Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From July 09,2012 to September 01,2012
Reliability:
2 (reliable with restrictions)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012

Materials and methods

Test guideline
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 material form:
liquid: viscous
Details on test material:
- Name of test material: SSP-SAMPLE 1
- Substance type: liquid
- Physical state: Amber-yellowish/brown undefined liquid
- Analytical purity:no data
- Storage condition of test material: Room temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:Charles River Italia
- Age at study initiation:
- Weight at study initiation:200 to 225 g for males and 151 to 175 g for females
- Fasting period before study:
- Housing:The animals were housed in a limited access rodent facility.5 of one sex to a cage, in polisulphone solid bottomed cages measuring 59.5x38x20 cm. Nesting material was provided inside suitable bedding bags and changed at least twice a week.
During mating, animals were housed one male to one female in clear polycarbonate cages measuring approximately 43x27x18 cm with a stainless steel mesh lid and floor. Each cage tray held absorbent material which was inspected and changed daily. After mating, the males were re-caged as they were before mating, the females were transferred to individual solid bottomed cages for the gestation period, birth and lactation. Suitable nesting material was provided and changed as necessary.
- Diet: A commercially available laboratory rodent diet was offered ad libitum throughout the study
- Water : Drinking water was supplied ad libitum to each cage via water bottles.
- Acclimation period:2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22°C ± 2°C
- Humidity (%):55% ± 15%
- Air changes (per hr):15 to 20 air changes per hour
- Photoperiod (hrs dark / hrs light):artificial light for 12 hours each day

OTHER: 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.
Dated and signed records of activities relating to the day to day running and maintenance of the study in the animal house were recorded.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was administered orally by gavage at a dose volume of 5 mL/kg body weight.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before the treatment period (with the exception of the stability performed during the treatment period), analysis was performed to confirm that the proposed formulation procedure was acceptable and that the stability of the formulation was satisfactory. Samples of the formulations prepared during the study were also analysed to check the homogeneity and concentration.
Chemical analysis was carried out by the Analytical Chemistry Department
Duration of treatment / exposure:
28 days
Frequency of treatment:
MALE
Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter through the day before necropsy (after 28-29 days of treatment).
Dose volumes were adjusted once per week for each animal according to the last recorded body weight.

Females
Animals were dosed once a day, 7 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 3 post partum or the day before sacrifice. Dose volumes were adjusted once per week for each animal according to the last recorded body weight.
During the post coitum period, dose volumes were calculated according to individual body weight on Days 0, 7, 14, 20 and on Day 1 post partum. Thereafter individual dose volumes remained constant.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 30, 180, 500 mg/mL
Basis:
actual ingested
No. of animals per sex per dose:
10 x sex x doses

Group Treatment Level Rat numbers
Number (mg/kg/day)+ M F
(even) (odd)
1 0 control 2-20 1-19
2 30 Low 22-40 21-19
3 180 Medium 42-60 41-59
4 500 High 62-80 61-79
+: in terms of active components (corrected for water content)
Control animals:
yes, concurrent no treatment
Details on study design:
The formulation was prepared daily at concentrations of 6, 36 and 100 mg/mL. Concentrations were calculated and expressed in terms of active components (excluding water content, 15%).

Examinations

Observations and examinations performed and frequency:
MORTALITY:yes, Throughout the study, all animals were checked early in each working day in the morning and in the afternoon. A complete necropsy was performed in all cases

CLINICAL SIGNS:Yes. Observations were performed at approximately the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions.

Once before commencement of treatment and at least once a week thereafter, each animal was given a detailed clinical examination. Each animal was removed from the home cage and observed in an open arena. The tests included observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern). All observations were recorded for individual animals but data reported as summary table.

Observed parameters, described by an evaluation scale, are indicated below:
Removal (from cage): Easy, Difficult, Very difficult
Handling reactivity: Normal, Slow, Moderate, Marked
Lachrymation: Absent, Slight, Marked
Palpebral closure: Absent, Slight, Moderate, Marked
Salivation: Absent, Slight, Marked
Piloerection: Absent, Present
Rearing: Absent, Intervals of number of times (i.e. 1-3, 4-7, 8-10)
Spasms: Absent, Tonic spasms, Clonic spasms, Tonic-clonic spasms
Myoclonia: Absent, Present
Mobility impairment: Absent, Slight, Moderate, Marked
Arousal (animal activity): Very slow, Slow, Normal, Moderate, Marked
Vocalisation: Absent, Present
Stereotypies: Absent, Present
Unusual respiratory pattern: Absent, Present
Bizarre behaviour: Absent, Present
Urination: Absent, Intervals of number of times (i.e. 1-3, 4-6)
Defecation: Absent, Intervals of number of times (i.e. 1-3, 4-6)
Tremors: Absent, Present
Gait (one of the following options):
Normal:
Ataxia (Slight, Moderate, Marked)
Hunched posture (Slight, Moderate, Severe)
Pronation
Forelimbs drag (Slight, Moderate, Marked)
Hindlimbs drag (Slight, Moderate, Marked)

All observed parameters are reported in a group incidence table. Individual data are not included in this report. Data of females were reported until week 6 of the study. All other data are not tabulated in this report but archived together with all raw data.

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes
Males were weighed weekly from allocation to termination.
Females were weighed weekly from allocation to positive identification of mating (or to sacrifice) and on gestation Days 0, 7, 14 and 20. Dams and litters were also weighed on Days 1 and 4 post partum.

FOOD CONSUMPTION:The weight of food consumed by each cage of males and females was recorded weekly (whenever possible) during the pre-mating period starting from allocation. Individual food consumption for the females was measured on gestation Days 7, 14 and 20 starting from Day 0 and on Day 4 post partum starting from Day 1.

OTHER:
Grip strength and sensory reactivity to stimuli
Once during the study, towards the end of treatment, 5 males and 5 females (on post partum Day 3) were randomly selected from each group for evaluation of sensory reactivity to stimuli of different modalities (approach, touch, clicker, tail pinch, pupil response, righting reflex, grip strength, landing foot splay). The measurements were performed using a computer generated random order.

Motor activity assessment (MA)
Once during the study, towards the end of treatment, 5 males and 5 females (on Day 3 post partum) were randomly selected from each group and the motor activity was measured (for approximately 5 minutes) by an automated activity recording device. Measurements were performed using a computer generated random order.

Vaginal smears
Vaginal smears were taken daily in the morning starting two weeks before pairing until a positive identification of copulation was made. The vaginal smear data were examined to determine the following:
a) anomalies of the oestrous cycle;
b) pre-coital interval (i.e., the number of nights paired prior to the detection of mating).

Mating
Mating was monogamous (one male to one female). A vaginal smear was taken from the day after the start of pairing until positive identification of copulation (sperm identification, vaginal plug in situ or copulation plugs found in the cage tray), when animals were considered mated. The vaginal smears were taken up to the maximum allowed pairing period (14 days).

Parturition and gestation length
A parturition check was performed from Day 20 post coitum. 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.

Pups identification, weight and observation
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.
Pups dying during the lactation period were weighed before the despatch to necropsy.
All litters were observed daily.

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 with viable litters, randomly selected from each group, under condition of food deprivation.
The blood samples collected were divided into tubes as follows:
EDTA anticoagulant for haematological investigations
Heparin anticoagulant for biochemical tests
Citrate anticoagulant for coagulation tests

Haematology

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 tests: Prothrombin time

Clinical chemistry

Alkaline phosphatase
Alanine aminotransferase
Aspartate aminotransferase
Gamma-glutamyltransferase
Urea
Creatinine
Glucose
Triglycerides
Bile acids
Inorganic Phosphorus
Total bilirubin
Total cholesterol
Total protein
Albumin
Globulin
A/G Ratio
Sodium
Potassium
Calcium
Chloride
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
The clinical history of the males and females of the parental generation 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 (excluding females found dead) and the required tissue samples preserved in fixative and processed for histopathological examination.
Females:
All females were examined also for the following:
a) number of visible implantation sites (pregnant animals);
b) number of corpora lutea (pregnant animals).
Uteri of females with no visible implantations were immersed in a 20% solution of ammonium sulphide to reveal evidence of implantation.
Pups
All pups found dead in the cage were examined for external and internal abnormalities.
All live pups sacrificed at termination were examined for external abnormalities and the sex confirmed by gonadal inspection.

Organ weights
From all animals completing the scheduled test period, the organs indicated in section 4.5.6 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 listed in section 4.5.6 were fixed and preserved in 10% neutral buffered formalin (except eyes, optic nerves and Harderian glands, testes and epididymides which were fixed in modified Davidson's fluid and preserved in 70% ethyl alcohol).

HISTOPATHOLOGY: Yes
The tissues required for histopathological examination are listed in section 4.5.6. After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometer thickness and stained with haematoxylin and eosin.
In addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS). A detailed qualitative evaluation of testes was performed. The evaluation took into account the tubular stages of the spermatogenic cycle, 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.
In the first instance the examination was restricted as detailed below:
a)Tissues specified in section 4.5.6 from 5 males and 5 females randomly selected (animals evaluated for clinical pathology) in the control and high dose group killed at term.
b)Tissues specified in section 4.5.6 from females dying during the treatment period.
c)All abnormalities in all groups.
The examination was extended to the stomach (both sexes) and liver (both sexes) from the remaining 5 males and 5 females (animals not evaluated for clinical pathology) of the control and the high dose group.
Other examinations:
Euthanasia
Parental animals and those that had completed the scheduled test period were killed by exsanguination under isofluorane anaesthesia.
Pups were euthanised at Day 4 post partum by intraperitoneal injection of Thiopenthal.
Parental males:
The males were killed on study Days 29 and 30 (after 28-29 days of treatment).
Parental females:
The females were killed on Day 4 post partum.
Female no. 91100061 of the high dose group, showing no evidence of copulation (did not mate), was killed after 27 days from the end of the paring period
Statistics:
The computerised system used in this study was the Xybion Path/Tox System, Version 4.2.2.
Means and standard deviations were calculated as appropriate. For body weight, body weight gain, food consumption, terminal body weight and organ weights (absolute and relative) 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.
Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test if n was more than 5.
The non-parametric Kruskal-Wallis analysis of variance was used for all the other parameters. The criterion for statistical significance was p<0.05.

Results and discussion

Results of examinations

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
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
at high dose
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
high dose
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
Mortality:
During the post coitum period, female no. 91100043 of the mid-dose group receiving 180 mg/kg/day was found dead on Day 4 and female no. 91100001 of the control group was sacrificed for humane reasons on Day 5. The presence of oily fluid in the thoracic cavity of both animals suggested a mis-dosing as a cause of death

Clinical signs:
Cold to touch, dyspnoea, hunched posture and semiclosed eyes were recorded in the control female no. 91100001 which was sacrificed for humane reasons.
No toxicological significance was attributed to the other clinical signs recorded during the study, since the signs were not dose related, recorded on occasions or commonly observed in animals of this species under this experimental condition.

Clinical observations (Functional Observation Battery Tests)
Neurotoxicity assessment (removal of animals from the home cage and open arena)
Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test item.
The only signs recorded were those of the animal in the control group sacrificed for humane reasons.

BODY WEIGHT AND WEIGHT GAIN
The body weight of treated animals of both sexes was not affected by treatment.
No relevance was attributed to the statistically significant increase in body weight gain noted in mid-dose males on Day 14 of the mating phase.
Body weight gain of females was unaffected by treatment.

FOOD CONSUMPTION :
Food consumed was comparable between the control and the treated groups.


HAEMATOLOGY: Treated males showed moderate to severe neutropenia (2.7 to 4-fold decrease). Lymphocytosis was also observed in some animals treated with 180 and 500 mg/kg/day (approximately 1.4-fold increase). On the contrary, females dosed with 500 mg/kg/day showed slight neutrophilia (1.7-fold increase). Due to the inconsistency between sexes, the above findings cannot be conclusively attributed to treatment.
The other statistically significant differences between control and treated animals (erythrocytes, mean corpuscular haemoglobin concentration , monocytes , basophils and large unstained cells in males; mean corpuscular volume , mean corpuscular haemoglobin concentration and platelets in females) were of minimal severity and/or recorded in the low or medium dose groups, therefore considered incidental.

Coagulation
Prothrombin time was increased in treated males, being statistically significant in those receiving 30 and 500 mg/kg/day. Due to the low severity (approximately 10%) and the inconsistency between sexes, the above change was considered of no toxicological relevance.

CLINICAL CHEMISTRY:
Treated males showed decrement of aspartate aminotransferase, total bilirubin, cholesterol, triglycerides, globulin and protein and increment of glucose and potassium. Females treated with 500 mg/kg/day showed increase of cholesterol, triglycerides and decrease of total bilirubin. Fluctuation of bile acids were also observed.

GROSS PATHOLOGY:
Unscheduled death
Two cases of premature death occurred during the study.
A single female animal from the control group (no. 91100001) was humanely killed during the study. Macroscopic findings observed in this animal included: enlarged adrenals; presence of adhesions in the heart, aorta, diaphragm, lungs and thymus; presence of fluid in the thoracic cavity and presence of an open area in the oesophagus.
At histopathology, acute inflammation involved the pleura, pericardium, aorta, diaphragm, thymus and oesophagus. In addition, the thymus was atrophic and inflammatory cell foci were noted in the liver. The cause of death of this animal was due to mis-dosing .
A single female rat from Group 3 (no. 91100043) was found dead during the treatment period.
Macroscopic findings observed in this animal included areas of discoloration in the liver; presence of adhesions in the lungs; presence of fluid in the thoracic cavity; red ovaries and thymus; and stomach distended with gas .

HISTOPATHOLOGY: NON-NEOPLASTIC
At histopathology, acute inflammation involved the pleura, pericardium, thymus, congested liver; and atrophic thymus. The cause of death of this animal was due to mis-dosing
Treatment-related findings were seen in the stomach and in the liver as specified below:
Stomach (non-glandular): the treatment-related changes, seen in the non-glandular stomach of the high and intermediate dosed groups of both sexes, were generally dose-related in incidence and/or severity, and consisted of mild to moderate diffused hyperplasia of the squamous epithelium, which was associated with mild thickening (i.e., hyperkerathosis) of the keratin layer. Sporadically, subchronic inflammation of the submucosa and focal erosion were also noted.

Liver: treatment-related findings were seen in the male of the high and intermediate dosed groups animals, and in the females of all treated groups. These were dose-related in incidence and/or severity, and consisted of hepatocytic hypertrophy associated with cytoplasmic eosinophilia.

In a single female rat from the high dosed group, mild atrophy of the thymus was observed. As this change was seen only in a single animal from this group, it is not considered as related to treatment. In two male animals of the low dose group, focal erosion was noted in the glandular stomach. As this change was seen only sporadically in the low dosage group, and as such a change is sporadically seen in untreated animals, the present cases are considered as incidental findings.
All other observed changes had comparable incidence in the control and treated groups and/or are known to occur spontaneously in untreated Sprague Dawley rats of the same age, under our experimental conditions.

OTHER FINDINGS:
Motor activity and sensory reactive to stimuli:
No differences in grip strength, landing foot splay or motor activity were noted between the treated and control animals.

Oestrus cycle and mating performance:
Oestrus cycles were unaffected by treatment.
All females mated with the exception of female no. 91100061 of the high dose group. This female was found in dioestrus for the entire mating period.
Although they mated, the control female no. 91100001 and the mid-dose female no. 91100043 were found not pregnant.
No differences in pre-coital interval or number of copulation plugs were noted between the control and treated groups.

Effect levels

Dose descriptor:
NOAEL
Effect level:
ca. 30 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Macroscopic observations

In a single male rat treated with the intermediate dose, in 4 males and a single female rat treated with the high dose, the non-glandular mucosa of the stomach was thickened. In addition, only in the female high dosed rat, the non-glandular mucosa was also oedematous. The macroscopic changes correlated with histopathological findings.

Microscopic observations

Stomach (non-glandular): the changes seen in the non-glandular stomach of the high and intermediate dosed groups of both sexes, were generally dose-related in incidence and/or severity, and consisted of mild to moderate diffused hyperplasia of the squamous epithelium, which was associated with mild thickening (i.e., hyperkerathosis) of the keratin layer. Sporadically, subchronic inflammation of the submucosa and focal erosion were also noted.

Liver :some changes were seen in the male of the high and intermediate dose groups and in the females of all treated groups. They appeared dose-related in incidence and/or severity, and consisted of hepatocytic hypertrophy associated with cytoplasmic eosinophilia.

Applicant's summary and conclusion

Conclusions:
In conclusion, no relevant effects were observed at the low dose of 30 mg/kg/day (NOAEL). Doses of 180 and 500 mg/kg/day were well tolerated but effects on stomach and liver were found at the histopathological examination.
Reproductive parameters were unaffected by treatment up to 500 mg/kg/day (NOAEL).
Executive summary:

The toxic effects on rats after repeated dosing with Paraffin waxes and Hydrocarbon waxes C14-17, chloro, sulfochlorinated, low sulphonated, saponified, as well as any effects of the test item on male and female reproductive performance, such as gonadal function, conception, parturition and early lactation of the offspring were investigated.

The dosage groups were as follows:

Group Treatment Number of animals
Number (mg/kg/day)
1 0 10M+10F
2 30 10M+10F
3 180 10M+10F
4 500 10M+10F

All doses were administered at a constant volume of 5 mL/kg body weight.

The vehicle was corn oil.

Males were treated for 2 weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 28/29 days.

Females were treated for 2 weeks prior to pairing, during pairing and throughout the gestation and lactation periods until Day 3 post partum or the day before necropsy.

The following investigations were performed in all groups: body weight, clinical signs (including neurotoxicity assessment, motor activity and sensory reaction to stimuli), 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 five males (five animals/sex/group).

Mortality and fate of females

During the post coitum period, one female in the control group was sacrificed for humane reasons and one mid-dose female was found dead. The presence of oily fluid in the thoracic cavity of both animals suggested a mis-dosing as a cause of death. One control female and one mid-dose female were found not pregnant.

All pregnant females littered and had live pups on post partum Day 4. One high dose female did not mate (confirmed at necropsy).

The number of females with live pups on Day 4 post partum was 9 in the control, 10 in the low dose group (30 mg/kg/day), 9 in the mid-dose group (180 mg/kg/day) and 9 in the high dose group (500 mg/kg/day).

Clinical signs and clinical observations (Functional Observation Battery Tests)

No clinical signs related to treatment were observed.

Neurotoxicity assessment (removal of animals from the home cage and open arena)

Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test item.

Body weight and body weight gain

No difference in body weights was recorded in animals of both sexes compared to the control group, throughout the study.

Food consumption

No effects on food consumption were observed in either males or females.

Motor activity and sensory reaction to stimuli

No differences were noted in all parameters investigated between control and treated groups.

Haematology

The principal alteration noted in male animals receiving 180 and 500 mg/kg/day was neutropenia . On the contrary, females dosed with 500 mg/kg/day showed slight neutrophilia. Due to the inconsistency between sexes, the above findings cannot be conclusively attributed to treatment .

Due to the low severity (approximately 10%) and the inconsistency between sexes, the increase noted in prothrombin time was considered of no toxicological relevance. 

Clinical chemistry

Treated males showed decrement of aspartate aminotransferase, total bilirubin, cholesterol, triglycerides, globulin and protein and increment of glucose and potassium.

Females treated with 500 mg/kg/day showed increase of cholesterol, triglycerides and decrease of total bilirubin.

 Almost all of the above changes were not dose-related and of low magnitude , therefore they were considered of no toxicological significance and could represent a metabolic adaptation.

Oestrus cycle, reproductive parameters, pairing combination and mating performance

Oestrus cycle, pre-coital intervals, copulatory index and fertility index did not show intergroup differences.

Implantation, pre-birth loss data and gestation length of females

No significant differences were observed for these parameters between the treated groups and the controls.

All dams gave birth between Days 21 and 23 post coitum.

Litter data and sex ratio of pups

Litter data and sex ratio were unaffected by treatment.

Clinical signs of pups

Clinical signs were comparable between treated and control groups.

Necropsy findings in decedent pups and in pups sacrificed on Day 4 post partum

Necropsy findings in decedent pups and in pups sacrificed on Day 4 post partum did not reveal any treatment-related effect.

Terminal body weight and organ weights

Terminal body weight was unaffected by treatment in both sexes.

Absolute and relative liver weight was significantly higher at statistical analysis compared to controls for mid-dose males and high dose males and females. 

Macroscopic observations

Unscheduled death

Two cases of premature death occurred during the study (i.e., a single female animal from the control group was humanely killed during the study, and a single female rat from Group 3 was found dead during the treatment period). In both cases the cause of death was a mis-dosing.

Final sacrifice

Treatment-related changes were noted in the stomach. In a single male rat treated with the intermediate dose, in 4 males and a single female rat treated with the high dose, the non-glandular mucosa was thickened. In addition, only in the female high dosed rat, the non-glandular mucosa was also edematous. The macroscopic changes correlated with histopathological findings.

Microscopic observations

Treatment-related findings were seen in the stomach of the high and intermediate dosed groups of both sexes, and in the liver of the male high and intermediate dosed animals, and in the females of all treated groups.

Stomach (non-glandular): the treatment-related changes, seen in the non-glandular stomach of the high and intermediate dosed groups of both sexes, were generally dose-related in incidence and/or severity, and consisted of mild to moderate diffused hyperplasia of the squamous epithelium, which was associated with mild thickening (i.e., hyperkerathosis) of the keratin layer. Sporadically, subchronic inflammation of the submucosa and focal erosion were also noted. 

Liver: treatment-related findings were seen in the male high and intermediate dosed animals, and in the females of all treated groups. The changes were dose-related in incidence and/or severity, and consisted of hepatocytic hypertrophy associated with cytoplasmic eosinophilia.

Spermatogenic cycle

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.

Conclusions

No relevant effects were observed at the low dose of 30 mg/kg/day (NOAEL). Doses of 180 and 500 mg/kg/day, were well tolerated but effects on stomach and liver were found at the histopathological examination .

Reproductive parameters were unaffected by treatment up to 500 mg/kg/