Willow, Salix alba, ext.

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 JUL 2019 - DEC 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 JUL 2019 - DEC 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

version from 2016

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, (Sandhofer Weg 7, D-97633, Sulzfeld, Germany) from SPF colony
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Young adult rats, at least 8 weeks old at starting and at least 12 weeks at mating.
- Weight at study initiation: (P) Males: 401-468 g; Females: 249-303 g;
- Fasting period before study: Not specified
- Housing: group-housed, up to 4 animals of the same sex and dose group/cage, with the exception of the mating and gestation/delivery/lactation period. Group housing allowed social interaction and the deep wood sawdust bedding allows digging and other normal rodent activities. Nest building material allowed normal nesting behaviour.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: At least 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 – 70 %
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

VEHICLE
- Concentration in vehicle:
0, 20, 60, 200 mg/mL
- Amount of vehicle (if gavage):
5 mL/kg bw

Details on mating procedure:

- M/F ratio per cage: 1 male and 1 female per cage
- Length of cohabitation: until copulation occurs or a maximum of 2 weeks
- Proof of pregnancy: presence of vaginal plug or sperm in the vaginal smear is considered as evidence of copulation referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: not specified, sufficient number or sperm positive females per group was achieved

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test item concentration and homogeneity of the formulated samples was analysed based on salicin content as marker by a validated HPLC-UV method. All test item formulations were shown to be homogeneous. The measured active ingredient concentrations of White willow bark extract evaluated for each test item-dose group varied between 90 % and 102% of the nominal concentrations. The relative standard deviation (RSD) was below 5% in each case. No test item was detected in the control samples. These results were within the acceptable ranges (85% - 115%) and were considered suitable for the study purposes.

Duration of treatment / exposure:

males: 28 days (14 days pre-emating, 14 days during mating/post-mating period)
females: 14 days pre-mating, for up to 14 days mating period, through gestation and up to and including the day before necropsy (at least 13 days post-partum dosing)

Frequency of treatment:

daily, 7 days/week

Details on study schedule:

Males were dosed for at least 28 days (14 days pre-mating and 14 days mating/post-mating period plus an optional extended post-mating period), then will be euthanized and subjected to necropsy examination or alternatively will be retained and continued to be dosed for the possible conduction of a second mating if considered appropriate.

Males were dosed for at least 28 days (14 days pre-mating and 14 days mating/post-mating period plus an optional extended post-mating period), then will be euthanized and subjected to necropsy examination or alternatively will be retained and continued to be dosed for the possible conduction of a second mating if considered appropriate


Females were dosed for 14 days pre-mating, for up to 14 days mating period, through gestation and up to and including the day before necropsy (at least 13 days post-partum dosing). The day of birth (when parturition is complete) is defined as Day 0 post-partum. Females showing no evidence of copulation will be sacrificed as practical (e. g. 24-26 days after the last day of the mating period).

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12 animals/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels were selected based on the results of a dose range finding (DRF) study, with the aim of inducing toxic effects but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose. No signs of toxicity were observed up to and including 1000 mg/kg bw/day.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: General clinical observations were made once a day, during the pre-treatment and treatment period in the afternoon (pm) or after treatment at approximately the same time with minor variations as practical during the working day.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: More detailed examinations werebe made at the start of the pre-exposure period and once before the first exposure (to allow for within-subject comparisons), then at least weekly, in the morning (am) or before treatment.

BODY WEIGHT: Yes
- Time schedule for examinations: All adult animals were weighed with accuracy of at least 1 g at least weekly during the pre-exposure period, on Day -1 for randomisation purposes, then on Day 0, and afterwards at least weekly, and at termination.

Oestrous cyclicity (parental animals):

Oestrus cycles were monitored by vaginal smears daily during the pre-exposure period before the treatments starts. Vaginal smears were also checked daily from the beginning of the treatment period until evidence of mating.
Additionally, vaginal smears were prepared and examined for each female on the day of necropsy to determine the stage of oestrus cycle and allow correlation with histopathology of the reproductive organs.

Sperm parameters (parental animals):

Parameters examined in male parental generation:
testis & epididymis weight.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no, all pups were culled on PND13.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
[number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies including genitalia, weight gain, physical or behavioural abnormalities, anogenital distance (AGD) at the da yof first weighing (PND0), presence of nipples/areolae in male pups (PND13).

GROSS EXAMINATION OF DEAD PUPS: All pups which were found dead before culling on PND13 were cannibalized or autolysed. No necropsy was possible.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were euthanized at day 28.
- Maternal animals: All surviving animals were euthanized at PND 14, after one night of fasting. Females showing no-evidence of copulation were sacrificed as practical, on 25 days after the day of presumed mating.

GROSS NECROPSY
- Gross necropsy was performed on all animals, irrespective of the date of death. After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate. Special attention was paid to the organs of the reproductive system.
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY / ORGAN WEIGHTS
At the time of termination, body weight and the weight of the following organs from all surviving adult animals were determined:
- With a precision of 0.01 g: uterus (including cervix), testes, epididymides, prostate, seminal vesicles with coagulating glands, brain, heart, kidneys, liver, spleen and thymus
- With a precision of 0.001 g: adrenals, ovaries, thyroids with parathyroids
Testes and epididymides were weighed individually. Individual and/or paired absolute organ weight was reported for each animal and adjusted for the body and brain weights. Paired organ weights as applicable were summarised. Relative organ weight (to body and brain weight) were calculated and reported.
The organ weights of the preterminally euthanized animal were measured similarly to the terminal animals.

A list with retained organs for histopathology is included under "any other information on materials and methods incl. tables"

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at PND13.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external examination for gross abnormalities.

HISTOPATHOLOGY / ORGAN WEIGHTS
- Thyroid gland weights were recorded for all pups.

Statistics:

Group means and standard deviations were calculated from numerical data obtained in the study.
The statistical evaluation of data was be performed with the program package SPSS PC+4.0 (SPSS Hungary Kft, Budapest) or SAS 9.2 (when using Provantis) as documented in the raw data and reported.

Details on statistical procedures are described in "Any other information on materials and methods incl tables".

Reproductive indices:

Male mating index, female mating index, male fertility index, female fertility index, gestation index. Formulas are given in "Any other information on materials and methods incl tables".

Offspring viability indices:

Survival index, Pre-implantation mortality, intratarauterine mortality, total mortality, sex ratio. Formulas are given in "Any other information on materials and methods incl tables".

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The following individual observations were not attributed to systemic effects of the test item: hunched back and piloerection in 1 out of 12 control females, alopecia in 1 out of 12 mid dose females, red discharge from the eye in 1 out of 12 low dose males, and scar turning into wheal in 1 out of 12 high dose male animals.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food efficiency:

no effects observed

Description (incidence and severity):

No test item related effect was observed on the food consumption of the test item treated animals when compared to the controls.

Significantly higher mean food consumption was observed in the high dose animals compared to the controls when the whole treatment period was considered – this difference was not considered to be a test item related effect.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no toxicologically differences between groups for Haematology parameters. Sporadic statistical differences in the Low and Mid groups were without relationship with treatment. In the High dose, slight differences were observed in the some RBC parameters, but all results were within the historic data range and are not considered to reflect an adverse effect of treatment. There were no adverse treatment related effects observed.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no statistical differences between groups for Clinical Chemistry parameters. There were no treatment related effects observed.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no differences between the control and test item treated groups with regard to reproductive ability, mating or gestation indices, and no effects considered adverse or toxicologically significant in correlation with the administration of the test item. The mating indices were 100% in all groups. The male and female fertility indices were 100% in the Control, 92% in the Low dose group, 92% in the Mid dose group and 83% in the High dose group. Based on this data, it is concluded that the test item did not have an effect on the reproductive parameters.

Test item administration was considered to have no impact on the duration of the mating period. Successful coitus (sperm positive vaginal smears and/or vaginal plugs) occurred within 5 days of pairing (cohabitation) for all females.

The mean duration of mating was 2.4, 3.5, 3.5 and 2.9 days in the Control, Low,
Mid and High dose groups, respectively.

There was no effect on the pre-implantation and gestation periods in the test item treated groups when compared to the controls.

Key result

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 effects observed at highest tested dose

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no test item related differences in the offspring body weights or weight gains in any test item treated group when compared to the controls. The measured values were within the range commonly recorded for this strain and age.
When evaluated per litter basis, the mean litter body weights and/or body weight gain on PND0, 4 and 13 showed no toxicologically significant differences compared to controls in the F1 generation.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Significantly lower thyroid gland weights were measured in the Mid (p < 0.05) and High (p < 0.01) dose pups, and, consequently, the thyroid gland weights relative to the pup body weight was also significantly lower in the female Mid and High dose animals (p < 0.05).

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

not specified

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed at highest tested dose

Key result

Reproductive effects observed:

no

This study is a combined repeated dose toxicity study with a screening test for reproductive/developmental toxicity.

For details on results regarding repeated dose toxicity please see in the cross referenced study record for repeated dose toxicity.

Summary of reproductive parameters (males)

Parameters	Group / Concentration (mg/kg bw/day)
	Control (0)	Low (100)	Mid (300)	High (1000)
Number of treated animals	12	12	12	12
Number of males used for mating	12	12	12	12
Number of pre-terminal death / euthanasia before successful mating	0	0	0	0
Number of successful mating	12	11	11	10
Number of infertile animals	0	1	1	2
Male mating index (%)	100	100	100	100
Male fertility index (%)	100	92	92	83

 

 

 

Summary of reproductive parameters (females)

Parameters	Group /Concentration (mg/kg bw/day)
	Control (0)	Low (100)	Mid (300)	High (1000)
Number of days Mating	2.4	3.5	3.4	2.9
Number of treated animals	12	12	12	12
Number of females used for mating	12	12	12	12
Number of sperm positive females	12	12	12	12
Number of females with no implantation sites	0	1	1	2
Number of pregnant females	12	11	11	10
Number of pre-terminal death during gestation	0	0	0	0
Number of pregnant females with live born(s)	12	11	11	10
Number of pre-terminal death during lactation	0	0	0	0
Female mating index (%)	100	100	100	100
Female fertility index (%)	100	92	92	83
Female gestation index (%)	100	100	100	100

 

 

 

 

Summary of the pre-implantation and gestation periods

Parameters	Group /Concentration (mg/kg bw/day)
	Control (0)	Low (100)	Mid (300)	High (1000)
Mean number of implantations	14.92	14.92	14.58	14.50
Gestation length, mean (days)	22.75	22.45	22.55	22.50
Pre-natal mortality, mean	1.08	0.91	0.73	1.00
Pre-natal mortality (%), mean	9.15	7.50	4.75	5.74
Number of pups born, mean	13.83	15.36	15.18	16.40
Number of live born pups, mean	13.83	15.36	15.18	16.40

 

 

Summary of the pre-natal and post-natal periods with survival indices

Parameters	Group /Concentration (mg/kg bw/day)
	Control (0)	Low (100)	Mid (300)	High (1000)
Mean number of implantations	13.83	15.36	15.18	16.40
Number of pups born, mean	13.83	15.36	15.18	16.40
Number of live born pups, mean	13.83	15.36	15.18	16.40
Pre-natal mortality, mean	1.08	0.91	0.73	1.00
Pre-natal mortality (%), mean	9.15	7.50	4.75	5.74
Post-natal mortality on PND0-4, mean	0.75	0.40	0.55	0.20
Post-natal mortality on PND0-4 (%), mean	10.93	2.30	3.24	1.18
Total mortality on PND4, mean	1.83	1.40	1.27	1.20
Total mortality on PND4 (%), mean	15.74	10.51	7.79	6.82
Post-natal mortality on PND0-13, mean	0.45	0.40	0.55	0.30
Post-natal mortality on PND0-13 (%), mean	2.83	2.30	3.24	1.76
Total mortality on PND13, mean	1.27	1.40	1.27	1.30
Total mortality on PND13 (%), mean	8.08	10.51	7.79	7.32
Survival index on PND0(%)	100.00	100.00	100.00	100.00
Sex ratio (% of females) on PND0		48.12	46.98	48.45
Survival index on PND4(%)	89.07	97.70	96.76	98.82
Sex ratio (% of females) on PND4	50.81	47.82	48.71	50.32
Survival index on PND13(%) (from PND4 after culling)	100.00	100.00	100.00	99.29
Sex ratio (% of females) (PND13)	50.94	48.24	49.13	50.70

 

Conclusions:

Daily administration of the test item by oral gavage to Wistar rats at dose levels of 100, 300 or 1000 mg/kg bw/day, under the conditions of this study, did not result in test item related mortality.

No test item related clinical signs were observed in any of the animals.

No changes at neurological assessment were seen at the end of the treatment period.

No test item related effect on the body weight parameters or on the food consumption was observed in the study.

At clinical pathology, there were no test item-related findings.

At necropsy, no test item related macroscopic changes were seen. Slightly higher liver and kidney weights were recorded in the male High dose group. These findings were considered, adaptive, non-adverse changes.

No test item related changes were noted in the reproductive parameters during mating and gestation, delivery and post-partum/lactation period until PPD14. There were no adverse effects on the F1 offspring viability, clinical signs or at observations following euthanasia.
Lower thyroid gland weights were measured in the Mid (p < 0.05) and High (p < 0.01) dose pups, and, consequently, the thyroid gland weights relative to the pup body weight was also significantly lower in the female Mid and High dose animals (p < 0.05). The measured thyroid gland weights are well within the historical control range. Further, there were no effects on thyroid hormone concentration levels recorded in any of the PND13 pup dose groups. In light of these results, the thyroid gland weight decrease is considered to be a non-adverse, incidental finding, not related to the test item. No other develppmental or endocrine changes were seen in the pups at any of the dose levels (anogenital distance, etc.).

The NOAEL for Reproductive effects is 1000 mg/kg bw/day.

The NOAEL for Pup development and survival is 1000 mg/kg bw/day.

Executive summary:

The purpose of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Rats was to obtain information on the toxicity of the test item White willow bark extract following repeated daily administration by oral gavage to Wistar rats. The study included a reproductive/ developmental toxicity-screening test, intended to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition and development of the F1 offspring from conception to Day 13 post-partum.

Male and female Wistar rats were treated for 2 weeks pre-mating and then during the mating/post-mating periods. This was 28 days in total for males. Females were treated throughout gestation and up to and including postpartum/lactation day (PPD) 13. The Experimental Design was as follows:

Parameters measured during the study included signs of morbidity and mortality twice daily, daily and/or weekly detailed observation of clinical signs, neurological assessment, weekly body weight and food consumption, and clinical pathology evaluation, including haematology, coagulation, clinical chemistry and urinalysis. Neurological assessment including functional observation battery (FOB) and measurements of the landing foot splay, grip strength and motor activity were performed during the last week of the treatment. In addition, the reproductive performance, pregnancy, parturition and postpartum/lactation period were monitored in the adult animals, and viability, clinical signs and development were evaluated in their F1 offspring until PND13. At termination, necropsy with macroscopic examination was performed. Weights of selected organs were recorded and representative tissues/organs were sampled and preserved in appropriate fixatives from the adult animals. The thyroxine (T4) levels in the Day 13 pups and adult males were also assessed.

For the adult animals, a detailed histological examination was performed on the selected list of retained organs in the Control and High dose groups.

Under the conditions of this study, daily administration of the test item did not result in test item related mortality. No test item related clinical signs were observed in any of the animals. No changes at neurological assessment were seen at the end of the treatment period. No test item related effect on the body weight parameters or on the food consumption was observed in the study. At clinical pathology, there were no test item-related findings. At necropsy, no test item related macroscopic changes were seen. Slightly higher liver and kidney weights were recorded in the male High dose group. These findings were considered, adaptive, non-adverse changes. No test item related changes were noted in the reproductive parameters during mating and gestation, delivery and post-partum/lactation period until PPD14. There were no adverse effects on the F1 offspring viability, clinical signs or at observations following euthanasia. Lower thyroid gland weights were measured in the Mid (p < 0.05) and High (p < 0.01) dose pups, and, consequently, the thyroid gland weights relative to the pup body weight was also significantly lower in the female Mid and High dose animals (p < 0.05). The measured thyroid gland weights are well within the historical control range. Further, there were no effects on thyroid hormone concentration levels recorded in any of the PND13 pup dose groups. In light of these results, the thyroid gland weight decrease is considered to be a non-adverse, incidental finding, not related to the test item. No other develppmental or endocrine changes were seen in the pups at any of the dose levels (anogenital distance, etc.). The NOAEL for Reproductive effects is 1000 mg/kg bw/day. The NOAEL for Pup development and survival is 1000 mg/kg bw/day. The NOAEL for Systemic toxicity for the adults is 1000 mg/kg bw/day.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

version from 2016

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

α-hydroxy-o-tolyl β-D-glucopyranoside

EC Number:

205-331-6

EC Name:

α-hydroxy-o-tolyl β-D-glucopyranoside

Cas Number:

138-52-3

Molecular formula:

C13H18O7

IUPAC Name:

(2R,3S,4S,5R,6S)-2-(hydroxymethyl)-6-[2-(hydroxymethyl)phenoxy]oxane-3,4,5-triol

Test material form:

solid: particulate/powder

Details on test material:

Name: White willow bark extract
CAS number: 84082-82-6

Specification:
Purity: 100% (UVCB substance)
Appearance: Brown powder
Odour: Characteristic
Sieve analysis: 100% pass 80 mesh

Loss on drying: 5.0 % max
Residue on ignition: 5.0 % max
Bulk density: 40 – 55 g/100ml
Extract solvent: Alcohol & water
Heavy metal: 10 ppm max
Lead (Pb): 2 ppm max
Arsenic (As): 2 ppm max
Cadmium (Cd): 2 ppm max
Mercury (Hg): 2 ppm max
Residual solvents: Complies with Eur.Pharm.


Microbiological:
===============
Total Plate Count: 1000 cfu/g max
Yeast & Mould: 100 cfu/g max
E. coli: Negative
Salmonella: Negative

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, (Sandhofer Weg 7, D-97633, Sulzfeld, Germany) from SPF colony
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Young adult rats, at least 8 weeks old at starting and at least 12 weeks at mating.
- Weight at study initiation: (P) Males: 401-468 g; Females: 249-303 g;
- Fasting period before study: Not specified
- Housing: group-housed, up to 4 animals of the same sex and dose group/cage, with the exception of the mating and gestation/delivery/lactation period. Group housing allowed social interaction and the deep wood sawdust bedding allows digging and other normal rodent activities. Nest building material allowed normal nesting behaviour.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: At least 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 – 70 %
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:


VEHICLE

- Concentration in vehicle: 0, 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 test item concentration and homogeneity of the formulated samples was analysed based on salicin content as marker by a validated HPLC-UV method. All test item formulations were shown to be homogeneous. The measured active ingredient concentrations of White willow bark extract evaluated for each test item-dose group varied between 90 % and 102% of the nominal concentrations. The relative standard deviation (RSD) was below 5% in each case. No test item was detected in the control samples. These results were within the acceptable ranges (85% - 115%) and were considered suitable for the study purposes.

Duration of treatment / exposure:

males: 28 days (14 days pre-emating, 14 days during mating/post-mating period)
females: 14 days pre-mating, for up to 14 days mating period, through gestation and up to and including the day before necropsy (at least 13 days post-partum dosing)

Frequency of treatment:

daily, 7 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12 animals/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of a dose range finding (DRF) study, with the aim of inducing toxic effects but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose. No signs of toxicity were observed up to and including 1000 mg/kg bw/day.

Positive control:

No

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at the start of the pre-exposure period and once before the first exposure (to allow for within-subject comparisons), then at least weekly, in the morning (am) or before treatment

BODY WEIGHT: Yes
- Time schedule for examinations: All adult animals were weighed with accuracy of at least 1 g at least weekly during the pre-exposure period, on Day -1 for randomisation purposes, then on Day 0, and afterwards at least weekly, and at termination.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, food consumption determined by weighing the non-consumed diet with a precision of at least 1 g at least weekly (on body weight measurements day).

- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No, exposure by gavage

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No


OPHTHALMOSCOPIC EXAMINATION:No

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
- Anaesthetic used for blood collection: collection by cardiac puncture under pentobarbital anaesthesia, immediately prior to scheduled necropsy.
- Animals fasted: Yes, overnight
- How many animals: 5 males, 5 females


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: same as haematology
- Animals fasted: same as haematology
- How many animals: same as haematology


URINALYSIS: Yes / No / Not specified
- Time schedule for collection of urine: prior to necropsy by placing the selected animals in metabolic cages for approximately 16 hours
- Animals fasted: Not specified


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during last exposure week
- Dose groups that were examined: 5 males / 5 females from all dosed groups
- Battery of functions tested: sensory activity, grip strength, motor activity

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes, on high dose group

Statistics:

Group means and standard deviations were calculated from numerical data obtained in the study.
The statistical evaluation of data was be performed with the program package SPSS PC+4.0 (SPSS Hungary Kft, Budapest) or SAS 9.2 (when using Provantis) as documented in the raw data and reported.

Details on statistical procedures are described below.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The following individual observations were not attributed to systemic effects of the test item: hunched back and piloerection in 1 out of 12 control females, alopecia in 1 out of 12 mid dose females, red discharge from the eye in 1 out of 12 low dose males, and scar turning into wheal in 1 out of 12 high dose male animals.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food efficiency:

no effects observed

Description (incidence and severity):

No test item related effect was observed on the food consumption of the test item treated animals when compared to the controls.

Significantly higher mean food consumption was observed in the high dose animals compared to the controls when the whole treatment period was considered – this difference was not considered to be a test item related effect.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no toxicologically differences between groups for Haematology parameters. Sporadic statistical differences in the Low and Mid groups were without relationship with treatment. In the High dose, slight differences were observed in the some RBC parameters, but all results were within the historic data range and are not considered to reflect an adverse effect of treatment. There were no adverse treatment related effects observed.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no statistical differences between groups for Clinical Chemistry parameters. There were no treatment related effects observed.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

There were no test item-related adverse effects on organ weights.

A slight increase of the liver and kidney weights in the High dose group, reaching statistical significance (p<0.05) in the male animals, was considered to be a non-adverse, adaptive response to the treatment

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

No test item related effect was observed on the adult male thyroid hormone levels.

Effect levels

Key result

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 effects observed at highest tested dose

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Daily administration of the test item by oral gavage to Wistar rats at dose levels of 100, 300 or 1000 mg/kg bw/day, under the conditions of this study, did not result in test item related mortality.
No test item related clinical signs were observed in any of the animals.
No changes at neurological assessment were seen at the end of the treatment period.
No test item related effect on the body weight parameters or on the food consumption was observed in the study.
At clinical pathology, there were no test item-related findings.
No test item related effect was observed on the adult male thyroid hormone levels.
At necropsy, no test item related macroscopic changes were seen. Slightly higher liver and kidney weights were recorded in the male High dose group. These findings were considered, adaptive, non-adverse changes.
The NOAEL for repeated dose toxicity is 1000 mg/kg bw/day.

Executive summary:

The purpose of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Rats was to obtain information on the toxicity of the test item White willow bark extract following repeated daily administration by oral gavage to Wistar rats. The study included a reproductive/ developmental toxicity-screening test, intended to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition and development of the F1 offspring from conception to Day 13 post-partum.

Male and female Wistar rats were treated for 2 weeks pre-mating and then during the mating/post-mating periods. This was 28 days in total for males. Females were treated throughout gestation and up to and including postpartum/lactation day (PPD) 13. The Experimental Design was as follows:

Parameters measured during the study included signs of morbidity and mortality twice daily, daily and/or weekly detailed observation of clinical signs, neurological assessment, weekly body weight and food consumption, and clinical pathology evaluation, including haematology, coagulation, clinical chemistry and urinalysis. Neurological assessment including functional observation battery (FOB) and measurements of the landing foot splay, grip strength and motor activity were performed during the last week of the treatment. In addition, the reproductive performance, pregnancy, parturition and postpartum/lactation period were monitored in the adult animals, and viability, clinical signs and development were evaluated in their F1 offspring until PND13. At termination, necropsy with macroscopic examination was performed. Weights of selected organs were recorded and representative tissues/organs were sampled and preserved in appropriate fixatives from the adult animals. The thyroxine (T4) levels in the Day 13 pups and adult males were also assessed.

For the adult animals, a detailed histological examination was performed on the selected list of retained organs in the Control and High dose groups.

Under the conditions of this study, daily administration of the test item did not result in test item related mortality. No test item related clinical signs were observed in any of the animals. No changes at neurological assessment were seen at the end of the treatment period. No test item related effect on the body weight parameters or on the food consumption was observed in the study. At clinical pathology, there were no test item-related findings. At necropsy, no test item related macroscopic changes were seen. Slightly higher liver and kidney weights were recorded in the male High dose group. These findings were considered, adaptive, non-adverse changes. No test item related effect was observed on the adult male thyroid hormone levels. (For investigations on reproductive parameters and observations in offspring see the study record under reproductive toxicity).

The NOAEL for systemic toxicity for the adults is 1000 mg/kg bw/day.