Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 March, 2014 to 15 October, 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 March, 2014 to 15 October, 2014
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:
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
Specific details on test material used for the study:
Name: FAT 36034/W
Physical State: solid
Storage Conditions: room temperature, protected from light
Colour: red
pH (specify): 10.2 (2 % (w/w) aqu. suspension), RT
Active Components: >90 %
Purity (qualitative and quantitative): 91.7 % (w/w)
Solubility in water: 3.26 mg/l
Date of Analysis: 07/08/2013
Expiry Date: 08/08/2018
Safety Precautions: The routine hygienic procedures procedures were sufficient to assure personnel health and safety
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 140271):
males: 253 - 281 g (mean: 268.93 g, ± 20 % = 215.14 – 322.71 g)
females: 171 - 191 g (mean: 180.34 g, ± 20 % = 144.27 – 216.41 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. 131113)
- 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
Type of inhalation exposure (if applicable):
not specified
Vehicle:
corn oil
Details on exposure:
The test item was ground to a fine powder with the help of a mortar and pestle. Afterwards it was weighed into a tared plastic vial on a precision balance and suspended in corn oil. The formulation was homogenized using an ultra turrax (homogenizer). The test item formulations were prepared once in every ten days and stored at 2-8 °C. Every day before the dose administration the formulation samples were allowed to reach the room temperature and the homogeneity was ensured by votexing the sample on vortex machine.

The following doses were evaluated:
Control: 0 mg/kg bw/d
Low Dose: 100 mg/kg bw/d
Medium Dose: 300 mg/kg bw/d
High Dose: 1000 mg/kg bw/d

The highest dose level was chosen with the aim of inducing toxic effects, but not death or severe suffering. This dose also happens to be a limit dose. 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 corn oil using the same volume as used for the test dose groups. The test item formulation 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 body weight. For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.
Details on mating procedure:
Mating was performed using a ratio of 1:1 (male to female). The vaginal smear of the females was checked every morning after the start of the mating period to confirm the evidence of mating. If the vaginal smear of a particular female was not found to be sperm-positive, the actual stage of the estrus cycle on that day was documented. The day of the vaginal plug and/or sperm was considered as day 0 of gestation. The cages were arranged in such a way that possible effects due to cage placement were minimized.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Each dosing concentration 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 formulation samples were analysed on the day of sample collection and were stored at -20° C. These samples were analysed after the completion
of the toxicity study at BSL BIOSERVICE Scientific Laboratories GmbH under the BSL study no. 140262.
Duration of treatment / exposure:
The animals were treated with the test item formulation or vehicle on 7 days per week for a period of 54 days,
i.e. during 14 days of pre-mating and 14 days of mating in both males and females, during the gestation period and
up to post-natal day 3 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.
Details on study schedule:
Arrival of the Test Item: 07 January 2014
Study Initiation Date: 24 March 2014
1st Amendment to Study Plan: 11 April 2014
2nd Amendment to Study Plan: 30 April 2014
Delivery of Animals: 01 April 2014
Acclimatisation Period: 01 April 2014 – 07 April 2014
Experimental Starting Date: 07 April 2014
Date of Behavioural Observation: 07 April 2014- 22 May 2014
Treatment Period Males: 10 April 2014 – 08 May 2014
Treatment Period Females: 10 April 2014 – 02 June 2014
Necropsies Males: 08 May 2014 and 09 May 2014
Necropsies Females: 20 May 2014 – 03 June 2014
Experimental Completion Date: 03 June 2014
Completion Date of Delegated Phase (Histopathology): 25 September 2014
Completion Date of Delegated Phase (Formulation Analysis): 29 August 2014
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
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 140271.
Control animals:
yes, concurrent vehicle
Details on study design:
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 corn oil, the vehicle used in this study. The 4 groups comprised 10 male and 10 female Wistar rats. The test item formulation was prepared once in every ten days and stored at 2-8 °C. The test item was suspended in corn oil and dose volumes were adjusted individually based on weekly body weight measurements. The administration volume was 5 mL/kg body weight.
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. Except 1/5 selected females of LD group was non pregnant and the FOB in the last week of treatment was not performed. 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.
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 male animals were sacrificed after the completion of the mating period (total dosing period: 28 days) on study day 29 or 30, while female animals were sacrificed on post-natal day 4 using an anaesthesia (ketamine/xylazin, 2:1, Pharmanovo, lot no: 24664 (expiry date: 06/2015) and lot no. 24863 (expiry date : 10/2015) and Serumwerk, lot no: 01213 (expiry date: 10/2015) and lot no.: 00513 (expiry date: 05/2015). All surviving pups were killed by decapitation on post natal day 4.
Dead pups and pups sacrificed on day 4 post-partum were carefully examined externally for gross abnormalities. Non-pregnant females (females 42 and 71) were sacrificed on day 26 from day of sperm positive vaginal smear or from the last day of mating period. 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 fixed in modified Davidson’s Solution for 24 hours and then 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. 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 histopathology was carried out on the preserved organs and tissues (see above) of 5 randomly selected male and female animals (only lactating females were evaluated) of the control and high dose groups which were sacrificed at the end of the treatment period. All gross lesions from all groups, were examined by light microscopy. 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. Because of possible treatment-related findings noted in the high dose group, spleen and bone marrow (sternum) from 5 selected male and female animals of the low and medium dose groups (groups 2 and 3, respectively) were examined to establish a no-effect level. Testes, epididymides, ovaries, uterus with cervix, vagina, accessory sex organs (prostate, seminal vesicle with coagulating gland) were trimmed,
embedded into paraffin, cut at an approximated thickness of 2-4 µm and stained with hematoxylin and eosin and were examined in all animals. For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle for the evaluation
of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides. Histological processing of tissues to microscope slides was performed at the GLP-certified contract laboratory AnaPath GmbH, AnaPath Services, Hammerstrasse 49, 4410 Liestal, Switzerland (test site for tissue processing). Histopathological evaluation was performed at the GLP-certified contract laboratory AnaPath GmbH, Buchsweg 56, 4625 Oberbuchsiten, Switzerland (test site for histopathology). The study phases from test site 1 and 2 was performed in compliance with the Swiss Ordinance relating to Good Laboratory Practice adopted 18 May 2005 [SR 813.112.1] (Status as of 01 December 2012). Blocking, embedding, cutting, H&E staining and scientific slide evaluation were performed according to the corresponding SOP’s of the test sites. The principal investigator for histopathological tissue processing sent all raw data (including blocks, slides, paper raw data, statement of compliance and quality assurance statement) to the study director. The principal investigator for histopathological evaluation provided the histopathology results to the study director by e-mail and sent a pathology phase report to the study director upon the completion of the study.
Postmortem examinations (offspring):
Pups sacrificed on 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 effect on the copulation, fertility and delivery indices of dose groups when compared to controls. A fertility index (number of pregnant females/ number of copulated females X 100) observed in the control and the dose groups were C 90 %, LD 100 %, MD 100 % and HD 100 %.
Offspring viability indices:
There was no effect on viability index of dose groups when compared to control. However, there was slightly lower viability index in the LD group, but in the absence of dose related response, the finding was considered incidental.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
no adverse effects
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
no adverse effects
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
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: oestrous cycle:
not examined
Description (incidence and severity):
Except for confirmation of mating
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Mortality:
No mortality occurred in the control or any of the dose groups during the study period.

Clinical Observations:
There were no clinical signs of toxicological relevance in the dose groups when compared to control. However, there were red feces in HD group and signs of moving the bedding in males and/ or females of MD and/ or HD groups. The red feces were due to test item colour and
the moving the bedding was considered due to local effect of test item. There were other clinical signs including alopecia, slight abnormal breathing, nasal discharge, salivation and regurgitation in a few animals of control and/ or dose groups. These were considered incidental. During the weekly detailed clinical observation, no changes or differences between the groups and the baseline values.

Functional Observations:
No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period. There were no biologically relevant differences in body temperature between the groups.

Body Weight Development:
In males and female, there were no adverse changes of toxicological relevance on body weight and body weight gain. However, In males there was statistically significantly lower weight gain in HD groups after premating days 7-14. There was also statistically significantly lower weight
gain in MD group after mating/ post matings days 7-14. There were also lower weight gain in LD and HD groups after the 1st and last week of mating/postmating period. These changes were either transient and/ or without dose response relation. Therefore, the findings was not
considered to be an adverse effect of treatment. In females, there was statistically significantly higher weight gain after lactation days 0-4 in HD group. This higher weight gain was within the
range of historical control data and was not considered likely to be an effect of treatment.

Food Consumption:
In males and females, there were no adverse changes of toxicological relevance for food consumption in dose groups when compared to control. There were no statistically significant differences between the dose and control group values. However, in males there was very marginal decrease in food consumption after premating period 7-14 in HD group and in females the food consumption was higher in HD group after lactation days 0-4. These changes in food consumption in males or females correlated with changes in weight gain. In the absence of statistically significant differences, the lower or higher food intake in males and females were not considered an adverse effect of treatment.

Pathology:
Enlarged of spleen, which correlated microscopically with increased extramedullary erythrocytic hemopoiesis, was recorded in three males of MD and 4 males and one female of HD group. This was considered to be treatment-related change (see 12.11). “Discoloured pale” or “Discoloured” of the kidney was recorded in all male groups including the control group and in one female of MD group. However, there were no corresponding microscopic findings in any animals. All other gross lesions recorded were within the range of normal background alterations which may be recorded in animals of this strain and age.

Organ Weight:
In males and females, there was statistically significantly higher absolute and relative (to terminal body weight) spleen weight in MD and HD groups. These findings were associated with the increase in mean severity of erythrocytic extramedullary hemopoiesis in the spleen recorded in both sexes of MD and HD groups. These findings were considered to be adverse event attributable to treatment with the test item. There was also statistically significantly lower absolute and relative (to terminal body weight) pituitary gland weight in MD group of male animals. There was also statistically significantly higher relative kidney weight in HD group. In the absence of dose response relation, the finding was not considered likely to be related to treatment.

Histopathology:
Microscopically, the treatment-related findings were recorded in the spleen and bone marrow.
The increase in mean severity of erythrocytic extramedullary hemopoiesis in the spleen was recorded in both sexes of MD and HD groups, along with an increase in incidence and/or severity of hemosiderin deposition in the spleen and erythropoiesis in the bone marrow. These findings
are most likely to be the histomorphologic indicator of hemolytic anemia and were considered to be adverse event attributable to treatment with the test item. The sperm stages of testes were checked on completeness of cell populations, completeness of stages and degenerative changes. Nothing particular was observed in testes of animal no. 2 (control), which was mated with female no. 42 not giving birth. Slight spermatic granulomas and minimal mononuclear cell foci in epididymides and minimal alveolitis in lungs were recorded in male no. 2. However, similar minimum to slight lesions are often observed in male animals that can produce pregnancy to the pairing females, and therefore, it is unlikely that these findings were related to the infertility.
In male no. 31 (HD group), marked tubular atrophy (ie. so-called sertoli cell-only tubules) in testes and marked oligospermia (ie. azoospermia) in epididymides were observed, and this was considered to be the cause why the paired female no. 71 did not give birth. Complete tubular atrophy sometimes happens spontaneously in the male rats of this strain and age, and sporadic occurrence with only one case is unlikely to be related to treatment with the test item. Nothing particular was observed in the female reproductive organs (ovaries, uterus with cervix and vagina) of the paired female nos. 42 (control) and 71 (HD group) which did not give birth. As a result of the detailed examination of the testis, there were no abnormalities on the completeness of stages and cell populations, except for male no. 31 (HD group). In the testis of male no. 31, it was impossible to evaluate the stages and cell population because of complete tubular atrophy.
However, this was judged to be spontaneous change as described above. There was no dose response relationship in any findings recorded in the male reproductive organs including testes, epididymides, prostate glands, coagulating glands and seminal vesicles which were examined in this study, and therefore, all histologic findings were considered to be spontaneous changes which may be recorded in animals of this strain and age. The remainder of findings recorded was within the range of normal background lesions which may be recorded in animals of this strain and age, or were considered to be accidental changes which can occur in the toxicology studies using the rodent models.

Litter Data
There were no effect of toxicological relevance on the litter data including no. of pups born, no. of live pups, no. of still birth and no. of runts on PND 0 and no. of male and female pups and sex ratio on PND 0 and PND 4. There were no statistically significant changes between the dose and control groups.

Litter Weight Data
There were no effect of toxicological relevance on litter weight data including pup mean weight, total litter weight, male litter weight and female litter weight measured on PND 0 and PND 4. There were no statistically significant changes between the dose and control groups.

Precoital Interval and Duration of Gestation
There were no effect on the duration of precoital interval and the duration of gestation in dose group animals when compared to the controls.

Pre- and Post-Natal Data
There were no effect on the pre and post natal data including the no. of corpora lutea, no. of implantation sites, no. of live pups (PND 0 and 4), percent pre and post implantation loss.

Reproductive Indices
There were no effect on the copulation, fertility and delivery indices of dose groups when compared to controls. A fertility index (number of pregnant females/ number of copulated females X 100) observed in the control and the dose groups were C 90%, LD 100 %, MD 100% and HD 100%. There was no effect on viability index of dose groups when compared to control. However, there was slightly lower viability index in the LD group, but in the absence of dose related response, the finding was considered incidental.

Pup Survival Data
There were no effect on the survival of the pups from PND 0 to PND 4 was observed in any dose group when compared to controls. One pup (pup no. 4) from female no. 59 of LD group was missing on PND 1. This missing pup was assumed to be cannibalized by dam. This being in single isolated female of LD group, the finding was considered incidental.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (nominal)
System:
haematopoietic
Organ:
bone marrow
spleen
other: Haemolytic anaemia was observed in both genders at 300 and 1000 mg/kg/d. Microscopically, this was evident by extramedullary haematopoiesis in bone marrow and spleen
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
not specified
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Litter Data:
There were no effect of toxicological relevance on the litter data including no. of pups born, no. of live pups, no. of still birth and no. of runts on
PND 0 and no. of male and female pups and sex ratio on PND 0 and PND 4. There were no statistically significant changes between the dose and
control groups.

Litter Weight Data:
There were no effect of toxicological relevance on litter weight data including pup mean weight, total litter weight, male litter weight and female
litter weight measured on PND 0 and PND 4. There were no statistically significant changes between the dose and control groups.

Precoital Interval and Duration of Gestation:
There were no effect on the duration of precoital interval and the duration of gestation in dose group animals when compared to the controls.

Pre- and Post-Natal Data:
There were no effect on the pre and post natal data including the no. of corpora lutea, no. of implantation sites, no. of live pups (PND 0 and 4),
percent pre and post implantation loss.

Reproductive Indices:
There were no effect on the copulation, fertility and delivery indices of dose groups when compared to controls. A fertility index
(number of pregnant females/ number of copulated females X 100) observed in the control and the dose groups were C 90 %, LD 100 %,
MD 100 % and HD 100 %.
There was no effect on viability index of dose groups when compared to control. However, there was slightly lower viability index in the LD group,
but in the absence of dose related response, the finding was considered incidental.

Pup Survival Data:
There were no effect on the survival of the pups from PND 0 to PND 4 was observed in any dose group when compared to controls.
One pup (pup no. 4) from female no. 59 of LD group was missing on PND 1. This missing pup was assumed to be cannibalized by dam.
This being in single isolated female of LD group, the finding was considered incidental.

Pup External Findings:
There were no gross external findings of toxicological relevance in any of the dose groups when compared to the controls.
Few incidences of external findings were observed in the pups of HD group on PND 0. The findings were dark snout (pup no. 3 of animal 74),
dry skin (pups no. 8 and 9 of animal 74) and cold and pale pup (pup no. 11 of animal 77). These external findings did not persist through PND 4
and hence were not considered to have treatment relevance.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Conclusions:
Based on the data generated from this combined repeated dose oral toxicity and reproduction/ developmental toxicity screening test with FAT 36034/W, the no observed adverse effect level (NOAEL) is considered to be 100 mg/kg/ d for systemic toxicity in males and females and 1000 mg/kg/d for reproduction/ developmental toxicity.
Executive summary:

The aim of this study was to assess the possible effects of FAT 36034/W on male and female fertility and embryofetal development after repeated oral dose administrationin 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 corn oil, the vehicle used in this study. The 4 groups comprised 10 male and 10 femaleWistarrats. The control group was shared with BSL study 140271. 


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 2-8 °C. The test item was suspended in corn oil and dose volumes were adjusted individually based on weekly body weight measurements. The administration volume was 5 mL/kg body weight. During the period of administration, the animals were observed each day for signs of toxicity. At the conclusion of the test, 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 and females 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 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 (females 42 and 71) were sacrificed on day 26 from day of sperm positive vaginal smear or from the last day of mating period. Pups sacrificed on post natal day 4 and those found dead, 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. 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. Because of possible treatment-related findings noted in the high dose group, spleen and bone marrow (sternum) from 5 selected male and female animals of the low and medium dose groups were examined to establish a no-effect level.


 


Summary Results


No mortality occurred in the control or any of the dose groups during the study period. There were no clinical signs of toxicological relevance in the dose groups when compared to control. However, there were red feces in HD group and signs of moving the bedding in males and/ or females of MD and/ or HD groups. The red feces were due to test item colour and the moving the bedding was considered 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 female, there were no adverse changes of toxicological relevance on body weight, body weight gain and food consumption in dose groups when compared to the concurrent control. In males and females, there were findings of toxicological relevance on haematological parameters in MD and HD groups. In males, there was statistically significantly lower mean RBC, HCT and Hb in MD and HD groups; statistically significantly higher mean MCV and reticulocyte count in MD and HD group; statistically significantly higher mean MCH in HD group. In females, there was statistically significantly lower mean RBC, Hb, HCT value in HD group and statistically significantly higher mean MCV, MCH and reticulocyte count in HD group. In males and females, there was no effect on the blood coagulation ,clinical biochemistry and urinary parameters of dose groups when compared to the control group. Enlarged spleen, which correlated microscopically with increased extramedullary erythrocytic hemopoiesis, was recorded in 3/10 males of MD group, in 4/10 males and one female of HD group, was considered to be treatment-related change. Discoloured pale or discoloured of the kidney was observed macroscopically in all male groups including the control group and in one female of MD group. However, there were no corresponding microscopic findings in any animals. All other gross lesions recorded were within the range of normal background alterations which may be recorded in animals of this strain and age. In males and females, there was statistically significantly higher absolute and relative (to terminal body weight) spleen weight in MD and HD groups, when compared to controls.The change in spleen weight correlated with the increase in mean severity of erythrocytic extramedullary hemopoiesis in the spleen recorded in both sexes of MD and HD groups. These findings are considered to be adverse events attributable to treatment with the test item.


Microscopically, the treatment-related findings were recorded in the spleen and bone marrow. The increase in mean severity of erythrocytic extramedullary hemopoiesis in the spleen was recorded in both sexes of MD and HD groups, along with an increase in incidence and/or severity of hemosiderin deposition in the spleen and erythropoiesis in the bone marrow. These findings are most likely to be the histomorphologic indicator of hemolytic anemia and were considered to be adverse event attributable to treatment with the test item. There were no effects on the duration of precoital interval and the duration of gestation in dose group animals when compared to the controls. There were no effects on the pre and post natal data including the no. of corpora lutea, no. of implantation sites, no. of live pups (PND 0 and 4), percent pre and post implantation loss. There were no effects on the copulation, fertility and delivery indices of dose groups when compared to controls. A fertility index (number of pregnant females/ number of copulated females X 100) observed in the control and the dose groups were C 90 %, LD 100 %, MD 100 % and HD 100 %. There was no effect on viability index of dose groups when compared to control. There were no effects of toxicological relevance on the litter data including no. of pups born, no. of live pups, no. of still birth and no. of runts on PND 0 and no. of male and female pups and sex ratio on PND 0 and PND 4. There were no effects of toxicological relevance on litter weight data including pup mean weight, total litter weight, male litter weight and female litter weight measured on PND 0 and PND 4. No effects on the survival of the pups from PND 0 to PND 4 were observed in any dose group when compared to controls. There were no gross external findings of pups of toxicological relevance in any of the dose groups when compared to the controls.


 


Conclusion


On the basis of this reproduction/developmental toxicity screening test with FAT 36034/W in male and female Wistar rats with dose levels of 100, 300, and 1000 mg/kg/d the following conclusions can be made: Haemolytic anaemia was observed in both genders at 300 and 1000 mg/kg/d. Microscopically, this was evident by extramedullary haematopoiesis in bone marrow and spleen. This also correlated with dose-dependently lower levels of red blood cell parameters (RBC, Hb and Hct) and a concurrent increase in reticulocytes and mean cell haemoglobin and mean cell volume. Whereas the effects were moderate at 1000 mg/kg/d, only a slight anaemia was found at 300 mg/kg/d.mThere were no effect on the reproduction/ developmental parameters.nBased on the data generated from this combined repeated dose oral toxicity and reproduction/ developmental toxicity screening test with FAT 36034/W, the no observed adverse effect level (NOAEL) is considered to be 100 mg/kg/ d for systemic toxicity in males and females and 1000 mg/kg/d for reproduction/ developmental toxicity.

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:
24 March, 2014 to 15 October, 2014
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
Specific details on test material used for the study:
Name: FAT 36034/W TE
Physical State: solid
Storage Conditions: room temperature, protected from light
Colour: red
pH (specify): 10.2 (2%(w/w) aqu. Suspension), RT
Active Components: >90%
Purity (qualitative and quantitative): 91.7 % (w/w)
Solubility in water: 3.26 mg/l
Date of Analysis: 07/08/2013
Expiry Date: 08/08/2018
Safety Precautions: The routine hygienic procedures procedures were sufficient to assure personnel health and safety
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 140271):
males: 253 - 281 g (mean: 268.93 g, ± 20 % = 215.14 – 322.71 g)
females: 171 - 191 g (mean: 180.34 g, ± 20 % = 144.27 – 216.41 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. 131113)
- 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:
corn oil
Details on exposure:
The test item was ground to a fine powder with the help of a mortar and pestle. Afterwards it was weighed into a tared plastic vial on a precision balance and suspended in corn oil. The formulation was homogenized using an ultra turrax (homogenizer).
The test item formulations were prepared once in every ten days and stored at 2-8 °C. Every day before the dose administration the formulation samples were allowed to reach the room temperature and the homogeneity was ensured by votexing the sample on vortex machine.

The following doses were evaluated:
Control: 0 mg/kg bw/d
Low Dose: 100 mg/kg bw/d
Medium Dose: 300 mg/kg bw/d
High Dose: 1000 mg/kg bw/d

The highest dose level was chosen with the aim of inducing toxic effects, but not death or severe suffering. This dose also happens to be a limit dose. 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 corn oil using the same volume as used for the test dose groups.

The test item formulation 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 body weight.
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 formulation samples were analysed on the day of sample collection and were stored at -20° C. These samples were analysed after the completion
of the toxicity study at BSL BIOSERVICE Scientific Laboratories GmbH under the BSL study no. 140262.
Details on mating procedure:
Mating was performed using a ratio of 1:1 (male to female). The vaginal smear of the females was checked every morning after the start of the mating period to confirm the evidence of mating. If the vaginal smear of a particular female was not found to be sperm-positive, the actual stage of the estrus cycle on that day was documented. The day of the vaginal plug and/or sperm was considered as day 0 of gestation.
The cages were arranged in such a way that possible effects due to cage placement were minimized.
Duration of treatment / exposure:
The animals were treated with the test item formulation or vehicle on 7 days per week for a period of 54 days,
i.e. during 14 days of pre-mating and 14 days of mating in both males and females, during the gestation period and
up to post-natal day 3 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.
Duration of test:
Arrival of the Test Item: 07 January 2014
Study Initiation Date: 24 March 2014
1st Amendment to Study Plan: 11 April 2014
2nd Amendment to Study Plan: 30 April 2014
Delivery of Animals: 01 April 2014
Acclimatisation Period: 01 April 2014 – 07 April 2014
Experimental Starting Date: 07 April 2014
Date of Behavioural Observation: 07 April 2014- 22 May 2014
Treatment Period Males: 10 April 2014 – 08 May 2014
Treatment Period Females: 10 April 2014 – 02 June 2014
Necropsies Males: 08 May 2014 and 09 May 2014
Necropsies Females: 20 May 2014 – 03 June 2014
Experimental Completion Date: 03 June 2014
Completion Date of Delegated Phase (Histopathology): 25 September 2014
Completion Date of Delegated Phase (Formulation Analysis): 29 August 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 140271.
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. Except 1/5 selected females of LD group was non pregnant and the FOB in the
last week of treatment was not performed.
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.
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.
Fetal examinations:
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.
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:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
no effects observed
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
Details on results:
Mortality:
No mortality occurred in the control or any of the dose groups during the study period.

Clinical Observations:
There were no clinical signs of toxicological relevance in the dose groups when compared to control. However, there were red feces in HD
group and signs of moving the bedding in males and/ or females of MD and/ or HD groups. The red feces were due to test item colour and
the moving the bedding was considered due to local effect of test item.
There were other clinical signs including alopecia, slight abnormal breathing, nasal discharge, salivation and regurgitation in a few animals of
control and/ or dose groups. These were considered incidental.
During the weekly detailed clinical observation, no changes or differences between the groups and the baseline values.

Functional Observations:
No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period.
There were no biologically relevant differences in body temperature between the groups.

Body Weight Development:
In males and female, there were no adverse changes of toxicological relevance on body weight and body weight gain. However, In males there
was statistically significantly lower weight gain in HD groups after premating days 7-14. There was also statistically significantly lower weight
gain in MD group after mating/ post matings days 7-14. There were also lower weight gain in LD and HD groups after the 1st and last week of
mating/postmating period. These changes were either transient and/ or without dose response relation. Therefore, the findings was not
considered to be an adverse effect of treatment.
In females, there was statistically significantly higher weight gain after lactation days 0-4 in HD group. This higher weight gain was within the
range of historical control data and was not considered likely to be an effect of treatment.

Food Consumption:
In males and females, there were no adverse changes of toxicological relevance for food consumption in dose groups when compared to control.
There were no statistically significant differences between the dose and control group values. However, in males there was very marginal
decrease in food consumption after premating period 7-14 in HD group and in females the food consumption was higher in HD group after
lactation days 0-4. These changes in food consumption in males or females correlated with changes in weight gain. In the absence of statistically
significant differences, the lower or higher food intake in males and females were not considered an adverse effect of treatment.

Pathology:
Enlarged of spleen, which correlated microscopically with increased extramedullary erythrocytic hemopoiesis, was recorded in three males of
MD and 4 males and one female of HD group. This was considered to be treatment-related change (see 12.11).
“Discoloured pale” or “Discoloured” of the kidney was recorded in all male groups including the control group and in one female of MD group.
However, there were no corresponding microscopic findings in any animals.
All other gross lesions recorded were within the range of normal background alterations which may be recorded in animals of this strain and age.

Organ Weight:
In males and females, there was statistically significantly higher absolute and relative (to terminal body weight) spleen weight in MD and HD groups. These findings were associated with the increase in mean severity of erythrocytic extramedullary hemopoiesis in the spleen recorded in both
sexes of MD and HD groups. These findings were considered to be adverse event attributable to treatment with the test item.
There was also statistically significantly lower absolute and relative (to terminal body weight) pituitary gland weight in MD group of male animals.
There was also statistically significantly higher relative kidney weight in HD group. In the absence of dose response relation, the finding was
not considered likely to be related to treatment.

Histopathology:
Microscopically, the treatment-related findings were recorded in the spleen and bone marrow.
The increase in mean severity of erythrocytic extramedullary hemopoiesis in the spleen was recorded in both sexes of MD and HD groups,
along with an increase in incidence and/or severity of hemosiderin deposition in the spleen and erythropoiesis in the bone marrow. These findings
are most likely to be the histomorphologic indicator of hemolytic anemia and were considered to be adverse event attributable to treatment with the
test item.
The sperm stages of testes were checked on completeness of cell populations, completeness of stages and degenerative changes. Nothing particular was observed in testes of animal no. 2 (control), which was mated with female no. 42 not giving birth. Slight spermatic granulomas and minimal mononuclear cell foci in epididymides and minimal alveolitis in lungs were recorded in male no. 2. However, similar minimum to slight lesions are often observed in male animals that can produce pregnancy to the pairing females, and therefore, it is unlikely that these findings were related to the infertility.
In male no. 31 (HD group), marked tubular atrophy (ie. so-called sertoli cell-only tubules) in testes and marked oligospermia (ie. azoospermia) in
epididymides were observed, and this was considered to be the cause why the paired female no. 71 did not give birth. Complete tubular atrophy
sometimes happens spontaneously in the male rats of this strain and age, and sporadic occurrence with only one case is unlikely to be related to treatment with the test item.
Nothing particular was observed in the female reproductive organs (ovaries, uterus with cervix and vagina) of the paired female nos. 42 (control)
and 71 (HD group) which did not give birth.
As a result of the detailed examination of the testis, there were no abnormalities on the completeness of stages and cell populations, except for male no. 31 (HD group). In the testis of male no. 31, it was impossible to evaluate the stages and cell population because of complete tubular atrophy.
However, this was judged to be spontaneous change as described above.
There was no dose response relationship in any findings recorded in the male reproductive organs including testes, epididymides, prostate glands,
coagulating glands and seminal vesicles which were examined in this study, and therefore, all histologic findings were considered to be spontaneous changes which may be recorded in animals of this strain and age.
The remainder of findings recorded was within the range of normal background lesions which may be recorded in animals of this strain and age, or were considered to be accidental changes which can occur in the toxicology studies using the rodent models.
Other effects:
no effects observed
Description (incidence and severity):
Litter Data
There were no effect of toxicological relevance on the litter data including no. of pups born, no. of live pups, no. of still birth and no. of runts on PND 0 and no. of male and female pups and sex ratio on PND 0 and PND 4. There were no statistically significant changes between the dose and control groups.

Litter Weight Data
There were no effect of toxicological relevance on litter weight data including pup mean weight, total litter weight, male litter weight and female litter weight measured on PND 0 and PND 4. There were no statistically significant changes between the dose and control groups.

Precoital Interval and Duration of Gestation
There were no effect on the duration of precoital interval and the duration of gestation in dose group animals when compared to the controls.

Pre- and Post-Natal Data
There were no effect on the pre and post natal data including the no. of corpora lutea, no. of implantation sites, no. of live pups (PND 0 and 4), percent pre and post implantation loss.

Reproductive Indices
There were no effect on the copulation, fertility and delivery indices of dose groups when compared to controls. A fertility index (number of pregnant females/ number of copulated females X 100) observed in the control and the dose groups were C 90%, LD 100 %, MD 100% and HD 100%.
There was no effect on viability index of dose groups when compared to control. However, there was slightly lower viability index in the LD group, but in the absence of dose related response, the finding was considered incidental.

Pup Survival Data
There were no effect on the survival of the pups from PND 0 to PND 4 was observed in any dose group when compared to controls.
One pup (pup no. 4) from female no. 59 of LD group was missing on PND 1. This missing pup was assumed to be cannibalized by dam. This being in single isolated female of LD group, the finding was considered incidental.
Dose descriptor:
LOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
gross pathology
haematology
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Abnormalities:
no effects observed
Developmental effects observed:
no
Conclusions:
Based on the data generated from this combined repeated dose oral toxicity and reproduction/ developmental toxicity screening test with FAT 36034/W TE, the no observed adverse effect level (NOAEL) is considered to be 100 mg/kg/ d for systemic toxicity in males and females and 1000 mg/kg/d for reproduction/ developmental toxicity.
Executive summary:

The aim of this study was to assess the possible effects of FAT 36034/W TE on male and female fertility and embryofetal development after repeated oral dose administrationin 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 corn oil, the vehicle used in this study. The 4 groups comprised 10 male and 10 femaleWistarrats. The control group was shared with BSL study 140271.

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 2-8 °C. The test item was suspended in corn oil and dose volumes were adjusted individually based on weekly body weight measurements. The administration volume was 5 mL/kg body weight.

During the period of administration, the animals were observed each day for signs of toxicity. At the conclusion of the test, 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 and females 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 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 (females 42 and 71) were sacrificed on day 26 from day of sperm positive vaginal smear or from the last day of mating period.

Pups sacrificed on post natal day 4 and those found dead, 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. 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.

Because of possible treatment-related findings noted in the high dose group, spleen and bone marrow (sternum) from 5 selected male and female animals of the low and medium dose groups were examined to establish a no-effect level.

Summary Results

No mortality occurred in the control or any of the dose groups during the study period.

There were no clinical signs of toxicological relevance in the dose groups when compared to control. However, there were red feces in HD group and signs of moving the bedding in males and/ or females of MD and/ or HD groups. The red feces were due to test item colour and the moving the bedding was considered 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 female, there were no adverse changes of toxicological relevance on body weight, body weight gain and food consumption in dose groups when compared to the concurrent control. 

In males and females, there were findings of toxicological relevance on haematological parameters in MD and HD groups. In males, there was statistically significantly lower mean RBC, HCT and Hb in MD and HD groups; statistically significantly higher mean MCV and reticulocyte count in MD and HD group; statistically significantly higher mean MCH in HD group. In females, there was statistically significantly lower mean RBC, Hb, HCT value in HD group and statistically significantly higher mean MCV, MCH and reticulocyte count in HD group.

In males and females, there was no effect on the blood coagulation ,clinical biochemistry and urinary parameters of dose groups when compared to the control group. Enlarged spleen, which correlated microscopically with increased extramedullary erythrocytic hemopoiesis, was recorded in 3/10 males of MD group, in 4/10 males and one female of HD group, was considered to be treatment-related change. Discoloured pale or discoloured of the kidney was observed macroscopically in all male groups including the control group and in one female of MD group. However, there were no corresponding microscopic findings in any animals.

All other gross lesions recorded were within the range of normal background alterations which may be recorded in animals of this strain and age.

In males and females, there was statistically significantly higher absolute and relative (to terminal body weight) spleen weight in MD and HD groups, when compared to controls.The change in spleen weight correlated with the increase in mean severity of erythrocytic extramedullary hemopoiesis in the spleen recorded in both sexes of MD and HD groups. These findings are considered to be adverse events attributable to treatment with the test item.

Microscopically, the treatment-related findings were recorded in the spleen and bone marrow. The increase in mean severity of erythrocytic extramedullary hemopoiesis in the spleen was recorded in both sexes of MD and HD groups, along with an increase in incidence and/or severity of hemosiderin deposition in the spleen and erythropoiesis in the bone marrow. These findings are most likely to be the histomorphologic indicator of hemolytic anemia and were considered to be adverse event attributable to treatment with the test item.

There were no effects on the duration of precoital interval and the duration of gestation in dose group animals when compared to the controls.

There were no effects on the pre and post natal data including the no. of corpora lutea, no. of implantation sites, no. of live pups (PND 0 and 4), percent pre and post implantation loss.

There were no effects on the copulation, fertility and delivery indices of dose groups when compared to controls. A fertility index (number of pregnant females/ number of copulated females X 100) observed in the control and the dose groups were C 90 %, LD 100 %, MD 100 % and HD 100 %.

There was no effect on viability index of dose groups when compared to control.

There were no effects of toxicological relevance on the litter data including no. of pups born, no. of live pups, no. of still birth and no. of runts on PND 0 and no. of male and female pups and sex ratio on PND 0 and PND 4.

There were no effects of toxicological relevance on litter weight data including pup mean weight, total litter weight, male litter weight and female litter weight measured on PND 0 and PND 4.

No effects on the survival of the pups from PND 0 to PND 4 were observed in any dose group when compared to controls.

There were no gross external findings of pups of toxicological relevance in any of the dose groups when compared to the controls.

Conclusion

On the basis of this reproduction/developmental toxicity screening test with FAT 36034/W TE in male and female Wistar rats with dose levels of

100, 300, and 1000 mg/kg/d the following conclusions can be made:

Haemolytic anaemia was observed in both genders at 300 and 1000 mg/kg/d. Microscopically, this was evident by extramedullary haematopoiesis in bone marrow and spleen. This also correlated with dose-dependently lower levels of red blood cell parameters (RBC, Hb and Hct) and a concurrent increase in reticulocytes and mean cell haemoglobin and mean cell volume. Whereas the effects were moderate at 1000 mg/kg/d, only a slight anaemia was found at 300 mg/kg/d.

There were no effect on the reproduction/ developmental parameters.

Based on the data generated from this combined repeated dose oral toxicity and reproduction/ developmental toxicity screening test withFAT 36034/W TE, the no observed adverse effect level (NOAEL) is considered to be 100 mg/kg/ d for systemic toxicity in males and females and 1000 mg/kg/d for reproduction/ developmental toxicity.

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

Constituent 1
Chemical structure
Reference substance name:
N-(4-amino-9,10-dihydro-3-methoxy-9,10-dioxo-1-anthryl)-4-methylbenzenesulphonamide
EC Number:
201-369-2
EC Name:
N-(4-amino-9,10-dihydro-3-methoxy-9,10-dioxo-1-anthryl)-4-methylbenzenesulphonamide
Cas Number:
81-68-5
Molecular formula:
C22H18N2O5S
IUPAC Name:
N-(4-amino-9,10-dihydro-3-methoxy-9,10-dioxo-1-anthryl)-4-methylbenzenesulphonamide
Test material form:
other: solid
Specific details on test material used for the study:
Name: FAT 36034/W TE
Physical State: solid
Storage Conditions: room temperature, protected from light
Colour: red
pH (specify): 10.2 (2 % (w/w) aqu. suspension), RT
Active Components: >90%
Purity (qualitative and quantitative): 91.7 % (w/w)
Solubility in water: 3.26 mg/l
Date of Analysis: 07/08/2013
Expiry Date: 08/08/2018
Safety Precautions: The routine hygienic procedures procedures were sufficient to assure personnel health and safety.

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 140271):
males: 253 - 281 g (mean: 268.93 g, ± 20 % = 215.14 – 322.71 g)
females: 171 - 191 g (mean: 180.34 g, ± 20 % = 144.27 – 216.41 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.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
The test item was ground to a fine powder with the help of a mortar and pestle. Afterwards it was weighed into a tared plastic vial on a precision balance and suspended in corn oil. The formulation was homogenized using an ultra turrax (homogenizer). The test item formulations were prepared once in every ten days and stored at 2-8 °C. Every day before the dose administration the formulation samples were allowed to reach the room temperature and the homogeneity was ensured by votexing the sample on vortex machine.

The dose range finding study (14 days repeated dose, BSL study no. 140259) was performed at dose levels 100, 300 and 1000 mg/kg/d with no overt toxicity observed up to the highest dose tested. According to these results and in consultation with the sponsor the following doses
are selected for the 3 dose groups (LD = low dose, MD = medium dose, HD = high dose) and 1 control group (C).

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

The highest dose level was chosen with the aim of inducing toxic effects, but not death or severe suffering. This dose also happens to be a limit dose. 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 corn oil using the same volume as used for the test dose groups. The test item formulation 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 body weight. 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 formulation samples were analysed on the day of sample collection and were stored at -20 °C. These samples were analysed after the completion
of the toxicity study at BSL BIOSERVICE Scientific Laboratories GmbH under the BSL study no. 140262.
Duration of treatment / exposure:
The animals were treated with the test item formulation or vehicle on 7 days per week for a period of 54 days,
i.e. during 14 days of pre-mating and 14 days of mating in both males and females, during the gestation period and
up to post-natal day 3 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.
Doses / concentrationsopen allclose all
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 140271.
Control animals:
yes, concurrent vehicle
Details on study design:
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 corn oil, the vehicle used in this study. The 4 groups comprised 10 male and 10 female Wistar rats. The test item formulation was prepared once in every ten days and stored at 2-8 °C. The test item was suspended in corn oil and dose volumes were adjusted individually based on weekly body weight measurements. The administration volume was 5 mL/kg body weight.

Examinations

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

Functional Observations
Multiple detailed behavioural observations were made in the week before the first treatment and during the last week of the treatment in 5 randomly selected males and on day 3 of the lactation period in 5 randomly selected females (only lactating females were evaluated) outside the home cage using a functional observational battery of tests. Except 1/5 selected females of LD group was non-pregnant and the FOB in the last week of treatment was not performed. 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.
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 were examined from 5 randomly selected males and females (only lactating females were evaluated) of each group at the end of the treatment prior to or as part of the sacrifice of the animals. Except 1/5 selected females of LD group was non-pregnant and hematology parameters was not examined on this animal. 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) of each group were examined at the end of the treatment prior to or as part of the sacrifice of the animals. Except 1/5 selected females of LD group was non-pregnant and blood coagulation parameters was not examined on this animal. 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) of each group were examined at the end of the treatment prior to or as part of the sacrifice of the animals. Except 1/5 selected females of LD group was non pregnant and clinical biochemistry parameters was not examined on this animal. 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 prior to or as part of the sacrifice of the animals. Additionally, urine colour/ appearance were recorded. Urinalysis was also performed on 4 females of control and dose groups. 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 male animals were sacrificed after the completion of the mating period (total dosing period: 28 days) on study day 29 or 30, while female animals were sacrificed on post-natal day 4 using an anaesthesia (ketamine/xylazin, 2:1, Pharmanovo, lot no: 24664 (expiry date: 06/2015) and lot no. 24863 (expiry date : 10/2015) and Serumwerk, lot no: 01213 (expiry date: 10/2015) and lot no.: 00513 (expiry date: 05/2015). All surviving pups were killed by decapitation on post natal day 4.
Dead pups and pups sacrificed on day 4 post-partum were carefully examined externally for gross abnormalities. Non-pregnant females (females 42 and 71) were sacrificed on day 26 from day of sperm positive vaginal smear or from the last day of mating period. 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 fixed in modified Davidson’s Solution for 24 hours and then 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. 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 histopathology was carried out on the preserved organs and tissues (see above) of 5 randomly selected male and female animals (only lactating females were evaluated) of the control and high dose groups which were sacrificed at the end of the treatment period. All gross lesions from all groups, were examined by light microscopy. 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. Because of possible treatment-related findings noted in the high dose group, spleen and bone marrow (sternum) from 5 selected male and female animals of the low and medium dose groups (groups 2 and 3, respectively) were examined to establish a no-effect level. Testes, epididymides, ovaries, uterus with cervix, vagina, accessory sex organs (prostate, seminal vesicle with coagulating gland) were trimmed,
embedded into paraffin, cut at an approximated thickness of 2-4 µm and stained with hematoxylin and eosin and were examined in all animals. For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle for the evaluation
of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides. Histological processing of tissues to microscope slides was performed at the GLP-certified contract laboratory AnaPath GmbH, AnaPath Services, Hammerstrasse 49, 4410 Liestal, Switzerland (test site for tissue processing). Histopathological evaluation was performed at the GLP-certified contract laboratory AnaPath GmbH, Buchsweg 56, 4625 Oberbuchsiten, Switzerland (test site for histopathology). The study phases from test site 1 and 2 was performed in compliance with the Swiss Ordinance relating to Good Laboratory Practice adopted 18 May 2005 [SR 813.112.1] (Status as of 01 December 2012) [9]. Blocking, embedding, cutting, H&E staining and scientific slide evaluation were performed according to the corresponding SOP’s of the test sites. The principal investigator for histopathological tissue processing sent all raw data (including blocks, slides, paper raw data, statement of compliance and quality assurance statement) to the study director. The principal investigator for histopathological evaluation provided the histopathology results to the study director by e-mail and sent a pathology phase report to the study director upon the completion of the study.
Statistics:
A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical
biochemistry and absolute and relative organ weights were performed for each gender by comparing values of dosed with control animals of the
main groups using a one-way ANOVA and a post-hoc Dunnett Test. These statistics were performed with GraphPad Prism V.6.01 software
(p<0.05 was considered as statistically significant).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
no adverse effects
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
no adverse effects
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
Clinical biochemistry findings:
no effects observed
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:
No mortality occurred in the control or any of the dose groups during the study period.

Clinical Observations:
There were no clinical signs of toxicological relevance in the dose groups when compared to control. However, there were red feces in HD group and signs of moving the bedding in males and/ or females of MD and/ or HD groups. The red feces were due to test item colour and the moving the bedding was considered due to local effect of test item. There were other clinical signs including alopecia, slight abnormal breathing, nasal discharge, salivation and regurgitation in a few animals of control and/ or dose groups. These were considered incidental. During the weekly detailed clinical observation, no changes or differences between the groups and the baseline values.

Functional Observations:
No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period. There were no biologically relevant differences in body temperature between the groups.

Body Weight Development:
In males and female, there were no adverse changes of toxicological relevance on body weight and body weight gain. However, In males there was statistically significantly lower weight gain in HD groups after premating days 7-14. There was also statistically significantly lower weight
gain in MD group after mating/ post matings days 7-14. There were also lower weight gain in LD and HD groups after the 1st and last week of mating/postmating period. These changes were either transient and/ or without dose response relation. Therefore, the finding was not
considered to be an adverse effect of treatment. In females, there was statistically significantly higher weight gain after lactation days 0-4 in HD group. This higher weight gain was within the range of historical control data and was not considered likely to be an effect of treatment.

Food Consumption:
In males and females, there were no adverse changes of toxicological relevance for food consumption in dose groups when compared to control. There were no statistically significant differences between the dose and control group values. However, in males there was very marginal decrease in food consumption after premating period 7-14 in HD group and in females the food consumption was higher in HD group after lactation days 0-4. These changes in food consumption in males or females correlated with changes in weight gain. In the absence of statistically significant differences, the lower or higher food intake in males and females were not considered an adverse effect of treatment.

Haematology and Coagulation:
In males and females, there were findings of toxicological relevance in MD and HD groups. In males, there was statistically significantly lower mean RBC, HCT and Hb in MD and HD groups; statistically significantly higher mean MCV and reticulocyte count in MD and HD group; statistically significantly higher mean MCH in HD group. In females, there was statistically significantly lower mean RBC, Hb, HCT value in HD group and statistically significantly higher mean MCV in MD and HD group, MCH and reticulocyte count in HD group. There findings were associated with microscopically observed erythrocytic extramedullary hemopoiesis in the spleen of both sexes of MD and HD groups, along with an increase in incidence and/or severity of hemosiderin deposition in the spleen and erythropoiesis in the bone marrow. These findings are most likely to be the histomorphologic indicator of hemolytic anemia and were considered to be adverse event attributable to treatment with the test item. There was also statistically significantly lower MCHC value in male MD group and statistically significantly higher Eosinophil and statistically significantly lower LUC count in female MD group. These were not in dose relation and were not considered related to treatment. In males and females, there were no effect on the blood coagulation parameters.


Clinical Biochemistry:
In males and females, there were no effect on the measured clinical biochemistry parameters of dose groups when compared to the control.

Urinalysis:
In males and females, there were no effect on urine parameters of dose groups when compared to the control.

Pathology:
Enlarged of spleen, which correlated microscopically with increased extramedullary erythrocytic hemopoiesis, was recorded in three males of
MD and 4 males and one female of HD group. This was considered to be treatment-related change (see 12.11).
“Discoloured pale” or “Discoloured” of the kidney was recorded in all male groups including the control group and in one female of MD group.
However, there were no corresponding microscopic findings in any animals.
All other gross lesions recorded were within the range of normal background alterations which may be recorded in animals of this strain and age.


Organ Weight:
In males and females, there was statistically significantly higher absolute and relative (to terminal body weight) spleen weight in MD and HD groups. These findings were associated with the increase in mean severity of erythrocytic extramedullary hemopoiesis in the spleen recorded in both sexes of MD and HD groups. These findings were considered to be adverse event attributable to treatment with the test item. There was also statistically significantly lower absolute and relative (to terminal body weight) pituitary gland weight in MD group of male animals. There was also statistically significantly higher relative kidney weight in HD group. In the absence of dose response relation, the finding was not considered likely to be related to treatment.

Histopathology:
Microscopically, the treatment-related findings were recorded in the spleen and bone marrow.
The increase in mean severity of erythrocytic extramedullary hemopoiesis in the spleen was recorded in both sexes of MD and HD groups, along with an increase in incidence and/or severity of hemosiderin deposition in the spleen and erythropoiesis in the bone marrow. These
findings are most likely to be the histomorphologic indicator of hemolytic anemia and were considered to be adverse event attributable to treatment with the test item. The sperm stages of testes were checked on completeness of cell populations, completeness of stages and degenerative changes. Nothing particular was observed in testes of animal no. 2 (control), which was mated with female no. 42 not giving birth. Slight spermatic granulomas and minimal mononuclear cell foci in epididymides and minimal alveolitis in lungs were recorded in male no. 2. However, similar minimum to slight lesions are often observed in male animals that can produce pregnancy to the pairing females, and therefore, it is unlikely that these findings were related to the infertility. In male no. 31 (HD group), marked tubular atrophy (ie. so-called sertoli cell-only tubules) in testes and marked oligospermia (ie. azoospermia) in epididymides were observed, and this was considered to be the cause why the paired female no. 71 did not give birth. Complete tubular atrophy sometimes happens spontaneously in the male rats of this strain and age, and sporadic occurrence with only one case is unlikely to be related to treatment with the test item.
Nothing particular was observed in the female reproductive organs (ovaries, uterus with cervix and vagina) of the paired female nos. 42 (control) and 71 (HD group) which did not give birth.
As a result of the detailed examination of the testis, there were no abnormalities on the completeness of stages and cell populations, except for male no. 31 (HD group). In the testis of male no. 31, it was impossible to evaluate the stages and cell population because of complete tubular atrophy. However, this was judged to be spontaneous change as described above.
There was no dose response relationship in any findings recorded in the male reproductive organs including testes, epididymides, prostate glands, coagulating glands and seminal vesicles which were examined in this study, and therefore, all histologic findings were considered to be spontaneous changes which may be recorded in animals of this strain and age. The remainder of findings recorded was within the range of normal background lesions which may be recorded in animals of this strain and age, or were considered to be accidental changes which can occur in the toxicology studies using the rodent models.

Effect levels

Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the data generated from this combined repeated dose oral toxicity and reproduction/ developmental toxicity screening test with FAT 36034/W TE, the no observed adverse effect level (NOAEL) is considered to be 100 mg/kg/ d for systemic toxicity in males and females and 1000 mg/kg/d for reproduction/ developmental toxicity.
Executive summary:

The aim of this study was to assess the possible effects of FAT 36034/W TE on male and female fertility and embryofetal development after repeated oral dose administrationin 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 corn oil, the vehicle used in this study. The 4 groups comprised 10 male and 10 femaleWistarrats. The control group was shared with BSL study 140271. 


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 2-8 °C. The test item was suspended in corn oil and dose volumes were adjusted individually based on weekly body weight measurements. The administration volume was 5 mL/kg body weight. During the period of administration, the animals were observed each day for signs of toxicity. At the conclusion of the test, 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 and females 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 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 (females 42 and 71) were sacrificed on day 26 from day of sperm positive vaginal smear or from the last day of mating period. Pups sacrificed on postnatal day 4 and those found dead, 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 groupsas well as all gross lesions from all groups, were examined by light microscopy. 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. Because of possible treatment-related findings noted in the high dose group, spleen and bone marrow (sternum) from 5 selected male and female animals of the low and medium dose groups were examined to establish a no-effect level.


 


Summary Results


No mortality occurred in the control or any of the dose groups during the study period. There were no clinical signs of toxicological relevance in the dose groups when compared to control. However, there were red feces in HD group and signs of moving the bedding in males and/ or females of MD and/ or HD groups. The red feces were due to test item colour and the moving the bedding was considered 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 female, there were no adverse changes of toxicological relevance on body weight, body weight gain and food consumption in dose groups when compared to the concurrent control. In males and females, there were findings of toxicological relevance on haematological parameters in MD and HD groups. In males, there was statistically significantly lower mean RBC, HCT and Hb in MD and HD groups; statistically significantly higher mean MCV and reticulocyte count in MD and HD group; statistically significantly higher mean MCH in HD group. In females, there was statistically significantly lower mean RBC, Hb, HCT value in HD group and statistically significantly higher mean MCV, MCH and reticulocyte count in HD group. In males and females, there was no effect on the blood coagulation, clinical biochemistry and urinary parameters of dose groups when compared to the control group. Enlarged spleen, which correlated microscopically with increased extramedullary erythrocytic hemopoiesis, was recorded in 3/10 males of MD group, in 4/10 males and one female of HD group, was considered to be treatment-related change. Discoloured pale or discoloured of the kidney was observed macroscopically in all male groups including the control group and in one female of MD group. However, there were no corresponding microscopic findings in any animals.


All other gross lesions recorded were within the range of normal background alterations which may be recorded in animals of this strain and age. In males and females, there was statistically significantly higher absolute and relative (to terminal body weight) spleen weight in MD and HD groups, when compared to controls.The change in spleen weight correlated with the increase in mean severity of erythrocytic extramedullary hemopoiesis in the spleen recorded in both sexes of MD and HD groups. These findings are considered to be adverse events attributable to treatment with the test item.


Micoscopically, the treatment-related findings were recorded in the spleen and bone marrow. The increase in mean severity of erythrocytic extramedullary hemopoiesis in the spleen was recorded in both sexes of MD and HD groups, along with an increase in incidence and/or severity of hemosiderin deposition in the spleen and erythropoiesis in the bone marrow. These findings are most likely to be the histomorphologic indicator of hemolytic anemia and were considered to be adverse event attributable to treatment with the test item. There were no effects on the duration of precoital interval and the duration of gestation in dose group animals when compared to the controls. There were no effects on the pre and post natal data including the no. of corpora lutea, no. of implantation sites, no. of live pups (PND 0 and 4), percent pre and post implantation loss. There were no effects on the copulation, fertility and delivery indices of dose groups when compared to controls. A fertility index (number of pregnant females/ number of copulated females X 100) observed in the control and the dose groups were C 90 %, LD 100 %, MD 100% and HD 100 %. There was no effect on viability index of dose groups when compared to control. There were no effects of toxicological relevance on the litter data including no. of pups born, no. of live pups, no. of still birth and no. of runts on PND 0 and no. of male and female pups and sex ratio on PND 0 and PND 4. There were no effects of toxicological relevance on litter weight data including pup mean weight, total litter weight, male litter weight and female litter weight measured on PND 0 and PND 4. No effects on the survival of the pups from PND 0 to PND 4 were observed in any dose group when compared to controls. There were no gross external findings of pups of toxicological relevance in any of the dose groups when compared to the controls.


 


Conclusion


On the basis of this reproduction/developmental toxicity screening test with FAT 36034/W TE in male and female Wistar rats with dose levels of 100, 300, and 1000 mg/kg/d the following conclusions can be made: Haemolytic anaemia was observed in both genders at 300 and 1000 mg/kg/d. Microscopically, this was evident by extramedullary haematopoiesis in bone marrow and spleen. This also correlated with dose-dependently lower levels of red blood cell parameters (RBC, Hb and Hct) and a concurrent increase in reticulocytes and mean cell haemoglobin and mean cell volume. Whereas the effects were moderate at 1000 mg/kg/d, only a slight anaemia was found at 300 mg/kg/d. There were no effect on the reproduction/ developmental parameters.


Based on the data generated from this combined repeated dose oral toxicity and reproduction/ developmental toxicity screening test with FAT 36034/W, the no observed adverse effect level (NOAEL) is considered to be 100 mg/kg/ d for systemic toxicity in males and females and 1000 mg/kg/d for reproduction/ developmental toxicity.