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Description of key information

A sub-acute repeated dose toxicity study, including screening for reproduction/ developmental effects, performed with Sakura Salicylate according to OECD/EC guidelines and GLP principles is available. The results of two preliminary studies are included as supplementary data.

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
Remarks:
Combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 March 2016 - 18 January 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
A total of 90 Hsd: Sprague Dawley SD rats (45 males and 45 virgin females), 6 to 7 weeks old and weighing 175 to 200 g for males and 150 to 175 g for females, were ordered from and supplied by Charles River Italia S.p.A., Calco (Lecco), Italy.
After arrival, on 3 March 2016, the weight range for each sex was determined (201-218 g for males, 162-186 g for females, both outside the range at order) 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 13 days 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.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
The required amount of SAKURA SALICYLATE was dissolved/suspended in the vehicle. The formulations were prepared daily at concentrations of 10, 20 and 40 mg/mL. Concentrations were calculated and expressed in terms of test item as supplied.
The test item was administered orally by gavage at a dose volume of 5 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.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
A separate 28 hour and 8 day stability at room temperature was established in the range from 5 to 50 mg/mL.
The proposed formulation procedure for the test item was checked in the range from 5 to 50 mg/mL by chemical analysis (concentration) to confirm that the method was suitable. Samples of the formulations prepared on Day 1 and Week 5 were analysed to check the concentration. Results of the analyses were within the acceptability limits stated in RTC SOPs for concentration of solutions (90-110 %). The software used for this activity was the Empower® 2 Build No. 2154.
Duration of treatment / exposure:
MAIN GROUPS (GROUPS 1 TO 4)
- Males: Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing and daily thereafter until the day before necropsy (Days 29 and 30 of study). They were treated for a total of 28 or 29 days. 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 2 consecutive weeks prior to pairing and thereafter during pairing, postcoitum and postpartum periods until Day 3postpartum (for 42 to 52 days). Dose volumes were adjusted once per week for each animal according to the last recorded body weight. During the gestation period, dose volumes were 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 remained constant.
Frequency of treatment:
Once daily
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
Group 2
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Group 3
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Remarks:
Group 4
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
1) Clinical signs: once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions.

2) Clinical Observations (Functional Observation Battery Tests): 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).

3) Grip strength and sensory reactivity to stimuli: once during the study, towards the end of treatment, 5 males and 5 females were randomly selected from each group for evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli) and for assessment of grip strength. Measurements were performed using a computer generated random order. For males, the tests were performed during Week 4 of the study and for females on Day 3 post partum (from Week 6 to Week 8).

4) Motor activity assessment (MA): once during the study, towards the end of treatment, 5 males and 5 females 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. For males, the tests were performed during Week 4 of the study and for females on Day 3 postpartum (from Week 6 to Week 8).

5) Body weight: males were weighed weekly from allocation to termination. Females were weighed weekly from allocation to positive identification of mating and on gestation Days 0, 7, 14 and 20. Dams were also weighed on Days 1 and 4 post partum.

6) 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 post coitum and on Day 4 post partum starting from Day 1 post partum.

7) Clinical pathology investigations: During the necropsy procedure, samples of blood were withdrawn under isofluorane anaesthesia from the abdominal vena cava from 5 males and 5 females (females with viable litters) randomly selected from each group under conditions of food deprivation.
Main Groups Males Females
1 2 6 8 10 12 3 5 11 13 19
2 24 30 32 34 38 25 27 31 35 39
3 44 46 52 54 60 41 43 53 55 57
4 62 70 72 76 80 63 65 69 71 75
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
The measurements performed on blood samples are listed below:
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, Lymphocites, Eosinophils, Basophils, Monocytes, Large unstained cells), Platelets
Coagulation: 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.
Hormone determination: Prior to necropsy, blood samples were taken from the abdominal vena cava under isofluorane anaesthesia of 5 males and 5 females (with viable litters) randomly selected for clinical pathology investigation. Blood samples were collected into tubes containing lithium heparin as anticoagulant, centrifuged and plasma samples were frozen at approximately -80°C for possible future analysis of T3, T4 and THS evaluation. These samples were not analysed.
Sacrifice and pathology:
1) Euthanasia: one parental animal in extremis and those that had completed the scheduled test period were killed by exsanguination under isofluorane anaesthesia. Pups killed for humane reasons or those that had completed the scheduled test period (Day 4 post partum) were euthanised by intraperitoneal injection of Thiopenthal.
- Parental males: the males were killed after the mating of all females or after at least 28 days of treatment period.
- Parental females: the females with live pups were killed on Day 4 post partum. The females with total litter loss were killed on the day of the occurrence of total litter loss or shortly after. The females showing no evidence of copulation were killed after 25 days of the last day of the mating session. The females which did not give birth 25 days after positive identification of mating were killed shortly after.

2) Necropsy: 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 and the required tissue samples preserved in fixative and processed for histopathological examination.
- Females: all females were examined also for the following: number of visible implantation sites (pregnant animals) and number of corpora lutea (pregnant animals). Uteri of females with no visible implantations were immersed in a 10-20 % solution of ammonium sulphide to reveal evidence of implantation.

3) Organ weights: from all animals completing the scheduled test period, the following organs were dissected free of fat and weighed: adrenal glands, brain (cerebrum, cerebellum, medulla/pons), epididymides, heart, kidneys, liver, ovaries with oviducts, parathyroid glands, prostate glands, seminal vesicles with coagulating glands, spleen, testes, thymus, thyroid, uterus including cervix. The ratios of organ weight to body weight were calculated for each animal.

4) Tissues fixed and preserved: Samples of all the following tissues were fixed and preserved in 10 % neutral buffered formalin (except eyes, optic nerves, testes and epididymides which were fixed in Modified Davidson’s fluid and preserved in 70 % ethyl alcohol): abnormalities, adrenal glands, bone marrow (from sternum), brain (cerebrum, cerebellum, medulla/pons), caecum, clitoral gland, colon, duodenum, epididymides, heart, ileum, jejunum (including Peyer’s patches), kidneys, liver, lungs (including mainstem bronchi), lymph nodes – cervical, lymph nodes – mesenteric, nasal cavity *, oesophagus *, ovaries with oviducts, parathyroid glands, pituitary gland, penis, prostate gland, rectum, sciatic nerve, seminal vesicles with coagulating glands, spinal column *, spinal cord (cervical, thoracic, lumbar), spleen, stomach, testes, thymus, thyroid, trachea, urinary bladder, uterus – cervix, vagina.
* Not examined as no signs of toxicity or target organ involvement were observed.

5) Histopathological examination: 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). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed.
The examination was restricted as detailed below: tissues specified above from 5 males and 5 females randomly selected (animals evaluated for clinical pathology) in the control and high dose group killed at term; tissues specified above from all animals killed or dying during the treatment period; all abnormalities in all groups.
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. Inter group differences between the control and treated groups were assessed by the non-parametric version of the Williams test. The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.
Clinical signs:
no effects observed
Description (incidence and severity):
Observed clinical signs were limited to hair loss, seen in 2 high dose females. A palpable mass was observed in 1 female animal (no. 39). This data was described at post mortem as an adenocarcinoma of the mammary gland (spontaneous pathology).
Mortality:
mortality observed, treatment-related
Description (incidence):
One female of the high dose group, receiving 200 mg/kg/day, was killed for humane reasons on Day 22 of the gestation phase. One male (Group 2) dosed at 50 mg/kg/day was found dead on Day 6 of the premating phase, while one female (Group 4) dosed at 200 mg/kg/day, was found dead on Day 23 of the gestation phase, after parturition. With the exception of the male, the observed deaths were considered treatment-related.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
A very slight reduction (- 6 %) was observed in the high dose females on Day 4 post partum only.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Reductions in food consumption (- 29 %) were observed in the high dose females on Day 4 post partum only.
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):
Macrocytic anaemia was recorded in many females dosed at 200 mg/kg/day, animal no. 63 being mostly affected. These animals showed reduction of erythrocytes (21%), haemoglobin (14%), haematocrit (4%), and increase of mean corpuscular volume (22%), mean corpuscular haemoglobin (9%) and mean corpuscular haemoglobin concentration (11%). Reticulocytosis was recorded in two females, those showing the lowest haemoglobin levels (nos. 63 and 75). Concerning females dosed at 100 mg/kg/day, only two animals showed similar changes (nos. 41 and 43). Males showed only minimal decrease of erythrocytes, increase of mean corpuscular volume and mean corpuscular haemoglobin (changes were 6%).
Regarding the coagulation, the statistically significant increase of prothrombin time, recorded in males dosed at 50 mg/kg/day (11%), was considered incidental.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Fluctuations of some biochemical parameters were recorded in some treated animals. In particular, creatinine was decreased in males dosed at 200 mg/kg/day (26%), high alkaline phosphatase was increased in those receiving 50 and 100 mg/kg/day (29% and 31%, respectively) and chloride was increased in males dosed at 100 mg/kg/day (4%) and in females receiving 200 mg/kg/day (5%). Due to the low severity and/or the absence of dose-relation, the above changes were considered of no toxicological significance.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Observation of animals at removal from the cage and in an open arena did not reveal changes attributable to the test item.
Increases in motor activity (+ 48 %) and grip strength (2.9 folds) were observed in the high dose females (dosed at 200 mg/kg/day), when compared to controls at measurements performed at the end of treatment.
No differences between treated animals and controls, which could be considered treatment-related, were observed in the other functional tests (sensory reactivity, landing footsplay) performed at the end of treatment.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Terminal body weight of males did not show any difference between treated animals and controls. A slight reduction of terminal body weight was observed in the females dosed at 200 mg/kg/day. An increase of the absolute and relative weight of the spleen (+ 15 % and 26 %, respectively) was observed in the high dose females in comparison with controls. A statistically significant decrease of the absolute and relative weights of the adrenals (- 21 % and - 14 %, respectively) was also seen in these females (Group 4). In addition, the weight of the uterus (+ 62 % and + 78 %, absolute and relative, respectively) was increased in the 3 non pregnant high dose females (nos. 67, 77 and 79). The increase in the uterus weight was due to hydrometra which was considered to be a physiological change and, as such, not treatment-related. The other above changes, supported by histopathological findings, were considered to be treatment-related.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment-related changes were observed at post mortem examination in treated animals, when compared with controls. The single instances of hairloss in the forelimb region of the skin, dark and/or red colour of thymus and subcutaneous mass in the cutis were considered as incidental pathology.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related changes, consisting in increased severity of extramedullary haemopoiesis (minimal to moderate), associated or not with yellow / brown pigmentation (mild to marked), were observed in the spleen of all females treated at 200 mg/kg/day (high dose) sacrificed at the end of the study.
The extramedullary haemopoiesis (EMH), also called compensatory reactivation or reactive EMH, is considered an upregulation of haemopoiesis in the bone marrow resulting in stem cell mobilization and subsequent upregulation of haemopoiesis in embryonic sites, mainly the spleen and liver and sometimes in other tissues like the lungs.
In addition, yellow/brown pigment laden macrophages, presumably haemosiderin like, could be considered as the result of macrophage engulfment of red blood cells (usually effected or damaged) or free haemoglobin. The extramedullary haemopoiesis, as well as the yellow brown pigmentation, reflected a physiological effect or a consequence, respectively, of the haematological alterations registered mainly in the high dose females. Therefore the extramedullary haemopoiesis could be considered an adaptative change to the haematological alterations observed in high dose females. Minimal presence of yellow / brown pigment, as well as extramedullary haematopoiesis are commonly observed in rodents as a normal component of the splenic red pulp.
The remanining lesions reported in control and treated animals were considered to be an expression of spontaneous and / or incidental pathology and / or physiological oestrous cyclic changes (i.e. changes in uterus), commonly seen in this species and age under our experimental conditions.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
A male dosed at 50 mg/kg/day was found dead on Day 6 of premating phase (due to a malignant leukemia), while two females dosed at 200 mg/kg/day were sacrificed for humane reasons or found dead on Day 22 of gestation phase and Day 0 post partum, respectively. The factor contributory to the death of the early decedent animal could be attributed mainly to hepatic, renal and pancreatic injuries and as consequence of thymus atrophy, while the poor health conditions of the second female (humanely sacrificed) could be stress related, maybe associated to the difficulty in parturition.

No clinical signs of toxicological relevance or signs of neurotoxicity (weekly observations at removal from the cage and in an open arena, alterations in motor activity, grip strength and sensory reaction to stimuli)were observed during the study in males at any of the investigated dose levels. Increases in motor activity and grip strength were observed in the females dosed at 200 mg/kg/day.

No differences in body weight and food consumption were observed in treated male animals compared to the control group, while very slight reductions were seen in the females dosed at 200 mg/kg/day on Day 4 post partum. No changes of toxicological significance were recorded at haematological, clinical chemistry investigations or at coagulation tests in male animals sacrificed at the end of the study.

Macrocytic anaemia and reticulocytosis were seen in the females dosed at 200 mg/kg/day.

No relevant changes were detected at post mortem examination (terminal body weight, organ weights, macroscopic and microscopic examinations) in treated male animals, when compared with controls. An increase of the absolute and relative weight of the spleen and decrease of the absolute and relative weight of the adrenals were seen in the females dosed at 200 mg/kg/day. In addition, the weight of the uterus was increased in the 3 non-pregnant high dose females. This change, due to hydrometra, was also observed in 1 control animal and is related to physiological changes, therefore, was not considered treatment-related.

No significant differences were observed in the remaining parameters between the treated groups and the controls.

Treatment-related changes, consisting in increased severity of extramedullary haemopoiesis (minimal to moderate), associated or not with yellow / brown pigmentation (mild to marked), were observed in the spleen of all females treated at 200 mg/kg/day (high dose) sacrificed at the end of the study. These changes were considered to be adaptative, due to the alterations observed in haematological parameters.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 100 - < 200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
mortality
haematology
organ weights and organ / body weight ratios
other: Microscopic observation
Dose descriptor:
NOAEL
Effect level:
>= 200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
200 mg/kg bw/day (nominal)
System:
haematopoietic
Organ:
kidney
liver
pancreas
spleen
thymus
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
Based on the results of the present study, the NOAEL (No Observed Adverse Effect Level) for general toxicity, reproductive and developmental toxicity was considered to be 100 mg/kg/day for females and ≥ 200 mg/kg bw/day for males.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
One reliable study available (Klimisch 1 study). In addition, two dose range finding studies are available (supporting data).
System:
haematopoietic
Organ:
kidney
liver
spleen
thymus

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 classification or non-classification