Reaction mass of Cobaltate(3-), bis[2-[[[3-[2-[1-[[(2-chlorophenyl)amino]carbonyl]-2-(oxo-κO)propyl]diazenyl-κN1]-4-(hydroxy-κO)phenyl]sulfonyl]amino]benzoato(3-)]-, sodium (1:3) and tetrasodium bis[2-[[[3-[[1-[(2-chloroanilino)carbonyl]-2-oxopropyl]azo]-4-hydroxyphenyl]sulphonyl]amino]benzoato(3-)]cobaltate(4-)

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-04-02 to 2014-10-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of Trisodium bis[2-({[3-({1-[(2-chlorophenyl)amino]-3-(hydroxy-kO)-1-oxobut-2-en-2-yl}diazenyl-kN1)-4-(hydroxy-kO)phenyl]sulfonyl}amino)benzoato(3-)]cobaltate(3-) and Trisodium [2-({[3-({1-[(2-chlorophenyl)amino]-3-(hydroxy-kO)-1-oxobut-2-en-2-yl}diazenyl-kN1)-4-(hydroxy-kO)phenyl]sulfonyl}amino)benzoato(3-)][2-({[3-({1-[(2-chlorophenyl)amino]-3-(hydroxy-kO)-1-oxobut-2-en-2-yl}diazenyl-kN2)-4-(hydroxy-kO)phenyl]sulfonyl}amino)benzoato(3-)]cobaltate(3-)

EC Number:

941-792-6

Cas Number:

not given

Molecular formula:

C46H32Cl2CoN8O14S2.3Na

IUPAC Name:

Reaction mass of Trisodium bis[2-({[3-({1-[(2-chlorophenyl)amino]-3-(hydroxy-kO)-1-oxobut-2-en-2-yl}diazenyl-kN1)-4-(hydroxy-kO)phenyl]sulfonyl}amino)benzoato(3-)]cobaltate(3-) and Trisodium [2-({[3-({1-[(2-chlorophenyl)amino]-3-(hydroxy-kO)-1-oxobut-2-en-2-yl}diazenyl-kN1)-4-(hydroxy-kO)phenyl]sulfonyl}amino)benzoato(3-)][2-({[3-({1-[(2-chlorophenyl)amino]-3-(hydroxy-kO)-1-oxobut-2-en-2-yl}diazenyl-kN2)-4-(hydroxy-kO)phenyl]sulfonyl}amino)benzoato(3-)]cobaltate(3-)

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test System:
Species/strain: 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: males: 11-12 weeks old, females: 11-12 weeks old.
Body weight at the allocation of the animals to the experimental groups (together with study 140351):
males: 272 - 307 g (mean: 289.09 g, ±20 % = 231.27 – 346.9 g)
females: 179 - 206 g (mean: 193.84 g, ±20 % = 155.07 – 232.61 g)
The animals were derived from a controlled full barrier-maintained breeding system (SPF). According to Art. 9.2, No. 7 of the German Act on
Animal Welfare the animals were bred for experimental purposes.
The animal study was authorized by local government under file no. 55.2-1-54-2532.2-11-11.

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 (lot no. 1526)
- 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 2 animals/ sex/ cage in IVC cages (type III H, polysulphone cages) during the premating period in both males and females and during post mating period in 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 and males were returned to its original cage. Each cage was provided with Altromin saw fibre bedding (lot no. 131113)
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- 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 animals showed significant clinical signs before the study initiation.
Before the first administration all animals to be used for the study were weighed. Mean body weight of the group housed animals were used to assign all animals to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females, respectively, while ensuring to keep each animal with its initial cage partner if possible.
Each animal was marked with its identification number by individual ear tattoo.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

The test item FAT 92368/Y was weighed into a tarred plastic vial on a precision balance and suspended in aqua ad injectable. A suspension with the vehicle was prepared by subjecting them to ultrasonic bath for 10 min at room temperature. Homogeneity of the test items in the vehicle was maintained by vortexing.
The test item formulations were prepared once in every ten days and stored at room temperature. The homogeneity was ensured by vortexing the sample on vortex machine.

The following doses were evaluated:
Control: 0 mg/kg/d
Low Dose: 100 mg/kg/d
Medium Dose: 300 mg/kg/d
High Dose: 750 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 aqua ad injectable using the same volume as used for the dose group. The test item and vehicle were administered at a single dose to the animals by oral gavage with disposable polypropylene feeding tubes for rats (78 mm long; 3.0 mm diameter; Instech Laboratories Inc). The application volume for all groups was 5 mL/kg. For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.

Details on mating procedure:

Mating was performed using a ratio of 1:1 (male to female). The vaginal smear of the females was checked every morning after the start of the mating period to confirm the evidence of 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 minimized.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Each dosing concentration were analysed with respect to the target nominal concentration. Stability and homogeneity of the test item in the vehicle were analysed for the low and high dose concentrations. Samples for the nominal concentration verification were taken in study week 1 (first week of pre-mating period), 3 (first week of mating), 5 (gestation) and in the last week of the study (gestation/lactation) from all groups (16 samples). Samples for homogeneity analysis were taken from the top, middle and bottom of the high dose and the low dose formulation in study week 1 and 5 (12 samples). Samples for stability analysis will be taken in the first week of the study [0 hours and 10 days after the preparation (stored at Room Temperature)] from high and low dose formulations (6 samples). All analytical samples collected on the day of sample collection and were stored at -20 °C. These samples were analysed after the completion of the toxicity study at BSL BIOSERVICE Scientific Laboratories GmbH under the BSL study no. 140386.

Duration of treatment / exposure:

The animals were treated with the test item formulation or vehicle on 7 days per week for a period of 54 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 3 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days were completed.

Frequency of treatment:

daily

Details on study schedule:

Arrival of the Test Item: 07 January 2014
Study Initiation Date: 02 April 2014
1st Amendment to Study Plan: 11 April 2014
2nd Amendment to Study Plan: 28 May 2014
Delivery of Animals: 03 April 2014 and 15 April 2014
Acclimatisation Period: 03 April 2014 23 April 2014
Experimental Starting Date: 23 April 2014
Date of Behavioural Observation: 16 April 2014- 05 June 2014
Treatment Period: 23 April 2014 – 06 June 2014
Necropsies: 21 May 2014 - 06 June 2014
Experimental Completion Date: 06 June 2014
Proposed Completion Date of Delegated Phase (Histopathology): 20 October 2014
1st Amendment to Phase report (Histopathology): 27 October 2014
Proposed completion date of delegated phase
(Formulation Analysis): 09 September 2014

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

750 mg/kg bw/day (nominal)

No. of animals per sex per dose:

80 animals (40 males and 40 females) were included in the study (10 male and 10 female animals per group). This included the control animals (10 males and 10 females) which were shared with BSL study 140351.

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

Clinical Observations:
General clinical observations were made once a day, preferably at the same time each day after dosing. 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 were made once daily. Once before the first exposure, and once a week thereafter, detailed clinical observations were made in all animals outside the home cage in a standard arena. Clinical observations included 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 on day 3 of the lactation period in 5 randomly selected females (only lactating females were evaluated) 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, 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 body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment period 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) as well as day 4 post-partum along with pups. Any animals prematurely sacrificed were weighed prior to the sacrifice. Food consumption was measured weekly 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.

Litter observations:

The duration of the gestation was recorded and was calculated from day 0 of the pregnancy. 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. Twice daily all pregnant females were checked for parturition except on weekends and public holidays when this was checked once daily. Live pups were counted and sexed, and litters weighed within 24 hours of parturition (day 0 post-partum) and on day 4 post-partum. Live pups were identified by tattoo mark on paw. In addition to the observations of parent animals, any abnormal behavior of the offspring was recorded.

Postmortem examinations (parental animals):

Pathology:
All surviving male animals were sacrificed after the completion of the mating period (total dosing period: 28 days) on study day 29 or 30, while female animals were sacrificed on post-natal day 4 using an anaesthesia (ketamine/xylazin, 2:1, Pharmanovo, lot no: 24863, expiry date: 10/2015 and Serumwerk, lot no: 00513, expiry date: 05/2015). All surviving pups were killed on PND 4 by decapitation. Dead pups and pups sacrificed on day 4 post-partum were carefully examined externally for gross abnormalities. Non-pregnant females were sacrificed on study day 26 using the sperm-positive vaginal smear as evidence of mating. All 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), 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 before being transferred to 70 % ethanol. The number of implantation sites and corpora lutea was recorded for each parental female at necropsy.


Organ Weights:
The wet weight of the organs (liver, uterus with cervix, kidneys, thymus, adrenals, thyroid/parathyroid glands, testes, spleen, epididymides, brain, prostate, seminal vesicles and coagulating glands, pituitary gland, ovaries, heart) of 5 sacrificed males and 5 females (only lactating females will be evaluated) randomly selected 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. In addition, reproductive organs of all animals were weighed. The following tissues (brain (cerebrum, cerebellum and pons), heart, spinal cord, ovaries (females), liver, uterus with cervix (females), kidneys, vagina (females), adrenal glands, testes (males), stomach, epididymides (males), small and large intestines (including Peyer´s patches), prostate and seminal vesicles with coagulating glands as a whole (males), thymus, urinary bladder, thyroid, lymph nodes (mesentric and axillary), spleen, peripheral nerve (e.g. sciatic nerve) with skeletal muscle, lung and trachea, bone with bone marrow (sternum), mammary glands, pituitary gland, gross lesions, oesophagus, skin) of the same selected animals from each group were preserved in 10 % neutral buffered formalin except for testes and epididymides that were fixed in Modified Davidson’s Fixative for approximately 24 hours before they were transferred to 70 % ethanol.

Histopathology:
Sections from the organs and tissues from 5 selected male and female animals of control, as well as all gross lesions from all groups, were examined by light microscopy. In this study, sections of the full list of organs and tissue were examined in 5 males and 5 females from the survivors of MD group and 5 males and 1 female from the survivors of HD group in addition to the decedents from MD and HD groups, because animals’ morbidity that were considered to be related to treatment with the test item was observed in many animals of HD group. Furthermore, testes, epididymides, prostate, seminal vesicles with coagulating glands, ovaries, uterus with cervix and vagina were examined in all animals, and, on the testes, special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure. 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 were examined in all animals. For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle for the evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides. 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). Histopathological evaluation was performed at the GLP-certified contract laboratory AnaPath GmbH, Buchsweg 56, 4625 Oberbuchsiten, Switzerland (test site for histopathology). The study phases from test site 1 and 2 was 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.

Postmortem examinations (offspring):

Pups sacrificed on post natal day 4 and those found dead, were carefully examined for gross external abnormalities.

Statistics:

A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical biochemistry and absolute and relative organ weights were performed for each gender by comparing values of dosed with control animals of the main groups using a one-way ANOVA and a post-hoc Dunnett Test. These statistics were performed with GraphPad Prism V.6.01 software (p <0.05 was considered as statistically significant).

Reproductive indices:

There was no effect on copulation index in LD, MD and HD groups. There was no effect on fertility and delivery indices in LD and MD groups and fertility index in HD group. The delivery index in HD group was not used for comparison as there were not enough animals available for a meaningful comparison.
There was a lower viability index in MD group (76.67 % compared to 100 % of control) which was due to viability of pups of female 121 and 125. These animals displayed significant reductions in body weight gain and adverse clinical observations. This was considered to be an adverse effect of test item treatment from the prospect of its effect on the viability of pups. In HD group the viability index was also lower but was not used for comparison.

Offspring viability indices:

There was a lower viability index in MD group (76.67 % compared to 100 % of control) which was due to viability of pups of female 121 and 125. These animals displayed significant reductions in body weight gain and adverse clinical observations. This was considered to be an adverse effect of test item treatment from the prospect of its effect on the viability of pups. In HD group the viability index was also lower but was not used for comparison.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, non-treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Description (incidence and severity):

Except for confirmation of mating

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

There was no effect on the precoital interval. There was no effect on the duration of gestation up to the MD level and also on two surviving females of HD group. There was higher percent post- implantation loss in MD group (16.96% compared to 4.96 % of control). These reductions were associated with 2 animals (female no's 121 and 125) that displaying significant reductions in body weight gain and adverse clinical observations. Given the lower no. of pups in MD group (8.67 compared to 10.50 of control), the higher percent post implantation loss was considered to be of toxicological relevance. There was no effect on copulation index in LD, MD and HD groups. There was no effect on fertility and delivery indices in LD and MD groups and fertility index in HD group. There was lower viability index in MD group (76.67 % compared to 100 % of control) which was considered to be an adverse effect of test item treatment.

Details on results (P0)

Mortality:
There were mortalities in male and female dose groups during the study period. In males 1/10 in LD group (male no. 85) and 1/10 in MD group (male no. 100) and 4/10 in HD group (males 103, 105, 107 and 108). In Females 1/10 in MD group (female no. 130) and 8/10 in HD group (female nos. 131, 132, 133, 134, 136, 138, 139 and 140). These animals were either found dead or euthanized for ethical reasons during the course of the study. The cause(s) of morbidity and mortality in LD male no. 85 and MD male no. 100 were aspiration pneumonia and peracute pulmonary necrosis due to aspiration, respectively, which happened accidentally, and it was judged that these events were not related to systemic toxicity of FAT 92368/Y TE. The cause of the remaining morbidity and mortality were attributed to inflammation in small and/or large intestines and tubular degeneration/ necrosis/ regeneration in the kidney. The remainder of animals survived the scheduled study period.

Clinical Observations:
The clinical sings in control and LD group males and/ or females were abnormal breathing, crust, moving the bedding, aggressive, alopecia. In male MD group there were signs of moving the bedding and regurgitation. In female MD group there were moving the bedding, regurgitation, alopecia, diarrhoea, nasal discharge and salivation. These findings were transient and/or isolated with no other relevant toxicological findings. In female MD groups there were also abnormal breathing, dehydration, discoloured feces, wasp waist and piloerection. These were considered to be related to test item treatment. In male HD group, there were abnormal breathing, diarrhoea, moving the bedding and salivation. In female HD group, there were abnormal breathing, diarrhea, discoloured feces, moving the bedding, piloerection, salivation, half eye lid closure, hypothermia, kyphosis and reduced spontaneous activity. These findings were considered to be due to test item treatment. In both males and females, abnormal breathing and piloerection was associated with aspiration and moving the bedding, salivation and diarrhoea to local effect of the test item. Discoloured feces were due to colour of the test item. Two females of MD group (female 121 and 125) showed test- item treatment related clinical signs during the lactation days which contributed to the litter losses of these animals. The clinical signs were slight to moderate piloerection, abnormal breathing and dehydration in female no. 121 and dehydration, slight to moderate piloerection, diarrhoea, red nasal discharge, discoloured feces and slight wasp waist in female no. 125. Clinical signs of decedents (HD males 103, 105, 107; LD female 130 and HD females 131, 132, 133, 134, 136, 138, 139, 140) were mostly moving the bedding, diarrhea and abnormal breathing, salivation, piloerection and discoloured feces. In addition, in few decedents reduced spontaneous activity and/or kyphosis and/or half eye lid closure and/or hypothermia were also observed. During the weekly detailed clinical observation, there were no changes or differences between the groups and the baseline values in males up to HD level and in females up to MD level. In the remaining female animals of the HD group (female no. 135 and 137) clinical signs of toxicity were not observed during detailed clinical observations during the last week of treatment.

Functional Observations:
No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period in males. 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 up to MD level. There was no effect on body temperature in both males and females. The female HD group values were not used for statistical analysis and data comparison due to the low number of surviving animals. However, in the remaining HD group female (female 137) there was no effect of test item treatment observed during the last week of treatment.

Body Weight Development:
In males of the HD group, there was a test article related reduced mean body weight gain from the 1st week onwards when compared to the control group, which was statistically significantly after premating days 1-7 and premating day 1 to post mating day 14. This resulted in a statistically significant reduction in mean body weight gain when the entire dosing period is considered (Premating Day 1 through terminal sacrifice). The reduction in weight gain in HD group resulted in absolute body weight during the study period 3 to 6% lower when compared to control. This effect of FAT 92368/Y on body weight of HD group was considered to be adverse.
There was statistically significantly lower body weight gain in LD group after premating days 1-7. This change in LD group was transient and as mean absolute body weight was 1 to 3 % above controls during the study period, this finding was not considered test item related. There was no effect on body weight and body weight gain in the MD group when compared to the control group. In females, there was a statistically significantly lower body weight on lactation day 4 in MD group (12 % below control), which was a result of a transient but statistically significantly lower weight gain after GD 0-7 in MD group and reduced mean weight gain from lactation day 0-4. The lower body weight gain during lactation days 0-4 in MD group is attributed to 2/9 isolated females (animals 121 and 125) that lost 31 and 32 g, respectively during this period. The finding in MD group was not considered to be a systemic adverse effect of FAT 92368/Y, as both animals showed abnormal clinical signs associated with local effects of test substance aspiration. The remaining 7/9 animals in this group gain weight comparable to controls. There was statistically significantly lower body weight gain during the gestation days, especially GD 14-20 in HD group and in the cumulative weight gain of gestation days (GD 0-20) in HD group, which resulted in a 15% reduction in absolute body weight on Gestation Day 20. The effect of FAT 92368/Y on body weight of HD group females is considered to be adverse. The female HD group values of weight gain collected during the lactation days 0 and 4 were not used for statistical analysis and data comparison as there were not enough animals available for the meaningful comparison.

Food Consumption:
In males, there was a test item related slightly lower food intake in HD group (16 % below control) after the first week of treatment, which during the 2nd week returned to normal level. This correlated with the lower body weight gain in HD group during this period. In females, the food intake in the HD group was generally lower through the dosing period with statistical significance achieved (13 % below controls) during the premating day 1-7. The lower food intake in the HD group was associated with lower weight gain. These changes are considered to be an adverse effect of test item treatment. There was also statistically significantly lower food consumption in the MD group after lactation days 0-4, which correlated with lower body weight and body weight gain.

Pathology:
Macroscopic Findings in Decedents
There were several test item treatment related macroscopic findings recorded at necropsy of decedents.
Small sized spleen and thymus enlarged adrenals and discoloured dark stomach correlated microscopically with lymphoid atrophy, thymic atrophy, diffuse cortical hypertrophy and glandular stomach erosion/ulcer, respectively. These microscopic findings were considered non-specific and secondary changes to a stressful condition. Macroscopic yellow/yellowish discoloration recorded in some organs and tissues was considered to be due to the presence of the test item as luminal contents or due to distribution of the test item (or its metabolites) into the adipose tissues present in and around each organ, or due to the presence of it in the lympho-vascular system. However, these organs and tissues showed no histologic alteration.
Other macroscopic findings showed no corresponding histologic abnormality. Gaseous distention of GI tract was likely to be due to postmortem alteration which could be often observed in the decedents, but intestinal inflammatory changes that were recorded microscopically in some animals were related partially to gaseous distension. The remainder of macroscopic alterations was the non-specific, agonal or postmortem changes and these incidental macroscopic alterations which did not correlate with microscopic changes or was within the range of physiologic responses.

Macroscopic Findings at terminal sacrifice:
The macroscopic findings considered to be treatment-related were a small sized spleen and/or thymus, and/or enlarged adrenal glands recorded in females’ no. 125 and no. 126 of MD group. These correlated microscopically with (lymphoid) atrophy of spleen and thymus, and diffuse cortical hypertrophy of adrenal glands, and were considered to be secondary changes to a stressful condition. Macroscopic yellow/yellowish discoloration recorded in in tongue and stomach of female 125 was considered to be due to the presence of the test item as oral or gastric luminal contents. In these organs and tissues, there was no histologic alteration correlated with macroscopic findings.Paleness or greenish discolouration recorded sporadically in kidneys, thyroid glands and/or parathyroid glands in some animals were not associated with microscopic alteration. The remainder of findings recorded in this study was within the range of normal background lesions which may be recorded in animals of this strain and age or was considered to be incidental macroscopic appearances that did not correlate with microscopic changes.

Organ Weight:
In males, there was statistically significantly lower absolute thyroid gland (18 % below control) and pituitary gland (14 % below control) weight in HD group, statistically significantly higher absolute and relative pituitary gland weight (17 % and 18 % above control, respectively) in MD group, statistically significantly higher relative brain (11 % above control), kidney (10 % above control), spleen (31 % above control) in HD group. There was also higher absolute spleen weight (20% above control) in HD group, but without statistical significance. In females, there was statistical significantly lower absolute liver weight (15 % below control) in MD group, statistically significantly lower absolute heart weight (12 % and 15 % below control) in LD and MD group, statistically significantly lower absolute uterus weight (15 % and 22 % below control) in LD and MD group, statistically significantly lower absolute ovary weight (20% below control) in MD group. There was also lower absolute kidney (12 % below control) and spleen weight (27 % below control) in MD group, lower absolute thymus (40 % below control) and pituitary gland (17 % below control) weight in MD group without attaining statistical significance. There were also changes in brain (19 % above control), adrenal gland (24 % above control), spleen (15 % above control) and thymus (32 % below control) weight relative to body weight in MD group. The changes in kidney weight were microscopically attributed to tubular degeneration, necrosis and/or regeneration and these findings were considered to be an adverse effect of test item treatment. The weight changes on spleen, thymus and adrenal gland were attributed to micrcoscopic changes which were further considered secondary changes to stressful condition.
All other variations in organ weights in the absence of microscopic findings were not associated with adverse effect of test item treatment. The female HD group values were not used for statistical analysis and data comparison as there were not enough animals available for the meaningful comparison.

Histopathology:
Histology revealed no histomorphologic evidence of toxicological properties in the male reproductive organs including testes, epididymides, prostate glands, coagulating glands and seminal vesicles, and the female reproductive organs including ovaries, uterus with cervix and vagina. In addition, as a result of the detailed examination of the testis, it was judged that there were no treatment-related effects on the testicular histomorphology including spermatogenesis and interstitial cell structure. Meanwhile the treatment-related histomorphologic adverse changes were observed in kidneys, ileum, cecum, colon, and rectum. Those consisted of tubular degeneration, necrosis and/or regeneration in the pars recta of the kidney, and inflammation and diffuse mucosal hyperplasia in ileum, cecum, colon and/or rectum. Tubular degeneration, necrosis and/or regeneration was recorded in all test-item treatment groups in a dose dependent manner, except for the HD females in which the severity was a little less than that of the medium-dose females because many animals died or were euthanized prematurely. This lesion was observed in the tubules of pars recta and was characterized by cytoplasmic vacuolation (vacuolar degeneration), cellular swelling and/or cytoplasmic basophilia, with/without nuclear enlargement with vesicular chromatin, single cell deaths and/or mitotic figures. Inflammation and diffuse mucosal hyperplasia were recorded in ileum, cecum, colon and/or rectum in one female (survivor) of MD group and in 5 males and 7 females of HD group. These lesions were particularly prominent in cecum and colon. The mucosal hyperplasia was considered to be regenerative responses to mucosal injuries, in which cellular or mucosal structural atypia was not recognized under the condition of this study. In this study, one male of the LD group, one male and one female of the MD group, and 4 males and 8 females of the HD were found dead or euthanized for ethical reasons during the course of the study. Among them, the cause(s) of morbidity in one LD and one MD male were aspiration pneumonia and peracute pulmonary necrosis due to aspiration, respectively, which happened accidentally, and it was judged that these events were not related to the toxicological properties of the test item. In the remainder of decedents, inflammation in small and/or large intestines and tubular degeneration/ necrosis/ regeneration in the kidney were considered to be involved in the animals’ morbidity. In some animals, pulmonary and tracheal lesions were also observed indicating that the incidental regurgitation or accidental influx of the dosing solution into the respiratory tracts somewhere in the gastrointestinal tracts resulting in perforation and acute peritonitis. These accidental events might have been involved in animals’ morbidity in addition to the toxicological events in the kidney and intestines. Other findings that were related to treatment were recorded in trachea, lungs, stomach, spleen, thymus, mesenteric lymph node and/or adrenal glands in both of survivors and decedents. All of these were incidental or accidental changes (trachea and lungs), physiological responses for scavenging the exogenous substances (mesenteric lymph node), or secondary changes to a stressful condition, and hence, were considered not to be adverse.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

750 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

2

Dose descriptor:

LOAEL

Remarks:

Systemic toxicity

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

Target system / organ toxicity (P0)

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

gross external examination

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

gross external examination

Histopathological findings:

not examined

Details on results (F1)

Litter Data:
There was a statistically significantly lower no. of live pups per litter on PND 4 in the MD group (7 compared to 10.50 of control). The no. of still births per litter was also higher in MD group (0.67 compared to none of control). There were also lower no. of pups per litter born (9.78 compared to 10.50 of control) and lower no. of live pups per litter (8.67 compared to 10.50 of control) on PND 0 and lower no. of male pups (4.67 and 3.22 compared to 4.88 and 4.88 of control on PND 0 and 4, respectively) and female pups (4.67 and 3.78 compared to 5.63 and 5.63 of control on PND 0 and 4, respectively) recorded in MD groups, but without attaining statistical significance. These reductions were associated with 2 animals (no.s 121 and 125) that displaying adverse clinical observations and significant reductions in body weight gain (see section 12.2 and 12.4 respectively). These findings were considered to be adverse effects of FAT 90368/Y. The litter data of HD group were not used for statistical analysis and data comparison as there were not enough animals available for a meaningful comparison. There was no test item treatment related effect on litter data in LD group when compared to control and all values were within the range of historical control data.

Litter Weight Data:
The litter weight data in the MD group were similar to the litter survival data discussed in Section 12.15. There was lower pup mean weight, total litter weight and male and female litter weight on PND 0 and 4. The total litter weight of MD group was statistically significantly lower in the MD group [50.58 g (PND 0) and 78.26 g (PND 4) in MD when compared to 64.20 g (PND 0) and 105.86 g (PND 4) in control, respectively], when compared to controls. Although the mean values were within the range of historical control data, the findings were dose responsive and were considered to be an adverse effect of test item treatment. The litter weight data of the HD group were not used for statistical analysis and data comparison as there were not enough animals available for a meaningful comparison.

Precoital Interval and Duration of Gestation:
There was no effect on the precoital interval. There was no effect on the duration of gestation up to the MD level and also on two surviving females of HD group.

Pre- and Post-Natal Data:
In the HD group, there were slightly lower numbers of corpora lutea (12.13 compared to 13.38 of control) and implantation sites (10.56 compared to 11.13 of control) but without statistical significance. There was no effect on percent preimplantation loss. The no. of live pups and percentage of post- implantation loss of HD group were not used for statistical analysis and data comparison as there were not enough animals available for the meaningful comparison.
In the MD group there were slightly lower number of live pups per litter (8.67 compared to 10.50 in control) and higher percent post implantation loss (16.96 compared to 4.96 in control), but without statistical significance. These reductions were associated with 2 animals (female no.s 121 and 125) that displaying significant reductions in body weight gain and adverse clinical observations. Given the lower no. of pups in MD group compared to control, the higher percentage of post implantation loss was considered to be of toxicological relevance.
In LD group the no. of corpora lutea, no. of implantation sites and live pups were comparable to controls and were within the historical control range, but there was a slightly increased post-implantation loss (11.25 vs 4.96 in the controls). Given that the no. of CL, no. of implantation sites, and number of live pups were comparable to controls and that the post-implantation loss were within historical controls, these changes were not associated with treatment of the test item.

Reproductive Indices:
There was no effect on copulation index in LD, MD and HD groups. There was no effect on fertility and delivery indices in LD and MD groups and fertility index in HD group. The delivery index in HD group was not used for comparison as there were not enough animals available for a meaningful comparison. There was a lower viability index in MD group (76.67 % compared to 100 % of control) which was due to viability of pups of female 121 and 125. These animals displayed significant reductions in body weight gain and adverse clinical observations. This was considered to be an adverse effect of test item treatment from the prospect of its effect on the viability of pups. In HD group the viability index was also lower but was not used for comparison.

Pup Survival Data:
The pup survival was highly affected in MD group due to test item treatment. In MD group the higher mortality was seen in pups, which was associated with 2 animals (nos. 121 and 125) that displaying adverse clinical observations and significant reductions in body weight gain (see section 12.2 and 12.4 respectively). These findings were considered to be adverse effects of FAT 90368/Y TE. In LD group 2 pups of female no. 113 were found dead between days 0 and 1. No further pup mortality was observed during the post-natal days. This was within the range of historical control data and was considered incidental. In the HD group there were only 1 surviving female and the data was not used for statistical analysis and data comparison as there were not enough animals available for a meaningful comparison.

Pup External Findings:
There were external findings in control -hematoma on neck of pups 1, 5, 10 of animal 41); LD group - yellowish skin in most pups of animal 113;
dry skin of pup 2, animal 115; MD group - yellow to orange skin in all pups of animals 127, 128 and 129, dark snout of pup 2, animal 123 and HD group- yellow to orange skin all pups of animals 137 and 138. The yellow or orange coloured pup skin was assumed to be due to licking by dam post dose administration. All these findings were considered incidental or non-adverse. Potential test item related pup external findings in the MD group include hypothermia, no indication of suckling and poor health condition (all pups, animal 121), orange skin and poor condition (all pups, animal 124), hypothermic and weak (pup 5, animal 125). These could be related to poor nursing secondary to general health condition of females 121, 124 and 125 during the post-natal days.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: see 'Remark'

Remarks on result:

other: maternal toxicity seen at 100 mg/kg bw/day as well

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

Table 11 Macroscopic Findings in Decedents, and Corresponding Microscopic Findings

Animal No. [Group]	Macroscopic findings	Corresponding microscopic findings
Male 85 [group 2]	Lung: Focus(es), several, dark red, d = 10 mm; Not collapsed Lymph node, axillary: Discoloured reddish/red/dark red	Diffuse aspiration pneumonia(with hypertrophy of tracheal mucosal epithelium) Congestion [agonal/postmortem change]
		[possible cause(s) of morbidity] Aspiration pneumonia in the lung
Male 100 [group 3]	Lung: Discoloured, yellowish/yellow; Not collapsed Tongue: Discoloured, yellowish/yellow Thymus: Focus(es), several , dark red, d = 1 mm	Diffuse peracute necrosis due to aspiration   NCF/NAD * Agonal hemorrhage
		[possible cause(s) of morbidity] Peracute necrosis in the lung Trachea: Inflammation
Female 130 [group 3]	Stomach: Discoloured, dark Spleen: Small size (Reduced in size) Kidneys: Discoloured, reddish/red/dark red Adrenal glands: Enlarged Uterus with Cervix: Enlarged Oviducts: Enlarged   Tongue, Adipose tissue: Discoloured, yellowish/yellow	Glandular stomach erosion/ulcer# Lymphoid atrophy (splenic atrophy)# Congestion [agonal/postmortem change] Diffuse cortical hypertrophy# Endometrial proliferation with fluid retention in the lumen [alteration within the range of physiologic response(s)] NCF/NAD *
		[possible cause(s) of morbidity] Lungs: Alveolar fluid contents, bronchiolar-alveolar duct region Kidneys: Tubular degeneration/ necrosis/regeneration, pars recta

Animal No. [Group]	Macroscopic findings	Corresponding microscopic findings
Male 103 [group 4]	Skin/subcutis: Discoloured, yellowish/yellow	NCF/NAD *
		[possible cause(s) of morbidity] Cecum, colon: Inflammation Kidneys: Tubular degeneration/ necrosis/regeneration, pars recta
Male 105 [group 4]	No necropsy observations noted	[possible cause(s) of morbidity] Trachea: Hypertrophy of mucosal epithelium and Foreign body granuloma Kidneys: Tubular degeneration/ necrosis/regeneration, pars recta
Male 107 [group 4]	Stomach, duodenum, jejunum, ileum, cecum, colon, rectum, pancreas: Gaseous distension/Gased. Skin/subcutis: Discoloured, yellowish/yellow	NCF/NAD *, §     NCF/NAD *
		[possible cause(s) of morbidity] Cecum, colon, rectum: Inflammation Kidneys: Tubular degeneration/ necrosis/regeneration, pars recta
Male 108 [group 4]	Lung: Discoloured, reddish/red/dark red; Focus(es), dark Thymus: Discoloured, reddish/red/dark red Lymph node, axillary: Discoloured, reddish/red/dark red Kidneys: Enlarged Stomach, duodenum, jejunum, ileum, cecum, colon: Gaseous distension/Gased Epididymides: Spot(s), yellow Liver: Enlarged, caudate process	Diffuse aspiration pneumonia   Agonal hemorrhage Congestion [agonal/postmortem change]   Congestion [agonal/postmortem change] NCF/NAD *   Spermatic granuloma(s) NCF/NAD *
		[possible cause(s) of morbidity] Aspiration pneumonia in the lung Kidneys: Tubular degeneration/ necrosis/regeneration, pars recta
Female 131 [group 4]	Skin/subcutis: Discoloured, yellowish/yellow	NCF/NAD *
		[possible cause(s) of morbidity] Lungs: Multiple foreign body granuloma at/around the bronchiolar/alveolar duct region, and with Hypertrophy of tracheal mucosal epithelium Cecum: Inflammation Kidneys: Tubular degeneration/ necrosis/regeneration, pars recta
Female 132 [group 4]	Adrenal glands: Enlarged	Diffuse cortical hypertrophy#
		[possible cause(s) of morbidity] Ileum, cecum, colon, rectum: Inflammation Kidneys: Tubular degeneration/ necrosis/regeneration, pars recta
Female 133 [group 4]	Adrenal glands: Enlarged Lungs: Discoloured, dark	Diffuse cortical hypertrophy# Congestion [agonal/postmortem change]
		[possible cause(s) of morbidity] Ileum, cecum, colon: Inflammation Abdominal cavity: Peritonitis Kidneys: Tubular degeneration/ necrosis/regeneration, pars recta

Animal No. [Group]	Macroscopic findings	Corresponding microscopic findings
Female 134 [group 4]	Adrenal glands: Enlarged Stomach: Discoloured, dark;                Gaseous distension/Gased Duodenum: Discoloured, dark;                Gaseous distension/Gased Jejunum, ileum, cecum, colon, rectum: Discoloured, dark and Gaseous distension / Gased Peyer’s patches: Discoloured, dark	Diffuse cortical hypertrophy# Glandular stomach erosion/ulcer# NCF/NAD * Congestion [agonal/postmortem change] NCF/NAD * NCF/NAD *     NCF/NAD *
		[possible cause(s) of morbidity] Lungs: Aspiration pneumonia Trachea: Inflammation (necrotizing) Kidneys: Tubular degeneration/ necrosis/regeneration, pars recta
Female 136 [group 4]	Lungs: Fluid distension Adrenal glands: Enlarged     Stomach: Discoloured, yellowish/yellow and Gaseous distension/Gased Duodenum, jejunum, ileum, cecum, colon: Gaseous distension/Gased	Aspiration pneumonia Diffuse cortical hypertrophy#
		NCF/NAD *
		[possible cause(s) of morbidity] Aspiration pneumonia in the lung [other possible cause(s) of morbidity] Trachea: Hypertrophy of mucosal epithelium Colon: Inflammation Kidneys: Tubular degeneration/ necrosis/regeneration, pars recta
Female 138 [group 4]	Tongue, stomach (gastric mucosa), duodenum, jejunum, ileum, cecum, pancreas, mesenteric lymph node, Peyer’s patches: Discoloured, yellowish/yellow Lymph node, axillary: Small size Adrenal glands: Enlarged; Discoloured, pale	NCF/NAD * NCF/NAD * Diffuse cortical hypertrophy # NCF/NAD *
		[possible cause(s) of morbidity] Cecum, colon: Inflammation Kidneys: Tubular degeneration/ necrosis/regeneration, pars recta
Female 139 [group 4]	Pancreas: Discoloured, reddish/red/dark red	Congestion [agonal/postmortem change]
		[possible cause(s) of morbidity] Cecum, colon, rectum: Inflammation Kidneys: Tubular degeneration/ necrosis/regeneration, pars recta
Female 140 [group 4]	Adrenal glands: Enlarged Spleen: Small size Thymus: Small size Stomach, duodenum, jejunum, ileum, cecum, colon: Gaseous distension/Gased	Diffuse cortical hypertrophy# Lymphoid atrophy (splenic atrophy)# (Not submitted for examination) NCF/NAD *, §
		[possible cause(s) of morbidity] Ileum, cecum, colon: Inflammation Kidneys: Tubular degeneration/ necrosis/regeneration, pars recta
*=NCF/NAD: No corresponding microscopic findings/Nothing histologic abnormal discovered. §=It is most likely to be due to postmortem change(s), but the intestinal inflammatory changes might havebeen related partially to the macroscopic alteration.

Table 12 Summary table of organs weights with statistically significant different in dose groups when compared to control combined

with histopathology

Gender	Organs	Absolute organ weight (g): Groups with statistically significantly different compared to control	Relative organ weight (%): Groups with statistically significantly different compared to control	Histopathological findings
Male	Thyroid/Parathyroid glands	HD (-17.8 %)	-	-
	Brain	-	HD (+10.7 %)	-
	Kidneys	-	HD (+10.2 %)	Tubular degeneration, necrosis and/or regeneration
	Spleen	-	HD (+31.95 %)	Lymphoid atrophy in spleen
	Pituitary Gland	MD (+16 %),      HD (-14.7 %)	MD (+19.0 %)	-
Female	liver	MD (-15.37 %)	-	-
	Heart	LD (-11.5 %),    MD (-15.1%)	-	-
	Uterus including cervix and oviducts	LD (-14.8 %),  MD(-21.6 %)	-	-
	Ovaries	MD (-19.98 %)	-	-

Applicant's summary and conclusion

Conclusions:

Due to the lack of histomorphological changes (including spermatogenesis and interstitial cell structure) and other reproductive parameters the NOAEL for reproductive toxicity was considered to be 750 mg/kg/d. the NOAEL for developmental toxicity was considered to be 100 mg/kg/d.

Executive summary:

The aim of this study was to assess the possible effects of FAT 92368/Y on male and female fertility and embryo fetal 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 54 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 3 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days were completed. Animals of an additional control group were handled identically as the dose groups but received aqua ad iniectabile (water for injection), the vehicle used in this study. The 4 groups comprised 10 male and 10 female Wistar rats.

The following doses were evaluated:

Control: 0 mg/kg/d

Low Dose: 100 mg/kg/d

Medium Dose: 300 mg/kg/d

High Dose: 750 mg/kg/d

The test item formulations were prepared once in every ten days and stored at room temperature. The test item was suspended in aqua ad iniectabile and administered daily during 14 days of pre-mating and 14 days of mating in both male and female animals, during the gestation period and up to post-natal day 3 in females. Males were dosed for 28 days. Dose volumes were adjusted individually based on weekly body weight measurements. The administration volume was 5 mL/kg. During the period of administration, the animals were observed each day for signs of toxicity. Animals that died were examined macroscopically and at the conclusion of the test, surviving animals were sacrificed and observed macroscopically. Body weight and food consumption were measured weekly, except for food consumption measurements which were not taken during the mating period in female animals and the mating and post-mating period in male animals. Haematological and clinical biochemistry evaluations were performedon blood samples collected at terminal sacrifice from five males and five randomly selected females from each group. Urinalysis was performed on samples collected at terminal sacrifice from five randomly selected males from each group. Functional observations including sensory reactivity to different stimuli, grip strength, motor activity assessments and other behavior observations were performed in the week before the treatment and at the end of the study. After 14 days of treatment to both male and female, 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 the 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 and on day 4 post-partum. The males were sacrificed after completion of the mating period on treatment days 29 and 30 and the females along with their pups were sacrificed on post natal day 4. Non-pregnant females were sacrificed on day 26. Pups sacrificed on postnatalday 4 and those found dead, were carefully examined for gross external abnormalities. Sections from the organs and tissues from 5 selected male and female animals of control, as well as all gross lesions from all groups, were examined bylight microscopy. In this study, sections of the full list of organs and tissue were examined in 5 males and 5 females from the survivors of MD group and 5 males and 2 females from the survivors of HD group in addition to the decedents from MD and HD groups, because animals’ morbidity that were considered to be related to treatment with the test item was observed in many animals of HD group. Furthermore, testes, epididymides, prostate, seminal vesicles with coagulating glands, ovaries, uterus with cervix and vagina were examined in all animals, and, on the testes, special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.

 

Summary Results:

There were mortalities in male and female dose groups during the study period. In males 1/10 in LD group (male no. 85) and 1/10 in MD group (male no. 100) and 4/10 in HD group (males 103, 105, 107 and 108). In Females 1/10 in MD group (female no. 130) and 8/10 in HD group (female nos. 131, 132, 133, 134, 136, 138, 139 and 140). The cause(s) of morbidity and mortality in LD male no. 85 and MD male no. 100 were aspiration pneumonia and peracute pulmonary necrosis due to aspiration, respectively, which happened accidentally, and it was judged that these events were not related to systemic toxicity of FAT 92368/Y TE. The cause of morbidity and mortality were mainly due to inflammation in small and/or large intestines and tubular degeneration/ necrosis/ regeneration in the kidney. There were clinical signs considered to be related to test item treatment in males and/or females of MD and/or HD groups. The clinical signs of test item treatment relevance were abnormal breathing, dehydration, diarrhea, moving the bedding, wasp waist, half eye lid closure, hypothermia, kyphosis and reduced spontaneous activity, piloerection, discoloured feces and salivation in males and/or females of MD and/or HD groups. Abnormal breathing and piloerection was associated with aspiration and moving the bedding and salivation to local effect of the test item. Discoloured feces were due to colour of the test item. The clinical sings including slight to moderate piloerection, abnormal breathing and dehydration were observed in female 121 and clinical signs dehydration, slight to moderate piloerection, diarrhoea, red nasal discharge, discoloured feces and slight wasp waist in female 125. These two females of MD group also displayed significant reductions in body weight gain and contributed to the litter losses. During the weekly detailed clinical observation, there were no changes or differences between the groups and the baseline values in males up to HD level and in females up to MD level. No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period in males. 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 up to MD level. There was no effect on body temperature in both males and females. In males, there was an adverse effect of test item treatment on body weight, body weight gain and food consumption at in only the HD. In females, there was an effect of test-item treatment on body weight, body weight gain and food consumption in MD and HD group. There was no effect at the LD level. In males, there were no effects of test-item treatment on hematology, blood coagulation and urinary parameters in dose groups when compared to control. There were statistically significantly higher mean AP value (357.24 U/L compared to 204.68 U/L of control) and statistically significantly lower mean GLU value (11.81 mmol/L compared 16.54 mmol/L of control) in HD group. There were no associated microscopic changes in liver and in addition the means of AP and GLU were within the historical control range. These findings were not considered to be an adverse effect of test item treatment. In females, there were no effects up to MD level. At necropsy several test item related macroscopic findings were observed in the decedents and in the terminally sacrificed females. The macroscopic findings small sized spleen and thymus, enlarged adrenals and discoloured dark stomach correlated microscopically with lymphoid atrophy, thymic atrophy, diffuse cortical hypertrophy and glandular stomach erosion/ulcer, respectively. Yellow/yellowish discoloration was recorded in some organs and tissues correlated to the presence of the test item as luminal contents or due to distribution of the test item (or its metabolites) into the adipose tissues present in and around each organ, or due to the presence of it in the lympho-vascular system. These finidngs were not considered to be adverse. The remainder of macroscopic alterations was the non-specific, agonal or postmortem changes and these incidental macroscopic alterations which did not correlate with microscopic changes, or was within the range of physiologic responses. There were effects of test item treatment on absolute and/or relative kidney, spleen, thymus and adrenal glands weight in males and/or females of MD and/or HD groups. Microscopically the kidney was associated with tubular degeneration, necrosis and/or regeneration and this was considered an adverse effect. The changes in spleen, thymus and adrenal weights were associated with micrcoscopic changes which were considered secondary changes to stressful condition. Histology revealed no histomorphologic evidence of toxicological properties in the male reproductive organs including testes, epididymides, prostate glands, coagulating glands and seminal vesicles, and the female reproductive organs including ovaries, uterus with cervix and vagina. In addition, there were no treatment-related effects on the testicular histomorphology including spermatogenesis and interstitial cell structure.Treatment-related histomorphologic adverse changes were observed in kidneys, ileum, cecum, colon, and rectum. Those consisted of tubular degeneration, necrosis and/or regeneration in the pars recta of the kidney, and inflammation and diffuse mucosal hyperplasia in ileum, cecum, colon and/or rectum. Tubular degeneration, necrosis and/or regeneration was recorded in all test-item treatment groups in a dose dependent manner, except for the HD females in which the severity was a little less than that of the medium-dose females because many animals died or were euthanized prematurely. Inflammation and diffuse mucosal hyperplasia were recorded in ileum, cecum, colon and/or rectum in one female (survivor) of MD group and in 5 males and 7 females of HD group. These lesions were particularly prominent in cecum and colon. In the decedents, inflammation in small and/or large intestines and tubular degeneration/ necrosis/ regeneration in the kidney were considered to be involved in the animals’ morbidity. In some animals, the pulmonary and tracheal lesions indicating that the incidental regurgitation or accidental influx of the dosing solution into the respiratory tracts happened and the acute peritonitis indicating that the perforation had happened somewhere in the gastrointestinal tracts were also observed, and these accidental events might have been involved in animals’ morbidity in addition to the toxicological events in the kidney and intestines. Other findings that were related to treatment were recorded in trachea, lungs, stomach, spleen, thymus, mesenteric lymph node and/or adrenal glands in both of survivors and decedents. All of these were incidental or accidental changes (trachea and lungs), physiological responses for scavenging the exogenous substances (mesenteric lymph node), or secondary changes to a stressful condition, and hence, were considered not to be adverse. There was no effect on the precoital interval. There was no effect on the duration of gestation up to the MD level and and also on two surviving females of HD group. There was higher percent post- implantation loss in MD group (16.96 % compared to 4.96 % of control). These reductions were associated with 2 animals (female no.s 121 and 125) that displaying significant reductions in body weight gain and adverse clinical observations. Given the lower no. of pups in MD group (8.67 compared to 10.50 of control), the higher percent post implantation loss was considered to be of toxicological relevance. There was no effect on copulation index in LD, MD and HD groups. There was no effect on fertility and delivery indices in LD and MD groups and fertility index in HD group. There was lower viablity index in MD group (76.67 % compared to 100 % of control) which was considerd to be an adverse effect of test item treatment. There were adverse effects of test-item treatment on litter data, litter weight data and pup survival data at MD group. There was a statistically lower no. of live pups per litter on PND 4 in MD group (7 compared to 10.50 of control). The no. of still births per litter was also higher in MD group (0.67 compared to none of control). There were also lower no. of pups born per litter (9.78 compared to 10.50 of control) and lower no. of live pups per litter (8.67 compared to 10.50 of control) on PND 0 and lower no. of male pups per litter (4.67 and 3.22 compared to 4.88 and 4.88 of control on PND 0 and 4, respectively) and female pups per litter (4.67 and 3.78 compared to 5.63 and 5.63 of control on PND 0 and 4, respectively) in the MD group. These reductions were associated with 2 animals (no.s 121 and 125) that displaying significant reductions in body weight gain and adverse clinical observations. These findings were considered to be adverse effects of FAT 90368/Y. Similar to litter data there was lower pup mean weight, total litter weight and male and female litter weight in MD group on PND 0 and 4. The total litter weight of the MD group was statistically significantly lower (50.58 g and 78.26 g in MD when compared to 64.20 g and 105.86 g in control, respectively), when compared to controls. These findings were considered to be an adverse effect of test item treatment. The pup survival was highly effected in MD group due to test-item treatment. In the HD group there were only 1 surviving females and the data was not used for statistical analysis and data comparison. 

Conclusion:

On the basis of this combined repeated dose oral toxicity and reproduction/ developmental toxicity screening test with FAT 92368/Y in male and female Wistar rats with dose levels of 100, 300, and 750 mg/kg/d the following conclusions can be made:

There was mortality of male and/ or female animals at 300 mg/kg/d and at 750 mg/kg/d. There was an also adverse effect of FAT 92368/Y treatment on body weight and food consumption in females at 300 and 750 mg/kg/d and males at 750 mg/kg/d. The increased (in males) and decreased (in females) kidney weight was associated with tubular degeneration, necrosis and/or regeneration in the pars recta of the kidney in all test-item treatment groups in a dose dependent manner, except for the HD females in which the severity was a little less than that of the medium-dose females because many animals died or were euthanized prematurely. There were no histomorphological changes in male and female reproductive organs at any dose level. There were adverse effects of test item treatment on litter data, litter weight data, percent post implantation loss and pup survival at 300 mg/kg/d which was associated with the maternal systemic toxicity. There were no treatment related on fetal development and survival at 100 mg/kg/d. Due to the lack of histormorphological changes (including spermatogenesis and interstitial cell structure) and other reproductive parameters the NOAEL for reproductive toxicity was considered to be 1000 mg/kg/d. the NOAEL for for developmental toxicity was considered to be 100 mg/kg/d. A NOAEL of systemic toxicity could not be obtained due to the presence of adverse lesions in the kidney of both sexes. The lowest-observed-adverse effect level (LOAEL) under the condition of this study was given at 100 mg/kg/day in both sexes.