[No public or meaningful name is available]

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study; RA from supporting substance

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

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test System:
Species/strain: Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Sex: 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: males: 248 - 281 g (mean: 267.83 g, ± 20 % = 214.26 – 321.39 g) females: 171 - 202 g (mean: 184.33 g, ± 20 % = 147.46 – 221.19 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. 1526)
Certificates of food, water and bedding are filed at BSL BIOSERVICE- Adequate acclimatisation period (at least 5 days) under laboratory conditions
Preparation of the Animals: Prior to the start of the treatment period a detailed clinical observation outside the home cage was made. None of the animals showed any adverse clinical signs before the study initiation. Before the first administration, all animals to be used for the study were weighed. Mean body weight of the group housed animals was used to assign all animals to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females, respectively, while ensuring to keep each animal with its initial cage partner if possible. Each animal was marked with its identification number by individual ear tattoo.

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

The test item was weighed into a tared plastic vial on a precision balance and suspended in aqua ad injectionem. Homogeneity of the test item in the vehicle was maintained by using an ultra turrax. 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:100mg/kg bw/d
Medium Dose: 300mg/kg bw/d
High Dose: 1000mg/kg bw/d
Since little or no toxicity was anticipated for the test substance, the highest dose level was set at 1000 mg/kg bw/d corresponding to a limit dose for this study. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dose-related response and a NOAEL. The animals in the control group were handled in an identical manner to the test group subjects and received aqua ad injectionem using the same volume as used for the dose groups.For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured. The administration volume was 5 mL/kg body weight.

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. 140307. The results are reported in the annex of the final report.

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.

Frequency of treatment:

None

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Low Dose

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

Medium Dose

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

High Dose

No. of animals per sex per dose:

80 animals (40 males and 40 females) were included in the study (10 male and 10 female animals per group).

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes - Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
Time schedule: 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. 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 lactation days in 5 randomly selected females (only lactating females were evaluated) outside the home cage using a functional observational battery of tests.

BODY WEIGHT: Yes
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: 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.

FOOD EFFICIENCY: Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: yes

HAEMATOLOGY: Yes
Time schedule for collection of blood: at the end of the treatment period
Anaesthetic used for blood collection: Yes (ketamine/xylazin, 2 :1)
Animals fasted: No
How many animals: five randomly selected males and females of each group
Parameters 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), prothrombin time (PT) and activated partial thromboplastin time (aPTT)

CLINICAL CHEMISTRY: Yes
Time schedule for collection of blood: at the end of the treatment period
Animals fasted: No
How many animals: five randomly selected males and females of each group
Parameters examined: alanine aminotransferase (ALAT), aspartate-aminotransferase (ASAT), alkaline phosphatase (AP), creatinine (Crea), total protein (TP), albumin (Alb), urea, total bile acids (TBA), total cholesterol (Chol), glucose (Gluc), sodium (Na) and potassium (K)

URINALYSIS: Yes
Time schedule for collection of urine: 5 randomly selected males and females at necropsy.
Metabolism cages used for collection of urine: No
Animals fasted: No
Parameters were examined: urine colour/ appearance, specific gravity, nitrite, ph-value (pH), protein, glucose, ketone bodies (ketones), urobilinogen (ubg), bilirubin, blood, leukocytes

NEUROBEHAVIOURAL EXAMINATION: Yes
Time schedule for examinations: in the week before the first treatment and during the last week of the treatment
Dose groups that were examined: all- Battery of functions tested: sensory activity / grip strength / motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
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.


HISTOPATHOLOGY: Yes
A full histopathology was carried out on the preserved organs and tissues 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. These examinations were not extended to animals of all other dosage groups as no treatment-related changes were observed in any organ of the high dose group. 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. All gross lesion macroscopically identified was examined microscopically 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.

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:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

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:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

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: In males and females, there were no clinical signs of toxicological relevance in any of the dose groups when compared to the control. However, there were isolated incidences of nasal discharge, alopecia on various body parts, moving the bedding, regurgitation, slight to moderate piloerection, aggressiveness, chromodacryorrhea at the right eye, slightly reduced spontaneous activity and emisis (blue mouth and nose) in a few control and/or dose group animals. There were also discoloured blue faeces observed in male and female animals of high dose group which could be attributed to the colur of the test item. During the weekly detailed clinical observation, no significant changes or differences between the groups were observed.

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. However, in males statistically significantly lower body temperature was observed in the last week of treatment in MD and HD group when compared with the controls. As this difference was marginal and overall there was no effect on the general health condition, body weight development and food consumption of these animals this effect on body temperature is not considered to be adverse.

Body Weight Development: In males, no adverse effects of toxicological relevance of FAT 20033/L were observed throughout the study period on body weight and body weight gain when compared to the controls. A statistical analysis of data revealed no statistically significant difference between the dose groups and corresponding control group. In females, no statistically significant difference between the dose groups and corresponding control group was observed on body weight and body weight gain. However, marginally lower (2-6 %) group mean body weight during gestation and lactation period and body weight gain during gestation period was observed in high dose group when compared to the controls. As this difference was marginal, values were within the range of biological variation and this effect on body weight development was attributed to low body weight values from single high dose female (no. 80) in which no live pups were born, no toxicological relevance could be attributed to this effect on female body weight development.

Food Consumption:Throughout the study period, there were no statistically significant effect of FAT 20033/L on food consumption of males and females of the dose groups when compared to the control group. However, marginally lower group mean food consumption from gestation day 7 to post-natal day 4 was observed in high dose group when compared to the controls. As this difference was correlated with body weight development it is not considered to be no toxicologically relevant.Haematology and Coagulation: In males and females, there were no adverse effects of toxicological relevance on parameters of haematology and blood coagulation parameters in dose groups when compared with corresponding control. However, in males there was marginally but statistically significantly higher reticulocyte count in LD and HD group when compared with the controls. As the mean value was in the range of historical control data and in the absence of dose dependency, the finding was not considered to be toxicological relevant.In females, statistical analysis of haematology and blood coagulation data revealed statistically significantly higher eosinophils in LD group (0.62 %) when compared with the controls (0.16 %). As the mean value was in the range of historical control data and due to lack of dose dependency, this effect was not considered to be toxicologically relevant.

Clinical Biochemistry:In males and females, there were no adverse changes of toxicological relevance for clinical biochemistry in dose groups when compared to the controls. However, statistically significantly higher creatinine in HD males (26.80) and statistically significantly lower potassium in HD females (3.45) was observed when compared with the corresponding controls. Since all mean and most individual values of clinical biochemistry of males and females were within the historical control range, the effect on creatinine in male HD group and potassium in female HD group was not considered to be an adverse effect due to the treatment.Urinalysis: There were no signs of toxicological relevance in urine parameters of males and females dose groups when compared to the controls.

Pathology: No gross lesions were observed in this study that could be attributed to treatment with the test item. All gross lesions recorded in male animals (yellow spots and soft nodule on epididymides, dilated renal pelvis of kidneys, increase in size of thymus, reduced size of thyroid and parathyroid, lobus cadatus (liver) herniation, increase in size of seminal vesicles with coagulating glands, prostate gland and heart, slightly reduced size of spleen, testes and pituitary gland in control and/ or dose group animals) and in female animals (kidneys doscoloured pale and enlarged adrenals in LD group ) were within the range of normal background alterations which may be recorded in animals of this strain and age, or were considered incidental macroscopic findings that did not correlate with microscopic changes.Bluish or greenish doscolouration of various organs (esophagus, stomach, small and large intestines, Peyer’s patches, mesenteric lymph node, and/or trachea) of the high-dose was observed in males and was considered to reflect the colour of the test item or the color being formed by the test item mixing with the intestinal contents. However, there was no histologic alteration correlated with macroscopic appearances.Greenish/green discoloration was also recorded in the kidney of some animals including the control animals. The expression of the colour was the same as the above mentioned alteration. However, the coloration of this case is unlikely to be derived from the test item, because this was observed in the control animals too, and probably this is due to the status of blood removal at the necropsy. For example, it is empirically known that the kidney appears greenish/green after the whole body or renal blood perfusion was performed with the use of saline or fixative. In any event, there were no histologic abnormalities which might be related to macroscopic coloration. All other gross lesions recorded were within the range of normal background alterations which may be recorded in animals of this strain and age, or were considered to be incidental macroscopic appearances that did not correlate with microscopic changes. Organ Weight:In males and females, there were no statistically significant differences on absolute and relative organ weights of the dose groups when compared with the corresponding controls and all group mean and individual values were comparable with the controls.

Histopathology: Macroscopic alteration of various organs due to bluish /greenish discolouration was considered to be the coloration derived from the unique colour of the test item and/or the colour being formed by the test item mixing with the intestinal contents. However, there was no histologic alteration correlated with macroscopic appearances.Discoloured kidney of some animals including the control animals was probably due to the status of blood removal at the necropsy. There were no histologic abnormalities which might be related to macroscopic coloration. All other gross lesions recorded were within the range of normal background alterations which may be recorded in animals of this strain and age, or were considered to be incidental macroscopic appearances that did not correlate with microscopic changes. Micropscopically, no histomorphologic evidence of toxicological properties observed in any organs and tissues including the male reproductive organs (testes, epididymides, prostate and seminal vesicles) and the female reproductive organs (ovaries, uterus with cervix and vagina). In addition, as a result of the detailed examination of the testis, it was judged that there were no treatment-related effects on the testicular histomorphology including spermatogenesis and interstitial cell structure. All findings recorded in the organs and tissues examined in this study were within the range of normal background lesions which may be recorded in animals of this strain and age.In conclusion, no histomorphological evidence of toxicity in any organs and tissues of male and females was established up to 1000 mg/kg bw/d.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Thus, the no observed adverse effect level (NOAEL) of FAT 20033/L TE is considered to be 1000 mg/kg/d for systemic toxicity and also for reproduction/ developmental toxicity in males and females.

Critical effects observed:

not specified

Conclusions:

Based on this combined repeated dose oral toxicity and reproduction/developmental toxicity screening test with FAT 20033/L in male and female Wistar rats with dose levels of 100, 300, and 1000 mg/kg/d the following conclusions can be made: There were no adverse toxicological effects in the adult males and females or significant reproductive and developmental effects in pups at any administered dose. Thus, the no observed adverse effect level (NOAEL) of FAT 20033/L is considered to be 1000 mg/kg/d for systemic toxicity and also for reproduction/ developmental toxicity in males and females.

Executive summary:

The study was conducted to assess the possible effects of FAT 20033/L at the dose level of 100, 300 and 1000 mg/kg repeated dose administration to male and female Wistar rats according to OECD 422 guideline. 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 or 29 days were completed. Animals of an additional control group were handled identically as the dose groups but received aqua ad injectionem, the vehicle used in this study. The 4 groups comprised 10 male and 10 female Wistar rats.

 

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 once in every ten days and stored at 2-8 °C. 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, surviving animals were sacrificed and observed macroscopically. Body weight and food consumption were measured weekly. 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. Haematological, clinical biochemistry and urine analysis evaluations were performed on blood samples collected at terminal sacrifice from five males and five randomly selected lactating females from each group. A full histopathological evaluation of the tissues was performed of 5 randomly selected male and female animals of the control and high dose groups. Gross lesions from all groups were examined by light microscopy. No treatment related adverse effects were found in any of the dose group.No gross lesions were observed in this study those could be attributed to treatment with the test item. All gross lesions were within the range of normal background alterations which may be recorded in animals of this strain and age, or were considered to be incidental macroscopic findings that did not correlate with microscopic changes. There were no adverse toxicological effects in the adult males and females at any administered dose. Thus, the no observed adverse effect level (NOAEL) of FAT 20033/L is considered to be 1000 mg/kg/d for systemic toxicity in males and females.

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)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 1-amino-9,10-dihydro-4-[5-[(2-hydroxyethyl)sulphamoyl]-3,4-xylidino]-9,10-dioxoanthracene-2-sulphonate

EC Number:

247-269-2

EC Name:

Sodium 1-amino-9,10-dihydro-4-[5-[(2-hydroxyethyl)sulphamoyl]-3,4-xylidino]-9,10-dioxoanthracene-2-sulphonate

Cas Number:

25797-81-3

Molecular formula:

C24H23N3O8S2.Na

IUPAC Name:

sodium 1-amino-4-({3-[(2-hydroxyethyl)sulfamoyl]-4,5-dimethylphenyl}amino)-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate

Test material form:

other: solid

Details on test material:

None

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: AT-PD13-359A1
- Expiration date of the lot/batch: 07 January 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, protected from light

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:
males: 248 - 281 g (mean: 267.83 g, ± 20 % = 214.26 – 321.39 g)
females: 171 - 202 g (mean: 184.33 g, ± 20 % = 147.46 – 221.19 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. 1526)
- 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

Type of inhalation exposure (if applicable):

not specified

Vehicle:

water

Details on exposure:

The test item was weighed into a tared plastic vial on a precision balance and suspended in aqua ad injectionem. Homogeneity of the test item in the vehicle was maintained by using an ultra turrax. 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

Since little or no toxicity was anticipated for the test substance, the highest dose level was set at 1000 mg/kg bw/d corresponding to a limit dose for this study. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dose-related response and a NOAEL. For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured. The administration volume was 5 mL/kg body weight.

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 pregnancy. If the vaginal smear of a particular female was not found to be sperm-positive, the actual stage of the estrus cycle on that day was documented. The day of the vaginal plug and/or sperm was considered as day 0 of gestation. The cages were arranged in such a way that possible effects due to cage placement were minimized.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Each dosing concentration was analysed with respect to the target nominal concentration. Stability and homogeneity of the test item in the vehicle were analysed for the low and high dose concentrations. Samples for the nominal concentration verification were taken in study week 1 (first week of pre-mating period), 3 (first week of mating), 5 (gestation) and in the last week of the study (gestation/lactation) from all groups (16 samples). Samples for homogeneity analysis were taken from the top, middle and bottom of the high dose and the low dose formulation in study week 1 and 5 (12 samples). Samples for stability analysis were taken in the first week of the study [0 hours, 6 hours (at RT) and 10 days after the preparation (stored at 2-8 °C)] from high and low dose formulations (6 samples). All 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. 140307.

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.

Frequency of treatment:

daily

Details on study schedule:

Arrival of the Test Item: 22 January 2014
Study Initiation Date: 22 April 2014
1st Amendment to Study Plan: 25 June 2014
2nd Amendment to Study Plan: 11 September 2014
Delivery of Animals: 29 April 2014
Acclimatisation Period: 29 April 2014 – 05 May 2014
Experimental Starting Date: 05 May 2014
Date of Behavioural Observation: 07 May 2014- 30 June 2014
Treatment Period Males: 08 May 2014 – 06 June 2014
Treatment Period Females: 08 May 2014 – 01 July 2014
Necropsies Males: 05 June 2014 and 06 June 2014
Necropsies Females: 17 June 2014 – 01 July 2014
Experimental Completion Date: 01 July 2014
Completion Date of Delegated
Phase (Histopathology): 29 September 2014
Completion Date of Delegated
Phase (Formulation Analysis): 29 September 2014

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Low Dose

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

Medium Dose

Doses / concentrations 3

Dose / conc.:

3 000 mg/kg bw/day (actual dose received)

Remarks:

High Dose

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: 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. 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 lactation days in 5 randomly selected females (only lactating females were evaluated) outside the home cage using a functional observational battery of tests.

BODY WEIGHT: Yes
- 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:
- 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.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: yes

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: [yes]
- If yes, maximum of [...] pups/litter ([...]/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in offspring:
[number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities]

GROSS EXAMINATION OF DEAD PUPS:
[yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.]

Postmortem examinations (parental animals):

SACRIFICE
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 along with pups using an anaesthesia (ketamine/xylazin, 2:1, Pharmanovo, lot no: 24863, expiry date: 10/2015 and Serumwerk, lot no: 00513, expiry date: 05/2015) was used for the females and decapitation for the pups.

GROSS NECROPSY
Pups sacrificed on day 4 post-partum were carefully examined externally for gross abnormalities.
Non pregnant (79) and non mated females (41 and 78) were sacrificed on study day 26 from the day of confirmed mating and day 26 from the end of 2 weeks mating period, respectively. 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. The number of corpora lutea and implantation sites was recorded for any females sacrificed 26 days after the end of the pairing period with no evidence of mating and for any females sacrificed on day 26 post-coitum due to non-delivery.


HISTOPATHOLOGY / ORGAN WEIGHTS
A full histopathology was carried out on the preserved organs and tissues 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. These examinations were not extended to animals of all other dosage groups as no treatment-related changes were observed in any organ of the high dose group. 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. All gross lesion macroscopically identified was examined microscopically 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.

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).

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Except for confirmation of mating

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

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

Clinical Observations:
In males and females, there were no clinical signs of toxicological relevance in any of the dose groups when compared to the control. However, there were isolated incidences of nasal discharge, alopecia on various body parts, moving the bedding, regurgitation, slight to moderate piloerection, aggressiveness, chromodacryorrhea at the right eye, slightly reduced spontaneous activity and emisis (blue mouth and nose) in a few control and/or dose group animals. There were also discoloured blue faeces observed in male and female animals of high dose group which could be attributed to the colur of the test item.
During the weekly detailed clinical observation, no significant changes or differences between the groups were observed.


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. However, in males statistically significantly lower body temperature was observed in the last week of treatment in MD and HD group when compared with the controls. As this difference was marginal and overall there was no effect on the general health condition, body weight development and food consumption of these animals this effect on body temperature is not considered to be adverse.

Body Weight Development:
In males, no adverse effects of toxicological relevance of FAT 20033/L TE were observed throughout the study period on body weight and body weight gain when compared to the controls. A statistical analysis of data revealed no statistically significant difference between the dose groups and corresponding control group.
In females, no statistically significant difference between the dose groups and corresponding control group was observed on body weight and body weight gain. However, marginally lower (2-6%) group mean body weight during gestation and lactation period and body weight gain during gestation period was observed in high dose group when compared to the controls. As this difference was marginal, values were within the range of biological variation and this effect on body weight development was attributed to low body weight values from single high dose female (no. 80) in which no live pups were born, no toxicological relevance could be attributed to this effect on female body weight development.

Food Consumption:
Throughout the study period, there were no statistically significant effect of FAT 20033/L TE on food consumption of males and females of the dose groups when compared to the control group. However, marginally lower group mean food consumption from gestation day 7 to post-natal day 4 was observed in high dose group when compared to the controls. As this difference was correlated with body weight development it is not considered to be no toxicologically relevant.

Pathology:
No gross lesions were observed in this study that could be attributed to treatment with the test item. All gross lesions recorded in male animals (yellow spots and soft nodule on epididymides, dilated renal pelvis of kidneys, increase in size of thymus, reduced size of thyroid and parathyroid, lobus cadatus (liver) herniation, increase in size of seminal vesicles with coagulating glands, prostate gland and heart, slightly reduced size of spleen, testes and pituitary gland in control and/ or dose group animals) and in female animals (kidneys doscoloured pale and enlarged adrenals in LD group ) were within the range of normal background alterations which may be recorded in animals of this strain and age, or were considered incidental macroscopic findings that did not correlate with microscopic changes.
Bluish or greenish doscolouration of various organs (esophagus, stomach, small and large intestines, Peyer’s patches, mesenteric lymph node, and/or trachea) of the high-dose was observed in males and was considered to reflect the colour of the test item or the color being formed by the test item mixing with the intestinal contents. However, there was no histologic alteration correlated with macroscopic appearances.
Greenish/green discoloration was also recorded in the kidney of some animals including the control animals. The expression of the colour was the same as the above mentioned alteration. However, the coloration of this case is unlikely to be derived from the test item, because this was observed in the control animals too, and probably this is due to the status of blood removal at the necropsy. For example, it is empirically known that the kidney appears greenish/green after the whole body or renal blood perfusion was performed with the use of saline or fixative. In any event, there were no histologic abnormalities which might be related to macroscopic coloration.
All other gross lesions recorded were within the range of normal background alterations which may be recorded in animals of this strain and age, or were considered to be incidental macroscopic appearances that did not correlate with microscopic changes.


Organ Weight:
In males and females, there were no statistically significant differences on absolute and relative organ weights of the dose groups when compared with the corresponding controls and all group mean and individual values were comparable with the controls.

Histopathology:
Macroscopic alteration of various organs due to bluish /greenish discolouration was considered to be the coloration derived from the unique colour of the test item and/or the colour being formed by the test item mixing with the intestinal contents. However, there was no histologic alteration correlated with macroscopic appearances.
Discoloured kidney of some animals including the control animals was probably due to the status of blood removal at the necropsy. There were no histologic abnormalities which might be related to macroscopic coloration. All other gross lesions recorded were within the range of normal background alterations which may be recorded in animals of this strain and age, or were considered to be incidental macroscopic appearances that did not correlate with microscopic changes.
 
Micropscopically, no histomorphologic evidence of toxicological properties observed in any organs and tissues including the male reproductive organs (testes, epididymides, prostate and seminal vesicles) and the female reproductive organs (ovaries, uterus with cervix and vagina).
In addition, as a result of the detailed examination of the testis, it was judged that there were no treatment-related effects on the testicular histomorphology including spermatogenesis and interstitial cell structure.
All findings recorded in the organs and tissues examined in this study were within the range of normal background lesions which may be recorded in animals of this strain and age.
In conclusion, no histomorphological evidence of toxicity in any organs and tissues of male and females was established up to 1000 mg/kg bw/d.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Thus, the no observed adverse effect level (NOAEL) of FAT 20033/L TE is considered to be 1000 mg/kg/d for systemic toxicity and also for reproduction/ developmental toxicity in males and females.

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

Litter Data:
No treatment related effect of FAT 20033/L was observed on total number of pups born, number of males, number of male and females, sex ratio, live pups, still birth and runt on PND 0 and number of males, number of females, sex ratio and total number of live pups on PND 4. However, there was slightly lower litter size observed in HD group without achieving statistical significance compared to the control group. This decrease was attributed to incidental finding that one female (no. 80) which showed implantation sites at necropsy and there were no live pups born. As this mean litter size was within the historical control data range (5.91-15.46), it is not considered to be an adverse effect of treatment with test item.

Litter Weight Data:
Statistical analysis of litter weight data revealed no treatment related effect of FAT 20033/L on group mean litter weight, total litter weight, male litter weight and female litter weight on PND 0 and PND 4 when compared with the controls.

Precoital Interval and Duration of Gestation:
No test item related effect was observed on precoital interval and duration of gestation and values were comparable between the dose groups and controls. All pregnancies resulted in normal births except female No. 80 from HD group was not littered and showed implantation sites noted at the time of necropsy on Gestation day 26.
Majority of females in control and treatment groups showed evidence of copulation during 14 days mating period except two females from MD group (No. 63 and 67) and 1 female from HD group (76) did not show evidence of mating through vaginal smear. However, the animals were pregnant and littered normally. There were also two females (41 and 78) which did not show evidence of mating during 14 day mating period and were sacrificed on day 26 from the end of 2 weeks mating period.
Successful mating resulted in 9/10 pregnancies in the control group, in 10/10 pregnancies in LD and MD and in 8/10 pregnancies in the HD group.
Pre- and Post-Natal Data:
The group mean number of corpora lutea, number of implantation sites, number of live pups born on PND 0 and 4, percentage of pre-implantation loss remained unaffected due to the treatment with test item when compared with the control group. However, there was higher post-implantation loss observed in HD group without achieving statistical significance compared to the control group. This increase was attributed to incidental finding that one female (no. 80) which showed implantation sites at necropsy and there were no live pups born. As this post implantation loss was within the historical control data range, the higher percent post implantation loss was not considered to be an adverse effect of treatment with test item

Reproductive Indices:
There were no effects of FAT 20033/L on the copulation, fertility and delivery indices of the dose groups when compared to the control. The copulation index and fertility index in control and dose groups were 90 % in control, 100 % in LD and MD 100 and 80 % in HD group. However, there was slight reduction in the delivery index of the HD group (87.5%) when compared with the controls. This reduction in delivery index of the HD group was attributed to one female (no. 80) without litter but a number of implantation sites noted at necropsy. This finding was considered to be incidental and without toxicological significance. There was no effect on the viability index in dose group when compared to the control group.

Pup Survival Data:
There was no test item related effect of FAT 20033/L on the survival of the pups from PND 0 through PND 4 in dose groups when compared to the control. However, one pup of control group female no. 49 (pup no. 6) and one pup from LD group female no. 57 (pup no. 4) was missing on PND 1 and 2, respectively. These missing pups were presumably cannibalised by the respective dam. Since a control animal was affected in 1 of 2 incidences, this cannibalism is not considered to be test item related.

Pup External Findings:
No treatment related gross external findings were observed in the pups of any of the dose groups on PND 0 and 4. However, following few external findings were observed:
In control females, one pup from female 45 (pup No. 1) was observed for dark head and two pups from female 48 (pup No. 1 and 2) were found cold to touch at the time of observation on PND 0. In LD females, one pup from female 56 (pup No. 4) had a dark head on PND 0. In MD females, one pup from female 62 and 63 (pup no. 8 and 1, respectively) had a dark snout, 4 pups from female 65 (pup no. 1-4) had dry skin and one pup of this dam had a dark snout on PND 0. All these findings were considered to be spontaneous in nature and not related to the treatment with test item.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (actual dose received)

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

In absence of significant reproductive and developmental effects in pups in this combined repeated dose oral toxicity and reproduction/developmental toxicity screening test with FAT 20033/L in male and female Wistar rats with dose levels of 100, 300, and 1000 mg/kg/d the no observed adverse effect level (NOAEL) of FAT 20033/L is considered to be 1000 mg/kg/d for reproduction/ developmental toxicity.

Executive summary:

The study was conducted to assess the possible effects of FAT 20033/L after repeated dose administration to male and female Wistar rats according to OECD 422 guideline. After 14 days of treatment to both male and female, animals were mated (1:1) for a maximum of 14 days. The subsequent morning onwards the vaginal smears of females were checked to confirm the evidence of mating. After the confirmation of the mating, females were separated and housed individually. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted, sexed and litters weighed within 24 hours of parturition and on day 4 post-partum. The males were sacrificed after completion of the mating period on treatment days 29 and 30 and the females along with their pups were sacrificed on post natal day 4. Non-pregnant females were sacrificed on day 26. Pups sacrificed on post natal day 4 were carefully examined for gross external abnormalities. A full histopathological evaluation of the tissues was performed of 5 randomly selected male and female animals of the control and high dose groups. No treatment related effect was observed on total number of pups born, number of male and females, sex ratio, live pups, still birth and runt on PND 0 and number of males, number of females, sex ratio and total number of live pups on PND 4. Statistical analysis of litter weight data revealed no treatment related effect on group mean litter weight, total litter weight, male litter weight and female litter weight on PND 0 and PND 4 when compared with the controls. There were no test item related effects observed on the precoital interval and the duration of gestation in dose groups when compared to the controls. The group mean number of corpora lutea, number of implantation sites, number of live pups born on PND 0 and 4, percentage of pre-implantation loss remained unaffected after treatment with test item, when compared with the control group. There was higher post-implantation loss observed in HD group that is not considered as test item related. No effects of FAT 20033/L were observed on the copulation, fertility and delivery indices of the dose groups when compared to the control. The copulation index and fertility index in control and dose groups were 90 % in control, 100 % in LD and MD 100 and 80 % in HD group. However, there was slight reduction in delivery index of HD group (87.5 %) was observed when compared with the controls. This finding was considered to be incidental and without toxicological significance. No test item related effect of FAT 20033/L was observed on the survival of the pups from PND 0 through PND 4 in dose groups when compared to the control.

No treatment related gross external findings were observed in the pups of any of the dose groups on PND 0 and 4. Dose formulation analysis for nominal concentration, stability and homogeneity revealed that all dose formulations were stable and homogenous at the time of application throughout the study period. There were no adverse toxicological effects in the adult males and females or significant reproductive and developmental effects in pups at any administered dose. Thus, the no observed adverse effect level (NOAEL) of FAT 20033/L is considered to be 1000 mg/kg/d for reproduction/ developmental toxicity in males and females rats. Thus, the no observed adverse effect level (NOAEL) of FAT 20033/L is considered to be 1000 mg/kg/d for systemic toxicity in males and females.