Registration Dossier

Administrative data

Endpoint:
reproductive toxicity, other
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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
OECD Guideline no. 422 adopted on 22March 1996.
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- State of aggregation: Clear colorless liquid
- Storage Conditions: Room temperature, in the dark

Test animals

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.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on 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.
Details on mating procedure:
Mating was monogamous (one male to one female). A vaginal smear was taken from the day after the start of pairing until positive identification of copulation (spermidentification, vaginal plug in situ or copulation plugs found in the cage tray). The female was paired with the same male until positive identification occurred or 14 days had elapsed.
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 3 post partum (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
Doses / concentrationsopen allclose all
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

Examinations

Parental animals: Observations and examinations:
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) 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.

Oestrous cyclicity (parental animals):
Vaginal smears were taken daily in the morning starting from two weeks before pairing until a positive identification of copulation was made. The vaginal smear data were examined to determine the following:
1. anomalies of the oestrous cycle
2. the pre-coital interval (i.e., the number of nights paired prior to the detection of mating)
Sperm parameters (parental animals):
During the necropsy procedure, one cauda from one epididymis of each animal completing the scheduled test period in the high dose and the control group was taken for sperm count and evaluation of motility, morphology and viability. The animals of the mid- and low dose groups were dosed until the evaluation of the control and high dose animals had been performed. They were not examined.
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 and a regular layering in the germinal epithelium was described.
Litter observations:
A parturition check was performed from Day 20 to Day 25 post coitum. Female no. 61 (Group 4) was sacrificed for humane reasons on Day 22 post coitum and it was found to be pregnant. Gestation length was calculated as the time between the day of successful mating (Day 0 post coitum) and the day of commencement of birth (i.e. first detected presence of offspring in the cage). The day that offspring were first detected in the cage was considered Day 0 post partum.
As soon as possible after parturition was considered complete (Day 0 or 1 post partum), all pups (live and dead) were counted, sexed and only live pups were identified. Live pups were individually weighed on Days 1 and 4 post partum. Pups dying during the lactation period were weighed before the despatch to necropsy. Observations were performed once daily for all litters.
Postmortem examinations (parental animals):
One parental animal in extremis and those that had completed the scheduled test period were killed by exsanguination under isofluorane anaesthesia.

1) Parental males: the males were killed after the mating of all females or after at least 28 days of treatment period.

2) 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 (see section 4.3.9). The females which did not give birth 25 days after positive identification of mating were killed shortly after.

3) 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); – 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.

4) 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 vescicles with coagulating glands, spleen, testes, thymus, thyroid, uterus including cervix. The ratios of organ weight to body weight were calculated for each animal.

5) 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.

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 follows: 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.
The examination was then extended to include the liver and spleen of the remaining 5 females (animals not evaluated for clinical pathology) of the control and the high dose group and to female animals of the low and mid-dose groups, since changes were observed in these organs in the examined females.
Postmortem examinations (offspring):
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.
All pups found dead in the cage or sacrificed for humane reasons were examined for external and internal abnormalities. All live pups sacrificed at termination were killed and examined for external abnormalities and sex confirmation by gonadal inspection. One pup with abnormalities was retained in an appropriate fixative.
Statistics:
Standard deviationswere 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 non-parametric version of theWilliams test. The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.
Reproductive indices:
Group mean values were calculated for all parameters. Data from females which did not mate, with total resorption or non-pregnant and from dams without live young were excluded from group mean calculations.

1) Males:
- Copulation Index (%) = no. of animals mated x 100/ no. of animals paired
- Fertility Index (%) = no. of males which induced pregnancy x 100/ no. of animals paired

2) Females:
- Copulatory Index (%) = no. of animals mated x 100/ no. of animals paired
- Fertility Index (%) = no. of pregnant females x 100/ no. of females paired
Offspring viability indices:
1) Males and females:
- Copulatory Interval = Mean number of days between pairing and mating

2) Females:
- Pre-implantation loss was calculated as a percentage from the formula:
(no. of corpora lutea − no. of visible implantations / no. of corpora lutea) ×100
- Pre-birth loss was calculated as a percentage from the formula:
(no. of visible implantations − total litter size at birth / no. of visible implantations) ×100
- Pup loss at birth was calculated as a percentage from the formula:
(Total litter size − live litter size / Total litter size) ×100
- Cumulative pup loss on Day 4 post partum was calculated as a percentage from the formula:
(Total litter size at birth − live litter size at Day 4 post partum / Total litter size at birth) ×100

Sex ratios were calculated at birth and on Day 4 post partum and were presented as the percentage of males per litter.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 dosed at 50 mg/kg bw/day. This data was described 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 bw/day, was killed for humane reasons on Day 22 of the gestation phase. One male dosed at 50 mg/kg bw/day was found dead on Day 6 of the premating phase, while one female dosed at 200 mg/kg bw/day, was found dead on Day 23 of the gestation phase, after parturition.
Body weight and weight changes:
effects observed, non-treatment-related
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, non-treatment-related
Description (incidence and severity):
Reductions in food consumption ( - 29 %) were observed in the high dose females on day 4 post partum only.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Macrocytic anaemia was recorded in many females dosed at 200 mg/kg bw/day. 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. Concerning females dosed at 100 mg/kg bw/day, only two animals showed similar changes. Males showed only minimal decrease of erythrocytes, increase of mean corpuscular volume and mean corpuscular haemoglobin (changes were 6%).
Clinical biochemistry findings:
effects observed, 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 bw/day (26%), high alkaline phosphatase was increased in those receiving 50 and 100 mg/kg bw/day (29% and 31%, respectively) and chloride was increased in males dosed at 100 mg/kg bw/day (4%) and in females receiving 200 mg/kg bw/day (5%). Due to the low severity and/or the absence of dose-relation, the above changes were considered of no toxicological significance.
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
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 effete 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 specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
No differences were observed at sperm analysis including sperm motility and concentration, and cauda weight between the control and the high dose group at the end of treatment
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
The mean pre-coital interval and the number of copulation plugs were similar between control and treated groups. All females mated with the exception of female no. 15 of the control group. However, 3 females in the high dose group (nos. 67, 77 and 79) were found not pregnant. Female no. 61 (Group 4) did not give birth and was humanely killed on Day 22 post coitum.
The copulatory index was 100 % for both sexes from Groups 2, 3 and 4 and 90 % for Group 1.
The fertility indices were 90 %, 100 %, 100 % and 70 %. A reduction in the number of females with live pups on Day 4 post partum was observed at the high dose (5), when compared to the other groups.
The reduction in fertility index observed in animals dosed at 200 mg/kg/day was considered to be treatment-related.

Details on results (P0)

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. A total of 3 females of the high dose group were found not pregnant. One female from the control group did not mate.

The number of females with live pups on Day4postpartum was: 9 in the control group, 10 in the low and mid-dose groups (50 and 100mg/kg/day) and 5 in the high dose group (200 mg/kg/day).
No treatment-related anomalies were noted in the oestrous cycle. The copulatory index was 100 % for both sexes from Groups 2, 3 and 4 and 90 % for Group 1. The fertility indices were 90 %, 100 %, 100 % and 70 %.

Reduction in total litter size at birth, a severe increase in pre-birth loss and a reduction in the number of females with live pups on Day 4 post partum were observed at the high dose of 200 mg/kg/day. No significant differences were observed in the remaining parameters between the treated groups and the controls. Necropsy findings in pups did not reveal any treatment-related effect, with the exception of 1 pup, found dead in the uterus of the dam killed for humane reasons, which showed anencephaly, spina bifida, short trunk and curled tail.

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 and a regular layering in the germinal epithelium was described. 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.

Effect levels (P0)

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
reproductive performance

Target system / organ toxicity (P0)

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

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
not examined

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Reduction in total litter size at birth, a severe increase in pre-birth loss and a reduction in the number of females with live pups on Day 4 post partum were observed at the high dose of 200 mg/kg/day. Anencephaly, spina bifida, short trunk and curled tail were described in the only decedent pup found in uterus of the female no. 61 (Group 4), humanely killed on Day 22 post coitum.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Litter weight was also significantly reduced, at statistical analysis, in animals dosed at 200 mg/kg/day on Days 1 and 4 post partum (-29% and -31%, respectively), while mean pup weight was comparable among all treated groups and controls on Days 1 and 4 post partum.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not specified
Histopathological findings:
not examined
Other effects:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Developmental effects observed at 200 mg/kg bw/day.

Target system / organ toxicity (F1)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
200 mg/kg bw/day (actual dose received)
Organ:
not specified
Treatment related:
yes
Dose response relationship:
yes

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
200 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
not specified
Dose response relationship:
yes
Relevant for humans:
not specified

Applicant's summary and conclusion

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 males and females.