Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 20 February 2017 (Allocation) to 27 April 2017 (last day of necropsy)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
3 October 2008
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
(EC) No. 440/2008
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The Sprague Dawley rat was the species and strain of choice because it is accepted by many regulatory authorities and there is ample experience and background data on this species and strain.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Italia, S.p.A., Calco (Lecco), Italy.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 8 to 9 weeks
- Weight at study initiation: 267 to 273 g for males and 190 to 233 g for females
- Fasting period before study: not reported
- Housing: clear polysulfone solid bottomed cages. Nesting material was provided inside suitable bedding bags and changed at least twice a week.
- Diet: commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019 Settimo Milanese (MI), Italy), ad libitum
- Water: drinking water ad libitum
- Acclimation period: 5 weeks before start of the treatment
DETAILS OF FOOD AND WATER QUALITY: 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.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C ± 2 °C
- Humidity (%): 55 % ± 15 %
- Air changes (per hr): 15 to 20
- Photoperiod (hrs dark / hrs light): 12/12
A total of 100 Sprague Dawley SD rats (45 males and 55 virgin females), 8 to 9 weeks old and weighing 250 to 275 g for males and 175 to 200 g for females, were ordered from Charles River Italia, S.p.A., Calco (Lecco), Italy. After arrival the weight range for each sex was determined (267 to 273 g for males and 190 to 233 g for females; slightly different from the requested for females) and the animals were temporarily identified within the cage by means of a coloured mark on the tail. A health check was then performed by a veterinarian. An acclimatisation period of approximately 5 weeks was allowed before the start of treatment, during which time the health status of the animals was assessed by thorough observations.
The animals were housed in a limited access rodent facility. Animal room controls were set to maintain temperature and relative humidity at 22°C ± 2°C and 55% ± 15% respectively; actual conditions were monitored, recorded and the records retained. No relevant deviations from these ranges were recorded during the study. There were approximately 15 to 20 air changes per hour and the rooms were lit by artificial light for 12 hours each day.
The animals were housed up to 5 of one sex to a cage, in clear polysulfone solid bottomed cages. Nesting material was provided inside suitable bedding bags and changed at least twice a week. Drinking water was supplied ad libitum to each cage via water bottles, except in the case of urinalysis investigations. A commercially available 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.

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The test item was administered orally by gavage at a dose volume of 10 mL/kg body weight. Control animals received the vehicle alone at the same dose volume. The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.
Vehicle:
water
Remarks:
softened by reverse osmosis
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS: The required amount of test item was dissolved in the vehicle. The formulations were prepared daily (concentrations of 10 and 30 mg/mL for the entire treatment period, 100 mg/mL for the first 14 days and 50 mg/mL for the remaining treatment period).
- VEHICLE
- Concentration in vehicle: 10 – 100 mg/mL
- Amount of vehicle: 10 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis was performed to confirm that the proposed formulation procedure was acceptable and that the stability of the formulation was satisfactory. The analytical method was validated in the range from 5 to 120 mg/mL. A 26 hour stability at room temperature and an 8 day stability at +5 °C ± 3 °C were verified in the same range of concentrations. In the present study, samples of the formulations prepared during the study were also analysed to check the concentration (the first and week 4 of treatment).
Chemical analysis was carried out by the Analytical Chemistry Department at the study site. The software used for this activity was Empower® 2 Build No. 2154.
Validation of the analytical method
The analytical method was validated in the range from 5 to 120 mg/mL. Linearity, accuracy and precision were within the limits stated for solutions (r > 0.98; accuracy 90-110 %; precision CV < 5 %).
Formulation sampling
Formulations of the test item were prepared as solutions in water. Concentration was assessed for all levels by taking two analytical aliquots (approximately 1 mL). Each analytical aliquot was analysed separately. Concentration was evaluated as the mean of the two determinations.
Stability
A 26 hour stability at room temperature and an 8 day stability at +5 °C ± 3 °C were verified in the range from 5 to 120 mg/mL. Solutions are considered to be stable if concentration, after the defined period of storage, is still acceptable (90-110 %).
Assessment of formulation procedure
The proposed formulation procedure for the test item was checked in the range from 5 to 120 mg/mL by chemical analysis (concentration) to confirm that the method was suitable.
Final results for all levels were within the acceptability limits stated (90-110 %).
Formulation analysis of dosing solutions
Samples of the formulations prepared on Weeks 1 and 4 were analysed to check the concentration. Results of the analyses were within the acceptability limits stated for concentration of solutions (90-110 %).
Formulation analysis – Week 1 – Content check
Concentration mg/mL Recovery (%)
Group Sex Intended Found Found Limits
1 M+F 0 0 - 90 - 110
2 M+F 10 9.431 94.31 90 - 110
3 M+F 30 28.92 96.39 90 - 110
4 M+F 100 96.20 96.20 90 - 110
Formulation analysis – Week 4 – Content check
Concentration mg/mL Recovery (%)
Group Sex Intended Found Found Limits
1 M+F 0 0 - 90 - 110
2 M+F 10 9.840 98.40 90 - 110
3 M+F 30 28.91 96.36 90 - 110
4 M+F 50 48.73 97.45 90 - 110


Duration of treatment / exposure:
All animals were dosed once a day, 7 days a week, for a minimum of 4 consecutive weeks followed by a recovery period of 4 weeks for the last surviving 5 males and 5 females from all groups. Animals scheduled for the final sacrifice after 4 weeks of treatment, were dosed up until the day before necropsy. No treatment was given during the recovery period.
Frequency of treatment:
Once a day
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Group 1
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Group 2
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Group 3
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Group 4 (dose level from Day 1 to Day 14 of treatment)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
Group 4 (new dose level starting from Day 15 of treatment)
No. of animals per sex per dose:
10 animals/sex/dose
Control animals:
yes
yes, concurrent vehicle
Details on study design:
- Rationale for animal assignment: rats were allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights.
- Fasting period before blood sampling for clinical biochemistry: yes

Initially, the aim of this study was to obtain information on the possible toxic effects on Sprague Dawley 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, conception, parturition and early lactation of the offspring. The animals, 10 per gender, were assigned to four groups and administered orally, via gavage, at dose levels of 100, 300 and 1000 mg/kg/day. Control animals received the vehicle (softened water by reverse osmosis) at the same dose volume of 10 mL/kg.
Due to unexpected mortality and overall adverse effects during the first and the second week of treatment, it seemed evident that pairing and subsequent gestation and parturition of animals could be affected by the observed toxicity.
Therefore, it was decided to reduce the high dose level from 1000 mg/kg/day to 500 mg/kg/day, starting from Day 15 of treatment and to investigate repeated dose toxicity and recovery from any treatment-related effects, changing the current study design to a 4 week oral toxicity study followed by 4 weeks of recovery.
Positive control:
none

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: each working day in the morning and 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: Yes
- Time schedule: Once before commencement of treatment and at least once a week thereafter, each animal was given a detailed clinical examination. Once during Week 4 of treatment and once during Week 4 of recovery, an evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli) and an assessment of grip strength were also performed in all animals. The motor activity (MA) of all animals was measured once during Week 4 of treatment and once during Week 4 of recovery by an automated activity recording. Measurements were performed using a computer generated random order.
BODY WEIGHT: Yes
- Time schedule for examinations: each animal was weighed on the day of allocation to treatment groups, on the day that treatment commenced, weekly thereafter and just prior to necropsy. An additional body weight measurement was performed on day 14 of treatment to assess the health status of the animals.
FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: Yes
- Time schedule for collection of blood: Prior to necropsy. At the end of Week 4 of the recovery period.
- Anaesthetic used for blood collection: Yes (isofluorane)
- Animals fasted: Yes
- How many animals: at necropsy: all male and female animals selected for the final sacrifice. At the end of Week 4 of the recovery period: all surviving animals
- Parameters examined:
– 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
– Prothrombin time
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Prior to necropsy. At the end of Week 4 of the recovery period.
- Animals fasted: Yes
- How many animals: all male and female animals selected for the final sacrifice. At the end of Week 4 of the recovery period: all surviving animals
- Parameters examined:
– 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
URINALYSIS: Yes
- Time schedule for collection of urine: During the last week of treatment, individual overnight urine samples were collected from all male and female animals selected for the final sacrifice.
- Metabolism cages used for collection of urine: No
- Animals fasted: Yes
- Parameters examined:
– Appearance
– Volume
– Specific gravity
– pH
– Protein
– Glucose
– Ketones
– Bilirubin
– Urobilinogen
– Blood
The sediment, obtained from centrifugation at approximately 3000 rpm for 10 minutes, was examined microscopically for:
– Epithelial cells
– Leucocytes
– Erythrocytes
– Crystals
– Spermatozoa and precursors
– Other abnormal components
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity
IMMUNOLOGY: No

Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see below)
HISTOPATHOLOGY: Yes (see below)
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.
Organ weights
From all animals completing the scheduled test period, the following organs were dissected free of fat and weighed: adrenal glands, brain, epididymides, heart, kidneys, liver,ovaries and oviducts, parathiroid glands, prostate gland, seminal vescicles with coagulating glands, spleen, testes, thymus, thyroid gland, uterus with cervix. The ratios of organ weight to body weight were calculated for each animal.
Histopathological examination
The tissues required for histopathological examination are the following: adrenal glands, bone marrow (from sternum), brain, caecum, clitoral gland, colon, duodenum, epididymides, eyes, femur with joint, heart, ileum, jejunum (including Peyer's patches), kidneys, liver, lungs (including mainstem bronchi), cervical and mesenteric lymph nodes, mammary glands (males and females), ovaries and oviducts, parathiroid glands, pituitary gland, penis, prostate gland, rectum, sciatic nerve, seminal vescicles with coagulating glands, skeletal muscle, spinal cord, spleen, stomach, testes, thymus, thyroid gland, trachea, urinary bladder, uterus with cervix, vagina.
The examination was performed as follows:
i. Tissues specified from all animals in the control and high dose groups killed after 4 weeks of treatment.
ii. Tissues specified from all animals killed or dying during the treatment period.
iii. All abnormalities in all animals killed after 4 weeks of treatment.
Statistics:
Standard deviations were calculated as considered appropriate. For continuous variables the significance of the differences amongst groups was assessed by analysis of variance.Differences between each treated group and the control group were assessed by Dunnett’s test using a pooled error variance. The homogeneity of the data was verified by Bartlett’s test before Dunnett’s test. If the data were found to be inhomogeneous aModified t test (Cochran and Cox) was applied.
The mean values, standard deviations and statistical analysis were calculated from the actual values in the computer without rounding off. Statistical analysis of histopathological finding was carried out by means of a nonparametric Kolmogorov-Smirnov test if “n” was more than 5.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Dosing Phase
Decreased activity, cyphosis, emaciation and piloerection in one control male and hairloss on the head in another control male were seen during the last week of treatment, and were considered incidental.
Low dose animals - 100 mg/kg/day: salivation was seen in one female in each Weeks 1 and 2, staining on the neck was seen in one female at the end of the treatment.
Mid-dose animals - 300 mg/kg/day: single episodes of salivation were seen in few animals of both sexes in Week 1 and only in few females in Weeks 3 and 4, piloerection was seen on one occasion in one female in Week 1 and in one male in Week 2, hairloss (head) was seen in single females during the treatment.
High dose animals (completing the scheduled period) - 1000/500 mg/kg/day: during the first 2 weeks of treatment at 1000 mg/kg/day, salivation was seen in two males and few females, dyspnoea, rales and swollen/hard abdomen were seen in single females. After the reduction of the dose level to 500 mg/kg/day, salivation was seen in one male and in single females and an isolated case of pallor was recorded in one female during Week 3; recovery from all signs was seen during the fourth week of treatment, when salivation was observed in only one female.
Recovery Phase
No relevant clinical signs were seen during the recovery period.
Mortality:
mortality observed, treatment-related
Description (incidence):
A total of five treated animals were found dead during the study: one mid-dose female, one high dose male and three high dose females.
The high dose male was found dead on Day 14 of the study. Macroscopic findings observed in this animal included: dark/red colour of different organs and stomach distended with gas. At histopathology, the major findings observed were decreased cellularity of bone marrow, moderate atrophy of thymus and spleen.
The three high dose females were found dead from Day 7 to Day 18 of study. One female was found dead on Day 10 of treatment. Salivation, rales, dyspnoea, swollen abdomen, staining on the perigenital region and muzzle, cyphosis, piloerection and hairloss were seen. At macroscopic examination, small kidneys and spleen and thymus not evident were observed. At microscopic examination, decreased cellularity, mainly represented by fatty replacement of bone marrow, marked atrophy and moderate increased pigmentation (yellow/brown) of the spleen were noted.
One female was found dead on Day 18 of treatment. Salivation, rales, dyspnoea, pallor, staining on the perigenital region and piloerection were seen. At macroscopic examination, small spleen and thymus, most of the gastrointestinal tract distended with gas (stomach, jejunum, duodenum, ileum, caecum and colon) and incomplete collapse of lungs were seen. At microscopic examination, decreased cellularity of bone marrow, severe atrophy of thymus and marked atrophy and increased pigmentation (yellow/brown) of the spleen were noted.
One female was found dead on Day 7 of treatment. Salivation, dyspnoea, swollen abdomen, staining on the perigenital region and muzzle and piloerection were seen. At macroscopic examination, jejunum distended with gas, ruptured trachea and yellow staining of skin were observed. At microscopic examination, decreased cellularity of bone marrow, marked atrophy of thymus and spleen, increased pigmentation (yellow/brown) of the spleen and moderate necrosis of the liver were noted.
Atrophy of spleen, thymus and decreased cellularity of bone marrow were considered the factor contributory to the death of the high dose animals. These findings were considered the response to stress and related to the very high dose or being associated with agony of these animals.
The mid-dose female was found dead on Day 6 of the study. No clinical signs were noted. Macroscopically, the findings observed in this animal included: incomplete collapse and dark red colour of lungs, red fluid in the thoracic cavity, red thymus and red and swollen pituitary. Microscopically, the most relevant finding was moderate oedema of lungs (alveolar and peribronchial) and bronchi. The cause of death of this animal was due to a mis-dosing.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Dosing and Recovery Phases
No relevant changes were seen in treated animals, compared to controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Dosing Phase
Slight reduction in food consumption was seen in high dose females on Days 8 and 15 of study, during treatment at 1000 mg/kg/day, compared to controls. No relevant changes were seen in the high dose group animals after reduction of the dose level (500 mg/kg/day) and in the remaining treated animals during the study, compared to controls.
Recovery Phase
No relevant changes were seen in food consumption of treated animals, compared to controls.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Dosing Phase
Leucocytosis was recorded in a number of males from all treated groups, in one female
dosed at 100 mg/kg/day (no. X0610027) and one female receiving 1000/500 mg/kg/day
(no. X0610065). Compared with mean control data, changes were 20% to 73% higher,
with no dose-relation, and were mainly due to lymphocytosis. In addition, reticulocytes
were increased in females dosed at 1000/500 mg/kg/day (51% above mean control data).
However, no changes of the other red blood cell parameters were recorded, therefore this
finding was not considered to be adverse.
No changes were recorded in coagulation parameters.
Recovery Phase
Leucocytosis was still present in a number of animals of both sexes receiving 1000/500
mg/kg/day. Compared with mean control data, mean group values were 30% higher in
males and 55% in females. One male receiving 300 mg/kg/day also showed leucocytosis
(no. X0610052, 27% above mean control data). The statistically significant differences of
mean corpuscular volume and reticulocytes recorded between controls and males dosed
at 300 and 1000/500 mg/kg/day were not dose-related, therefore they were considered
unrelated to treatment.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Compared with mean control data, fluctuations of some biochemical parameters were recorded in a number of treated animals, such as: increase of glucose in males dosed at 300 and 1000/500 mg/kg/day and females receiving 100 and 1000/500 mg/kg/day (36 % to 77 %, with some dose-relation), decrease of cholesterol in males dosed at 1000/500 mg/kg/day (31 %), decrease of creatinine in males from all treated groups (16 % to 21 %, with some dose relation) and increase of calcium in males receiving 300 and 1000/500 mg/kg/day (4 %, both). The severity of the findings observed was not considered to be suggestive of tissue/organ injury.
One female (300 mg/kg/day) showed relevant increases of aspartate and alanine aminotransferases. Compared with mean control data, the increases were 3.8 and 3.21-fold, respectively. Due to the absence of dose-relation and the minimal incidence, these findings were not conclusively attributed to treatment.
Recovery Phase
In males, glucose, cholesterol and creatinine values were similar to those recorded at the Dosing Phase, while calcium showed complete reversibility. In females, glucose was still higher than controls in those receiving 1000/500 mg/kg/day. The other statistically significant differences between controls and treated animals (phosphorus, sodium and potassium in males, bilirubin and globulin in females) were not recorded at the Dosing Phase, therefore they were considered unrelated to treatment.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Dosing Phase
No relevant changes were observed.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Weekly detailed clinical signs (removal from cage and open field measurements)
Dosing and Recovery Phases
Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test item.
Neurotoxicity assessment (Functional Tests and Motor activity)
Dosing Phase
Increases in grip strength in all treated groups, statistically significant in the first trial (all treated male groups) and in the mean value (mid-dose male and high dose female groups) were seen at the end of treatment.
Measurements of motor activity and sensory reaction to stimuli were comparable between control and treated groups.
Recovery Phase
Similar trend was again seen in the grip strength at the end of the recovery period (statistical significance limited to second trial and mean value of low dose females). No relevant differences between treated and control groups were seen in the motor activity and sensory reaction to stimuli at the end of the recovery periods.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Final and recovery sacrifice
No relevant changes were observed on terminal body weight of treated animals, when compared to the controls. No relevant changes were observed in the absolute and relative organ weights of treatment groups of both sexes, when compared to control data, with the exception of the increased relative spleen weight of high dose male groups. However, since the histopathological evaluation of this organ was comparable to control animals, no toxicological significance could be attributed to this finding.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Final and recovery sacrifice
No remarkable differences were noted at post mortem examination in treated animals, when compared with controls, either in animals that completed the treatment or recovery period. Gross lesions such as pelvic dilatation of kidneys or distended and clear fluid contents in the uterus noted in few instances in treated animals, when compared to the controls, were considered to be related to a physiological variation in oestrous cycle or to a spontaneous/incidental pathology.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Final sacrifice
No treatment related changes were observed in treated animals, when compared to the controls. The sporadic lesions, reported in control and treated animals in the main phase, including nephropathy and/or pelvic dilatation in the kidneys of two high dose males or lumen dilatation of uterus in some instances in treated females, were considered to be an expression of spontaneous/or incidental pathology or physiological changes, seen in this species and age in this kind of studies in our experimental conditions.
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.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
mortality

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based on the results of the present study, the dose level of 500 mg/kg/day can be considered the NOAEL (No Observed Adverse Effect Level). This dose level could be used as high dose level for a subsequent OECD 421 study.
Executive summary:

In a repeated dose study according to OECD guideline 407, GLP, the test item “Sophorolipids: fermentation products of glucose and fatty acids, C18-unsatd., esters with glycerol with yeast Candida bombicola, partially hydrolysed” was administered to male and female Sprague Dawley rats via gavage for 4 weeks. The following recovery period was 4 weeks.

Study design

Initially, the aim of this study was to obtain information on the possible toxic effects on Sprague Dawley 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, conception,arturition and early lactation of the offspring.

The animals, 10 per sex, were assigned to four groups and administered orally, via gavage, at dose levels of 100, 300 and 1000 mg/kg/day. Control animals received the vehicle (softened water by reverse osmosis) at the same dose volume of 10 mL/kg.

Due to unexpected mortality and overall adverse effects during the first and the second week of treatment, it seemed evident that pairing and subsequent gestation and parturition of animals could be affected by the observed toxicity.

Therefore, it was decided to reduce the high dose level from 1000 mg/kg/day to 500 mg/kg/day, starting from Day 15 of treatment and to investigate repeated dose toxicity and recovery from any treatment-related effects, changing the current study design to a 4 week oral toxicity study followed by 4 weeks of recovery.

Mortality

A total of five treated animals were found dead during the study: one mid-dose female (300 mg/kg/day), one high dose male and three high dose females (1000 mg/kg/day). Atrophy of spleen, thymus and decreased cellularity of bone marrow were considered the factor contributory to the death of the high dose animals. These findings were considered the response to stress and related to the very high dose, or being associated with agony of these animals. In the female of the mid-dose group, the cause of death was due to a mis-dosing.

Clinical signs

Dosing Phase: Decreased activity, cyphosis, emaciated and piloerection were seen in one control male during the last week of treatment. At 100 mg/kg/day, salivation and staining on the neck were seen in single females. At 300 mg/kg/day salivation, piloerection on occasions in Weeks 1 and 2, hairloss/head in single males and females were seen during the treatment. At 1000/500 mg/kg/day, salivation, dyspnoea, rales and swollen/hard abdomen were seen in single animals, with higher incidence in females while receiving 1000 mg/kg/day. After reduction of dose level to 500 mg/kg/day, recovery from all signs was seen during the fourth week of treatment. No relevant signs were seen during the recovery period.

Neurotoxicity assessment (Functional Tests and Motor activity)

Dosing Phase: Increases in grip strength in all treated groups, statistically significant in the first trial and in the mean value of the mid-dose male and high dose female groups, were seen at the end of treatment. Measurements of motor activity and sensory reaction to stimuli were comparable between control and treated groups.

Recovery Phase: Similar trend was again seen in the grip strength at the end of the recovery period (statistical significance limited to second trial and mean value of low dose females). No relevant differences between treated and control groups were seen in the motor activity and sensory reaction to stimuli at the end of the recovery periods.

Body weight

No relevant changes were seen in treated animals, compared to controls.

Food consumption

Dosing Phase: Slight reduction in food consumption was seen in high dose females on Days 8 and 15 of the study, during treatment at 1000 mg/kg/day, compared to controls.

Recovery Phase: No relevant changes were seen in treated animals, compared to controls.

Haematology

Dosing Phase: Leucocytosis was recorded in a number of males from all treated groups, in one female dosed at 100 mg/kg/day and one female receiving 1000/500 mg/kg/day. Compared with mean control data, changes were with no dose-relation and mainly due to lymphocytosis. No changes were recorded in the coagulation parameter.

Recovery Phase: Leucocytosis was still present in a number of animals of both sexes receiving 1000/500 mg/kg/day and in one male receiving 300 mg/kg/day.

Clinical chemistry

Dosing Phase: Compared with mean control data, fluctuations of some biochemical parameters (glucose, cholesterol, creatinine, calcium) recorded in a number of animals treated at 300 and 500/1000 mg/kg/day were not considered to be suggestive of tissue/organ injury.

Recovery Phase: Changes observed were considered unrelated to treatment.

Urinalysis

No relevant changes were observed.

Terminal body weight and organ weights

No relevant changes were observed on terminal body weight, absolute and relative organ weight of treated animals, when compared to the controls.

Macroscopic observations

No remarkable differences were noted at post mortem examination in treated animals, when compared with controls, either in animals that completed the treatment or recovery period.

Microscopic observations

No treatment related changes were observed in treated animals, when compared to the

controls. 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.

Conclusion

Based on the results of the present study, the dose level of 500 mg/kg/day can be considered the NOAEL (No Observed Adverse Effect Level). This dose level could be used as high dose level for a subsequent OECD 421 study.