard pregnancy rate ofrat i.e. 80 % and therefore this effect on fertility index was considered as biological variation and not related to treatment with the test item administration.






Effect levels (P0)

open allclose all
Dose descriptor:
LOAEL
Effect level:
ca. 100 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other:
Remarks:
The LOAEL (Low Observed Adverse Effect Level) of 2,6-Di-tert-butyl-4-nonylphenol in this study for general toxicity is considered to be 100 mg/kg bw/day.
Dose descriptor:
NOAEL
Effect level:
ca. 300 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
reproductive performance
Remarks on result:
other:
Remarks:
The NOAEL (No Observed Adverse Effect Level) for reproductive toxicity screening is considered to be 300 mg/kg bw/day.

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
see Details on results F1
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
see Details on results F1
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
see Details on results F1
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
see Details on results F1
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
see Details on results F1
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
see Details on results F1

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

Litter Data
There were no test item treatment related or statistically significant effects observed on litter data parameters like number of male pups, number of female pups,
sex ratio, number of live pups, still birth, runt on PND 0 as well as number of male pups, number of female pups, number of live pups and sex ratio on PND 4 and
PND 13 when compared with the controls. However, group mean total number of pups born were statistically significantly lower in HD group when compared
with the controls.
This decrease in group mean total number of pups born in HD group was marginal but could be attributed to treatment with test item and considered to be
toxicologically relevant.

Litter Weight Data
There was no statistically significant effect on pup mean weight, total litter weight, male and female litter weight on PND 0, PND 4 and PND 13 observed in
treatment groups when compared with the controls. However, in correlation to low number of female pups, lower group mean female litter weight values
were observed on PND 0, 4 and 13 without achieving statistical significance. This decrease in female litter weight in HD group was attributed to low female
pups in just few females (74, 79 and 80) of the HD group. Therefore this effect on female litter weight was not considered to be test item related.
There were also statistically significantly higher pup mean weight observed on day 13 in LD group when compared with the controls. Due to lack of dose
dependency, this effect in LD group was considered to be incidental and not related to treatment with test item.

Pup Survival Data
A slightly higher mean mortality of pups between PND 0 and PND 4 was observed in the LD (1%) and MD (6.25%) group compared to the control group
(0.0 %). No effect on pup survival was observed in treatment groups during PND 4-13. This effect on pup survival in LD and MD group from PND 0-4 was not considered as test item related due to lack of dose dependency and consistency.

Anogenital Distance and Nipple Retention
In males, no test item related effect of toxicological relevance was observed on nipple retention and anogenital distance in the pups of any of the groups.
However, statistically significant higher pup weight and cube root of pup weight on the day of anogenital measurement was observed in male and female LD and HD group when compared with the controls. In absence of effect on anogenital distance in treatment group, this statistically significant effect on pup
weight and cube root of pup weight in LD and HD group was not considered as toxicologically relevant.

Thyroid Hormone (T4) Analysis and Pup Thyroid Weight on PND 13
No test item related effect of toxicological relevance was observed on pup thyroid weight, male and PND 13 pup thyroxine hormone (T4) in the treatment
groups when compared to the controls. However, statistically significantly higher group mean thyroid weight in female pups were observed in LD group
when compared with the controls. Due to lack of dose dependency, this effect on thyroid weight in LD group female was not considered to be test item
related.

Pup External Findings
No test item related gross external abnormalities of toxicological relevance on PND 0-12 were observed in the pups of any of the groups. Few specific findings like squashed abdomen (pup no. 15 from dam 47 of control group on PND 0, pup no. 8 from dam 50 of control group on PND 1, pup no. 11 from dam 59 of
LD group on PND 0, pup no. 10 from dam 62 of MD group on PND 0 and pups no. 15 from dam no. 70 of MD group), no indication of suckling and cold to touch on PND 0 (pup no. 3 from dam 77 of HD group), dark head on PND 0 (pup no. 10 from dam no. 62 of MD group) and partly cannibalism (pup no. 9 from dam
no. 50 of control group) were observed in all groups including control group.
The external finding like absent hair coat (pup no. 1-9 from dam no. 62 of MD group) at death was considered to be spontaneous and not related to test item
treatment.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
300 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: group mean total number of pups born
Remarks on result:
other:
Remarks:
The NOAEL (No Observed Adverse Effect Level) for reproductive toxicity screening is considered to be 300 mg/kg bw/day.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
On the basis of this combined repeated dose oral toxicity and reproduction/ developmental toxicity screening test with Art. 277455 (PY-2-B(OH)2) in male and female Wistar rats with dose levels of 25, 100 and 400 mg/kg body weight/day the following conclusions can be made:
At a dose level of 400 mg/kg body weight/day toxicologically relevant, unspecific clinical signs of reduced health condition (piloerection, reduced spontaneous activity, hunched posture and red nasal discharge) were noted. Two males and two females dosed with 400 mg/kg body weight/day died prematurely during the course of treatment. One male was found dead on study day 5 and the other male was sacrificed in a moribund condition on study day 24. Both decedent females were found dead on study day 28.
Significantly reduced body weight and food consumption were noted in adult males and females at 400 mg/kg body weight/day.
Test item-related, adverse effects were also observed for litter data at 400 mg/kg body weight/day. In relation to an increased number of stillbirths, gestation period was noted to be prolonged. The delivery index (no. of dams with live pups born / no. of pregnant dams x 100) was shown to be markedly reduced (50 %) when compared to 100 % in the control group. The mean number of total pups delivered (live and dead) was markedly reduced (4.67 compared to 11.90 in controls) and there was also an adverse effect on mean number of live pups born (2.83 compared to 11.90 in controls). Due to still births, non-littering and/or resorptions, post-implantation loss at 400 mg/kg body weight/day was markedly higher (76.81 %) compared to the control group (5.07 %). Furthermore, slightly lower mean weight of pups on PND 0 (15 % below controls) was also considered as biologically relevant. Total litter weight was markedly reduced at the same dose level due to the markedly lower number of live pups. No such effects on litter data were observed at the lower dose levels of 100 and 25 mg/kg body weight/day.
Test item-related, adverse effects were also noted for pup survival. At 400 mg/kg body weight/day a reduced viability index of 79.17 % was noted during post-natal days 0-4 and of 94.80 % at 100 mg/kg body weight/day when compared to 99.23 % in the control group. The dose dependently and biologically significantly increased mortality of pups at 100 and 400 mg/kg body weight/day was considered as an adverse effect of the treatment with the test item.
The toxicological relevance of slightly higher mean absolute anogenital distance in male but not female pups at 400 mg/kg body weight/day was unclear due to high variability and small sample size (7 live male pups).
Slightly lower mean percentage of reticulocytes was noted at 400 mg/kg body weight/day in adult males but not females (45 % below controls) which could be related to the microscopic finding of slight focal hypocellularity in the sternal bone marrow.
Under the conditions of this study, treatment with the test item induced a tubulopathy in the kidneys, characterized by minimal to moderate degrees of tubular degeneration/necrosis, tubular regeneration as well as tubular dilation in proximal tubules, distal tubules and/or collecting ducts at 400 mg/kg body weight/day. These findings were sometimes accompanied by slight cortical tubular vacuolation, slightly increased mononuclear cell infiltrates and minor degrees of papillary necrosis or urothelial hyperplasia. Minimal and/or slight cortical tubular vacuolation was also recorded in the kidneys of the lower dose groups, and minimal tubulopathy was present in one male at 100 mg/kg body weight/day.

The tubulopathy correlated with increased kidneys weights and grossly visible enlarged kidneys and was considered to be of adverse nature. Predominantly, morbidity and mortality of the adult males and females in this study was considered to be related to the observed tubulopathy.
No adverse effects of Art. 277455 (PY-2-B(OH)2) were noted at a dose level of 25 mg/kg body weight/day. Thus, the NOAEL for general systemic toxicity as well as for developmental toxicity could be established at 25 mg/kg body weight/day.
Executive summary:

The aim of this study was to assess the possible effects of Art. 277455 (PY-2-B(OH)2) on male and female fertility and development after repeated dose administration in Wistar rats.

The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of a maximum of 63 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed. Animals of an additional control group were handled identically as the dose groups but received 0.25 % aqueous hydroxypropyl methylcellulose, the vehicle used in this study. The 4 groups comprised 10 male and 10 femaleWistarrats each.

Before initiation of dosing, all females were screened for two weeks for regular estrous cyclicity and animals (10 females / group) with regular estrous cycle (4-5 day cycle) were used in the study.

The following doses were evaluated:

Control (C):                   0         mg/kg body weight/day

Low Dose (LD):             25       mg/kg body weight/day

Medium Dose (MD)    100     mg/kg body weight/day

High Dose (HD):         400     mg/kg body weight/day

The test item formulation was prepared with the vehicle, 0.25 % aqueous hydroxypropyl methylcellulose, at least once every ten days based on available stability data. The prepared formulation was stored at 2-8°C and protected from light.Formulates were kept under magnetic stirring during the daily administration.The vehicle was also used as control item. Dose volumes were adjusted individually based on the body weight most recently measured. Theadministration volume was 10 mL/kg body weight.

During the period of administration, the animals were observed each day for signs of toxicity. Animals that died or were euthanised for animal welfare reasons were examined macroscopically and at the conclusion of the test all surviving animals were sacrificed and observed macroscopically.

Functional observations including sensory reactivity to different stimuli, grip strength, motor activity assessments and other behavior observations were performed before the treatment period and in the last week of treatment infive randomly selected males and femalesof each group.

Body weight and food consumption were measured weekly, except for food consumption measurements which were not performed during the mating period in males and females and the post-mating period in males. During pregnancy, females were weighed on gestation days 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum), on PND 4 and PND 13 along with the pups.

After 14 days of treatment of males and females, animals were mated (1:1) for a maximum of 14 days. The subsequent morning onwards the vaginal smears of females were checked to confirm the evidence of mating. After the confirmation of mating, females were separated and housed individually. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted, sexed and litters weighed within 24 hours of parturition, on day 4 and day 13 post-partum.

The anogenital distance of each pup was measured on PND 0. The number of nipples/areolae in male pups was counted on PND 12. Blood samples from the day 13 pups and the adult males were assessed for serum levels for thyroid hormones (T4).

Males were sacrificed after completion of the mating period on day 29 and females along with their pups were sacrificed on post-natal day 13. Non-pregnant females were sacrificed on day 26. The number of implantation sites and corpora lutea was recorded for each parental female at necropsy.

Pups sacrificed on post-natal day 4 for interim sacrifice for blood sampling and those found dead were carefully examined for gross external abnormalities.

At the conclusion of the test, all surviving adult animals were sacrificed and observed macroscopically. The wet weight of a subset of tissues was taken and a set of organs/tissues was preserved.

Haematological and clinical biochemistry evaluations and coagulation measurements were performedon blood samples collected at terminal sacrifice fromfive randomly selected males and females from each group. Urinalysis wasperformedon samples collected at terminal sacrifice fromfive randomly selected males and females from each group.

A full histopathological evaluation of preserved organs/tissues was performed in 5 randomly selected males and females of the control and HD group and in all animals that died prematurely during the course of treatment. Due to test item-related morphologic changes detected in stomach, liver (males only), kidneys, adrenal glands, spleen, bone marrow (males only) and thymus of high dose animals, these organs were also examined from five randomly selected animals from the low and medium dose groups.Testes, epididymides, ovaries, uterus with cervix, vagina and accessory sex organs were examined in all control and HD animals and in non-pregnant females of the LD and MD group and their male mating partners. Any gross lesions macroscopically identified were examined microscopically in all animals.

Summary Results

Four animals of the HD group died prematurely during the course of treatment. Male no. 33 was sacrificed in a moribund condition for animal welfare reasons on study day 24. Microscopic findings related to its morbidity consisted ofmultifocal mucosal mineralization in the glandular stomach, multifocal hepatocellular necrosis in the liver, hemorrhagic necrosis in the prostate gland, minor degrees of lymphoid atrophy in the axillary lymph nodes, alveolar edema in the lung and tubulopathy of the kidneys.Male no. 35 (high dose) was found dead on day 5 of the premating period without any preceding signs of morbidity. Female no. 71 and 74 (high dose) were both found dead on study day 28. Before female no. 71 showed slight loss of body weight and piloerection. No signs of reduced health condition were observed in female no. 74 before death. At necropsy, blood was found in the uterus of female no. 71 and in the jejunum and ileum of female no. 74.Tubulopathy of the kidneys was considered to be the main cause of morbidity of male no. 33 and, along with papillary necrosis, to be the cause of death (renal failure) of female no. 71. Further microscopic findings related to the premature death of male no. 35 and female no. 71 and 74 consisted of minor degrees of alveolar edema in the lung, of lymphoid atrophy in the mesenteric lymph nodes (no. 35) and lymphocyte apoptosis in the Peyer’s Patches (no. 74). The exact cause of death of the male no. 35 and female no. 74 could not be established from the organs and tissues examined. No mortality of adult animals occurred in the control, the LD and the MD group during the treatment period of this study.

Various unspecific clinical signs of reduced health condition were noted in some males and females of the HD group mainly at the end of the treatment period: piloerection, reduced spontaneous activity, hunched posture and red nasal discharge. These signs in the HD group were considered as toxicologically relevant effects of the treatment with the test item.

Moving the bedding and salivation were observedtransiently and dose dependently in animals of the HD and the MD group. These slight clinical signs were mainly seen at the end of the treatment period in males and during gestation and lactation period in females. Both signs were observed in short timely relation to dose application or in anticipation thereof and thus were considered to be a sign of discomfort or a local reaction to the test item applied by gavage. These signs were not considered as adverse systemic effects.

None of the females showed signs of abortion or premature delivery.

During the weekly detailed clinical observation, no relevant differences between the groups were found except rarely salivation in some animals of the MD and HD group.

In males and females, no relevant effects were observed in any of the parameters of the functional observation battery at the end of the treatment period. There were no biologically or statistically significant differences in body temperature between the dose groups and the controls. There were no ophthalmoscopic findings in any of the animals of this study.

Test item-related, adverse effects were noted for body weight, body weight development and food consumption in the HD group:

After initiation of treatment, males of the HD group showed a moderate and statistically significant loss of body weight compared to a slight gain of body weight in controls. Except for the second week of the study, mean body weight further decreased slightly until the end of the study. Considering the whole treatment period, there was a biologically and statistically significant mean loss of -7.25 g body weight (equal to -1.9 % under exclusion of decedents) in the male HD group compared to a gain of +35.70 g (equal to +9.6 %) in the respective controls. Thus, mean body weight of the male HD group was moderately and statistically significantly lower throughout the mating/postmating period and at terminal sacrifice (up to 10 % below controls). A comparable test item-related effect was noted for the female HD group. A slight but statistically significant mean loss of body weight occurred in the first week of treatment in the female HD group. Although body weight increased subsequently, mean weight gain remained slightly lower in the female HD group throughout the whole study period when compared to controls. Statistical significance was achieved in the 1stweek of gestation, the last week of gestation and when related to the whole gestation period (day 0-20) (body weight gain 56 % below controls). Thus, mean body weight of the female HD group was moderately and statistically significantly reduced on day 7 and day 20 of gestation and day 4 and day 13 of lactation (up to 18 % below controls).

In males, in correlation to the body weight loss, mean food consumption per animal in the HD group was moderately lower in the first week of the premating period when compared to controls (27 % below controls).In females, a slight tendency towards lower food consumption was also observed in the first week of the premating period. In accordance to the attenuated body weight gain during gestation, food consumption of the HD group was also noted to be moderately and statistically significantly lower when compared to the control group during the whole gestation period (up to 30 % below controls). Food consumption was further reduced in the HD group during lactation with values being 38 % below controls in the first week and 54 % below controls in the second week of lactation.

No effects of toxicological relevance were noted in the male and female LD and MD groups for mean body weight, body weight gain and food consumption.

The test item had no biologically significant effect on the estrous cyclicity analyzed during the 2 weeks premating period after the first administration.

The copulation index was 100 % in all groups. Length of the precoital interval was within the normal range of variation for animals of this age and strain in the dose groups and the control group. However, a biologically and statistically significant effect was noted on the duration of gestation in the HD group (mean 23.80 days compared to 22.30 days in controls) with two females showing a gestation length of 24 days and one female 25 days. The observed prolonged gestation period in the HD group was considered to be related to the increased number of stillbirths.

The fertility index (number of pregnant females/number of copulated females x 100) was slightly reduced in the MD group (90 %) and the HD group (80 %) when compared to the control group (100 %): As histologically no morphological reason for non-mating of those females and their male mating partners was seen, this slightly reduced fertility index was considered as incidental in nature.

There were no biologically or statistically significant effects on the number of corpora lutea and number of implantation sites in the dose groups when compared to the control group.

Test item-related, adverse effects were observed for litter data.

Litters were delivered by 10 dams of the control, 10 of the LD, 9 of the MD and 5 of the HD group.The delivery index(no. of dams with live pups born / no. of pregnant dams x 100)was markedly reducedin the HD group (50 %) when compared to the control group (100 %). The delivery index was 100 % in the LD and the MD group. The mean number of total pups delivered (live and dead) was markedly and statistically significantly reduced in the HD group (4.67 compared to 11.90 pups in the control group). The number of live pups delivered in the HD group (2.83) was also markedly and statistically significantly lower when compared to the control group (11.90). These significant differences for number of pups between the HD group and the control group remained until the end of the study period (post-natal day 4 and 13). The number of still births was biologically and statistically significantly higher in the HD group when compared to the control group (1.83 compared to 0.00) based on two fully stillborn litters and two more stillborn pups in two HD females each. Only one litter of the HD group was observed without any still births. Marginally but not statistically significantly higher number of still births was observed in the LD group (0.40) and the MD group (0.22). Without dose dependency, this marginally higher number of still births in the LD and the MD group was not considered as an adverse effect of the test item. Due to still births, non-littering and/or resorptions, post-implantation loss in the HD group (76.81 %) was markedly and statistically significantly higher compared to the control group (5.07 %). No toxicologically relevant effect on post-implantation loss and litter data was noted for the LD and the MD group.Percentage of pre-implantation loss was within the normal range of biological variation for all groups.

There was no biologically or statistically significant effect on the sex ratio of pups on post-natal day 0, 4 or 13.

Slightly lower mean weight of pups on PND 0 was noted in the HD group (5.06 g) when compared to the control group (6.05 g). Though only three litters with live pups were delivered in the HD group, this biologically relevant 15 % difference of mean pup weight compared to controls was considered as an effect of the treatment with the test item. Mean pup weight remained slightly (but not statistically significantly) below controls on post-natal day 4 and day 13. Markedly and statistically significantly lower total litter weight was noted on post-natal day 0, 4 and 13 for the HD group which was mainly related to the markedly lower number of live pups.

Test item-related, adverse effects were also noted for pup survival.There was a moderately reduced viability index during post-natal days 0-4 (no. of live offspring at day 4 / no. of live offspring at birth x 100) of 79.17 % in the HD group when compared to 99.23 % in the control group. Viability index during post-natal days 0-4 was slightly reduced in the MD group (94.80 %). No effect was noted in the LD group (100 %).

The markedly and statistically significantly increased mortality of pups in the HD group with a total mortality (post-natal day 0 to 4) of 20.83 % was based on 3 missing pups attributed to cannibalism by the dam) from in total 2 litters on post-natal day 1. Observed mortality in the MD group was related to pups missing (on post-natal day 1 (3 pups), day 3 (1 pups) and day 4 (1 pup) from in total 3 litters. Though not statistically significant, this dose dependently and biologically significantly increased mortality in the MD group was considered as toxicologically relevant. All live pups on post-natal day 4 in the dose groups and the control group survived until terminal sacrifice on post-natal day 13.

Occasionally some of the pups of the MD and the HD group which did not survive until terminal sacrifice were seen without indication of suckling after birth. There were no remarkable, toxicologically relevant external abnormalities in the pups of any group.

Despite the lower pup weight in the HD group, the mean absolute anogenital distance of male pups was marginally but statistically significantly higher in the HD group (2.69 mm) when compared to the control group (2.36 mm) (14 % above controls). Statistical significance was also achieved for the relative (to cube root of pup weight) anogenital distance of male pups. Values for anogenital distance showed high variability throughout the groups and differences in the HD group to controls was mainly based on one single pup of the HD group with a comparably high value (absolute anogenital distance 3.36 mm). Due to the high variability and the small sample size (7 live male pups) it remains unclear if the observed effect was incidental or induced by the test item. No statistically significant differences were observed for anogenital distance of female pups.

No effect of toxicological relevance was observed for nipple retention in the pups of any group.

Serum analysis in adult males at terminal sacrifice revealed dose dependently and statistically significantly lower thyroxine (T4) hormone levels throughout all dose groups when compared to the control group (LD: 81.92 nmol/L (p < 0.05), MD: 55.97 nmol/L (p <0.001), HD: 37.91 nmol/L (p < 0.001) compared to 93.40 nmol/L in controls). However, there were no statistically or biologically significant weight changes in pituitary or thyroid/parathyroid glands which could have been further indicative of disturbances of the normal function of the hypothalamic-pituitary-thyroid axis (HPT axis). There were also no corresponding microscopic findings in the histopathologically examined thyroid/parathyroid glands of the male and female HD group such as increased cell proliferation. Thyroid-stimulating hormone (TSH) levels are regulated by negative feedback of thyroid hormones. In general, when thyroid hormone serum levels are decreased, thyroid-releasing hormone (TRH) and TSH release are stimulated. However, in this study, analysis of circulating TSH levels in serum samples of adult males showed no dose dependency and no statistically significant difference between the dose groups and the control group. No correlation between TSH- and T4-values was observed for the individual animals. Furthermore, there were no alterations to certain endpoints such as sperm maturation in males or estrous cycles in females which could be influenced by changed thyroid hormone levels though increased number of stillbirths was noted in the HD group. The reason for the statistically significantly lower T4 levels in the dose groups remains unclear. However, in the absence of changes to thyroid weight, histopathological findings, clinical manifestation and changes of TSH levels, the observed isolated finding of statistically significant changes to thyroxine levels was not considered as adverse.

There were no statistically or biologically significant differences for T4 level of pups measured on post-natal day 13 between the dose groups and controls. There was no effect of toxicological relevance on mean pup thyroid/parathyroid weight on post-natal day 13.

In adult males but not females at necropsy,slightly lower mean percentage of reticulocytes was noted in the HD group (0.92 %) when compared to the control group (1.68 %) (45 % below controls). A relationship with the treatment of the test item cannot be excluded for this biologically relevant observation and could be related to the microscopic finding of minimal to slight focal hypocellularity in the sternal bone marrow in the male HD group. There were no further toxicologically relevant effects on haematology, blood coagulation and clinical biochemistry in any group. Slight differences to controls were not assumed to be biologically relevant.Single deviating values in individual animals compared to controls were considered as incidental.

There were no toxicologically relevant effects on parameters of urinalysis in any group at the end of the treatment period.

Macroscopic findings that could be attributed to the treatment with the test item consisted of grossly enlarged kidneys (males: 1/10 of the MD, 5/10 of the HD group; females: 2/10 of the HD group) and enlarged adrenal glands (males: 3/10 of the HD group; females: 2/10 of the HD group). Kidneys of 1/10 males of the HD group were also seen with abnormal color (spotted). Furthermore, small spleen was noted in 4/10 males of the HD group and small thymus in 6/10 males of the HD group.

Statistically significant changes in organ weights correlating to macroscopic and microscopic findings were recorded in spleen, thymus and kidneys.

At the end of the treatment period, a toxicologically relevant effect was noted for absolute and relative (to body weight) spleen weight in the HD group. Spleen weight was moderately decreased in males of the HD group (absolute weight 42 % below controls) and females of the HD group (absolute weight 35 % below controls). Furthermore, the relative kidney weight to body weight ratio was moderately and dose dependently higher in males of the MD and the HD group (16 % and 28 % above controls) and in females of the HD group (15 % above controls) when compared to the respective controls. In males of the HD group, the absolute thymus weight was markedly reduced (45 % below controls), whereas no such effect was noted in females. Other slight but statistically significant changes in organ weights were considered incidental or secondary to body weight changes and did not correlate with microscopic findings.

Under the conditions of this study, treatment with the test item induced a tubulopathy in the kidneys, characterized by minimal to moderate degrees of tubular degeneration/necrosis, tubular regeneration as well as tubular dilation in proximal tubules, distal tubules and/or collecting ducts of animals of the HD group. These findings were sometimes accompanied by slight cortical tubular vacuolation, slightly increased mononuclear cell infiltrates and minor degrees of papillary necrosis or urothelial hyperplasia. Minimal and/or slight cortical tubular vacuolation was also recorded in the kidneys of the LD and MD group, and minimal tubulopathy was present in one male of the MD group. The tubulopathy correlated with increased kidneys weights and grossly visible enlarged kidneys (and in one case along with abnormal color, spotted kidneys), and was considered to be of adverse nature.

Further treatment-related microscopic findings were observed in the stomach,liver (males only), adrenal glands, spleen, thymus and bone marrow (males only) in the HD group.

In the stomach, they consisted of minimal to moderate epithelial hyperplasia along with parakeratosis and minimal submucosal inflammation. These findings were considered to be due to a local irritant effect of the test item when administered orally and therefore non-adverse.

In the liver, minimal centrilobular hepatocellular hypertrophy was recorded in males of the HD group. Since there were no indicators of liver injury (Kupffer cell proliferation, necrosis, apoptosis, fibrosis, other degenerative changes, etc.), this lesion was considered to be of adaptive nature, reversible and therefore non-adverse.

Macroscopically enlarged adrenal glands correlated microscopically with minimal to slight diffuse hypertrophy in the Zona fasciculata of the adrenal glands in animals of both sexes treated with 400 mg/kg/day. In addition, a moderate to marked cortical diffuse hemorrhage was present in both males that died prematurely. Minimal hemorrhage was also present in another high dose male. The reason for this change remains unclear. In the spleen, a slight to marked lymphoid atrophy, correlating with decreased organs weights and small spleens (males only) was recorded in animals of both sexes of the HD group. A further finding consisted of slight increased hemosiderin deposits in the spleen of these animals. Decreased thymus weights and small thymuses correlated microscopically with an increased severity grade of atrophy in high dose males. In high dose females, the severity grade for this finding was increased only in one female that was found dead. In the bone marrow, some male animals treated with 400 mg/kg/day showed minimal to slight focal hypocellularity. All findings recorded in adrenal glands, spleen, thymus and bone marrow were considered to be indirect effects secondary to body weight loss and/or stress, and as such, were considered to be not systemic effects of oral treatment with the test item and, therefore, non-adverse.

There were no microscopic findings in the male and female reproductive organs that could be attributed tothetreatment with the test item. The treatment with the test item did not reveal effects on the completeness ofspermstages or cell populations. There was no indication for maturation arrest, reabsorption of sperm or any other degenerative type.

Based on microscopic findings noted in the kidneys, a histopathological NOAEL could be established at 25 mg/kg/day; a histopathological NOEL could not be established.

Nominal concentrations of the test item were confirmed in analyzed dose formulations of all dose groups in study weeks 1, 3, 5 and the last week. All samples were homogenous.