Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From October 2013 to January 2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study is conducted on a read across test material. The complete read across justification is attached in section 13. The reliability of the original study is 1.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Animals: a total of 90 Wistar Hannover (Crl:WI(Glx/BRL/Han)IGSBR) rats (45 males and 45 virgin females) were used.
- Source: Charles River France Laboratories, Domaine des Oncins B.P. 0109, F 69592 L’Arbresle Cedex, France.
- Age at study initiation: at least 7 to 8 weeks old.
- Weight at study initiation: 226-250 g for males and 201-225 g for females.
- Housing: housed up to 5 of one sex to a cage, in polysulphone solid bottomed cages measuring approximately 59.5x38x20 cm (Techniplast Gazzada S.a.r.l., Buguggiate, Varese). Nesting material was provided inside suitable bedding bags and changed at least twice a week.
- Diet: a commercially available powdered laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI), Italy) was offered ad libitum throughout the study. There was no information available to indicate that any non-nutrient substance likely to influence the effect of the test item was present in the drinking water or the diet.
- Water: drinking water was supplied ad libitum to each cage via water bottles.
- Acclimation period: an acclimatisation period of at least 2 weeks was allowed before the start of treatment, during which time the health status of the animals was assessed by thorough observations.
- Health survegliance: after arrival the weight range for each sex was determined and the animals were temporarily identified within the cage by means of a coloured mark on the tail. A health check was performed by a veterinarian.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C; no relevant deviations from this range were recorded during the study.
- Humidity: 55 ± 15 °C; no relevant deviations from this range were recorded during the study.
- Air changes: approximately 15 to 20 air changes per hour.
- Photoperiod: rooms were lit by artificial light for 12 hours each day.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet: fresh diets were prepared at intervals for up to 5 days, according to the stability of the test item in the diet.
- Formulation: the test item was formulated, using powdered diet, by initial preparation of a pre-mix (using mortar and pestle) followed by dilution with further quantities of diet and mixing.
- Concentration: the formulation was prepared at fixed concentrations of 100, 300 and 1000 ppm. Concentrations were calculated and expressed in terms of test item as supplied.
Details on mating procedure:
- M/F ratio per cage: during the mating period, animals were housed on the basis of one male to one female in polysulphone cages measuring 43x27x18 cm with a stainless steel mesh lid and floor (Techniplast Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray held absorbent material which was inspected and changed daily. Thus, matings were monogamous.
- Length of cohabitation: the female was paired with the same male until positive identification of copulation occurred or 14 days had elapsed.
- Proof of pregnancy: vaginal smears were taken from the day after the start of pairing until positive identification of copulation (sperm identification, vaginal plug in situ or copulation plug found on the cage tray).
- After mating housing: the males were re-caged after mating as they were before mating. After mating, the females were transferred to individual polysulphone solid bottomed cages measuring approximately 43x27x18 cm (Techniplast Gazzada S.a.r.l., Buguggiate, Varese). Suitable nesting material was provided and changed at least 2 times a week.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
On Day 1 and Week 4, two replicates of 50 g were drawn from Group 1 and Group 3 preparations, while 6 replicates of 50 g were drawn from Group 2 and Group 4 preparations (2 from the top, 2 from the middle and 2 from the bottom of the formulated diet sample). Therefore, for each sampling date a total of 16 specimens were prepared for shipment. For each specimen an additional back up sample of 10 g was retained at the test facility. Additional back up samples of 50 g were preserved at room temperature in Week 1 and Week 4 analyses. These samples were destroyed at the end of analysis.

The specimens were transported by a dedicated courier and were subject to the latter’s standard insurance policy. Prior to shipment, the Principal Investigator was notified. Back up samples were frozen at -20 °C. They will be destroyed after the issue of the Final Report following the Study Director’s approval.

For the determination of Aluminium in the test item formulation, calibration was performed using a certified Aluminium reference solution. Six levels were measured in the range 0.1 – 1.5 mg/l Aluminium, following validation report.

The determination of the first two pre-treatment sample series showed considerably different test item concentrations in comparison to the nominal loadings. Therefore, a new extraction method was applied to the samples to account for the different behaviour of the test item using diluted HNO3.
Using the new method, more than twice the amount of aluminium was measured compared to the blank value which was prepared following the validated method. Therefore, the determination of the lowest nominal load of the test item in diet (100 ppm), using the new method, was less precise.
The respective blank values (using 1 g resp. 2 g unspiked rat food) were subtracted from the measured value in the test solution. The test samples were measured in the first and the fourth week of the test; six replicates of the nominal loads 1000 ppm and 100 ppm test item in diet, and two replicates of the blank mixture and 300 ppm test item in diet, each, were used.

The relative standard deviation of the measured values (six replicates) for 100 ppm test item in the diet was > 10 % with 83 % recovery in the first week.
These values, slightly outside the ranges of acceptance, were considered to be acceptable for this study and this route of administration. In the fourth week, all measured values for all nominal loads of the test item in the diet were in the required range: RSD < 10 %, recovery rate 90 – 110 %.
Duration of treatment / exposure:
MALES
Animals had access to their appropriate diets (treated or control diet, depending on treatment group) seven days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter for at least other two consecutive weeks until the day of necropsy, except for animals subjected to bleeding procedure.

FEMALES
Animals had access to their appropriate diets (treated or control diet, depending on treatment group) seven days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 4 post partum, (the day of sacrifice), except for animals subjected to bleeding procedure.
Frequency of treatment:
Daily
Details on study schedule:
PARTURITION CHECK and DURATION of GESTATION
A parturition check was performed from Day 20 to Day 25 post coitum.
Females which did not give birth after 25 days of post coitum period were sacrificed shortly after. Gestation length was calculated as the time between the day of successful mating (Day 0 post coitum) and the day of commencement of birth (i.e. first detected presence of offspring in the cage). The day that offspring were first detected in the cage was considered Day 0 post partum.
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm
Remarks:
corresponding to 0 mg/kg bw/day.
Dose / conc.:
100 ppm
Remarks:
corresponding to 9 mg/kg bw/day for males and 10-11 mg/kg bw/day for females.
Dose / conc.:
300 ppm
Remarks:
corresponding to 23 mg/kg bw/day for males and 25-31 mg/kg bw/day for females.
Dose / conc.:
1 000 ppm
Remarks:
corresponding to 77 mg/kg bw/day for males and 95 mg/kg bw/day for females.
No. of animals per sex per dose:
Each group comprised 10 male and 10 female rats.
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: doses was selected according to an existing study on repeated dose toxicity. Oral gavage was not feasible due to the low pH of a Phoslite IP-A solution. Full precipitation of Aluminium occurs when the pH is stabilised at the physiological pH (around 7). Higher doses in feed would have been problematic for palatability and analysis.
- Rationale for animal assignment: on the day of allocation (19 days prior to the start of treatment), all animals were weighed. Animals at the extremes of the weight distribution and one animal showing signs of ill health were excluded to leave the required number of animals. The rats were allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights.

Examinations

Parental animals: Observations and examinations:
MORTALITY
Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.

DETAILED CLINICAL OBSERVATIONS
Once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs were recorded.
Observations were performed approximately at the same time interval each day.

BODY WEIGHT
Males were weighed weekly from allocation to termination. Females were weighed weekly from allocation to positive identification of mating and on gestation Days 0, 7, 14 and 20. Dams were also weighed on Days 1 and 4 post partum. Body weight of females recorded during mating phase were not tabulated in the report.

FOOD CONSUMPTION AND COMPOUND INTAKE
Food consumption was recorded at least once weekly by each cage of rats from allocation to pairing. Pre-treatment data were not tabulated in this report. For female animals, food consumption was also recorded daily during post coitum period, starting from Day 0 post coitum and daily during post partum period from Day 6 post partum up to Day 20 post coitum. Due to a technical problem with the computerised system during post coitum period, food consumption was reported in a tabulated form only from Day 7 post coitum. The raw data are maintained within the study file.

ACHIEVED DOSAGE
The group mean achieved intake of test item was calculated for pregnant females and males from the group mean body weight and food consumption data and the dietary inclusion levels of the test item.

CLINICAL PATHOLOGY INVESTIGATIONS
As a part of the sacrificial procedure, samples of blood were withdrawn under isofluorane anaesthesia from the abdominal vena cava from 5 males and 5 females (females with viable litters) randomly selected from each group, under condition of overnight fasting.
The blood samples collected were divided into tubes as follows:
– EDTA anticoagulant for haematological investigations
– Heparin anticoagulant for biochemical tests

HAEMATOLOGY
- Parameters: Haematocrit, Haemoglobin, Red blood cell count, Reticulocyte count, Mean red blood cell volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, White blood cell count, Differential leucocyte count (Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes, Large unstained cells), Platelets.
- Coagulation test: prothrombin time

CLINICAL CHEMISTRY
- Parameters: Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Gamma-glutamyltransferase, Urea, Creatinine, Glucose, Triglycerides, Phosphorus, Total bilirubin, Total cholesterol, Bile acids, Total protein, Albumin, Globulin, A/G Ratio, Sodium, Potassium, Calcium, Chloride.
Oestrous cyclicity (parental animals):
VAGINAL SMEARS
Vaginal smears were taken daily in the morning from the first day of treatment and up to the end of the mating period, until a positive identification of mating was made. The vaginal smear data were examined to determine the following:
1. Anomalies of the oestrous cycle
2. The pre-coital interval (i.e., the number of nights paired prior to the detection of mating)
Litter observations:
PARAMETERS EXAMINED
As soon as possible, after parturition was considered complete (Day 0 or 1 post partum), all pups (live and dead) were counted, sexed and live pups were identified.
Live pups were individually weighed on Days 1 and 4 post partum.

GROSS EXAMINATION OF DEAD PUPS
Pups dying during the lactation period were weighed before the despatch to necropsy. Observation was performed once daily for all litters.
Postmortem examinations (parental animals):
SACRIFICE
Parental animals that had completed the scheduled test period were killed by exsanguination under isofluorane anaesthesia. Pups that has completed the scheduled test period (Day 4 post partum) were euthanised by intraperitoneal injection of Thiopenthal. All animals were subjected to necropsy, supervised by a pathologist.
- Male animals: the males were killed after the mating of all females or at least after 28 days of treatment period.
- Maternal animals: the females with live pups were killed on Day 4 post partum. The females with total litter loss were killed on the day of the occurrence of total litter loss or shortly after. The females showing no evidence of copulation were killed after 25 days of the last day of the mating session. The females which did not give birth 25 days after positive identification of mating were killed shortly after.

NECROPSY
The clinical history of the animals was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices). Changes were noted, the requisite organs weighed and the required tissue samples preserved in fixative and processed for histopathological examination.
All females were examined also for the following: external and internal abnormalities; number of visible implantation sites (pregnant animals); number of corpora lutea (pregnant animals).
Uteri of females with no visible implantations were immersed in a 10-20 % solution of ammonium sulphide to reveal evidence of implantation.

HISTOPATHOLOGY/ORGAN WEIGHTS
- Organ weights: from all animals completing the scheduled test period, the organs listed below were dissected free of fat and weighed. The ratios of organ weight to body weight were calculated for each animal.
- Tissues fixed and preserved: samples of all the tissues (all parental animals) were fixed and preserved in 10 % neutral buffered formalin (except testes and epididymides which were fixed in Modified Davidson’s fluid and preserved in 70 % ethyl alcohol).
- Histopathological examination: after dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometre thickness and stained with haematoxylin and eosin. In addition, the testes and epididymides were cut at 2-3 micrometre thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed. The examination was restricted as detailed below: tissues specified, from all animals in the control and high dose group killed at term; tissues specified, from one animal which dred during the study; all abnormalities in all groups.
- Organs/tissues: abnormalities, Adrenal glands, Bone marrow (from sternum), Brain (cerebrum, cerebellum, medulla/pons), Caecum, Clitoral gland, Colon, Duodenum, Epididymides, Heart, Ileum, Jejunum (including Peyer’s patches), Kidneys, Liver, Lungs (including mainstem bronchi), Lymph nodes – cervical, Lymph nodes – mesenteric, Nasal cavity, Oesophagus, Ovaries with oviducts, Parathyroid glands, Penis, Pituitary gland, Prostate gland, Rectum, Sciatic nerve, Seminal vesicles with coagulating, glands, Spinal column, Spinal cord (cervical, thoracic, lumbar), Spleen, Stomach, Testes, Thymus (where present), Thyroid, Trachea, Urinary bladder, Uterus – cervix, Vagina.
Postmortem examinations (offspring):
SACRIFICE
All pups found dead in the cage were examined for external and internal abnormalities.
All live pups were killed and examined for external abnormalities and sex confirmation by gonadal inspection.
Statistics:
Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the non-parametric version of the Williams test. The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off. Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov.
Reproductive indices:
Group mean values were calculated for all parameters. Data for not pregnant females were excluded from means. Data of one female, whose pregnancy was not detected, were not available during the gestation phase. Data relevant to the total resorption animals were included in the body weight means, as well as the body weight relevant to 1 dam total litter loss on Day 3 post partum were included in the mean calculated on Day 4 post partum. The following reproductive indices were calculated:
Males: Copulation Index, Fertility Index, Pre-coital Interval.
Females: Copulation Index, Fertility Index, Pre-implanation loss, Pre-birth loss, Pup loss at birth, Cumulative pup loss on Day 4 post partum.
Offspring viability indices:
Sex ratios were calculated at birth and on Day 4 post partum and were presented as the percentage of males per litter.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Hair loss and/or scab(s) in females at 0, 300 and 1000 ppm; piloerection and hunched posture in a single high dose female.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female from the control group was found dead on Day 23, during gestation phase.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Comparable between control and treated groups both in males and female.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Slightly higher than the expected ones for the treated males and moderately higher for the treated females.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Some females dosed with 300 and 1000 ppm showed decreases of neutrophils (approximately 23 %) and monocytes (57 %). Due to the low severity, these changes were not considered adverse.
In addition, a male showed moderate neutrophilia and lymphocytosis. Due to the incidence and the absence of dose-relation, this finding was considered incidental.
The statistically significant increase of prothrombin time, recorded in females receiving 100 ppm (7 %), was considered incidental.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Some statistically significant fluctuations of biochemical parameters were observed in females dosed with 100 and/or 300 ppm, such as: decrease of bilirubin (64 %) and increase of glucose (63 %) in animals dosed with 300 ppm, decrease of protein (approximately 8 %) and globulin (approximately 10 %) in those receiving 100 and 300 ppm.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
The histopathological changes reported in control and treated animals were considered to be an expression of spontaneous and/or incidental pathology, commonly seen in this species and age under our experimental conditions.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
The histopathological changes reported in control and treated animals were considered to be an expression of spontaneous and/or incidental pathology, commonly seen in this species and age under our experimental conditions.
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
No treatment-related anomalies were noted.
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
Copulatory index was 100 % for both sexes from Groups 0, 100 and 1000 ppm and 90 % for Group 300 ppm. The fertility indices were 90 %, 80 %, 77.8 % and 90 %.

Details on results (P0)

MORTALITY AND FATE OF FEMALES
One female from the control group was found dead on Day 23, during gestation phase. Staining around the uro-genital region was seen prior to death. At post mortem examination, multiple depressed areas in the glandular region of the stomach with pale colour of the liver and pancreas, multiple dark/red areas in the thymus and red colour in the skin of the urogenital region were observed.
The factor contributory to the illness status of this female was attributed to the marked atrophy of the thymus and to the minimal to mild tingible body macrophages in the cervical and in the mesenteric lymph nodes, while the multiple mild areas of mucosal erosion, detected in the glandular region of the stomach, could be considered as secondary effect to the immuno-depression.
A total of 7 females were proved not pregnant at necropsy: 1 female in the control group, 2 in the low dose group, 3 in the mid-dose group and 1 in the high dose group. One female from the high dose group showed unilateral total resorption, while total resorption was noted in a female from the low dose group. Two females from the low dose group showed unilateral implantation,while mating was not detected in female of the same group The number of females with live pups on Day 4 post partum was: 8 in the control group, 5 in the low dose group, 6 in the mid-dose group, 8 in the high dose group. Total litter loss was observed in 2 females from the low dose group and in 1 female from the mid-dose group.

CLINICAL SIGNS
Hair loss and/or scab(s) were observed in individual males from the control and high dose groups and in some females from Groups 1, 3 and 4. Piloerection and hunched posture were seen in a single high dose female on Day 15 of gestation.

BODY WEIGHT
Means of body weight and body weight gain were comparable between control and treated groups both in males and females throughout the study. Decreases in body weight and in body weight gain noted during the post partum phase in control and treated females were considered as a normal consequence of the parturition occurred in the females and/or the overnight fast for clinical pathology evaluation.

FOOD CONSUMPTION
Food consumption was unaffected by treatment in both sexes during the study.

TEST SUBSTANCE INTAKE
Using fixed dietary inclusion levels of the test item, the mean achieved dosages for pre-mating dosing period were found to be slightly higher than the expected ones (7, 20 and 65 mg/kg/day) for the treated males (+21.4 %, +12.5 % and +18.5 %, respectively) and moderately higher for the treated females (+35.7 %, +22.5 % and +46.2 %, respectively). The same trend was maintained in the females during the post coitum and post partum periods, when the achieved dosages were higher than expected (+57.1 %, +52.5 % and +46.15 %). The discrepancy is due to a measured food consumption higher than expected. The achieved dosages in mg/kg/day, for each group, are (mean and standard deviation values calculated from weekly data):
- group 2: nominal dosage of 7 mg/kg/day (100 ppm); achieved dosage males pre-mating period 8.5 ± 0.7 mg/kg/day, females pre-mating period 9.5 ± 0.7 mg/kg/day and females Post coitum /post partum 11.0 ± 0.0 mg/kg/day
- group 3: nominal dosage of 20 mg/kg/day (300 ppm); achieved dosage males pre-mating period 22.5 ± 0.7 mg/kg/day, females pre-mating period 24.5 ± 2.1 mg/kg/day and females Post coitum /post partum 31.3 ± 1.9 mg/kg/day
- group 2: nominal dosage of 65 mg/kg/day (1000 ppm); achieved dosage males pre-mating period 77.0 ± 7.1 mg/kg/day, females pre-mating period 95.0 ± 1.4 mg/kg/day and females Post coitum /post partum 96.3 ± 8.2 mg/kg/day

HAEMATOLOGY
Some females dosed with 300 and 1000 ppm showed decreases of neutrophils (approximately 23 %) and monocytes (57 %). Due to the low severity, these
changes were not considered adverse.
In addition, one male (300 ppm), showed moderate neutrophilia and lymphocytosis. Due to the incidence and the absence of dose-relation, this finding was considered incidental.
- Coagulation: the statistically significant increase of prothrombin time, recorded in females receiving 100 ppm (7 %), was considered incidental.

CLINICAL CHEMISTRY
Some statistically significant fluctuations of biochemical parameters were observed in females dosed with 100 and/or 300 ppm, such as: decrease of bilirubin (64 %) and increase of glucose (63 %) in animals dosed with 300 ppm, decrease of protein (approximately 8% ) and globulin (approximately 10 %) in those receiving 100 and 300 ppm, Due to the absence of dose-relation, the above changes were considered unrelated to treatment.

REPRODUCTIVE FUNCTION: SPERM MEASURES
A detailed qualitative examination of the testes was performed on all control and high dose group males. The evaluation, taking into account the tubular stages of the spermatogenic cycle, was conducted in order to identify treatment-related effects, such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Identification of the stages of the spermatogenic cycle was carried out as described by Leblond and Clermont, 1952 and referred to the comprehensive reviews on the subject Russell, 1990; Creasy, 1997; Creasy, 2002. The PAS-H stained sections were used to identify the spermatogenic stages.
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.

REPRODUCTIVE FUNCTION: OSTROUS CYCLE
No treatment-related anomalies were noted in the oestrous cycle of the treated females when compared to controls.

REPRODUCTIVE PERFORMANCE
Two females in the high dose group were found sperm positive after 13 and 10 days of paring, respectively. With the exception of these 2 animals, the mean pre-coital interval and the number of copulation plugs were similar between control and treated groups. Therefore, these isolated cases were considered incidental.
All females mated with the exception of one female of the mid-dose group. However, 1 female in the control group, 2 in the low dose group, 3 in the mid-dose grou and 1 in the high dose group were found not pregnant. One female (Group 4) showed unilateral total resorption, while total resorption was noted in another female (Group 2).
The copulatory index was 100 % for both sexes from Groups 1, 2 and 4 and 90 % for Group 3.

REPRODUCTIVE/GESTATION/IMPLANTATION PARAMETERS
The fertility indices were 90 %, 80 %, 77.8 % and 90 %. A reduction in the number of females with live pups on Day 4 post partum was observed in the low dose females (5) when compared to the other groups.
All the above findings were equally distributed among groups without any relation to the dose and as such, they were not considered treatment-related.

Gestation periods were similar in treated groups and controls. All dams, with the exception of 1 control and 1 low dose dam which gave birth on Day 24 post coitum, gave birth between Days 22 and 23 post coitum. Corpora lutea, implantations and pre implantation loss, total litter size and pre-birth loss (percentage) did not show dose-related or treatment-related differences.

ORGAN WEIGHTS
No changes were observed in the weight of the organs.

GROSS PATHOLOGY
Detailed macroscopic observations have been reported for male and female animals from all groups. Increased incidence of single or multiple depressed and red area/s in the glandular region of the stomach was noted at post mortem examination in some males of Groups 3 and 4, when compared to control. This was the only remarkable change observed in treated animals.

HISTOPATHOLOGY
Histopathological evaluation was performed on all control and high dose males and females, as well as on all abnormalities detected during post mortem observation. No treatment-related changes were noted. The histopathological changes reported in control and treated animals were considered to be an expression of spontaneous and/or incidental pathology, commonly seen in this species and age under our experimental conditions.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
77 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
histopathology: neoplastic
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance
Dose descriptor:
NOAEL
Effect level:
95 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
histopathology: neoplastic
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Cold to touch, apparently no food intake (milk) and small appearance noted in control and treated early decedent pups.
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Pup loss on Day 4 post partum was slightly higher in the high dose groups.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No significant differences in mean pup weights were observed among the surviving treated dams and the controls.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No significant abnormalities were recorded.
Histopathological findings:
not examined

Details on results (F1)

VIABILITY
Mean pup loss on Day 4 post partum was slightly higher in the high dose groups as all dams with 100 % litter loss in Groups 2 and 3 were excluded from the statistical analysis.

CLINICAL SIGNS
Cold to touch, apparently no food intake (milk) and small appearance were in general the clinical signs noted in control and treated early decedent pups.

BODY WEIGHT
No significant differences in total litter size, live litter size and mean pup weights were observed among the surviving treated dams and the controls.

NECROPSY FINDINGS
No milk in stomach was noted at necropsy in the decedent pups. No significant abnormalities were recorded in the pups sacrificed on Day 4 post partum.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 ppm
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
NOAEL for general toxicity and for fertility and reproduction parameters was considered to be 1000 ppm for males and females (77 and 95 mg/ks bw/day, respectively).
Executive summary:

The toxic effects on rats of both sexes after repeated dosing with the test item, as well as any effects of the test item on male and female reproductive performance, such as gonadal function, mating behaviour, conception, development of conceptus, parturition and early lactation of the offspring were investigated in this study.

Three groups, each of 10 male and 10 female Wistar rats, received the test item in the diet at fixed concentrations of 100, 300 and 1000 ppm, corresponding to theoretical dosages of 7, 20 and 65 mg/kg/day. A fourth similarly constituted group received the untreated diet and acted as a control.

Males were treated for 2 weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 29 days. Females were treated for 2 weeks prior to pairing, during pairing and throughout the gestation and lactation periods until Day 3 post partum.

The following investigations were performed in all groups: body weight, clinical signs, food consumption, oestrous cycle, mating performance, clinical pathology investigations (haematology and clinical chemistry), litter data, macroscopic observations, organ weights and histopathological examination. Clinical signs and macroscopic observations of pups were also performed. The histopathological examination was performed only on control and high dose groups. It included identification of the stages of the spermatogenic cycle in all males of Groups 1 and 4.

The toxic effects on rats of both sexes after repeated administration of the test item in the diet at fixed concentrations of 100, 300 and 1000 ppm, as well as any effects of the test item on male and female reproductive performance, such as gonadal function, mating behaviour, conception, development of conceptus, parturition and early lactation of the offspring were investigated in this study.

No significant changes and no treatment-related effects were detected in males and females during the in vivo phase.

Mating performance, gestation, parturition, lactation, implantation, litter data and sex ratio were unaffected by treatment. All changes observed in fertility (a number of non-pregnant females), implantation and litter data (pre-implantation, pre-birth and Day 4 post partum litter losses) were equally distributed among treated and control groups and as such, they were not considered treatment-related.

Necropsy findings in pups did not reveal any treatment-related effect. No relevant changes were detected at post mortem examination in treated animals, when compared with controls.

No treatment-related changes were seen in organs/tissues evaluated nor in the abnormalities detected in all groups at post mortem.

Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages and a regular layering in the germinal epithelium was described.

Conclusion

No treatment-related effects indicating systemic toxicity were observed in male or female animals at any of the dose levels investigated (fixed concentrations of 100, 300 and 1000 ppm, corresponding to mean achieved dose levels of 9, 23 and 77 mg/kg/day for males and 10-11, 25-31 and 95 mg/kg/day for females). No adverse effects on sexual function and fertility or in developmental parameters and lactation were observed at any of the dose levels investigated.

On the basis of the results obtained in this study, the NOAEL (No Observed Adverse Effect Level) for general toxicity and for fertility and reproduction parameters was considered to be 1000 ppm for males and females.