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Description of key information

NOEL oral rat subacute > 1000 mg/Kg bw

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
short-term repeated dose toxicity: oral
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January-May 2013
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study, well conducted
Reason / purpose:
reference to same study
according to
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Details on test animals and environmental conditions:
Animal supply and acclimatisation
A total of 90 Sprague Dawley rats (45 males and 45 virgin females), 6 to 7 weeks old and weighing 176 to 200 g for males and 151 to 175 g for females, have been ordered from Charles River Italia S.p.A., Calco (Lecco), Italy.
After arrival the weight range for each sex have been determined and the animals have been temporarily identified within the cage by means of a coloured mark on the tail. A health check has then been performed by a veterinarian.
An acclimatisation period of approximately 2 weeks has been allowed before the start of treatment, during which time the health status of the animals has been assessed by thorough observations. Rats considered unsatisfactory have been killed and were appropriate subjected to pathological examination.

Animal husbandry
The animals have been housed in a limited access rodent facility. Animal room controls have been set to maintain temperature and relative humidity at 22°C +/- 2°C and 55%+/- 15% respectively; actual conditions have been monitored, recorded and the records retained. There will be approximately 15 to 20 air changes per hour and the rooms will be lit by artificial light for 12 hours each day.
From arrival to pairing, animals have been housed up to 5 of one sex to a cage, in polisulphone solid bottomed cages measuring 59.5x38x20 cm (Techniplast Gazzada S.a.r.l., Buguggiate, Varese). Nesting material has been provided inside suitable bedding bags and changed at least twice a week.
During mating, animals have been housed one male to one female in clear polycarbonate cages measuring approximately 43x27x18cm with a stainless steel mesh lid and floor (Techniplast – Gazzada S.a.r.l.). Each cage tray holded absorbent material which has been inspected and changed daily.
After mating, the males have been recaged as they were before mating, the females have been transferred to individual solid bottomed cages (Techniplast Gazzada S.a.r.l.) for the gestation period, birth and lactation. Suitable nesting material has been provided and has been changed as necessary.
Drinking water has been 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) will be offered ad libitum throughout the study.
There is no information available to indicate that any non-nutrient substance likely to influence the effect of the test item is present in the drinking water or the diet. Records of analyses of water and diet are kept on file at RTC.
Dated and signed records of activities relating to the day to day running and maintenance of the study in the animal house have been recorded.

Route of administration:
oral: gavage
unchanged (no vehicle)
Analytical verification of doses or concentrations:
Duration of treatment / exposure:
Animals have been 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.
Dose volumes will be adjusted once per week for each animal according to the last recorded body weight.

Animals will be 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 will be adjusted once per week for each animal according to the last recorded body weight.
During the gestation period, dose volumes will be calculated according to individual body weight on Days 0, 7, 14 and 20 post coitum and on Day 1 post partum. Thereafter individual dose volumes will remain constant.
Frequency of treatment:
Once a day
Doses / Concentrations:
10 mg/Kg bw
actual ingested
Doses / Concentrations:
100 mg/Kg bw
actual ingested
Doses / Concentrations:
1000 mg/Kg bw
actual ingested
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
The test item have been 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 has been administered to each animal on the basis of the most recently recorded body weight and the volume administered has been recorded for each animal
Positive control:
Observations and examinations performed and frequency:
Early in each working day in the morning and in the afternoon. At weekends and Public Holidays a similar procedure will be followed except that the final check has been carried out at approximately mid-day.

Clinical signs
Once before commencement of treatment and at least once daily during the study,

Clinical observations (Functional Observation Battery Tests)
Once before commencement of treatment and at least once a week thereafter

Grip strength and sensory reactivity to stimuli
Once during the study, towards the end of treatment, on 5 males and 5 females randomly selected from each group

Motor activity assessment (MA)
Once during the study, towards the end of treatment, on 5 males and 5 females randomly selected from each group

Food consumption
Recorded weekly (whenever possible) during the pre-mating period starting from allocation. Individual food consumption for the females have been measured on gestation Days 7, 14 and 20 starting from Day 0 post coitum and on Day 4 post partum starting from Day 1 post partum.

Body weight
Males weekly from allocation to termination.
Females weekly from allocation to positive identification of mating and on gestation Days 0, 7, 14 and 20.
Dams Days 1 and 4 post partum.

Vaginal smears
Vaginal smears has been taken daily in the morning starting two weeks before pairing until a positive identification of copulation is made.


Parturition and gestation length
A parturition check has been performed from Day 20 to Day 25 post coitum. F

Pups identification, weight and observation
As soon as possible, after parturition is considered complete (Day 0 or 1 post partum), .
Once daily for all litters
Sacrifice and pathology:

Parental animals in extremis or killed for humane reasons and those that have completed the scheduled test period will be killed by exsanguination under isofluorane anaesthesia.
Pups in extremis or killed for humane reasons or those that have completed the scheduled test period (Day 4 post partum) will be euthanised by intraperitoneal injection of Thiopenthal.

Parental males:
The males will be killed after the mating of all females or after at least 28 days of treatment period.

Parental females:
The females with live pups will be killed on Day 4 post partum.
The females with total litter loss will be killed on the day of the occurrence of total litter loss or shortly after.
The females showing no evidence of copulation will be killed after 25 days of the last day of the mating session (see section 4.2.9).
The females which do not give birth 25 days after positive identification of mating will be killed shortly after.

4.4.2 Necropsy

The clinical history of the males and females of the parental generation will be studied and a detailed post mortem examination will be conducted (including examination of the external surface and orifices).
Changes will be noted, the requisite organs weighed (excluding animals sacrificed for humane reasons or found dead) and the required tissue samples preserved in fixative and processed for histopathological examination.

All females will be examined also for the following:

b) number of visible implantation sites (pregnant animals);
c) number of corpora lutea (pregnant animals).

Uteri of females with no visible implantations will be immersed in a 10-20% solution of ammonium sulphide to reveal evidence of implantation.

All pups found dead in the cage or sacrificed for humane reasons will be examined for external and internal abnormalities.
All live pups sacrificed at termination will be killed and examined for external abnormalities and sex confirmation by gonadal inspection.

4.4.3 Organ weights

From all animals completing the scheduled test period, the organs indicated in Annex 1 will be dissected free of fat and weighed.
The ratios of organ weight to body weight will be calculated for each animal.
At the discretion of the pathologist, organs may be weighed from animals dying or killed prior to terminal kill.

4.4.4 Tissues fixed and preserved

Samples of all the tissues listed in Annex 1 will be fixed and preserved in 10% neutral buffered formalin (except eyes, optic nerves and Harderian glands, testes and epididymides which will be fixed in modified Davidson's fluid and preserved in 70% ethyl alcohol).

4.4.5 Histopathological examination

The tissues required for histopathological examination are listed in Annex 1. After dehydration and embedding in paraffin wax, sections of the tissues will be cut at 5 micrometer thickness and stained with haematoxylin and eosin.
In addition, the testes and epididymides will be cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) will be performed.
If considered necessary, histological processing may be subcontracted to a GLP certified test site. In such cases, a protocol amendment will be issued, the Sponsor will be informed of the location of the test site and the complete address and name of the Principal Investigator will be presented in the Final Report.
In the first instance the examination will be restricted as detailed below:

a) Tissues specified in Annex 1 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 Annex 1 from all animals killed or dying during the treatment period.
c) All abnormalities in all groups.

The examination could then be extended to include the remaining 5 males and 5 females (animals not evaluated for clinical pathology) of the control and the high dose group (additional cost).
The examination could then be extended to animals of the other dose groups, if treatment-related changes are observed in the high dose group.
All histopathological activities which cannot be foreseen before the start of the study (i.e. processing of all abnormalities, tissues of unscheduled deaths and target tissues in the low, medium dose groups) will incur an additional cost.

4.4.6 Photomicrographs

Representative photomicrographs may be taken of any treatment-related lesions. Other photomicrographs may be taken as required by the Sponsor (additional cost).
Other examinations:
As a part of the sacrificial procedure, samples of blood will be withdrawn under isofluorane anaesthesia from the abdominal vena cava from 5 males and 5 females (females with viable litters, if possible) randomly selected from each group, under condition of food deprivation.
The blood samples collected will be divided into tubes as follows:

EDTA anticoagulant for haematological investigations
Heparin anticoagulant for biochemical tests
Citrate anticoagulant for coagulation tests

The measurements to be performed on blood samples are listed below:

4.3.1 Haematology

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

4.3.2 Coagulation tests

Prothrombin time

4.3.3 Clinical chemistry

Alkaline phosphatase
Alanine aminotransferase
Aspartate aminotransferase
Bile acids
Total bilirubin
Total cholesterol
Total protein
A/G Ratio

4.3.4 Urinalysis (Only males – optional, to be discussed see section 3.2)

At the same time interval (if required) of the clinical pathology investigations, individual overnight urine samples will also be collected from the same animals under the same conditions. Before starting urine collection water bottles will be removed from each cage and each animal will receive approximately 10 mL/kg of drinking water by gavage, in order to obtain urine samples suitable for analysis.

Specific gravity
Total reducing substances (only if Glucose is higher than 100 mg/dL)
The sediment, obtained from centrifugation at approximately 3000 rpm for 10 minutes, will be examined microscopically for:

Epithelial cells
Spermatozoa and precursors
Other abnormal components
Standard deviations will be calculated as appropriate. For continuous variables the significance of the differences amongst group means will be assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
Statistical analysis of histopathological findings will be carried out by means of the non-parametric Kolmogorov-Smirnov test if n is more than 5.
The non-parametric Kruskal-Wallis analysis of variance will be used for the other parameters. Intergroup differences between the control and treated groups will be assessed by the non-parametric version of the Williams test. The criterion for statistical significance will be p<0.05.
Further tests will be used as considered appropriate. Details of all tests used and the data to which they are applied will be included in the Final Report.
Clinical signs:
no effects observed
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
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:
no effects observed
Dose descriptor:
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Critical effects observed:
not specified
NOEL > 1000 mg/Kg bw
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
1 000 mg/kg bw/day
Study duration:

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:
Reliable GLP study

Justification for classification or non-classification

Repeated oral toxicity

The substance is not classified orally toxic because it doesn't meet the classification criteria of the CLP regulation n. 1272/2008 on repeated exposure:

Category 1: substances that have produced significant toxicity in humans or that, on the basis of evidence from studies in experimental animals, can be presumed to have the potential to produce significant toxicity in humans following repeated exposure.

Category 2: substances that, on the basis of evidence from studies in experimental animals can be presumed to have the potential to be harmful to human health following repeated exposure.

No oral repeated toxicity on organs was observed at the highest tested dose (1000 mg/Kg bw)