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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 APR 2009 to 21 AUG 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD 422)
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
: the exposure duration proir to pairing was extended to 10 weeks, an analysis of bleeding time was performed in addition to the usual examination range of the guideline
GLP compliance:
yes
Remarks:
Deviation: In agreement with the Sponsor no formulation analyses were performed at RTC for technical reasons (the test item was not analysable in the vehicle).
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Licowax S FL (alternative name: Licowax S)
- Substance type: Flakes-scales light yellow
- Physical state: solid
- Storage condition of test material: Room temperature, protected from light

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Italy S.r.l., San Pietro al Natisone (UD), Italy.
- Age at study initiation: 27-29 days old
- Weight at study initiation: weight range of 84-107g for males and 86-104g for females
- Housing: From arrival to pairing animals were housed 5 of one sex to a cage. During mating animals were housed one male to one female. Males were re-caged as they were before mating. Females werde housed individually during gestation period, birth and lactation. Housing was performed in clear polycarbonate cages.
- Diet (ad libitum): commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4,
20019, Settimo Milanese (MI), Italy)
- Water (ad libitum): Drinking water
- Acclimation period: 18 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 15%
- Air changes (per hr): 15 to 25
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: sesame oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulation procedure Group 2 (concentration of 1 mg/ml) and Group 3 (concentration of 10 mg/ml):
- the required volume of Sesame oil was added to the required amount of Licowax S FL.
- formulations were kept in water bath at 80-100°C until the test item was completely
dissolved in the vehicle (approximately 15 minutes).
- the obtained suspensions were then homogenised (by Silverson) for approximately 2-3
minutes and left on a magnetic stirrer at room temperature prior to use.

Formulation procedure for Group 4 (concentration of 100 mg/ml):
- the required volume of Sesame oil was added to the required amount of Licowax S FL.
- the formulation was kept in water bath at 80-100°C until the test item was completely
dissolved in the vehicle (approximately 15 minutes).
- the obtained suspension was then homogenised (by Silverson) for approximately 2-3
minutes.
- the formulation was kept in water bath at approximately 37°C.
- the formulation was stirred manually by means of a spatula, from Day 1 to Day 13 of
treatment.
- the formulation was homogenised (by Silverson) once again for approximately 1minutes,
before despatching to the rodent facility.
During the dosing procedure in the animal facility, the formulation was kept in water bath at
approximately 37°C.
Formulations were prepared daily and concentrations were calculated and expressed in terms
of the test item as supplied.


VEHICLE
- Amount of vehicle (if gavage): 10 ml/kg bw
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Due to technical reasons (test item was not analysable in the vehicle) prior to commencement of treatment, analysis was not performed to confirm that the proposed formulation procedure was acceptable (content check and homogeneity). In addition, in agreement with the Sponsor no formulation analyses were performed at RTC for technical reasons (the test item was not analysable in the vehicle).
Duration of treatment / exposure:
Both sexes: once a day, 7 days a week, for 10 consecutive weeks prior to pairing. Males were sacrificed after completion of the mating period. Females with live pups were sacrificed on Day 4 post partum. Females which did not give birth after 25 days of post coitum period were sacrificed shortly after. Females which did not mate were sacrifice on Day 36 of the mating phase.
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg bw/day
Basis:
other: vehicle control
Remarks:
Doses / Concentrations:
10 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected in consultation with the Sponsor.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: all animals were checked in each working day, early 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: All clinical signs were recorded for individual animals.
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 at the same time interval each day, the interval was selected
taking into consideration the presence of post-dose reactions.
Functional Observation Battery Tests: Once before commencement of treatment, and at least once per week from the start of
treatment until week 14 of treatment each animal was given a detailed clinical examination.
Each animal was removed from the home cage and observed in an open arena.


BODY WEIGHT: Yes
- Time schedule for examinations: Males were weighed weekly from allocation to termination.
Females were weighed weekly from allocation to pairing, on post coitum Days 0, 7, 14 and
20 and on Days 1 and 4 post partum

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Both eyes of all animals assigned to the study were examined just prior to the
commencement of treatment by means of an ophthalmoscope, and by a slit-lamp
microscope, after the instillation of 0.5% Tropicamide (Visumidriatic®, Visufarma, Rome,
Italy). Two females, one of group 2 (76740037) and one of group 4 (76740075) with nonresolving
lesions were replaced with spare animals showing no ocular abnormality, from the
batch initially ordered for the study.
- Dose groups that were examined: In the first instance the eyes of all animals from high
dose and control groups were re-examined during the last week of treatment. Since changes
were observed in high dose female groups, the examination was extended to the low and
mid- dose female groups.


HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy
- Anaesthetic used for blood collection: Yes (isofluorane)
- Animals fasted: No data
- How many animals: all survivors
- Parameters checked:
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
Prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at necropsy
- Anaesthetic used for blood collection: Yes (isofluorane)
- Animals fasted: No data
- How many animals: all survivors
- Parameters checked:
Alkaline phosphatase
Alanine aminotransferase
Aspartate aminotransferase
Gamma -glutamyltransferase
Urea
Creatinine
Glucose
Triglycerides
Phosphorus
Total bilirubin
Total cholesterol
Total protein
Albumin
Globulin
A/G Ratio
Sodium
Potassium
Calcium
Chloride

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once before commencement of treatment, and at least once per week from the start of
treatment until week 14 of treatment 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, sterotypies or bizarre behaviour and effects on the
autonomic nervous system (e.g. lachrymation, piloerection, unusual respiratory pattern).
All observations were recorded for individual animals. During the gestation periods the observations were carried out weekly
- Dose groups that were examined: all groups
- Battery of functions tested: Grip strength and sensory reaction to stimuli, motor activity assessment


OTHER: The weight of food consumed by each cage of males and females was recorded weekly
during the pre-pairing period starting from allocation. Individual food consumption for the
females were measured on gestation Days 7, 14 and 20 starting from Day 0 of gestation and
on Day 4 post partum starting from Day 1 post partum
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table)
HISTOPATHOLOGY: Yes (see table)

Abnormalities
Adrenal glands
Aorta
Bone marrow (from sternum)
Brain (cerebrum, cerebellum, medulla/pons)
Caecum
Colon
Duodenum
Epididymides
Eyes
One Epididymal caudac
Femur with joint
Heart
Ileum (including Peyer¿s patches)
Jejunum
Kidneys
Liver
Lungs (including mainstem bronchi)
Lymph nodes - cervical
Lymph nodes - mesenteric
Mammary area
Oesophagus
Ovaries
Oviductsa
Pancreas
Parathyroid glands
Pituitary gland
Prostate gland
Rectum
Salivary glands
Sciatic nerve
Seminal vesicles and coagulating glands
Skeletal muscle
Skin
Spinal column
Spinal cord
Spleen
Stomach
Testes
Thymus (where present)
Thyroid
Trachea
Urinary bladder
Uterus - cervix
Other examinations:
Bleeding time:
During week 4 of treatment (approximately 1 hour after treatment), 5 males and 5 females
selected from the control and high dose groups were anaesthetised with isofluorane.
At approximately 3 mm from the tip of the tail a stab wound was made by means of a needle
of 18 gauge.
The distal portion of the tail was immediately immersed in a tube containing physiological
saline kept at 37°C and the time between the immersion of the tail in the tube and cease of
the bleeding was measured. This additional evaluation was requested by the Sponsor.

Sperm analysis
Daily sperm production
Reproductive Parameters according to guideline OECD 422
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 nonparametric
version of the Williams test.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
At the daily clinical examination matted fur was noted in treated males receiving 100 and
1000 mg/kg/day, throughout the study. Treated females receiving 100 and 1000 mg/kg/day
of Licowax S FL showed also matted fur. In addition, one female receiving 100 mg/kg/day
and two females receiving 1000 mg/kg/day showed damaged eye. One of the latter females
also showed atrophic eye. All these signs were noted before mating.
No clinical signs were noted in control and treated females during the post coitum and post
partum periods.
Observation of animals at removal from the cage and in an open arena (neurotoxicity
assessment) did not reveal changes attributable to the test item.


HAEMATOLOGY
Increments of lymphocytes (28%) and large unstained cells (43%) were recorded in males
treated with 1000 mg/kg/day.
Females from the same group showed a slight decrement of erythrocytes and haemoglobin
(10% and 7%, respectively) and an increment of reticulocytes (50%). However, changes
were insufficient in magnitude to represent an appreciable anaemia.
Haemoglobin was slightly decreased also in the females receiving 10 mg/kg/day, with the
same severity.
All the above changes were considered devoid of toxicological relevance.

CLINICAL CHEMISTRY
Glucose and phosphorus were increased in all treated males, without a clear dose-relation
(changes were 59% to 96%). However, changes were not dose-related and data from the
control animals were lower compared with our historical data.
The other statistically significant changes observed (decrement of alkaline phosphatase,
bilirubin, cholesterol, triglycerides, protein, globulin, calcium and sodium in males treated
with 100 mg/kg/day, decrement of albumin in females dosed with 1000 mg/kg/day) were of
low severity or seen in the intermediate groups only. Therefore, they were considered to be
of no toxicological significance.

OTHER FINDINGS
No differences in bleeding time were noted between control and high dose groups during
week 4 of the study.

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: no treatment-related adverse effects were observed

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

The results of the reproductive/developmental screening test are presented in chapter 7.8.1

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, no treatment-related effects were observed up to 1000 mg/kg bw/day, the highest dose tested.
Executive summary:

The effects after repeated dosing with Licowax S FL, 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 in a study according to guideline OECD 422, with extended treatment prior to mating. Groups of 10 male and 10 female Sprague-Dawley rats received Licowax S FL by gavage at dosages of 10, 100 and 1000 mg/kg/day. A similarly constituted group of animals received the vehicle (sesame oil) and acted as a control. All doses were administered at a constant volume of 10 ml/kg body weight. Males were treated for 10 weeks prior to pairing and during pairing until the day before necropsy, for a total of approximately 15 weeks. Females were treated for 10 weeks prior to pairing, during pairing and throughout the gestation and lactation periods until Day 3 post partum.

Body weight, body weight gain, clinical signs (including neurotoxicity assessment, motor activity and sensory reaction to stimuli), food consumption, mating performance, ophthalmoscopy and bleeding time measurements were evaluated.Clinical pathology investigations (haematology and clinical chemistry) were also evaluated. At necropsy a detailed external and internal examination was performed. The histopathological examination, including identification of the stages of the spermatogenic cycle was carried out in five males of control and high dose groups, selected randomly. No relevant signs of toxicological significance were observed in any of the above parameters investigated during the in-life phase or at post mortem examination. There were no significant differences between treated and control groups. In an additional investigation, no differences in bleeding time were noted between control and high dose groups during

week 4 of the study.

On the basis of these results the NOAEL (No Observed Adverse Effect Level) for males and females could be considered 1000 mg/kg/day. Therefore, the test substance has not to be classified for repeated oral toxicity according to Regulation (EC) 1272/2008 and Council Directive 67/548/EEC.