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Diss Factsheets

Administrative data

Description of key information

Oral administration to Wistar rats at doses of 100, 300 and 1000 mg/kg/day, for 28 days resulted in no substance related deaths, no clinical symptoms (at daily or weekly observations), no clinical symptoms (at the functional observational battery), no effects on food consumption or body weight development, no effects upon hematology and clinical biochemistry parameters, no changes in mean absolute or relative organ weights, and no macroscopical or microscopical findings of toxicological relevance. 
Based on the results of this study, 1000 mg/kg body weight/day was established as the no-observed-adverse-effect-level (NOAEL) for the test substance.
Repeated dermal toxicity:
The dermal route was waived. The substance is considered not to exert adverse effects.
Repeated inhalation toxicity:
The inhalation route was waived. The substance is considered not to exert adverse effects.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: well performed GLP compliant OECD guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy
- Age at study initiation: approximately 7-8 weeks
- Weight at study initiation: 223-230 g
- Fasting period before study: no
- Housing: 5 sex/cage
- Diet (e.g. ad libitum): ad libitum, except for clinical pathology investigations
- Water (e.g. ad libitum): ad libitum, except for clinical pathology investigations
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C +/- 2°C
- Humidity (%): 55% +/- 15%
- Air changes (per hr): approximately 15 to 20
- Photoperiod (hrs dark / hrs light): 12 /12

IN-LIFE DATES: From: 15 June 2012 To: 03 August 2012
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): solubility test
- Concentration in vehicle: 20, 60, 200 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability was found to be 24 hours at room temperature in the concentration range of 20 to 200 mg/mL.
Samples of the formulations prepared on Day 1 and on Week 4 of the study were also analysed to check the concentration and homogeneity. Results of the analyses were within the limits of acceptance.
Chemical analyses were carried out by the Analytical Chemistry Department at RTC, using a validated spectrophotometric detection in the range from 1.0 to 250 mg/mL.
The software used for this activity was Empower Probuild No. 2154.
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected in agreement with the Sponsor based on information from a preliminary, non GLP compliant RTC dose range finding study.

The purpose of this study was to investigate the toxicity of the test substance in rats after daily oral administration for 4 weeks and recovery from any treatment-related effects during a recovery period of 2 weeks (conrol and high dose group).
Positive control:
not necessary
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: three times weekly
- Cage side observations checked in table yes

CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: on the day of allocation to treatment groups, on the day that treatment commenced, weekly thereafter and just prior to necropsy.

FOOD CONSUMPTION :
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, at weekly intervals following allocation


HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 4 of treatment and Week 2 of recovery, just prior to necropsy
- Anaesthetic used for blood collection: Yes, under isofluorane
- Animals fasted: Yes
- How many animals: all 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

Prothrombin time


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 4 of treatment and Week 2 of recovery, just prior to necropsy
- Animals fasted: Yes
- How many animals: all 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
Albumin/Globulin Ratio
Sodium
Potassium
Calcium
Chloride


URINALYSIS: Yes
- Time schedule for collection of urine: Week 4 of treatment, just prior to necropsy
- Metabolism cages used for collection of urine: Yes
- 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: Weekly
- Dose groups that were examined: All
- Battery of functions tested: sensory activity / grip strength / motor activity / open field observations

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Euthanasia
Animals that had completed the scheduled test period were killed by exsanguination under isofluorane anaesthesia.
All animals were subjected to necropsy, supervised by a pathologist, as detailed 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 organs indicated 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 listed below were fixed and preserved in 10% neutral buffered formalin (except eyes, testes and epididymides which were fixed in Modified Davidson's fluid and preserved in 70% ethyl alcohol).

Histopathological examination
The tissues required for histopathological examination are listed below. After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometre thickness and stained with haematoxylin and eosin.

The examination was restricted as detailed below:
a) Tissues specified in the list below from all animals in the control and high dose groups killed after 4 weeks of treatment
b) All abnormalities in all main phase groups



Statistics:
For continuous variables the significance of the differences amongst group means 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. If the data were found to be heterogeneous, a modified t test (Cochran and Cox) was applied.
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
Description (incidence and severity):
Dosing phase: An increase in monocytes value was observed in some females receiving 1000 mg/kg/day. Recovery phase At the end of the recovery period, the monocytes value of the treated females was comparable to the control group.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Dosing phase: An increase in phosphorus was recorded in a few animals from all treated groups. Recovery phase: After 2 weeks of recovery period, phosphorus value of treated animals was comparable with controls.
Urinalysis findings:
no effects observed
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: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not specified
Details on results:
No clinical signs of toxicological significance were observed during treatment or recovery phases.

During the treatment period, neurotoxicity assessment (including motor activity and sensory reaction to stimuli), body weight and food consumption of treated groups did not show relevant signs, when compared to the control group.
Also during the recovery period, all the above parameters of the treated groups were comparable to the control group.

At haematological evaluation performed at the end of the treatment period, monocytosis was recorded in some females receiving 1000 mg/kg/day. In the absence of other clinical pathology and/or histopathological related changes and due to the inconsistency between sexes, the above findings was considered of no toxicological importance. At the end of the recovery period, monocytosis was no longer observed.

At clinical chemistry evaluation the principal alteration was an increase in phosphorus value (14% to 35%) in a few animals from all treated groups. But changes were not dose-related and were no longer observed at the end of the recovery period. Therefore changes were considered of no toxicological importance.

Terminal body weights, absolute and relative organ weights of treated animals were comparable to the control group both in dosing and recovery phases.

No findings of toxicological significance were observed at macroscopic observations.

No treatment-related lesions were noted at microscopic observations.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed up to the highest dose tested
Critical effects observed:
not specified
Conclusions:
On the basis of the above mentioned results 1000 mg/kg/day of the test item was established as the NOAEL (No Observed Adverse Effect Level) for animals of both sexes.

Executive summary:

Study design

 

The toxicity of the test item was investigated, when given orally to rats for 4 weeks followed by a recovery period of 2 weeks. During the recovery period, no treatment was given to the recovery animals.

Each group comprised 5 male and 5 female rats, while control and high dose groups included 5 additional animals per sex to be sacrificed after 2 weeks of recovery.

 

The test item was administered by oral gavage at a dose volume of 5 mL/kg.

 

Group

Number

Treatment

(mg/kg/day)

Number of animals

1

2

3

4

0

100

300

1000

10M+10F

5M+5F

5M+5F

10M+10F

M = Males; F = Females

 

The following investigations were performed: daily clinical signs, neurotoxicity assessment (weekly detailed clinical examinations, motor activity and sensory reactivity to stimuli), body weight, food consumption, clinical pathology investigations, organ weights, macroscopic observations and histopathological examination.

 

Mortality

 

No animals died during the study.

 

Clinical signs and observation of the cage tray

 

Dosing phase and recovery phase

 

The clinical sign noted throughout the study (blue staining of the body surface) was clearly related to the blue colour of the test item.

 

Observation of the cage tray

 

As consequence of the colour of the test item, light blue staining was observed on the cage tray in animals of both sexes receiving the dose level ≥ 300 mg/kg/day.

 

1.4        Neurotoxicity assessment – Detailed clinical examination

 

Dosing phase and recovery phase

 

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


 

1.5        Body weight

 

Dosing phase and recovery phase

 

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

 

Food consumption

 

Dosing phase and recovery phase

 

Food consumption did not show any differences between control and treated groups, during both treatment and recovery periods.

 

Motor activity

 

Dosing phase and recovery phase

 

Motor activity, performed at the end of the treatment and recovery periods, did not show particular differences between the control and treated groups in both sexes.

 

Sensory reactivity to stimuli

 

Dosing phase and recovery phase

 

Sensory reactivity to stimuli, recorded at the end of treatment and recovery periods, was comparable between control and treated groups.

 

Haematology

 

Dosing phase

 

An increase in monocytes value was observed in some females receiving 1000 mg/kg/day. In the absence of other clinical pathology and/or histopathological related changes and due to the inconsistency between sexes, the above finding was considered of no toxicological importance.

 

Recovery phase

 

At the end of the recovery period, the monocytes value of the treated females was comparable to the control group.

 

Clinical chemistry

 

Dosing phase

 

An increase in phosphorus was recorded in a few animals from all treated groups. Changes were less severe, not dose-related and not evident in the recovery group. Therefore changes were considered of no toxicological importance.

 

Recovery phase

 

After 2 weeks of recovery period, phosphorus value of treated animals was comparable with controls.


 

Urinalysis

 

Dosing phase

 

No changes in urine parameters were observed in treated animals when compared to the control group.

 

Terminal body weight and organ weights

 

Final sacrifice and recovery sacrifice

 

Terminal body weight in treated animals did not show any differences between control and treated groups, both in dosing and recovery phases.

 

Final sacrifice

 

Absolute and relative organs weights

No intergroup differences in organ weights were observed in treated groups, when compared to controls.

 

Recovery sacrifice

 

Absolute and relative organs weights

No differences were observed between control and treated groups.

 

Macroscopic observations

 

Final sacrifice

 

The macroscopic findings observed at post mortem examination (blue staining in the mucosa of non glandular region of the stomach, in the caecum and on the skin and the tail surface) were clearly related to the colour of the test item administered.

 

Recovery sacrifice

 

No treatment-related macroscopic changes were noted.

 

Microscopic observations

 

Final sacrifice

 

No treatment-related lesions were noted.

 

Conclusions

 

On the basis of the above mentioned results, 1000 mg/kg/day of the test item was established as the NOAEL (No Observed Adverse Effect Level) for animals of both sexes.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
reliable without restrictions - well performed GLP compliant OECD guideline study

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
no alternative study available

Justification for classification or non-classification

The submission substance does not have to be classified regarding systemic and target organ toxicity after repeated oral exposure according to the criteria laid down in Directive 67/548/EEC and in Regulation (EC) No 1272/2008, because:

- The submission substance caused no relevant systemic effects and revealed an NOAEL of 1000 mg/kg/day (which is the highest dose tested) in a Repeated Dose Toxicity Test after oral application.

 

It can reasonably be deduced that the submission substance does not exert systemic toxic effects after repeated dermal application and thus does not have to be classified according to the criteria laid down in Directive 67/548/EEC and in Regulation (EC) No 1272/2008 and that testing is not scientifically necessary, because:

- The submission substance caused no relevant systemic effects and revealed an NOAEL of 1000 mg/kg/day in a Repeated Dose Toxicity Test after oral application,

- it is unlikely that the submission substance becomes systemically bioavailable to a relevant extend after dermal exposure due to its low solubility in water and n-octanol,

- the repeated dose toxicity after dermal application is not considered to be higher than after oral administration,

- testing for acute dermal toxicity revealed no signs of bioavailability or toxicity.

- the submission substance does not have to be classified as skin sensitizing or as skin or eye irritating, indicating that its chemical inertness and low solubility in water and n-octanol largely prevent interaction with living cells and tissues.

 

It can reasonably be deduced that the submission substance does not exert systemic toxic effects after repeated inhalation exposure and thus does not have to be classified according to the criteria laid down in Directive 67/548/EEC and in Regulation (EC) No 1272/2008 and that testing is not scientifically necessary, because:

- the submission substance caused no relevant systemic effects and revealed an NOAEL of 1000 mg/kg/day in a Repeated Dose Toxicity Test after oral application,

- it is unlikely that the submission substance becomes systemically bioavailable to a relevant extend after inhalation due to its low solubility in water and in n-octanol,

- the submission substance does not have to be classified as skin sensitizing or as skin or eye irritating, indicating that its chemical inertness and low solubility in water and in n-octanol largely prevent interaction with living cells and tissues,

- the submission substance, when aerosolized in respirable form, is likely to behave like an inert dust.