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-)

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-04-02 to 2014-10-27

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650

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

Specific details on test material used for the study:

Name: FAT 92368/Y
Batch No.: 1309001
Physical State / Colour: solid / orange brown
Storage Conditions: room temperature, protected from light
Expiry Date: 30.09.2018
Safety Precautions: gloves (butyl or neoprene), respiratory protection, safety glasses

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 – 346.90 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: 10x/hour
- Diet: Free access to Altromin 1324 maintenance diet for rats and mice (lot no. 1526)
- Water: Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls
at regular intervals)
- Housing: 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 postmating 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

Details on route of administration:

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.

Vehicle:

water

Details on oral exposure:

Preparation of the test item formulation:
The test item FAT 92368/Y was weighed into a tarred plastic vial on a precision balance and suspended in aqua ad iniectabile. 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.

Experimental Groups and Doses:
The dose range finding study (14 days repeated dose, BSL study no. 140383) was performed at dose levels 100, 300 and 1000 mg/kg/d. At 1000 mg/kg/d slightly attenuated body weight development and reduced food consumption were observed during the first treatment week
and at the end of the 14 days of treatment. One of 3 female animals treated at 1000 mg/kg/d was found dead on study day 12. According to these results and in consultation with the sponsor the following doses are selected for the 3 dose groups (LD = low dose, MD = medium dose, HD = high dose) and 1 control group (C):

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.

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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

low dose

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

mid dose

Doses / concentrations 4

Dose / conc.:

750 mg/kg bw/day (nominal)

Remarks:

high dose

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

Observations and examinations performed and frequency:

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.

Haematology:
Haematological parameters from 5 randomly selected males and females (only lactating females were evaluated) from 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 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), large unstained cells (Luc)


Blood Coagulation:
Coagulation parameters from 5 randomly selected males and females (only lactating females were evaluated) from 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-coated tubes. The following coagulation parameters were examined: prothrombin time (PT), activated partial thromboplastin time (aPTT).

Clinical Biochemistry:
Parameters of clinical biochemistry from 5 randomly selected males and females (only lactating females were evaluated) from 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). potassium (K).

Urinalysis:
A urinalysis was performed with samples collected from 5 randomly selected males and female 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 (Heiland Urine Stripes URI 10SL): specific gravity, nitrite, ph-value (pH), protein, glucose, ketone bodies (ketones), urobilinogen (ubg), bilirubin, blood, leukocytes

Sacrifice and pathology:

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. 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 hematoxyline 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.

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).

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

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 HDlevel 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.

Mortality:

mortality observed, treatment-related

Description (incidence):

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. 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.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

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 and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

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. But the lower food intake did not correlate with the body weight (except female no. 121) and therefore, was not considered to be an adverse effect of test item treatment.

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

Description (incidence and severity):

In males, there was a statistically significantly higher MCHC value in the MD group, compared to controls (36.14 g/dL compared to 34.80 g/dL of control). As the mean values were within the range of historical control data and did not show dose response relation, this effect is not assumed to be related to test item treatment. There was a statistically significantly higher reticulocyte count in HD group (3.77 % compared 2.07 % of control), Although the mean was slightly above the historical control data range, this is considered incidental due to the absence of effects on other red blood cell parameters. There was no effect on the coagulation parameters. In females, there were no statistically significant differences on hematology and coagulation parameters up to the MD level when compared to the control group. The HD group values were not used for statistical analysis and data comparison as there were not enough animals available in HD group for a meaningful comparison.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

In males, there was 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. Hence, the findings are not considered to be test item treatment related, but non adverse. In females, there was statistically significantly higher mean Chol value (2.20 mmol/L compared 1.35 mmol/L of control) in LD group. The mean and most individual values were within historical control range. There were no statistically significant differences of clinical biochemistry parameters up to MD level. In the absence of dose response relation this was not considered related to test item treatment. The HD group values were not used for statistical analysis and data comparison as there were not enough animals available in HD group for a meaningful comparison.

Urinalysis findings:

no effects observed

Description (incidence and severity):

The urinalysis performed in male animals revealed no effect of FAT 92368/Y at any of the dose levels tested, when compared to the control group. In females, the urinalysis revealed no significant effects up to MD level. In HD group there were not enough female animals available for a meaningful comparison.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

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 138) there was no effect of test item treatment observed during the last week of treatment.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

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 microscopic 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.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

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.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

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.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Effect levels

Dose descriptor:

LOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

not specified

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:

The lowest-observed-adverse effect level (LOAEL) under the condition of this study was given at 100 mg/kg bw/day in both sexes.

Executive summary:

The aim of this study was to assess the possible effects of FAT 92368/Y on male and female fertility and embryofetal 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

 

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 and 1000 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.