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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

The no observed adverse effect level (NOAEL) is considered to be 1000 mg/kg bw/d for systemic toxicity in males and females and also for reproduction.

Link to relevant study records
Reference
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
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
Inadvertently, the behavioural observations before the first treatment were not performed in few animals
GLP compliance:
yes (incl. QA statement)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)
Limit test:
no
Justification for study design:
This test is performed on the rat. Although several mammalian species may be used, the rat is the preferred rodent species. This study provides information on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. It further comprises a reproduction/developmental toxicity screening test and is therefore useful to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition, either at an early stage of assessing the toxicological properties of chemicals, or on chemicals of concern. This study emphasizes neurological effects as a specific endpoint and the identification of chemicals with neurotoxic potential, which may warrant further in-depth investigation of this aspect. In addition, this study method may also give a basic indication of immunological effects. The test item is orally administered daily, i.e. 7 days per week in graduated doses to several groups of test animals (male and female), one dose level per group with a maximum exposure of 54 days in total (at least 14 days pre-mating, up to 14 days mating, 22 days of gestation and 4 days of post-partum). During the administration period the animals are observed each day for signs of toxicity. All animals are necropsied at the end of the study. The litters are examined as soon as possible after birth. On day 4 of the lactation period, the pups are euthanized and externally examined for abnormalities.
Specific details on test material used for the study:
Name: FAT 36014/Z TE
Product: TERASIL YELLOW GWL CRUDE MOIST (LAB DRIED) Disperse Yellow 042
Batch No: Q30661AASY
Physical State: solid
Storage Conditions: room temperature, protected from light
Colour: yellow
pH (specify): 9.0 (2%(w/w) aqu. Suspension), RT
Molecular Weight: 396.40 g/mol
Melting Point: 158 °C
Active Components: > 90%
Purity (qualitative and quantitative): 94.0 % (w/w)
Solubility in water: 5.72 mg/l
Date of Analysis: 07/08/2013
Expiry Date: 08/08/2018
Safety Precautions: The routine hygienic 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 140258):
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.
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. A suspension with the vehicle was prepared by subjecting it to ultrasonic bath for 5 min at room temperature. Homogeneity of the test item in the vehicle was maintained by vortexing. 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 vortexing the sample on vortex machine. The dose range finding study (14 days repeated dose, BSL study no. 140272) 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

Since little or no toxicity was anticipated for the test substance, the highest dose level was set at 1000 mg/kg/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 corn oil using the same volume as used for the 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:
Animals were allowed to mate in a ratio of 1:1 (male to female). The vaginal smear of the females was checked every morning after the start of the mating period to confirm the pregnancy. If the vaginal smear of a particular female was not found to be sperm-positive, the actual stage of the estrus cycle on that day was documented. The day of the vaginal plug and/or sperm was considered as day 0 of gestation. The cages were arranged in such a way that possible effects due to cage placement were minimized.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Each dosing concentration was analysed with respect to the target nominal concentration. Stability and homogeneity of the test item in the vehicle were analysed for the low and high dose concentrations. Samples for the nominal concentration verification were taken in study week 1 (first week of pre-mating period), 3 (first week of mating), 5 (gestation) and in the last week of the study (gestation/lactation) from all groups (16 samples). Samples for homogeneity analysis were taken from the top, middle and bottom of the high dose and the low dose formulation in study week 1 and 5 (12 samples). Samples for stability analysis were taken in the first week of the study [0 hours, 6 hours (at RT) and 10 days after the preparation (stored at 2-8 °C)], from high and low dose formulations (6 samples). All 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. 140276.
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: 07 January 2014
Study Initiation Date: 24 March 2014
1st Amendment to Study Plan: 11 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 – 21 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): 29 September 2014
Completion Date of Delegated Phase (Formulation Analysis): 29 September 2014
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control (Group - 1)
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Test group (Low Dose - LD) - Group 2
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Test group (Mid Dose - MD) - Group 3
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Test group (High Dose - HD) - Group 4
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 140258.
Control animals:
yes, concurrent vehicle
Details on study design:
The dose range finding study (14 days repeated dose, BSL study no. 140272) 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).

Since little or no toxicity was anticipated for the test substance, the highest dose level was set at 1000 mg/kg/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 corn oil using the same volume as used for the dose groups.
Positive control:
Not available
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.

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.
Oestrous cyclicity (parental animals):
Not available
Sperm parameters (parental animals):
Not available
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. Pups sacrificed on day 4 post-partum were carefully examined externally for gross abnormalities. Non-pregnant females (females 42 and 118) 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. Except 1/5 selected females of LD group was non pregnant and organ weight of this animals was not measured. The following tissues (brain (cerebrum, cerebellum and pons), heart, spinal cord, ovaries (females), liver, uterus with cervix (females), kidneys, vagina (females), adrenal glands, testes (males), stomach, epididymides (males), small and large intestines (including Peyer´s patches), prostate and seminal vesicles with coagulating glands as a whole (males), thymus, urinary bladder, thyroid, lymphnodes (mesentric and axillary), spleen, peripheral nerve (e.g. sciatic nerve) with skeletal muscle, lung and trachea, bone with bone marrow (sternum), mammary glands,pituitary gland, gross lesions, oesophagus, skin) of the same selected animals from each group were preserved in 10% neutral buffered formalin except for testes and epididymides that were fixed in Modified Davidson’s fixative for approximately 24 hours before they were transferred to 70% ethanol.

Histopathology
A full 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. 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. Histological processing of tissues to microscope slides was performed at the GLP-certified contract laboratory AnaPath GmbH, AnaPath Services, Hammerstrasse 49, 4410 Liestal, Switzerland (test site for tissue processing). Histopathological evaluation was performed at the GLP-certified contract laboratory AnaPath GmbH, Buchsweg 56, 4625 Oberbuchsiten, Switzerland (test site for histopathology). The study phases from test site 1 and 2 was performed in compliance with the Swiss Ordinance relating to Good Laboratory Practice adopted 18 May 2005 [SR 813.112.1] (Status as of 01 December 2012). Blocking, embedding, cutting, H&E staining and scientific slide evaluation were performed according to the corresponding SOP’s of the test sites. The principal investigator for histopathological tissue processing sent all raw data (including blocks, slides, paper raw data, statement of compliance and quality assurance statement) to the study director. The principal investigator for histopathological evaluation provided the histopathology results to the study director by e-mail and sent a pathology phase report to the study director upon the completion of the study.
Postmortem examinations (offspring):
Pups sacrificed on day 4 post-partum were carefully examined externally for gross abnormalities.
Statistics:
A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical biochemistry and absolute and relative organ weights were performed for each gender by comparing values of dosed with control animals of the main groups using a one-way ANOVA and a post-hoc Dunnett Test. These statistics were performed with GraphPad Prism V.6.01 software (p <0.05 was considered as statistically significant).
Reproductive indices:
There were no effects on the copulation, fertility and delivery indices of the dose groups when compared to the control. The pregnancy rate in control and dose groups were, C 90 %, LD 80 %, MD 100 and HD 100 %. However, there was slight reduction in fertility index of LD group, in the absence of dose response relation, the finding was considered incidental.
Offspring viability indices:
There was no effect on the viability index in dose group when compared to the control.
Clinical signs:
no effects observed
Description (incidence and severity):
In males and females, there were no clinical signs of toxicological relevance in all dose groups when compared to control. However, there were isolated incidences of crust, alopecia, nasal discharge, regurgitation, injury, slight salivation and aggressiveness in a few control and/ or dose group animals. During the weekly detailed clinical observation, no significant changes or differences between the groups were found.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In males and females, there were no adverse effects of toxicological relevance on body weight and body weight gain in dose groups when compared to controls. In males there were no statistically significant difference between the dose and corresponding control group. However, there was marginally lower weight gain after the last week of treatment (mating/post mating days 7-14) in HD group. However, there was no change in cumulative weight gain (premating day 1 to post-mating day 14) in HD group. In the absence of statistical significance, the marginally lower weight gain was not likely to be adverse. In females, there was statistically significantly higher weight gain in MD group after GD 7-14. There was no dose response relation and therefore was not likely to be of toxicological relevance. There were also marginal and transient changes (increase and decrease) in weight gain in HD group during premating and gestation days. There was a severely lower weight gain at the end of treatment (lactation days 0-4) in HD group, which was due lower value in single isolated female (Female no. 136). This was reflected in lower food consumption of this animal at the end of treatment. Given the single isolated effected female, this finding was not of toxicological relevance.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
In males and females, there were no adverse effects of toxicological relevance observed for hematology and blood coagulation in dose groups when compared corresponding control. However, in males there was a very slightly but statistically significantly higher lymphocyte count in MD group. As the mean value was in the range of historical control data and in the absence of dose response relation, the finding was not considered of toxicological relevance. In females there was a slightly but statistically significantly lower WBC in LD and HD groups, but not in dose response relation. There was also a minimally but statistically significantly higher PT value in female MD and HD groups. Given the mean and most individual hematology and blood coagulation values within the historical control range, the changes were not considered to be an adverse effect of treatment.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
In males and females, there were no adverse changes of toxicological relevance for clinical biochemistry in dose groups when compared to the controls. In males there were no statistically significant differences between the dose and control groups. There was higher TBA in male HD group, which was due to higher value in single male (animal no. 106). In females there was statistically significantly lower ASAT in HD group. There was lower ALAT and higher AP value in dose groups, but without statistical significance and dose response relation. There was also slightly higher glucose value, without statistical significance, in HD group. Given the mean and most individual values of clinical biochemistry of males and females within the historical control range, the changes in ALAT, AP and GLU of female and TBA of males were not considered an adverse effect of treatment.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
All microscopic findings recorded were 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. The stages were checked on completeness of cell populations, completeness of stages and degenerative changes. As a result of the detailed examination of the testis, there were no abnormalities on the completeness of stages and cell populations. There was no dose response relationship in any findings recorded 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. Nothing was observed in testes of animal nos. 2 (Control) and 85 (LD), which were mated with female nos. 42 and 115 not giving birth, respectively. Slight spermatic granulomas and minimal mononuclear cell foci in epididymides and minimal alveolitis in lungs were recorded in male no. 2 and minimal inflammation in prostate glands was recorded in male no. 85, respectively. 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 like manner, it is unlikely that minimal-unilateral tubular degeneration/atrophy and sertoli cell vacuolation in testes, minimal-unilateral mononuclear cell foci in epididymides and minimal inflammation in prostate glands which were recorded in male no. 88 (LD) were related to the infertility. Nothing particular was observed in the female reproductive organs (ovaries, uterus with cervix and vagina) of female nos. 42 (Control), 115 and 118 (LD) which did not give birth. 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.
Histopathological findings: neoplastic:
no effects observed
Reproductive function: oestrous cycle:
not examined
Description (incidence and severity):
Except for confirmation of mating.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
There were no effects on the copulation, fertility and delivery indices of the dose groups when compared to the control. The pregnancy rate in control and dose groups were, C 90 %, LD 80 %, MD 100 and HD 100 %. However, there was slight reduction in fertility index of LD group, in the absence of dose response relation, the finding was considered incidental. There was no effect on the viability index in dose group when compared to the control.
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'
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
There were no adverse changes of toxicological relevance for litter data including total no. of pups born, no. of still birth and no. of runts on PND 0 and no. of live pups,no. of male pups and no. of female pups on PND 0 and PND 4. There were no statistically significant differences between the dose and control groups. However, there was marginally lower number of female pups in HD group, which also affected the sex ratio. Given the mean and most of the individual values of the litter data being within the historical control data range, the changes were not likely to be an adverse effect.
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
Precoital Interval and Duration of Gestation:
There were no effects on the precoital interval and the duration of gestation in dose groups when compared to the controls.

Pre- and Post-Natal Data:
There were no adverse changes of toxicological relevance for the no. of corpora lutea, no. of implantation sites, no. of live pups, percent pre and post implantation loss. There were no statistically significant differences between the dose and the control groups. However, there was a higher percent pre- implantation loss in HD group when compared to control. Given the number of corpora lutea, no. of implantation sites, live pups (PND 0 and 4) and percent pre- and post- implantation loss being within the historical control data range the marginally higher percent pre- implantation loss was not considered likely to an adverse effect of treatment.

Reproductive Indices:
There were no effects on the copulation, fertility and delivery indices of the dose groups when compared to the control. The pregnancy rate in control and dose groups were, C 90 %, LD 80 %, MD 100 and HD 100 %. However, there was slight reduction in fertility index of LD group, in the absence of dose response relation, the finding was considered incidental. There was no effect on the viability index in dose group when compared to the control.

Pup Survival Data:
There was no effect on the survival of the pups from PND 0 through PND 4 in dose groups when compared to the control.

Pup External Findings:
There were no treatment related gross external findings in the pups of any of the dose groups. There was a single incidence of dark snout (pup no. 1, female 134) in HD groups which was considered spontaneous in origin.
Dose descriptor:
NOAEL
Generation:
F1
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
Critical effects observed:
no
Reproductive effects observed:
no
Conclusions:
There were no adverse toxicological effects in the adult males and females or significant reproductive and developmental effects at any administered dose. Based on the data generated from this combined repeated dose oral toxicity and reproduction/ developmental toxicity screening test with FAT 36014/Z, the no observed adverse effect level (NOAEL) is considered to be 1000 mg/kg/d for systemic toxicity in males and females and also for reproduction/ developmental toxicity.
Executive summary:

The aim of this study was to assess the possible effects of FAT 36014/Z TE on male and female fertility and embryofetal development after repeated dose administration in Wistar rats. The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of 54 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 3 in females. Males were dosed after the mating period until the minimum total dosing period of 28-29 days were completed. Animals of an additional control group were handled identically as the dose groups but received corn oil, the vehicle used in this study. The 4 groups comprised 10 male and 10 female Wistar rats. The control group was shared with BSL study 140258.

The following doses were evaluated:

Control (C):                  0 mg/kg bw/d

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

Medium Dose (MD):     300 mg/kg bw/d

High Dose (HD):           1000 mg/kg bw/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. Theadministration 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, except for food consumption measurements which were not taken during the mating period in female animals and the mating and post-mating period in male animals. Haematological and clinical biochemistry evaluations were performedon blood samples collected at terminal sacrifice fromfive males and four females from each group. Urinalysis wasperformedon samples collected at terminal sacrifice fromfive males and four 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 118) were sacrificed on day 26 from day of sperm positive vaginal smear or from the last day of mating period. Pups sacrificed on postnatalday 4 were carefully examined for gross external abnormalities. A full histopathological evaluation of the tissues was performed of 5 randomly selected male and female animals of the control and high dose groups.As well as all gross lesions from all groups, were examined by light microscopy. 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. Organs and tissues listed above were preserved in 10 % neutral buffered formalin, except for testes and epididymides which were fixed in modified Davidson’s fixative for 24 hours before being transferred to 70 % ethanol.

Results

No mortality occurred in the control or any of the dose groups during the study period. In males and females, there were no clinical signs of toxicological relevance in dose group. During the weekly detailed clinical observation, no significant changes or differences between the groups were found. No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period. In males and females, there were no adverse effects of toxicological relevance on body weight and body weight gain in dose groups when compared to controls. There was no effect on food consumption in male and female dose groups when compared to the control group. In males and females, there were no adverse effects of toxicological relevance observed on hematology and blood coagulation parameters in dose groups when compared to the corresponding control. In males and females, there were no adverse effects of toxicological relevance on clinical biochemistry parameters in dose groups when compared to the controls. There were no effects of toxicological relevance on urine parameters of males and females dose groups when compared to the control. There were no gross lesions that 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. In males and females, there were no effects of toxicological relevance on absolute and relative organ weight in dose groups when compared to the control group. In females, there was statistically significantly higher absolute and relative ovary weight in the HD group when compared to the controls. In the absence of macroscopic and microscopic findings, the higher ovary weight was not considered to be an adverse effect of treatment. Under the conditions of this study, the test item FAT 36014/Z produced no histomorphologic evidence of toxicological properties 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). All microscopic 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, or were considered to be accidental changes which can occur in the toxicology studies using the rodent models. There were no adverse effects of toxicological relevance on litter data including total no. of pups born, no. of still birth and no. of runts on PND 0 and no. of live pups, no. of male pups and no. of female pups on PND 0 and PND 4. There were no statistically significant differences between the dose and control groups. There were no adverse effects of toxicological relevance on litter weight data including total litter weight, male litter weight, female litter weight and pup mean weight on PND 0 and PND 4. There were no statistically significant differences between the dose and control groups. There was no effect on the precoital interval and the duration of gestation in dose groups when compared to the controls. There were no effects on the copulation, fertility and delivery indices of the dose groups when compared to the control group. The pregnancy rate in control and dose groups were, C 90 %, LD 80 %, MD 100 and HD 100 %. There was no effect on the viability index in dose group when compared to the control. There was no effect on the survival of the pups from PND 0 through PND 4 in dose groups when compared to the control. There were no treatment related gross external findings in the pups of any of the dose groups.

Conclusion

There were no adverse toxicological effects in the adult males and females or significant reproductive and developmental effects at any administered dose. Based on the data generated from this combined repeated dose oral toxicity and reproduction/ developmental toxicity screening test with FAT 36014/Z TE, the no observed adverse effect level (NOAEL) is considered to be 1000 mg/kg/d for systemic toxicity in males and females and also for reproduction/ developmental toxicity.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

A study was performed to assess the possible effects of FAT 36014/Z on male and female fertility and embryofetal development after repeated 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. All groups comprised 10 male and 10 female Wistar rats. No mortality occurred in the control or any of the dose groups during the study period. In males and females, there were no clinical signs of toxicological relevance in dose group. During the weekly detailed clinical observation, no significant changes or differences between the groups were found. No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period. In males and females, there were no adverse effects of toxicological relevance on body weight and body weight gain in dose groups when compared to controls. There was no effect on food consumption in male and female dose groups when compared to the control group. In males and females, there were no adverse effects of toxicological relevance observed on hematology and blood coagulation parameters in dose groups when compared to the corresponding control. In males and females, there were no adverse effects of toxicological relevance on clinical biochemistry parameters in dose groups when compared to the controls. There were no effects of toxicological relevance on urine parameters of males and females dose groups when compared to the control. There were no gross lesions that 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. In males and females, there were no effects of toxicological relevance on absolute and relative organ weight in dose groups when compared to the control group. In females, there was statistically significantly higher absolute and relative ovary weight in the HD group when compared to the controls. In the absence of macroscopic and microscopic findings, the higher ovary weight was not considered to be an adverse effect of treatment. Under the conditions of this study, the test item FAT 36014/Z produced no histomorphologic evidence of toxicological properties 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). All microscopic 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 or were considered to be accidental changes which can occur in the toxicology studies using the rodent models. There were no adverse effects of toxicological relevance on litter data including total no. of pups born, no. of still birth and no. of runts on PND 0 and no. of live pups, no. of male pups and no. of female pups on PND 0 and PND 4. There were no statistically significant differences between the dose and control groups. There were no adverse effects of toxicological relevance on litter weight data including total litter weight, male litter weight, female litter weight and pup mean weight on PND 0 and PND 4. There were no statistically significant differences between the dose and control groups. There was no effect on the precoital interval and the duration of gestation in dose groups when compared to the controls. There were no effects on the copulation, fertility and delivery indices of the dose groups when compared to the control group. The pregnancy rate in control and dose groups were, C 90 %, LD 80 %, MD 100 and HD 100 %. There was no effect on the viability index in dose group when compared to the control. There was no effect on the survival of the pups from PND 0 through PND 4 in dose groups when compared to the control. There were no treatment related gross external findings in the pups of any of the dose groups. On the basis of this reproduction/developmental toxicity screening test with FAT 36014/Z in male and femaleWistar 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 at any administered dose. Based on the data generated from this combined repeated dose oral toxicity and reproduction/ developmental toxicity screening test with FAT 36014/Z, the no observed adverse effect level (NOAEL) is considered to be 1000 mg/kg/d for systemic toxicity in males and females and also for reproduction/ developmental Toxicity.

Effects on developmental toxicity

Description of key information

The no observed adverse effect level (NOAEL) is considered to be 1000 mg/kg/d for systemic toxicity in males and females and also for reproduction/ developmental Toxicity.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the absence of adverse effects in the reproduction/developmental toxicity screening test with FAT 36014/Z, Disperse Yellow 042 can be regarded as "not classified" for reproduction /developmental toxicity.

Additional information