Chromate(2-), [2,4-dihydro-4-[(2-hydroxy-4-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulfonato(3-)]-, disodium

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 2014-10-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

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

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

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: males and females. The female animals were non-pregnant and nulliparous.
Age at the start of the treatment period: males: 9-10 weeks old, females: 9-10 weeks old.
Body weight at the allocation of the animals to the experimental groups (together with study
140382): 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 [8] 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 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 any adverse 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 was 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.

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

The test item FAT 20060/F was weighed into a tarred plastic vial on a precision balance and suspended in aqua ad injectable (water for injection). 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 (RT). The homogeneity was ensured by vortexing the sample on vortex machine. The dose range finding study (14 days repeated dose, BSL study no. 140352) was performed at dose levels 100, 300 and 1000 mg/ kg/d with no overt toxicity were observed up to the highest dose tested. According to these results and in consultation with the sponsor the following doses were selected for the 3 dose groups (LD = low dose, MD = medium dose, HD = high dose) and 1 control group (C):

Control (C): 0 mg/kg/d
Low Dose (LD): 100 mg/kg/d
Medium Dose (MD): 300 mg/kg/d
High Dose (HD): 1000 mg/kg/d

Since little or no toxicity was anticipated for the test substance, the highest dose level was set at 1000 mg/kg bw/d corresponding to a limit dose for this study. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dose-related response and a NOAEL. The animals in the control group were handled in an identical manner to the test group subjects and received aqua ad injectable (water for injection) using the same volume as used for the dose groups. 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.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration analysis of formulation samples was determined in study weeks 1, 3, 5 and 7 for all dose groups. The mean recoveries observed in the low dose (LD), medium dose (MD) and high dose (HD) groups were 94.5 %, 89.0 % and 86.1 % of the nominal concentration, respectively. Nominal concentrations were confirmed for all dose groups, as measured mean concentration did not differ from nominal concentration by more than 20 %. Stability of formulation samples was investigated in study week 1 based on concentration in the LD and HD dose groups. After 10 days storage at room temperature recovery compared to starting value was 93.2 % and 108.1 %. All samples were stable, as concentration after storage did not differ from start value by more than 20 %. Homogeneity of formulation samples was determined in study weeks 1 and 5 for the LD and HD dose groups. The mean recovery observed for the LD dose group was 93.6 % and 97.7 % of the nominal value and 83.3 % and 91.9 % of the nominal value for HD dose group. The coefficients of variation of the different sampling locations (top, middle, bottom) were 18.3 % and 12.3 % and 6.3 % and 9.0 % in HD dose group. All samples were homogenous, as COV was below 20 %.

Duration of treatment / exposure:

The animals were treated with the test item formulation or vehicle 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 28-29 days is completed.

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 140382.

Control animals:

yes, concurrent vehicle

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, visual placing, fore and hind limb grip strength, tail pinch response, toe pinch reflex, extensor thrust/limb tone, hind limb reflex, righting reflex on the ground, air righting reflex, pupil response, body temperature and ophthalmoscopy (anterior chamber of the eye and fundus of eye).

Body Weight and Food Consumption:
The 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 females 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, Pharmanova, lot no: 24863, expiry date: 10/2015 and Serumwerk, lot no: 00513, expiry date: 05/2015). The surviving pups were killed by decapitation on PND 4. 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 from the day of 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 10 % neutral buffered formalin, 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 were evaluated) randomly selected from each group was recorded as soon as possible. Paired organs were weighed together. In addition, reproductive organs of all animals were weighed. Except 1/5 selected females of LD group was non pregnant and organ weight of this animals was not measured. 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, lymphnodes (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:
A full histopathological evaluation of the tissues was performed of 5 randomly selected male and female animals of the control and high dose groups. As well as all gross lesions from all groups, were examined by light microscopy. Sections of the full list of organs and tissue were examined in 8 males and all females of HD group, because macroscopic discoloration was recorded in all organs and tissues in these animals. With regard to female no. 71 of HD group, full list of organs was examined as the decedent in addition to the reason of discoloration in all organs and tissues. Furthermore, testes, epididymites, 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. In order to demonstrate glycogen accumulation in the hepatocytes, additional PAS stain sections from the representative animals (animal nos. 2, 33, 34, 36, 41, 73, 74 and 77) were prepared and examined by light microscopy. In addition, because of treatment-related findings that were considered to be implicated in the effects to reproductive and developmental performances in the high dose group, adrenal glands, thymus and liver from 5 selected animals/sex/group of the low and mid dose groups (LD and MD groups, respectively) were examined to establish a no-effect level. 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).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

no adverse effects

Mortality:

mortality observed, treatment-related

Description (incidence):

no adverse effects

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:

effects observed, treatment-related

Description (incidence and severity):

no adverse effects

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

Mortality:
There was no mortality of animals due to test- item treatment in dose groups during the study period. However, one female (female no. 71) of HD group was euthanized for ethical reasons during the course of the study. Microscopic examination revealed the cause of animal’s morbidity to an aspiration pneumonia which happened accidentally, and the event was not related to the toxicological properties of the test item. The remainder of animals survived the scheduled study period.

Clinical Observations:
There were test item treatment related clinical signs moving the bedding, salivation, diarrhoea, discoloured red feces and skin in male and female animals of MD and/ or HD groups. The discoloured red feces and skin were due to colour of the test item. Moving the bedding and salivation observed in the MD and HD groups and a single animal of the LD group was assumed to be due to local effect of the test item. Other clinical signs including alopecia, crust, piloerection, regurgitation, abnormal breathing, dehydration, hypothermia, prostration was observed in few control or dose group animals. These clinical signs were mostly transient in appearance. Clinical findings noted in animal no. 71 that was euthanized for ethical reasons were abnormal breathing, respiratory sounds, piloerection and severe diarrhea. During the weekly detailed clinical observation, no significant changes or differences between the groups were found.

Functional Observations:
There was an increased defecation count in male animals of the HD group during the last treatment week. The defecation counts in male HD group were statistically significant. In the absence of other autonomous effect, the higher defecation counts in HD group was considered incidental.

Body Weight Development:
From premating days 1 to 7 a slight test article related body weight loss was noted in males of the HD group which was statistically significantly different from the body weight gain observed in animals of the control group. A slight body weight loss was also observed in this group between pre-mating day 14 and mating day 7. Thus, the overall mean body weight gain observed at the end of the treatment period was also statistically significantly lower than in controls. The absolute body weight in HD group males was 4 % below control group at the end of the treatment period. There was also slightly reduced mean body weight gain after the last week of treatment in male MD group, which was elicited by single isolated male no. 30 (reduced weight gain of -39 g). In females of the HD group, there was a lower mean body weight gain during the gestation days 0-7 and 14-20, with statistical significance achieved between days 14 and 20, resulting in a significant reduction in mean body weight gain when the entire gestation period is considered (GD 0-20). On gestation day 7 and 20, absolute body weight of HD females was 7 and 13 % below controls, respectively. The effect on body weight during the last week of gestation days is likely due to lower litter weight. There was also statistically significantly lower absolute body weight in female HD group during premating day 1. The body weight on premating day 1 was measured before the initiation of test item administration and the changes observed in LD group was not dose responsive. These findings were not related to test item treatment. No effect of the test item on body weight gain was observed in the LD and the MD groups.

Food Consumption:
In males, there was statistically significantly lower food consumption in HD group after premating day 1-7 (approx. 16 % below controls). This change correlated with the changes in body weight. Compared to the controls, there is a slight increase in food consumption during the second week of treatment which is reflected in a slight body weight gain during this period. In females, there was statistically significantly lower food consumption in HD group after premating day 1 to 7 (approx. 26 % below controls). Non-significant but slightly lower food consumption was observed throughout the remaining treatment period. This decreased food consumption often did not correlate with body weight development observed in female animals.

Haematology and Coagulation:
In males and females, there were no statistically significant changes in hematology and blood coagulation parameters. However, in males there were slightly higher WBC, Neutrophil and monocyte count in MD and/ or HD groups and slightly lower aPTT value in HD group when compared to the corresponding control group. In females there was slightly higher monocyte and reticulocyte count in MD and/ or HD groups. These findings were not assumed to be toxicologically relevant. The mean monocyte of males and mean RBC, Hb, HCT and reticulocyte of females in HD groups were not within the range of historical control data. Given the absence of statistically significant differences and lack of other related pathological findings, these findings were not considered adverse. All other mean and most individual values of hematology and blood coagulation parameters were within the historical control data range.

Clinical Biochemistry:
In males, there was statistically significantly higher ASAT in HD group. This was mainly caused by animal no. 37 that displayed a comparably high value of 182 U/L, which was associated with high ALAT and lower AP values. There was also lower AP and TBA value in HD group without attaining statistical significance. In females there was a slight but statistically significantly higher Chol value in the LD group. This is not assumed to be toxicologically relevant. There were also higher ASAT and TBA values in LD, MD and HD groups and lower AP values in HD group without attaining the statistical significance. The differences were only slight and are not assumed to be toxicologically relevant. The mean ALAT of male and ASAT of female in HD group were not within the range of historical control data. There were no associated cellular/tissue injuries in liver, however, was related to stress. All other mean and most individual values of clinical biochemistry parameters were within the historical control data range.


Urinalysis:
In males, there were slightly higher levels of bilirubin in 2 males each of MD and HD group. There was also an elevated protein level in in 1/5 or 2/5 males of LD, MD and HD group. In the absence of cellular/tissue injuries in liver and kidneys, the findings were not considered to be an adverse effect test-item treatment. In females there were no significant changes in urinalysis.

Pathology:
At necropsy the animals showed reddish, red or dark red discoloration in one organ, several organs or all organs and tissues in one female of LD, 8 males and 6 females of MD and all males and females of HD. There was also enlargement of adrenal glands in some high dose females and in a single HD female (no. 71) there was distended cecum. These findings corroborated to microscopic findings (see 12.11) and were associated with secondary effects to a stressful condition. 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 were considered to be incidental macroscopic appearances that did not correlate with microscopic changes.


Organ Weight:
In males, there was a slightly but statistically significantly higher absolute testes weight in LD group (13 % above controls), a slight but statistically significantly higher relative adrenal weight in HD group (22 % above controls) and statistically significantly higher relative spleen weight in LD, MD and HD groups (24, 21 and 26 % above controls). In females, there was statistically significantly lower absolute heart weight (21 % below controls); pituitary gland weight (20 % below controls) and ovary weight (20 % below controls) in HD group. The organ weights were not statistically significant different from controls when related to body weight and they did not correlate with macroscopic alterations. The changes in adrenal gland and pituitary gland weights in males and/or females could be attributed to secondary event to stressful condition. The remaining slight organ weight changes were not associated with cellular/ tissue injury.

Histopathology:
Microscopic findings that were considered to be related to treatment with the test item were recorded in testes of males, ovaries and uterus with cervix of females, and kidneys, mesenteric lymph node, liver, thymus and adrenal gland of both sexes.

Microscopic Findings in Decedent:
In female no. 71 (HD group) which was euthanized for ethical reasons, aspiration pneumonia in the lung as well as reactive tracheal mucosal hypertrophy was observed, and these were the cause of animal’s morbidity. This occasionally happens due to regurgitation and/or accidental influx of the dosing solution into the respiratory tracts after or during the dosing procedure, and it was judged that the animal’s morbidity was not related to the toxicological properties of the test item. Thymic atrophy, adrenocortical diffuse hypertrophy and glandular stomach erosion were recorded in this animal, and these were considered to be secondary effects to a stressful condition associated with respiratory tracts' lesions. The remainder of findings is mentioned in the following sections along with the findings in the survivors.

Thymus, Adrenal Glands and Liver
Thymic atrophy as well as adrenocortical diffuse hypertrophy was recorded in both sexes of HD group, and increased hepatocytic glycogen accumulation, which was confirmed further by PAS staining in some representative animals, was recorded in both sexes of HD group as well. There was no further indicator of cellular injuries in adrenal glands and liver examined in the high-dose group, and the histologic alterations described in this section were not observed in animals of LD and MD groups. The above-mentioned findings were considered to be secondary to the stressful condition in the parent animals but judged to be adverse under the condition of this study from the comprehensive aspect of the reproductive/developmental toxicity assessment.

Kidneys
Gold, yellow and/or brown pigment deposition in the proximal tubules was recorded in one male of LD group and 8 males and 10 females of HD group, without any further indicator of cellular injuries. In females, eosinophilic substances similar to hyaline droplets that are often observed in the male rat were occasionally found in the proximal tubular epithelial cells. Pigment deposition, as well as hyaline droplets-like eosinophilic substance in females, was considered to be the histomorphologic representation indicating physiologic excretion processes that occurred by re-absorption of the test item or its metabolites and was considered to be present as the form of phagolysosomal cytoplasmic components as with spontaneous hyaline droplets in the male rats. There was no further indicator of cellular injuries, and therefore, these findings were considered not to be adverse.

Testes, Ovaries, Uterus with Cervix, Mesenteric Lymph Node, and Lungs
Yellow to brown pigment laden macrophages/histiocytes were observed in testes, ovaries, uterus (including cervix), mesenteric lymph node and lungs. There was no indicator of cellular and tissue injuries in macrophages/histiocytes and in intrinsic tissue components of each organ. Pigment was considered to be derived from the test item or its metabolites distributed to the whole body, and this finding indicates the physiological activities for scavenging the exogenous substances, and hence, was considered not to be adverse. Likewise, increased incidence and/or severity of sinus histiocytosis in the mesenteric lymph node, which were recorded in females of MD group and both sexes of HD group, were considered to be physiological activities for scavenging the exogenous substances, and hence, considered not to be adverse.

In the lung, accumulation of macrophages at/around the bronchiolar/alveolar duct region appears to have increased slightly in the incidence and severity in the high dose group compared to the control group. Thymus, Adrenal Glands and Liver”, however, animals in the high-dose group of this study were considered to have been under the stressful status. Such situation is prone to incidental aspiration or regurgitation caused by the animals' hypodynamic or by the evasion against the doing-procedure. Likewise, it was considered that the accumulation of macrophages had happened incidentally also in the lung of the present study, and therefore, this was concluded not to be a toxicological event. There were no abnormalities in the male and female reproductive organs, except for the presence of pigment-laden macrophages, and there were no noteworthy differences in histologic appearances between the control groups and the test item treated groups.

Testes - Detailed Examination Including Sperm Staging
The stages were checked on completeness of cell populations, completeness of stages and degenerative changes. There were no treatment-related effects on the completeness of stages or cell populations of testes. The presence of pigment laden macrophages/histiocytes did not affect the spermatogenesis and the interstitial structures, and hence, this was considered not to be adverse event. Nothing particular was observed in testes of animal nos. 2, 10 (control) and 36 (group 4), which were mated with female nos. 42, 50 and 76 not giving birth, respectively, and nothing particular was found in prostate glands, seminal vesicles and coagulating glands as well. Minimal mononuclear cell foci were recorded unilaterally in epididymides of male nos. 10 and 36. However, such a lesion is often observed in male animals that can produce pregnancy, and therefore, it is unlikely that such minimal finding was related to the infertility. There were no abnormalities in the female reproductive organs (ovaries, uterus with cervix and vagina) of the paired female nos. 42, 50 (control) and 76 (HD group) which did not give birth.

Other Findings
The remainder of findings recorded was within the range of normal background lesions which may be recorded in animals of this strain and age.

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

Conclusions:

The NOAEL for systemic as well as reproduction/ developmental toxicity was considered to be 300 mg/kg/d.

Executive summary:

The aim of this study was to assess the possible effects of FAT 20060/F 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 28-29 days were completed. Animals of an additional control group were handled identically as the dose groups but received aqua ad iniectabilia (water for injection), the vehicle used in this study. The 4 groups comprised 10 male and 10 female Wistar rats. The control group was shared with BSL study 140382.

 

The following doses were evaluated:

Control (C): 0 mg/kg/d

Low Dose (LD): 100 mg/kg/d

Medium Dose (MD): 300 mg/kg/d

High Dose (HD): 1000 mg/kg/d

 

The test item formulation was prepared once in every ten days and stored at room temperature. The test item was suspended inaqua ad iniectabile (water for injection) and 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. 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 performed on 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 post natal day 4 were carefully examined for gross external abnormalities. A full histopathological evaluation of the tissues was performed of 5 randomly selected male and female animals of the control and high dose groups. As well as all gross lesions from all groups, were examined by light microscopy. Sections of the full list of organs and tissue were examined in 8 males and all females of HD group, because macroscopic discoloration was recorded in all organs and tissues in these animals. With regard to female no. 71 of HD group, full list of organs were examined as the decedent in addition due to discoloration in all organs and tissues. 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. In order to demonstrate glycogen accumulation in the hepatocytes, additional PAS stain sections from the representative animals (animal nos. 2, 33, 34, 36, 41, 73, 74 and 77) were prepared and examined by light microscopy.

 

Summary Results:

There was no mortality of animals due to test-item treatment in dose groups during the study period. However, one female (female no. 71) of HD group was euthanized for ethical reasons during the course of the study. Microscopically the morbidity was not considered to be due to test item treatment. There were test item treatment related clinical signs including moving the bedding, salivation, diarrhoea, discoloured red feces and skin in male and female animals of MD and/ or HD groups. The discoloured red feces and skin were due to colour of the test item. Moving the bedding and salivation observed in the MD and HD groups and a single animal of the LD group was assumed to be due to local effect of the test item.During the weekly detailed clinical observation, no significant changes or differences between the groups were found. 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 and females, there were test item treatment related effect on body weight, body weight gain and food consumption in HD group when compared to controls. However, there were mild effects on body weight and food consumption. Considering the microscopic changes in adrenal and thymus weight, the changes were not primary/direct effects of the test item, but were non-specific secondary event to stressful condition. In males and females, there were no adverse effects of toxicological relevance on hematology and blood coagulation parameters in dose groups when compared corresponding control. There were slightly higher ALAT and ASAT values in male or female HD groups which was associated with stress response. There were no effects on urinary parameters in both males and females of dose groups when compared to the control. At necropsy reddish, red or dark red discoloration was observed in one organ, several organs or all organs and tissues in 1/10 females of LD, 8/10 males and 6/10 females of MD and all males and females of the HD group. There was also enlargement of adrenal glands in some HD females and in single female (no. 71) there was distended cecum. These findings corroborated microscopic findings and were attributed to secondary events to the stressful condition. There were statistically significant changes in absolute and/or relative organ weights including adrenal gland (male HD group), spleen (male LD, MD and HD groups), pituitary gland (female HD group), heart (female HD group) and ovaries (female HD group). These organ weights in males and/or females were without cellular/ tissue injuries, but were attributed to secondary event to stressful condition.

Microscopic findings that were considered to be related to treatment with the test item were recorded in testes of males (pigment laden macrophages/histiocytes), ovaries and uterus with cervix of females (pigment laden macrophages/histiocytes), and kidneys (Pigment deposition, as well as hyaline droplets-like eosinophilic substance), mesenteric lymph node (pigment laden macrophages/histiocytes), liver (increased hepatocytic glycogen accumulation), thymus (Thymic atrophy) and adrenal gland (adrenocortical diffuse hypertrophy) of both sexes. Under the condition of study the diffuse adrenocortical hypertrophy, thymic atrophy and increased hepatocytic glycogen accumulation were considered non specific secondary to stressful situation. There was a statistically significantly longer gestation in HD group (mean of 23.50 gestation days) when compared to concurrent control group (mean of 22.38 gestation days). This finding at the HD level was considered to be likely to be an adverse effect of test-item treatment. There was an effect of test item treatment on the no. of implantation sites, live pups and percent pre and post implantation loss and viability index(%) in HD group. There were adverse effects of test item treatment for no. of pups born, no. of still births, no. of live pups, and no. of male and female pups, pup mean weight, total litter weight, male litter weight and female litter weight on PND 0 and / or PND 4 in HD group. There were also an adverse effect of test item treatment for no. implantation sites, live pups and percent pre and post implantation loss in HD group. The viability index (%) was also adversely affected in HD group. There was statistically significantly higher % mortality between days 0-1 and between days 0-4 in HD group. There were test-item related external findings in several pups of HD group on PND 0 and/ or PND 4.

 

Discussion:

Under the conditions of this study, the test item FAT 20060/F TE produced adverse histomorphological changes in thymus, adrenal glands and liver of both sexes at 1000 mg/kg/day. These changes consisted of thymic atrophy as well as adrenocortical diffuse hypertrophy, and increased hepatocytic glycogen accumulation, which was confirmed further by PAS staining in some representative animals, was recorded in both sexes at this dose level. Further, results indicate slight effects on the body weight, food consumption and clinical biochemistry were observed in both sexes when compared to the control group. There were also treatment related clinical and macroscopic findings and slight organ weight changes in both sexes. The administration of higher dosage of the test agents can cause stress to animals in the toxicology studies especially using the rodents’ model, which is generally indicated as the effects to body weight and food consumption with varying degrees. In addition, high-dose administration can activate the hypothalamic-pituitary-adrenal axis resulting in diffuse adrenocortical hypertrophy as well as thymic atrophy which are non-specific histomorphologic appearances indicating a stressful situation in parent animals. Further, corticosteroids released from adrenal cortex prompt glycogen synthesis in the liver, and hence, the increased hepatocytic glycogen accumulation observed in this study was considered to be a secondary response to increased level of corticosteroids released from the adrenal cortex and is a non-specific response to the stressful condition. Meanwhile, possible treatment-related effects were observed in reproductive/developmental parameters in the 1000 mg/kg/day group, including percent pre and post implantation loss, litter weight, duration of gestation, pup survival and viability index at the high-dose group. The stressful condition in parent females can elicit the undesirable alterations on reproductive/developmental performances even though it was a non-specific, secondary response to the stress. Therefore, the above-mentioned findings recorded in parent animals of the high-dose group were judged to be adverse under the condition of this study from the comprehensive aspect of the reproductive/developmental toxicity assessment, even though those were secondary to the stressful condition in parent animals.

 

Conclusion:

On the basis of this reproduction/developmental toxicity screening test withFAT 20060/F in male and female Wistar rats with dose levels of 100, 300, and 1000 mg/kg/d the following conclusions can be made: There were mild and/or transient effects on body weight and food consumption at 1000 mg/kg/d associated with microscopic changes in adrenal gland (adrenocortical hypertrophy), thymus (thymic atrophy) and increased hepatocytic glycogen accumulation. These were not due to the primary/direct effects of the test item, rather they were non-specific secondary events to the stressful condition. The possible treatment-related effects were observed in reproduction/ developmetal parameters including percent pre and post implantation loss, litter weight, duration of gestation, pup survival and viability index at 1000 mg/kg/d. The stressful condition in parent females can elicit the undesirable alterations on reproductive/ developmental performances thought it was a non-specific, secondary response to stess. The above mentioned findings in parent animals at 1000 mg/kg/d although were secondary to the stressful condition, but were judged to be adverse from the comprehensive aspect of the reproductive/developmental toxicity assessment. Based on the data generated from this combined repeated dose oral toxicity and reproduction/ developmental toxicity screening test withFAT 20060/F, the NOAEL for systemic toxicity of adult males and females and for reproduction/ developmental toxicity the NOAEL is considered to be 300 mg/kg/d.

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

developmental toxicity

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

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

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: 9-10 weeks old, females: 9-10 weeks old.
Body weight at the allocation of the animals to the experimental groups (together with study 140382):
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: 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 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 any adverse 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 was 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.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

The test item FAT 20060/F was weighed into a tarred plastic vial on a precision balance and suspended in aqua ad injectable (water for injection). 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 (RT). The homogeneity was ensured by vortexing the sample on vortex machine. The dose range finding study (14 days repeated dose, BSL study no. 140352) was performed at dose levels 100, 300 and 1000 mg/ kg/d with no overt toxicity were observed up to the highest dose tested. According to these results and in consultation with the sponsor the following doses were selected for the 3 dose groups (LD = low dose, MD = medium dose, HD = high dose) and 1 control group (C):
Control (C): 0 mg/kg/d
Low Dose (LD): 100 mg/kg/d
Medium Dose (MD): 300 mg/kg/d
High Dose (HD): 1000 mg/kg/d
Since little or no toxicity was anticipated for the test substance, the highest dose level was set at 1000 mg/kg bw/d corresponding to a limit dose for this study. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dose-related response and a NOAEL. The animals in the control group were handled in an identical manner to the test group subjects and received aqua ad injectable (water for injection) using the same volume as used for the dose groups. 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.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Each dosing concentration was 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 were taken in the first week of the study [0 hours, 6 hours (at RT) and 10 days after the preparation (stored at 2-8 °C)], from high and low dose formulations (6 samples). All analytical samples collected on the day of sample collection 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. 140356.

Details on mating procedure:

Animals were allowed to mate in 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 pregnancy. 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.

Duration of treatment / exposure:

The animals were treated with the test item formulation or vehicle 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 28-29 days is completed.

Duration of test:

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 – 16 April 2014
Experimental Starting Date: 16 April 2014
Date of Behavioural Observation: 16 April 2014-05 June 2014
Treatment Period Males: 23 April 2014 – 20 May 2014
Treatment Period Females: 23 April 2014 – 13 June 2014
Necropsies Males: 22 May 2014
Necropsies Females: 02 June 2014 – 13 June 2014
Experimental Completion Date: 13 June 2014
Completion Date of Delegated Phase (Histopathology): 22 October 2014
Completion Date of Delegated Phase (Formulation Analysis): 01 October 2014

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 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 140382.

Control animals:

yes, concurrent vehicle

Maternal 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, visual placing, fore and hind limb grip strength, tail pinch response, toe pinch reflex, extensor thrust/limb tone, hind limb reflex, righting reflex on the ground, air righting reflex, pupil response, body temperature and ophthalmoscopy (anterior chamber of the eye and fundus of eye).

Body Weight and Food Consumption:
The 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. 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, Pharmanova, lot no: 24863, expiry date: 10/2015 and Serumwerk, lot no: 00513, expiry date: 05/2015). The surviving pups were killed by decapitation on PND 4. 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 from the day of 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 10 % neutral buffered formalin, 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 were evaluated) randomly selected from each group was recorded as soon as possible. Paired organs were weighed together. In addition, reproductive organs of all animals were weighed. Except 1/5 selected females of LD group was non pregnant and organ weight of this animals was not measured.

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:
A full histopathological evaluation of the tissues was performed of 5 randomly selected male and female animals of the control and high dose groups. As well as all gross lesions from all groups, were examined by light microscopy. Sections of the full list of organs and tissue were examined in 8 males and all females of HD group, because macroscopic discoloration was recorded in all organs and tissues in these animals. With regard to female no. 71 of HD group, full list of organs was examined as the decedent in addition to the reason of discoloration in all organs and tissues. 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. In order to demonstrate glycogen accumulation in the hepatocytes, additional PAS stain sections from the representative animals (animal nos. 2, 33, 34, 36, 41, 73, 74 and 77) were prepared and examined by light microscopy. In addition, because of treatment-related findings that were considered to be implicated in the effects to reproductive and developmental performances in the high dose group, adrenal glands, thymus and liver from 5 selected animals/sex/group of the low and mid dose groups (LD and MD groups, respectively) were examined to establish a no-effect level. 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.

Fetal examinations:

Observations:
Any abnormal behavior of the offspring was recorded.

Postmortem examinations (offspring)
Dead pups and pups sacrificed on day 4 post-partum were carefully examined externally for gross 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).

Clinical signs:

effects observed, treatment-related

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Haematological findings:

effects observed, non-treatment-related

Clinical biochemistry findings:

effects observed, non-treatment-related

Urinalysis findings:

effects observed, non-treatment-related

Behaviour (functional findings):

effects observed, non-treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Details on results:

Mortality:
There was no mortality of animals due to test- item treatment in dose groups during the study period. However, one female (female no. 71) of HD group was euthanized for ethical reasons during the course of the study. Microscopic examination revealed the cause of animal’s morbidity to an aspiration pneumonia which happened accidentally, and the event was not related to the toxicological properties of the test item. The remainder of animals survived the scheduled study period.

Clinical Observations:
There were test item treatment related clinical signs moving the bedding, salivation, diarrhoea, discoloured red feces and skin in male and female animals of MD and/ or HD groups. The discoloured red feces and skin were due to colour of the test item. Moving the bedding and salivation observed in the MD and HD groups and a single animal of the LD group was assumed to be due to local effect of the test item. Other clinical signs including alopecia, crust, piloerection, regurgitation, abnormal breathing, dehydration, hypothermia, prostration was observed in few control or dose group animals. These clinical signs were mostly transient in appearance. Clinical findings noted in animal no. 71 that was euthanized for ethical reasons were abnormal breathing, respiratory sounds, piloerection and severe diarrhea. During the weekly detailed clinical observation, no significant changes or differences between the groups were found.

Functional Observations:
There were an increased defecation counts in male animals of the HD group during the last treatment week. The defecation counts in male HD group were statistically significant. In the absence of other autonomous effect, the higher defecation counts in HD group was considered incidental.

Body Weight Development:
From premating days 1 to 7 a slight test article related body weight loss was noted in males of the HD group which was statistically significantly different from the body weight gain observed in animals of the control group. A slight body weight loss was also observed in this group between pre-mating day 14 and mating day 7. Thus, the overall mean body weight gain observed at the end of the treatment period was also statistically significantly lower than in controls. The absolute body weight in HD group males was 4 % below control group at the end of the treatment period. There was also slightly reduced mean body weight gain after the last week of treatment in male MD group, which was elicited by single isolated male no. 30 (reduced weight gain of -39 g). In females of the HD group, there was a lower mean body weight gain during the gestation days 0-7 and 14-20, with statistical significance achieved between days 14 and 20, resulting in a significant reduction in mean body weight gain when the entire gestation period is considered (GD 0-20). On gestation day 7 and 20, absolute body weight of HD females was 7 and 13 % below controls, respectively. The effect on body weight during the last week of gestation days is likely due to lower litter weight. There was also statistically significantly lower absolute body weight in female HD group during premating day 1. The body weight on premating day 1 was measured before the initiation of test item administration and the changes observed in LD group was non dose responsive. These findings were not related to test item treatment. No effect of the test item on body weight gain was observed in the LD and the MD groups.

Food Consumption:
In males, there was statistically significantly lower food consumption in HD group after premating day 1-7 (approx. 16 % below controls). This change correlated with the changes in body weight. Compared to the controls, there is a slight increase in food consumption during the second week of treatment which is reflected in a slight body weight gain during this period. In females, there was statistically significantly lower food consumption in HD group after premating day 1 to 7 (approx. 26 % below controls). Nonsignificant but slightly lower food consumption was observed throughout the remaining treatment period. This decreased food consumption often did not correlate with body weight development observed in female animals.

Pathology:
At necropsy the animals showed reddish, red or dark red discoloration in one organ, several organs or all organs and tissues in one female of LD, 8 males and 6 females of MD and all males and females of HD. There was also enlargement of adrenal glands in some high dose females and in a single HD female (no. 71) there was distended cecum. These findings corroborated to microscopic findings and were associated with secondary effects to a stressful condition. 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 were considered to be incidental macroscopic appearances that did not correlate with microscopic changes.

Organ Weight:
In males, there was a slightly but statistically significantly higher absolute testes weight in LD group (13 % above controls), a slight but statistically significantly higher relative adrenal weight in HD group (22 % above controls) and statistically significantly higher relative spleen weight in LD, MD and HD groups (24, 21 and 26 % above controls). In females, there was statistically significantly lower absolute heart weight (21 % below controls); pituitary gland weight (20 % below controls) and ovary weight (20 % below controls) in HD group. The organ weights were not statistically significant different from controls when related to body weight and they did not correlate with macroscopic alterations. The changes in adrenal gland and pituitary gland weights in males and/or females could be attributed to secondary event to stressful condition. The remaining slight organ weight changes were not associated with cellular/ tissue injury.

Histopathology:
Microscopic findings that were considered to be related to treatment with the test item were recorded in testes of males, ovaries and uterus with cervix of females, and kidneys, mesenteric lymph node, liver, thymus and adrenal gland of both sexes.

Microscopic Findings in Decedent
In female no. 71 (HD group) which was euthanized for ethical reasons, aspiration pneumonia in the lung as well as reactive tracheal mucosal hypertrophy was observed, and these were the cause of animal’s morbidity. This occasionally happens due to regurgitation and/or accidental influx of the dosing solution into the respiratory tracts after or during the dosing procedure, and it was judged that the animal’s morbidity was not related to the toxicological properties of the test item. Thymic atrophy, adrenocortical diffuse hypertrophy and glandular stomach erosion were recorded in this animal, and these were considered to be secondary effects to a stressful condition associated with respiratory tracts' lesions. The remainder of findings is mentioned in the following sections along with the findings in the survivors.

Thymus, Adrenal Glands and Liver
Thymic atrophy as well as adrenocortical diffuse hypertrophy was recorded in both sexes of HD group, and increased hepatocytic glycogen accumulation, which was confirmed further by PAS staining in some representative animals, was recorded in both sexes of HD group as well. There was no further indicator of cellular injuries in adrenal glands and liver examined in the high-dose group, and the histologic alterations described in this section were not observed in animals of LD and MD groups. The above-mentioned findings were considered to be secondary to the stressful condition in the parent animals, but judged to be adverse under the condition of this study from the comprehensive aspect of the reproductive/developmental toxicity assessment.

Kidneys
Gold, yellow and/or brown pigment deposition in the proximal tubules was recorded in one male of LD group and 8 males and 10 females of HD group, without any further indicator of cellular injuries. In females, eosinophilic substances similar to hyaline droplets that are often observed in the male rat were occasionally found in the proximal tubular epithelial cells. Pigment deposition, as well as hyaline droplets-like eosinophilic substance in females, was considered to be the histomorphologic representation indicating physiologic excretion processes that occurred by re-absorption of the test item or its metabolites and was considered to be present as the form of phagolysosomal cytoplasmic components as with spontaneous hyaline droplets in the male rats. There was no further indicator of cellular injuries, and therefore, these findings were considered not to be adverse.

Testes, Ovaries, Uterus with Cervix, Mesenteric Lymph Node, and Lungs
Yellow to brown pigment laden macrophages/histiocytes were observed in testes, ovaries, uterus (including cervix), mesenteric lymph node and lungs. There was no indicator of cellular and tissue injuries in macrophages/histiocytes and in intrinsic tissue components of each organ. Pigment was considered to be derived from the test item or its metabolites distributed to the whole body, and this finding indicates the physiological activities for scavenging the exogenous substances, and hence, was considered not to be adverse. Likewise, increased incidence and/or severity of sinus histiocytosis in the mesenteric lymph node, which were recorded in females of MD group and both sexes of HD group, were considered to be physiological activities for scavenging the exogenous substances, and hence, considered not to be adverse. In the lung, accumulation of macrophages at/around the bronchiolar/alveolar duct region appears to have increased slightly in the incidence and severity in the high dose group compared to the control group. Thymus, Adrenal Glands and Liver”, however, animals in the high-dose group of this study were considered to have been under the stressful status. Such situation is prone to incidental aspiration or regurgitation caused by the animals' hypodynamia or by the evasion against the doing-procedure. Likewise, it was considered that the accumulation of macrophages had happened incidentally also in the lung of the present study, and therefore, this was concluded not to be a toxicological event. There were no abnormalities in the male and female reproductive organs, except for the presence of pigment-laden macrophages, and there were no noteworthy differences in histologic appearances between the control groups and the test item treated groups.

Testes - Detailed Examination Including Sperm Staging
The stages were checked on completeness of cell populations, completeness of stages and degenerative changes. There were no treatment-related effects on the completeness of stages or cell populations of testes. The presence of pigment laden macrophages/histiocytes did not affect the spermatogenesis and the interstitial structures, and hence, this was considered not to be adverse event. Nothing particular was observed in testes of animal nos. 2, 10 (control) and 36 (group 4), which were mated with female nos. 42, 50 and 76 not giving birth, respectively, and nothing particular was found in prostate glands, seminal vesicles and coagulating glands as well. Minimal mononuclear cell foci were recorded unilaterally in epididymides of male nos. 10 and 36. However, such a lesion is often observed in male animals that can produce pregnancy, and therefore, it is unlikely that such minimal finding was related to the infertility. There were no abnormalities in the female reproductive organs (ovaries, uterus with cervix and vagina) of the paired female nos. 42, 50 (control) and 76 (HD group) which did not give birth.

Other Findings
The remainder of findings recorded was within the range of normal background lesions which may be recorded in animals of this strain and age.

Details on maternal toxic effects:

Precoital Interval and Duration of Gestation:
At the HD level there was a statistically significantly longer gestation observed. Most of the females in HD group had a gestation period of 23 or 24 days (mean gestation day of 23.50 days) whereas most concurrent control animals had gestation periods of 22 or 23 days (mean of 22.38 days). LD and MD groups were not affected. This finding was considered likely to be a non- specific effect secondary to stress response of parent females.

Pre- and Post-Natal Data:
There were statistically significantly lower implantation sites (7.67 in HD group compared to 11.13 in Control), lower live pups per litter (PND 0= 4.88 in HD group compared to 10.50 in controls, and PND 4= 3.63 in HD compared to 10.50 in controls) and higher percentage of post implantation loss in HD group (35 % compared to 5 % in controls). There was also higher percent preimplantation loss in HD group (31 %, compared to 16 % in controls). These changes corresponded to the changes in litter data (section 12.15). Altogether, the above-mentioned findings were considered to be a non-specific effect secondary to stress response of parent females. In MD group, there was also higher percent preimplantation loss (31 % compared to 16 % in controls) and higher percent post implantation loss (11.94 % compared to 4.96 % in control). The higher percent pre and post implantation loss was driven by female no. 66 with lower implantation and no births. There were no significant clinical observations during the dosing period, no maternal findings at necropsy and microscopically nothing abnormal was observed in uterus and cervix. This was a single isolated incidence, and all other values were within historical control range. Therefore, this finding was not considered to be related to test-item treatment.

Reproductive indices
There were no effects on copulation, fertility and delivery indices. However, there was lower viability index in HD group (58 % compared to 100 % in controls). These changes were considered to be an adverse effect elicited by non-specific effect secondary to stress response of parent females. Viability index was not affected in other dose groups.

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

gross pathology

histopathology: non-neoplastic

maternal abnormalities

organ weights and organ / body weight ratios

Remarks on result:

other: Systemic toxicity obsserved at 1000 mg/kg bw/day

Key result

Abnormalities:

effects observed, treatment-related

Details on embryotoxic / teratogenic effects:

Litter Data:
In the HD group there was a statistically significantly lower average of total no. of pups per litter born (6.25 compared to 10.5 in the control group). It was associated with a higher average no. of still births (1.38 compared to none in the control group) and lower no. of live pups on PND 0 (4.88 compared to 10.50 in the control group). On PND 4 mean no. of live pups even dropped to 3.63 compared to 10.50 in the control group). This was equally distributed between the genders (male pups 2.38 compared to 4.88 in the control group; female pups 2.50 compared to 5.63 in the control group). Again, both genders were equally affected. These changes were an adverse effect elicited by non-specific effect secondary to stress response of parent females.
Litter data was unaffected by test-item administration in the LD and MD groups.

Litter Weight Data:
In HD group there was statistically significantly lower pup mean weight, total litter weight, male litter weight and female litter weight on PND 0 (5.03 g compared to 6.18 g in controls, 25.3 g compared to 64.2 g in controls and 12.6 g compared to 30.6 g in controls and 12.8 g compared to 33.7 g in controls, respectively) and on PND 4 (8.78 g compared to 10.20 g in controls, 50.2 g compared to 105.9 g in controls, 24.5 g compared to 49.7 g in controls and 25.8 g compared to 56.2 g in controls, respectively) when compared to the concurrent control. These changes corresponded to the changes in litter data and were limited to HD group. These changes were an adverse effect elicited by non-specific effect secondary to stress response of parent females.
Litter weight was unaffected by test-item administration in the LD and MD groups.

Pup Survival Data:
There was statistically significantly higher % mortality in HD group between days 0-1 (21.6 % compared to none in controls) and between days 0-4 (41.6 % compared to none in controls). Higher % mortality was also observed between days 1-2 and 2-3 in HD group but without statistically significant difference. These changes were an adverse effect elicited by non-specific effect secondary to stress response of parent females. Pup survival was unaffected by test-item administration in the LD and MD groups.

Pup External Findings:
There were test item related external findings in several pups of HD group on PND 0 and/ or PND 4. The findings were dry skin and lean appearance (pup 1, female 72), swollen forepaw, dry skin and lean appearance (pup 2, female 72), cold and red skin (pup 1-7, female 73), weak, cold and red skin (pup 1, female 74; pup 1-5, female 75), red skin (pup 1, 3-8, female 78; pup 1-5, female 79), cold and red skin (pup 1-6, female 80). The red skin in most pups of HD group was likely to be the colour of the test item mostly due to licking by the dam. Other findings were not associated with developmental, but were related to nursing by dam and therefore, were due to secondary to stress response. There was also a hematoma on the neck of control pups (pup 1, 5 and 10) and of female 41 and a small-sized pup in the MD group (pup 13, female 68). These findings were either seen in control animals or in an isolated pup of the MD group and are not considered to be related to treatment with the test item. No significant findings were observed in the LD group.

Dose Formulation Analysis:
Concentration analysis of formulation samples was determined in study weeks 1, 3, 5 and 7 for all dose groups. The mean recoveries observed in the low dose (LD), medium dose (MD) and high dose (HD) groups were 94.5 %, 89.0 % and 86.1 % of the nominal concentration, respectively. Nominal concentrations were confirmed for all dose groups, as measured mean concentration did not differ from nominal concentration by more than 20 %. Stability of formulation samples was investigated in study week 1 based on concentration in the LD and HD dose groups. After 10 days storage at room temperature recovery compared to starting value was 93.2 % and 108.1 %. All samples were stable, as concentration after storage did not differ from start value by more than 20 %. Homogeneity of formulation samples was determined in study weeks 1 and 5 for the LD and HD dose groups. The mean recovery observed for the LD dose group was 93.6 % and 97.7 % of the nominal value and 83.3 % and 91.9 % of the nominal value for HD dose group. The coefficients of variation of the different sampling locations (top, middle, bottom) were 18.3 % and 12.3 % and 6.3 % and 9.0 % in HD dose group. All samples were homogenous, as COV was below 20 %.

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: Secondary to the stressful condition in parental animals

Abnormalities:

effects observed, treatment-related

Developmental effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

not specified

Dose response relationship:

not specified

None

Conclusions:

The NOAEL for systemic as well as reproduction/ developmental toxicity is considered to be 300 mg/kg/d.

Executive summary:

The aim of this study was to assess the possible effects of FAT 20060/F 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 28-29 days were completed. Animals of an additional control group were handled identically as the dose groups but receivedaqua ad iniectabile (water for injection), the vehicle used in this study. The 4 groups comprised 10 male and 10 female Wistar rats. The control group was shared with BSL study 140382.

The following doses were evaluated:

Control (C): 0 mg/kg/d

Low Dose (LD): 100 mg/kg/d

Medium Dose (MD): 300 mg/kg/d

High Dose (HD): 1000 mg/kg/d

The test item formulation was prepared once in every ten days and stored at room temperature. The test item was suspended inaqua ad iniectabile(water for injection) and 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. 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 performed on 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 post natal day 4 were carefully examined for gross external abnormalities. A full histopathological evaluation of the tissues was performed of 5 randomly selected male and female animals of the control and high dose groups.As well as all gross lesions from all groups, were examined by light microscopy. Sections of the full list of organs and tissue were examined in 8 males and all females of HD group, because macroscopic discoloration was recorded in all organs and tissues in these animals. With regard to female no. 71 of HD group, full list of organs were examined as the decedent in addition due to discoloration in all organs and tissues. 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. In order to demonstrate glycogen accumulation in the hepatocytes, additional PAS stain sections from the representative animals (animal nos. 2, 33, 34, 36, 41, 73, 74 and 77) were prepared and examined by light microscopy.

 

Summary Results:

There was no mortality of animals due to test-item treatment in dose groups during the study period. However, one female (female no. 71) of HD group was euthanized for ethical reasons during the course of the study. Microscopically the morbidity was not considered to be due to test item treatment. There were test item treatment related clinical signs including moving the bedding, salivation, diarrhoea, discoloured red feces and skin in male and female animals of MD and/ or HD groups. The discoloured red feces and skin were due to colour of the test item. Moving the bedding and salivation observed in the MD and HD groups and a single animal of the LD group was assumed to be due to local effect of the test item. During the weekly detailed clinical observation, no significant changes or differences between the groups were found. 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 and females, there were test item treatment related effect on body weight, body weight gain and food consumption in HD group when compared to controls. However, there were mild effects on body weight and food consumption. Considering the microscopic changes in adrenal and thymus weight, the changes were not primary/direct effects of the test item, but were non-specific secondary event to stressful condition. In males and females, there were no adverse effects of toxicological relevance on hematology and blood coagulation parameters in dose groups when compared corresponding control. There were slightly higher ALAT and ASAT values in male or female HD groups which was associated with stress reponse. There were no effects on urinary parameters in both males and females of dose groups when compared to the control. At necropsy reddish, red or dark red discoloration was observed in one organ, several organs or all organs and tissues in 1/10 females of LD, 8/10 males and 6/10 females of MD and all males and females of the HD group. There was also enlargement of adrenal glands in some HD females and in single female (no. 71) there was distended cecum. These findings corroborated microscopic findings and were attributed to secondary events to the stressful condition.

There were statistically significant changes in absolute and/or relative organ weights including adrenal gland (male HD group), spleen (male LD, MD and HD groups), pituitary gland (female HD group), heart (female HD group) and ovaries (female HD group). These organ weights in males and/or females were without cellular/ tissue injuries, but were attributed to secondary event to stressful condition. Microscopic findings that were considered to be related to treatment with the test item were recorded in testes of males (pigment laden macrophages/histiocytes), ovaries and uterus with cervix of females (pigment laden macrophages/histiocytes), and kidneys (Pigment deposition, as well as hyaline droplets-like eosinophilic substance), mesenteric lymph node (pigment laden macrophages/histiocytes), liver (increased hepatocytic glycogen accumulation), thymus (Thymic atrophy) and adrenal gland (adrenocortical diffuse hypertrophy) of both sexes. Under the condition of study the diffuse adrenocortical hypertrophy, thymic atrophy and increased hepatocytic glycogen accumulation were considered non specific secondary to stressful situation. There was a statistically significantly longer gestation in HD group (mean of 23.50 gestation days) when compared to concurrent control group (mean of 22.38 gestation days). This finding at the HD level was considered to be likely to be an adverse effect of test-item treatment.

There was an effect of test item treatment on the no. of implantation sites, live pups and percent pre and post implantation loss and viability index (%) in HD group. There were adverse effects of test item treatment for no. of pups born, no. of still births, no. of live pups, and no. of male and female pups, pup mean weight, total litter weight, male litter weight and female litter weight on PND 0 and / or PND 4 in HD group. There were also an adverse effect of test item treatment for no. implantation sites, live pups and percent pre and post implantation loss in HD group. The viability index (%) was also adversely affected in HD group. There was statistically significantly higher % mortality between days 0-1 and between days 0-4 in HD group. There were itest-item related external findings in several pups of HD group on PND 0 and/ or PND 4.

 

Discussion:

Under the conditions of this study, the test item FAT 20060/F produced adverse histomorphological changes in thymus, adrenal glands and liver of both sexes at 1000 mg/kg/day. These changes consisted of thymic atrophy as well as adrenocortical diffuse hypertrophy, and increased hepatocytic glycogen accumulation, which was confirmed further by PAS staining in some representative animals, was recorded in both sexes at this dose level. Further, results indicate slight effects on the body weight, food consumption and clinical biochemistry were observed in both sexes when compared to the control group. There were also treatment related clinical and macroscopic findings and slight organ weight changes in both sexes. The administration of higher dosage of the test agents can cause stress to animals in the toxicology studies especially using the rodents’ model, which is generally indicated as the effects to body weight and food consumption with varying degrees. In addition, high-dose administration can activate the hypothalamic-pituitary-adrenal axis resulting in diffuse adrenocortical hypertrophy as well as thymic atrophy which are non-specific histomorphologic appearances indicating a stressful situation in parent animals. Further, corticosteroids released from adrenal cortex prompt glycogen synthesis in the liver, and hence, the increased hepatocytic glycogen accumulation observed in this study was considered to be a secondary response to increased level of corticosteroids released from the adrenal cortex and is a non-specific response to the stressful condition. Meanwhile, possible treatment-related effects were observed in reproductive/developmental parameters in the 1000 mg/kg/day group, including percent pre- and post implantation loss, litter weight, duration of gestation, pup survival and viability index at the high-dose group. The stressful condition in parent females can elicit the undesirable alterations on reproductive/developmental performances even though it was a non-specific, secondary response to the stress. Therefore, the above-mentioned findings recorded in parent animals of the high-dose group were judged to be adverse under the condition of this study from the comprehensive aspect of the reproductive/developmental toxicity assessment, even though those were secondary to the stressful condition in parent animals.

 

Conclusion:

On the basis of this reproduction/developmental toxicity screening test with FAT 20060/F in male and female Wistar rats with dose levels of 100, 300, and 1000 mg/kg/d the following conclusions can be made:

There were mild and/or transient effects on body weight and food consumption at 1000 mg/kg/d associated with microscopic changes in adrenal gland (adrenocortical hypertrophy), thymus (thymic atrophy) and increased hepatic glycogen accumulation. These were not due to the primary/direct effects of the test item, rather they were non-specific secondary events to the stressful condition. The possible treatment-related effects were observed in reproduction/ developmental parameters including percent pre- and post-implantation loss, litter weight, duration of gestation, pup survival and viability index at 1000 mg/kg/d. The stressful condition in parent females can elicit the undesirable alterations on reproductive/ developmental performances thought it was a non-specific, secondary response to stress.The above mentioned findings in parent animals at 1000 mg/kg/d although were secondary to the stressful condition, but were judged to be adverse from the comprehensive aspect of the reproductive/developmental toxicity assessment. Based on the data generated from this combined repeated dose oral toxicity and reproduction/ developmental toxicity screening test with FAT 20060/F, the NOAEL for systemic as well as reproduction/ developmental toxicity is considered to be 300 mg/kg/d.

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:

Chromate(2-), [2,4-dihydro-4-[(2-hydroxy-4-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulfonato(3-)]-, disodium

EC Number:

274-386-6

EC Name:

Chromate(2-), [2,4-dihydro-4-[(2-hydroxy-4-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulfonato(3-)]-, disodium

Cas Number:

70209-87-9

Molecular formula:

C32H21CrN10Na2O11S

IUPAC Name:

Disodium-[2,4-dihydro-4-[(2-hydroxy-4-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulfonato(3-)]-chromate(2-)

Test material form:

other: solid/dark red

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: 9-10 weeks old, females: 9-10 weeks old.
Body weight at the allocation of the animals to the experimental groups (together with study 140382):
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: 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 any adverse 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 was 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 20060/F was weighed into a tarred plastic vial on a precision balance and suspended in aqua ad injectable (water for injection). 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 (RT). The homogeneity was ensured by vortexing the sample on vortex machine.
The dose range finding study (14 days repeated dose, BSL study no. 140352) was performed at dose levels 100, 300 and 1000 mg/ kg/d with no overt toxicity were observed up to the highest dose tested. According to these results and in consultation with the sponsor the following doses were selected for the 3 dose groups (LD = low dose, MD = medium dose, HD = high dose) and 1 control group (C):
Control (C): 0 mg/kg/d
Low Dose (LD): 100 mg/kg/d
Medium Dose (MD): 300 mg/kg/d
High Dose (HD): 1000 mg/kg/d

Since little or no toxicity was anticipated for the test substance, the highest dose level was set at 1000 mg/kg bw/d corresponding to a limit dose for this study. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dose-related response and a NOAEL. The animals in the control group were handled in an identical manner to the test group subjects and received aqua ad injectable (water for injection) using the same volume as used for the dose groups. 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:

Animals were allowed to mate in 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 pregnancy. 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 was 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 were taken in the first week of the study [0 hours, 6 hours (at RT) and 10 days after the preparation (stored at 2-8°C)], from high and low dose formulations (6 samples). All analytical samples collected on the day of sample collection 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. 140356.

Duration of treatment / exposure:

The animals were treated with the test item formulation or vehicle 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 28-29 days is completed.

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 – 16 April 2014
Experimental Starting Date: 16 April 2014
Date of Behavioural Observation: 16 April 2014-05 June 2014
Treatment Period Males: 23 April 2014 – 20 May 2014
Treatment Period Females: 23 April 2014 – 13 June 2014
Necropsies Males: 22 May 2014
Necropsies Females: 02 June 2014 – 13 June 2014
Experimental Completion Date: 13 June 2014
Completion Date of
Delegated Phase (Histopathology): 22 October 2014
Completion Date of
Delegated Phase
(Formulation Analysis): 01 October 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.:

1 000 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 140382.

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, visual placing, fore and hind limb grip strength, tail pinch response, toe pinch reflex, extensor thrust/limb tone, hind limb reflex, righting reflex on the ground, air righting reflex, pupil response, body temperature and ophthalmoscopy (anterior chamber of the eye and fundus of eye).

Body Weight and Food Consumption:
The 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. 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, Pharmanova, lot no: 24863, expiry date: 10/2015 and Serumwerk, lot no: 00513, expiry date: 05/2015). The surviving pups were killed by decapitation on PND 4. 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 from the day of 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 10 % neutral buffered formalin, 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 were evaluated) randomly selected from each group was recorded as soon as possible. Paired organs were weighed together. In addition, reproductive organs of all animals were weighed. Except 1/5 selected females of LD group was non pregnant and organ weight of this animals was not measured. 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, lymphnodes (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:
A full histopathological evaluation of the tissues was performed of 5 randomly selected male and female animals of the control and high dose groups. As well as all gross lesions from all groups, were examined by light microscopy. Sections of the full list of organs and tissue were examined in 8 males and all females of HD group, because macroscopic discoloration was recorded in all organs and tissues in these animals. With regard to female no. 71 of HD group, full list of organs was examined as the decedent in addition to the reason of discoloration in all organs and tissues. 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. In order to demonstrate glycogen accumulation in the hepatocytes, additional PAS stain sections from the representative animals (animal nos. 2, 33, 34, 36, 41, 73, 74 and 77) were prepared and examined by light microscopy. In addition, because of treatment-related findings that were considered to be implicated in the effects to reproductive and developmental performances in the high dose group, adrenal glands, thymus and liver from 5 selected animals/sex/group of the low and mid dose groups (LD and MD groups, respectively) were examined to establish a no-effect level. 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 day 4 post-partum were carefully examined externally for gross 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 were no effects on copulation, fertility and delivery indices. However, there was lower viability index in HD group (58 % compared to 100 % in controls). These changes were considered to be an adverse effect elicited by non-specific effect secondary to stress response of parent females. Viability index was not affected in other dose groups.

Offspring viability indices:

There was no effect on the viability index in dose group when compared to the control.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

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

Details on results (P0)

Mortality:
There was no mortality of animals due to test- item treatment in dose groups during the study period. However, one female (female no. 71) of HD group was euthanized for ethical reasons during the course of the study. Microscopic examination revealed the cause of animal’s morbidity to an aspiration pneumonia which happened accidentally and the event was not related to the toxicological properties of the test item. The remainder of animals survived the scheduled study period.

Clinical Observations:
There were test item treatment related clinical signs moving the bedding, salivation, diarrhoea, discoloured red feces and skin in male and female animals of MD and/ or HD groups. The discoloured red feces and skin were due to colour of the test item. Moving the bedding and salivation observed in the MD and HD groups and a single animal of the LD group was assumed to be due to local effect of the test item. Other clinical signs including alopecia, crust, piloerection, regurgitation, abnormal breathing, dehydration, hypothermia, prostration were observed in few control or dose group animals. These clinical signs were mostly transient in appearance. Clinical findings noted in animal no. 71 that was euthanized for ethical reasons were abnormal breathing, respiratory sounds, piloerection and severe diarrhea. During the weekly detailed clinical observation, no significant changes or differences between the groups were found.

Functional Observations:
There was an increased defecation counts in male animals of the HD group during the last treatment week. The defecation counts in male HD group were statistically significant. In the absence of other autonomous effect, the higher defecation counts in HD group was considered incidental.

Body Weight Development:
From premating days 1 to 7 a slight test article related body weight loss was noted in males of the HD group which was statistically significantly different from the body weight gain observed in animals of the control group. A slight body weight loss was also observed in this group between pre-mating day 14 and mating day 7. Thus, the overall mean body weight gain observed at the end of the treatment period was also statistically significantly lower than in controls. The absolute body weight in HD group males was 4 % below control group at the end of the treatment period. There was also slightly reduced mean body weight gain after the last week of treatment in male MD group, which was elicited by single isolated male no. 30 (reduced weight gain of -39 g).
In females of the HD group, there was a lower mean body weight gain during the gestation days 0-7 and 14-20, with statistical significance achieved between days 14 and 20, resulting in a significant reduction in mean body weight gain when the entire gestation period is considered (GD 0-20). On gestation day 7 and 20, absolute body weight of HD females was 7 and 13 % below controls, respectively. The effect on body weight during the last week of gestation days is likely due to lower litter weight (see 12.16).
There was also statistically significantly lower absolute body weight in female HD group during premating day 1. The body weight on premating day 1 was measured before the initiation of test item administration and the changes observed in LD group was non dose responsive. These findings were not related to test item treatment. No effect of the test item on body weight gain was observed in the LD and the MD groups.

Food Consumption:
In males, there was statistically significantly lower food consumption in HD group after premating day 1-7 (approx. 16 % below controls). This change correlated with the changes in body weight. Compared to the controls, there is a slight increase in food consumption during the second week of treatment which is reflected in a slight body weight gain during this period. In females, there was statistically significantly lower food consumption in HD group after premating day 1 to 7 (approx. 26 % below controls). Non-significant but slightly lower food consumption was observed throughout the remaining treatment period. This decreased food consumption often did not correlate with body weight development observed in female animals.

Pathology:
At necropsy the animals showed reddish, red or dark red discoloration in one organ, several organs or all organs and tissues in one female of LD, 8 males and 6 females of MD and all males and females of HD. There were also enlargement of adrenal glands in some high dose females and in a single HD female (no. 71) there was distended cecum. These findings corroborated to microscopic findings (see 12.11) and were associated withsecondary effects to a stressful condition. 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 were considered to be incidental macroscopic appearances that did not correlate with microscopic changes.

Organ Weight:
In males, there was a slightly but statistically significantly higher absolute testes weight in LD group (13 % above controls), a slight but statistically significantly higher relative adrenal weight in HD group (22 % above controls) and statistically significantly higher relative spleen weight in LD, MD and HD groups (24, 21 and 26 % above controls). In females, there was statistically significantly lower absolute heart weight (21 % below controls); pituitary gland weight (20 % below controls) and ovary weight (20 % below controls) in HD group. The organ weights were not statistically significant different from controls when related to body
weight and they did not correlate with macroscopic alterations. The changes in adrenal gland and pituitary gland weights in males and/or females could be attributed to secondary event to stressful condition. The remaining slight organ weight changes were not associated with cellular/ tissue injury.

Histopathology:
Microscopic findings that were considered to be related to treatment with the test item were recorded in testes of males, ovaries and uterus with cervix of females, and kidneys, mesenteric lymph node, liver, thymus and adrenal gland of both sexes.
Microscopic Findings in Decedent
In female no. 71 (HD group) which was euthanized for ethical reasons, aspiration pneumonia in the lung as well as reactive tracheal mucosal hypertrophy was observed, and these were the cause of animal’s morbidity. This occasionally happens due to regurgitation and/or accidental influx of the dosing solution into the respiratory tracts after or during the dosing procedure, and it was judged that the animal’s morbidity was not related to the toxicological properties of the test item.Thymic atrophy, adrenocortical diffuse hypertrophy and glandular stomach erosion were recorded in this animal, and these were considered to be
secondary effects to a stressful condition associated with respiratory tracts' lesions. The remainder of findings is mentioned in the following sections along with the findings in the survivors.
Thymus, Adrenal Glands and Liver
Thymic atrophy as well as adrenocortical diffuse hypertrophy was recorded in both sexes of HD group, and increased hepatocytic glycogen accumulation, which was confirmed further by PAS staining in some representative animals, was recorded in both sexes of HD group as well. There were no further indicator of cellular injuries in adrenal glands and liver examined in the high-dose group, and the histologic alterations described in this section were not observed in animals of LD and MD groups. The above-mentioned findings were considered to be secondary to the stressful condition in the parent animals, but judged to be adverse under the condition of this study from the comprehensive aspect of the reproductive/developmental toxicity assessment.
Kidneys
Gold, yellow and/or brown pigment deposition in the proximal tubules was recorded in one male of LD group and 8 males and 10 females of HD group, without any further indicator of cellular injuries. In females, eosinophilic substances similar to hyaline droplets that are often observed in the male rat were occasionally found in the proximal tubular epithelial cells. Pigment deposition, as well as hyaline droplets-like eosinophilic substance in females, was considered to be the histomorphologic representation indicating physiologic excretion processes that occurred by re-absorption of the test item or its metabolites, and was considered to be present as the form of phagolysosomal cytoplasmic components as with spontaneous hyaline droplets in the male rats. There were no further indicator of cellular injuries, and therefore, these findings were considered not to be adverse.
Testes, Ovaries, Uterus with Cervix, Mesenteric Lymph Node, and Lungs
Yellow to brown pigment laden macrophages/histiocytes were observed in testes, ovaries, uterus (including cervix), mesenteric lymph node and lungs. There was no indicator of cellular and tissue injuries in macrophages/histiocytes and in intrinsic tissue components of each organ. Pigment was considered to be derived from the test item or its metabolites distributed to the whole body, and this finding indicates the physiological activities for scavenging the exogenous substances, and hence, was considered not to be adverse.Likewise, increased incidence and/or severity of sinus histiocytosis in the mesenteric lymph node, which were recorded in females of MD group and both sexes of HD group, were considered to be physiological activities for scavenging the exogenous substances, and hence, considered not to be adverse.
In the lung, accumulation of macrophages at/around the bronchiolar/alveolar duct region appears to have increased slightly in the incidence and severity in the high dose group compared to the control group. Thymus, Adrenal Glands and Liver”, however, animals in the high-dose group of this study were considered to have been under the stressful status. Such situation is prone to incidental aspiration or regurgitation caused by the animals' hypodynamia or by the evasion against the doing-procedure. Likewise it was considered that the accumulation of macrophages had happened incidentally also in the lung of the present study, and therefore, this was concluded not to be a toxicological event.
There were no abnormalities in the male and female reproductive organs, except for the presence of pigment-laden macrophages, and there were no noteworthy differences in histologic appearances between the control groups and the test item treated groups.
Testes - Detailed Examination Including Sperm Staging
The stages were checked on completeness of cell populations, completeness of stages and degenerative changes. There were no treatment-related effects on the completeness of stages or cell populations of testes. The presence of pigment laden macrophages/histiocytes did not affect the spermatogenesis and the interstitial structures, and hence, this was considered not to be adverse event.
Nothing particular was observed in testes of animal nos. 2, 10 (control) and 36 (group 4), which were mated with female nos. 42, 50 and 76 not giving birth, respectively, and nothing particular was found in prostate glands, seminal vesicles and coagulating glands as well. Minimal mononuclear cell foci were recorded unilaterally in epididymides of male nos. 10 and 36.However, such a lesion is often observed in male animals that can produce pregnancy, and therefore, it is unlikely that such minimal finding was related to the infertility.There were no abnormalities in the female reproductive organs (ovaries, uterus with cervix and vagina) of the paired female nos. 42, 50 (control) and 76 (HD group) which did not give birth.
Other Findings
The remainder of findings recorded was within the range of normal background lesions which may be recorded in animals of this strain and age.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

other: see remarks;

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not examined

Urinalysis findings:

not specified

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 observation

Histopathological findings:

not examined

Other effects:

not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

no effects observed

Details on results (F1)

Litter Data:
In the HD group there was a statistically significantly lower average of total no. of pups per litter born (6.25 compared to 10.5 in the control group). It was associated with a higher average no. of still births (1.38 compared to none in the control group) and lower no. of live pups on PND 0 (4.88 compared to 10.50 in the control group). On PND 4 mean no. of live pups even dropped to 3.63 compared to 10.50 in the control group). This was equally distributed between the genders (male pups 2.38 compared to 4.88 in the control group; female pups 2.50 compared to 5.63 in the control group). Again, both genders were equally affected. These changes were an adverse effect elicited by non-specific effect secondary to stress response of parent females. Litter data was unaffected by test-item administration in the LD and MD groups.

Litter Weight Data:
In HD group there was statistically significantly lower pup mean weight, total litter weight, male litter weight and female litter weight on PND 0 (5.03 g compared to 6.18 g in controls, 25.3 g compared to 64.2 g in controls and 12.6 g compared to 30.6 g in controls and 12.8 g compared to 33.7 g in controls, respectively) and on PND 4 (8.78 g compared to 10.20 g in controls, 50.2 g compared to 105.9 g in controls, 24.5 g compared to 49.7 g in controls and 25.8 g compared to 56.2 g in controls, respectively) when compared to the concurrent control. These changes corresponded to the changes in litter data and were limited to HD group. These changes were an adverse effect elicited by non-specific effect secondary to stress response of parent females. Litter weight was unaffected by test-item administration in the LD and MD groups.


Precoital Interval and Duration of Gestation:
At the HD level there was a statistically significantly longer gestation observed. Most of the females in HD group had a gestation period of 23 or 24 days (mean gestation day of 23.50 days) whereas most concurrent control animals had gestation periods of 22 or 23 days (mean of 22.38 days). LD and MD groups were not affected. This finding was considered likely to be a non- specific effect secondary to stress response of parent females.

Pre- and Post-Natal Data:
There were statistically significantly lower implantation sites (7.67 in HD group compared to 11.13 in Control), lower live pups per litter (PND 0= 4.88 in HD group compared to 10.50 in controls, and PND 4= 3.63 in HD compared to 10.50 in controls) and higher percentage of post implantation loss in HD group (35 % compared to 5 % in controls). There was also higher percent preimplantation loss in HD group (31 %, compared to 16 % in controls). These changes corresponded to the changes in litter data (section 12.15). Altogether, the above-mentioned findings were considered to be a non-specific effect secondary to stress response of parent females. In MD group, there was also higher percent preimplantation loss (31 % compared to 16 % in controls) and higher percent post implantation loss (11.94 % compared to 4.96 % in control). The higher percent pre and post implantation loss was driven by female no. 66 with lower implantation and no births. There were no significant clinical observations during the dosing period, no maternal findings at necropsy and microscopically nothing abnormal was observed in uterus and cervix. This was a single isolated incidence, and all other values were within historical control range. Therefore, this finding was not considered to be related to test-item treatment.

Reproductive Indices:
There were no effects on copulation, fertility and delivery indices. However, there was lower viability index in HD group (58 % compared to 100 % in controls). These changes were considered to be an adverse effect elicited by non-specific effect secondary to stress response of parent females. Viability index was not affected in other dose groups. At necropsy of female no. 66 (MD group) pups were seen in the uterus and as mentioned above microscopically nothing abnormal was discovered. This was a single isolated incidence and was not considered to be related to test-item treatment.


Pup Survival Data:
There was statistically significantly higher % mortality in HD group between days 0-1 (21.6 % compared to none in controls) and between days 0-4 (41.6 % compared to none in controls). Higher % mortality was also observed between days 1-2 and 2-3 in HD group but without statistically significant difference. These changes were an adverse effect elicited by non-specific effect secondary to stress response of parent females. Pup survival was unaffected by test-item administration in the LD and MD groups.


Pup External Findings:
There were test item related external findings in several pups of HD group on PND 0 and/ or PND 4. The findings were dry skin and lean appearance (pup 1, female 72), swollen forepaw, dry skin and lean appearance (pup 2, female 72), cold and red skin (pup 1-7, female 73), weak, cold and red skin (pup 1, female 74; pup 1-5, female 75), red skin (pup 1, 3-8, female 78; pup 1-5, female 79), cold and red skin (pup 1-6, female 80). The red skin in most pups of HD group was likely to be the colour of the test item mostly due to licking by the dam. Other findings were not associated with development, but were related to nursing by dam and therefore, were due to secondary to stress response. There was also a haematoma on the neck of control pups (pup 1, 5 and 10) and of female 41 and a small-sized pup in the MD group (pup 13, female 68). These findings were either seen in control animals or in an isolated pup of the MD group and are not considered to be related to treatment with the test item. No significant findings were observed in the LD group.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

not specified

Relevant for humans:

not specified

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:

The NOAEL for systemic as well as reproduction/ developmental toxicity is considered to be 300 mg/kg/d.

Executive summary:

The aim of this study was to assess the possible effects of FAT 20060/F 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 28-29 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 control group was shared with BSL study 140382.

The following doses were evaluated:

Control (C): 0 mg/kg/d

Low Dose (LD): 100 mg/kg/d

Medium Dose (MD): 300 mg/kg/d

High Dose (HD): 1000 mg/kg/d

The test item formulation was prepared once in every ten days and stored at room temperature. The test item was suspended in aqua ad iniectabile (water for injection) and 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. 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 performed on 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 post natal day 4 were carefully examined for gross external abnormalities. A full histopathological evaluation of the tissues was performed of 5 randomly selected male and female animals of the control and high dose groups.As well as all gross lesions from all groups, were examined by light microscopy. Sections of the full list of organs and tissue were examined in 8 males and all females of HD group, because macroscopic discoloration was recorded in all organs and tissues in these animals. With regard to female no. 71 of HD group, full list of organs were examined as the decedent in addition due to discoloration in all organs and tissues. 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. In order to demonstrate glycogen accumulation in the hepatocytes, additional PAS stain sections from the representative animals (animal nos. 2, 33, 34, 36, 41, 73, 74 and 77) were prepared and examined by light microscopy.

 

Summary Results:

There was no mortality of animals due to test-item treatment in dose groups during the study period. However, one female (female no. 71) of HD group was euthanized for ethical reasons during the course of the study. Microscopically the morbidity was not considered to be due to test item treatment. There were test item treatment related clinical signs including moving the bedding, salivation, diarrhoea, discoloured red feces and skin in male and female animals of MD and/ or HD groups. The discoloured red feces and skin were due to colour of the test item. Moving the bedding and salivation observed in the MD and HD groups and a single animal of the LD group was assumed to be due to local effect of the test item.During the weekly detailed clinical observation, no significant changes or differences between the groups were found. 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 and females, there were test item treatment related effect on body weight, body weight gain and food consumption in HD group when compared to controls. However, there were mild effects on body weight and food consumption. Considering the microscopic changes in adrenal and thymus weight, the changes were not primary/direct effects of the test item, but were non-specific secondary event to stressful condition. In males and females, there were no adverse effects of toxicological relevance on hematology and blood coagulation parameters in dose groups when compared corresponding control. There were slightly higher ALAT and ASAT values in male or female HD groups which was associated with stress reponse. There were no effects on urinary parameters in both males and females of dose groups when compared to the control. At necropsy reddish, red or dark red discoloration was observed in one organ, several organs or all organs and tissues in 1/10 females of LD, 8/10 males and 6/10 females of MD and all males and females of the HD group. There was also enlargement of adrenal glands in some HD females and in single female (no. 71) there was distended cecum. These findings corroborated microscopic findings and were attributed to secondary events to the stressful condition.

There were statistically significant changes in absolute and/or relative organ weights including adrenal gland (male HD group), spleen (male LD, MD and HD groups), pituitary gland (female HD group), heart (female HD group) and ovaries (female HD group). These organ weights in males and/or females were without cellular/ tissue injuries, but were attributed to secondary event to stressful condition. Microscopic findings that were considered to be related to treatment with the test item were recorded in testes of males (pigment laden macrophages/histiocytes), ovaries and uterus with cervix of females (pigment laden macrophages/histiocytes), and kidneys (Pigment deposition, as well as hyaline droplets-like eosinophilic substance), mesenteric lymph node (pigment laden macrophages/histiocytes), liver (increased hepatocytic glycogen accumulation), thymus (Thymic atrophy) and adrenal gland (adrenocortical diffuse hypertrophy) of both sexes. Under the condition of study the diffuse adrenocortical hypertrophy, thymic atrophy and increased hepatocytic glycogen accumulation were considered non specific secondary to stressful situation. There was a statistically significantly longer gestation in HD group (mean of 23.50 gestation days) when compared to concurrent control group (mean of 22.38 gestation days). This finding at the HD level was considered to be likely to be an adverse effect of test-item treatment.

There was an effect of test item treatment on the no. of implantation sites, live pups and percent pre and post implantation loss and viability index(%) in HD group. There were adverse effects of test item treatment for no. of pups born, no. of still births, no. of live pups, and no. of male and female pups, pup mean weight, total litter weight, male litter weight and female litter weight on PND 0 and / or PND 4 in HD group. There were also an adverse effect of test item treatment for no. implantation sites, live pups and percent pre and post implantation loss in HD group. The viability index (%) was also adversely affected in HD group. There was statistically significantly higher % mortality between days 0-1 and between days 0-4 in HD group. There were itest-item related external findings in several pups of HD group on PND 0 and/ or PND 4.

 

Discussion:

Under the conditions of this study, the test item FAT 20060/F produced adverse histomorphological changes in thymus, adrenal glands and liver of both sexes at 1000 mg/kg/day. These changes consisted of thymic atrophy as well as adrenocortical diffuse hypertrophy, and increased hepatocytic glycogen accumulation, which was confirmed further by PAS staining in some representative animals, was recorded in both sexes at this dose level. Further, results indicate slight effects on the body weight, food consumption and clinical biochemistry were observed in both sexes when compared to the control group. There were also treatment related clinical and macroscopic findings and slight organ weight changes in both sexes. The administration of higher dosage of the test agents can cause stress to animals in the toxicology studies especially using the rodents’ model, which is generally indicated as the effects to body weight and food consumption with varying degrees. In addition, high-dose administration can activate the hypothalamic-pituitary-adrenal axis resulting in diffuse adrenocortical hypertrophy as well as thymic atrophy which are non-specific histomorphologic appearances indicating a stressful situation in parent animals. Further, corticosteroids released from adrenal cortex prompt glycogen synthesis in the liver, and hence, the increased hepatocytic glycogen accumulation observed in this study was considered to be a secondary response to increased level of corticosteroids released from the adrenal cortex and is a non-specific response to the stressful condition. Meanwhile, possible treatment-related effects were observed in reproductive/developmental parameters in the 1000 mg/kg/day group, including percent pre- and post implantation loss, litter weight, duration of gestation, pup survival and viability index at the high-dose group. The stressful condition in parent females can elicit the undesirable alterations on reproductive/developmental performances even though it was a non-specific, secondary response to the stress. Therefore, the above-mentioned findings recorded in parent animals of the high-dose group were judged to be adverse under the condition of this study from the comprehensive aspect of the reproductive/developmental toxicity assessment, even though those were secondary to the stressful condition in parent animals.

 

Conclusion:

On the basis of this reproduction/developmental toxicity screening test with FAT 20060/F in male and female Wistar rats with dose levels of 100, 300, and 1000 mg/kg/d the following conclusions can be made: There were mild and/or transient effects on body weight and food consumption at 1000 mg/kg/d associated with microscopic changes in adrenal gland (adrenocortical hypertrophy), thymus (thymic atrophy) and increased hepatic glycogen accumulation. These were not due to the primary/direct effects of the test item, rather they were non-specific secondary events to the stressful condition. The possible treatment-related effects were observed in reproduction/ developmental parameters including percent pre- and post-implantation loss, litter weight, duration of gestation, pup survival and viability index at 1000 mg/kg/d. The stressful condition in parent females can elicit the undesirable alterations on reproductive/ developmental performances thought it was a non-specific, secondary response to stress. The above mentioned findings in parent animals at 1000 mg/kg/d although were secondary to the stressful condition, but were judged to be adverse from the comprehensive aspect of the reproductive/developmental toxicity assessment. Based on the data generated from this combined repeated dose oral toxicity and reproduction/ developmental toxicity screening test with FAT 20060/F, the NOAEL for systemic as well as reproduction/ developmental toxicity is considered to be 300 mg/kg/d.