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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 11 December 2017 to 18 March 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2019

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:
2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
Saccharomyces cerevisiae, lysate
EC Number:
305-230-8
EC Name:
Saccharomyces cerevisiae, lysate
Cas Number:
94350-12-6
IUPAC Name:
Saccharomyces cerevisiae, lysate
Test material form:
solid: particulate/powder
Remarks:
light beige
Details on test material:
- Source and lot/batch No.of test material:
supplied by the sponsor, batch no. AC17F00560
- Expiration date of the lot/batch: February 2019
- Purity test date: 30 June 2017
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: supplied by the Sponsor, batch no. AC17F00560
- Expiration date of the lot/batch: 28 February 2019
- Purity test date: 30 June 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25ºC, ≤70% Relative Humidity); as the powder was hygroscopic, it should be stored appropriately (in a tightly closed container).
- Stability under test conditions: Stable under the test conditions
- Solubility and stability of the test substance in the solvent/vehicle: All test item formulation samples were found to be homogeneous. Formulations were considered to be adequately stable under the study conditions.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was formulated in the vehicle (as a visibly stable homogenous suspension). Formulations were prepared daily (fresh prior to administration to animals) at appropriate concentrations (according to the dose level and treatment volume selected)
- Preliminary purification step (if any): not applicable
- Final dilution of a dissolved solid, stock liquid or gel: 0, 20, 60, 200 mg/mL
- Final preparation of a solid: The calculated amount of test item was added into a beaker, then it was filled up with the vehicle up to the calculated final volume. The mixture was mixed vigorously by a magnetic stirrer to make a homogenous formulation and was kept mixed until the end of treatment.

FORM AS APPLIED IN THE TEST (if different from that of starting material)
In formulation in vehicle

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The rat is regarded as a suitable species for toxicology and reproduction toxicology studies. Wistar rat was selected due to experience with this strain of rat in toxicity and reproduction toxicity studies and known fertility. The same strain was used for the Dose Range Finding study
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: Young adult rats, approximately 12 weeks old at start and 14 weeks old at mating.
- Weight at study initiation: Young adult rats, approximately 12 weeks old at start and 14 weeks old at mating.
- Fasting period before study: No fasting period
- Housing: Type II polycarbonate
- Diet (e.g. ad libitum): ssniff® SM R/M “Autoclavable complete diet for rats and mice – breeding and maintenance” ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 13 days

DETAILS OF FOOD AND WATER QUALITY: The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. Water quality control analysis was performed at least once every three months and microbiological assessment is performed monthly.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.5-25.4°C
- Humidity (%): 21-48%
- Air changes (per hr): 15-20 air exchanges per hour
- Photoperiod (hrs dark / hrs light): 12h/12h

IN-LIFE DATES: From: 30 November 2017 To:24 January 2018

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS: :
The test item was formulated in the vehicle (as a visibly stable homogenous suspension). Formulations were prepared daily (fresh prior to administration to animals) at appropriate concentrations (according to the dose level and treatment volume selected)

- VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on the available information provided by the Sponsor as well as results of two studies performed at the Test Facility, distilled water was selected as vehicle for this study in agreement with the Sponsor. The same vehicle was used in the Dose Range Finding study
- Concentration in vehicle: 0, 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no. (if required): Distilled water
Manufacturer: Hungaro-Gal Ltd.
Batch number: 8130917
- Purity: pure
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The results of the dose formulation analysis was performed using a UV spectrophotometric method. Samples were collected three times during the study. Samples were kept on ice and analysed within the stability period. The measured test item concentrations of the individual test item containing dose formulations varied between 96.7% and 104.4% of their nominal concentrations, the mean values were in the 98.2-103.8% range. No test item was detected in the control samples. These results were within the acceptable ranges (90% - 110%) and were considered suitable for the study purposes. All test item formulation samples were found to be homogeneous. Formulations were considered to be adequately stable under the study conditions.
Duration of treatment / exposure:
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 (63 days)
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12 animals per sex per dose were used
Control animals:
yes
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on available data, including the results of an acute oral toxicity study in rats (according to OECD No. 423) performed at the Test Facility and a 7-day repeated Dose Range Finding (DRF) study in the rat performed at the Test Facility with the aim of inducing toxic effects but ideally no death or suffering at the highest dose, up to a limit of 1000 mg/kg/day, and a NOAEL at the lowest dose
- Rationale for animal assignment (if not random): All adult/parental (P) male and female animals were sorted according to body weight by computer and divided into weight ranges on the day of start of treatment. There were an equal number of animals from each weight group randomly assigned to each dose group to ensure that animals of all test groups were as nearly as practicable of a uniform weight. This process was controlled by the computer software PROVANTIS v.9, to verify the homogeneity/variability between/within the groups. Males and females were randomised separately.
- Rationale for selecting satellite groups: no satellite group
- Post-exposure recovery period in satellite groups: no satellite group
- Section schedule rationale (if not random): according to OECD No. 422 guideline

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- Cage side observations checked: Animals were inspected for signs of morbidity and mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule and parameters checked: More detailed examinations were performed once before the first exposure (to allow for within-subject comparisons), then at least weekly, in the morning or before treatment. These observations were performed outside the home cage in a standard arena, at similar day times as practical. The animals were monitored for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

BODY WEIGHT: Yes
- Time schedule for examinations: All adult animals were weighed with an accuracy of 1 g for randomisation purposes, then at least weekly during the pre-exposure period, on Day 0, 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: No : g/animal/day
- Compound intake 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: All animals selected for blood sampling were fasted (overnight period of food deprivation, after the litter had been culled). Blood samples were collected by cardiac puncture under pentobarbital anaesthesia, immediately prior to scheduled necropsy.
For terminal blood sampling in all selected animals (5 males and 5 females/group),
3 samples were taken from each animal: one for haematology (in tubes with K3-EDTA as anticoagulant, 1.6 mg/mL blood), one for blood clotting times (in tubes with sodium citrate as anticoagulant) and one to obtain serum (in tubes with no anticoagulant) for clinical chemistry.
- Anaesthetic used for blood collection: Yes (pentobarbital)
- Animals fasted: Yes
- How many animals: 40 animals
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
All animals selected for blood sampling were fasted (overnight period of food deprivation, after the litter had been culled). Blood samples were collected by cardiac puncture under pentobarbital anaesthesia, immediately prior to scheduled necropsy.
For terminal blood sampling in all selected animals (5 males and 5 females/group),
3 samples were taken from each animal: one for haematology (in tubes with K3-EDTA as anticoagulant, 1.6 mg/mL blood), one for blood clotting times (in tubes with sodium citrate as anticoagulant) and one to obtain serum (in tubes with no anticoagulant) for clinical chemistry.
- Anaesthetic used for blood collection: Yes (pentobarbital)
- Animals fasted: Yes
- How many animals: 40 animals
- Parameters checked in table 2 were examined.

URINALYSIS: Yes / No / Not specified
- Time schedule for collection of urine: Urine sampling was performed prior to necropsy by placing the selected animals in metabolic cages for approximately 16 hours
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table 3 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: last exposure week (Day 22-23 for males and PPD8-9 for females)
- Dose groups that were examined: 5 animals per sex per group were examined
- Battery of functions tested: sensory activity ; grip strength ; motor activity
other: Parameters including body position, locomotor activity, respiration rate, respiration type, piloerection, head searching compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and/or vocalisation were evaluated using a scoring system

Assessment of potential test item related neurotoxicity was performed during the last exposure week (males on Day 22-23; females on PPD8-9). Selected animals (5 per sex per group) were subjected to the functional observation battery including quantitative assessment of grip strength and measurement of landing foot splay and fore/hind limb grip strength.Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity was conducted and the general physical condition and behaviour of animals were tested. A modified Irwin test was performed Parameters including body position, locomotor activity, respiration rate, respiration type, piloerection, head searching compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and/or vocalisation were evaluated using a scoring system, where score “0” was given when the behaviour or reaction of the animal was considered normal, and -1 or -2, or +1 and +2 was given if the response was less than or more than expected in an untreated animal. Locomotor activity assessment was conducted using Automatic Monitoring System of rat locomotor activity SMART v. 2.5 (Harvard Apparatus, Germany). Locomotor activity was monitored by placing each animal individually into an open-field of
50 x 50 cm for a 1-hour observation time; a DVD recording of activity/movement was made. A recording was made for a duration of 60 minutes, under dim-light and undisturbed conditions for each evaluated animal. The DVD was analysed with “SMART” software after all recordings were made, to produce the appropriate parameters. The data from all groups was evaluated for distance travelled in 5-minute segments of the 1 hour. The data from the 5-minute segments were presented graphically with the intention of showing plateau activity in controls, and comparing the treatment groups.
During micoscopic evaluation, special attention was paid to the central and peripheral nervous system tissues for any evidence of neurotoxicity

IMMUNOLOGY: During micoscopic evaluation, special attention was paid to the organ weight, appearance and histopathology of immune-system tissues for any evidence of immunotoxicity (spleen, thymus, lymph nodes and bone marrow).

OTHER: For thyroid hormone analysis, blood samples were taken by cardiac puncture or venepuncture into tubes containing K3-EDTA as anticoagulant from all adult males at termination.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
At termination, the adult rats were euthanized under pentobarbital anaesthesia, followed by exsanguination.
Gross necropsy was performed on all animals. 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.

HISTOPATHOLOGY: Yes (see table 4)
Other examinations:
This study was performed according to OECD TG 422 method. Hence, additional parameters were evaluated as reproductive functions and organs and pups (detailed in the section 7.8.1 Toxicity to reproduction)
Statistics:
The statistical evaluation of data was performed with the program package SPSS PC+4.0 or SAS v9.2.
In case of the SPSS PC+4.0 software, the heterogeneity of variance between groups was checked by Bartlett's test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, then Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorow-Smirnow test. In the case of non-normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was applied. If a positive result was detected, the inter-group comparisons were performed using Mann-Whitney U-test. The Chi-squared test was used for non-continuous data. In case of the SAS v9.2 software the normality and heterogeneity of variance between groups was checked by Shapiro-Wilk and Levene tests. Where both tests showed no significant heterogeneity, an Anova / Ancova test was carried out. If the obtained result was positive, Dunnett’s test was used to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01.
If either of the Shapiro-Wilk or Levene tests showed significance on the data, a Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons were performed using Dunn test. For non-continuous data, the Cochran-Armitage test for trend was applied and the Chi-squared test was used for differences relative to control.
For pathology data, Chi-squared test was used to check for overall similarity of the relative frequencies, the system then checked the significance against a 0.05 value and also performed pairwise tests of the treatment groups versus the control group. The Fisher’s Exact Test was performed replacing the Chi-squared test if the group size was <5.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs were observed during the study.
Mortality:
no mortality observed
Description (incidence):
No mortality was observed in the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No test item effect on body weight or body weight gain was detected during the study. There were no statistically significant variations in body weight or body weight gain values in the test item treated groups of either sex when compared to the control at any occasion. The measured values were within the range commonly recorded for this strain and age. The slight increased total body weight gain value of the High dose females compared to control (calculated for the entire period of the study) was without statistical significance and without dose response, thus it was not considered as a test item related effect.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no test item related differences in the mean daily food consumption in any test item treated group when compared to the controls. The measured values were within the normal range for this strain and age. Occasional statistically significant differences were regarded as incidental and of no toxicological significance.
Haematological findings:
no effects observed
Description (incidence and severity):
When compared to the controls, there were no statistically significant differences or biologically relevant test item related changes in males and females of the test item treated groups. The relative amount of eosinophil leukocytes showed relatively large percentage differences in the test item treated groups when compared to control due to the low absolute values which is normal for this parameter. However, opposite trends were seen in males and females, there was no statistical significance and the observed values were within the historical control range. Thus, these differences were considered as animal variability, not being a test item related effect. No statistically significant changes were recorded for any other haematology or coagulation parameters in the test item treated males and females.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no significant changes or biologically relevant effects on the serum chemistry that could be ascribed to the test item administration. A relatively large percentage difference for Aspartate Aminotransferase (AST) activity was detected in test item treated females when compared to control, without a statistical difference. An apparent dose response was observed in females only, but the difference did not gain statistical significance in any dose groups. No similar trend was seen in males and all the observed values were within the historical control range. Thus the differences seen in females were considered as not being related to test item treatment. No statistically significant changes were recorded for any other parameters in the test item treated males and females.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No test item related changes were recorded in any of the test item treated groups when compared to the control.
Higher urine volume was collected in the test item treated females when compared to the control, slightly higher volume was also detected in High dose males. However, none of the observed values were statistically significant when compared to the relevant control data, and were within the historical control range. All the individual values obtained in this study were considered to be normal based on the detailed comparison of both sets (the collected urine volume is usually highly variable as indicated by the historical control data). Therefore, the numerical differences were not considered to reflect a test item related effect. This fact was confirmed by the lack of any supporting evidence of any changes in these animals (clinical chemistry or other urinary analysis parameters).
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no test item related effects in the neurological assessment as there were no observed differences in animal behaviour, general physical condition or in the reactions to different types of stimuli in the control or test item treated groups (summary of results is seen in Table 6). No statistically significant or biologically relevant differences were noted during the assessment of landing foot splay test or grip strength.



Immunological findings:
no effects observed
Description (incidence and severity):
Basic indication of immunological effects were observed during microscopic examination, no effects were reported.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No test item related effects on organ weights were observed in any dose groups (males and females). Terminal body weights of test item treated animals (males and females) were not significantly different from control animals. There were no statistically significant differences or toxicological relevant changes in organ weights (including thyroid weight) of any test item treated dose groups (males and females) when compared to control data. Thus, no test item effect on organ weights were concluded.

Gross pathological findings:
no effects observed
Description (incidence and severity):
No test item-related macroscopic findings were noted at necropsy.
The following incidental or background findings were seen in the terminally euthanized animals. Dilated right kidney pelvis was seen in one Control male (#1002). Focal discoloration (paleness) of right kidney was observed in one Mid dose male (#3010). Discoloured liver (pale, diffuse discoloration) in one Mid dose female (#3506). Dark red multifocal discoloration of left lobe of lung was recorded in one Control female (#1507). Small left seminal vesicle (with coagulating gland) was recorded in one Mid dose male (#3010). Small testis (right) was detected in a Low dose male (#2008), soft testis (left) was noted in one Control male (#1010). Adrenal gland on the left side was not presented in one control male (#1003).
Neuropathological findings:
no effects observed
Description (incidence and severity):
There were no test item related effects in the neurological assessment as there were no observed differences in animal behaviour, general physical condition or in the reactions to different types of stimuli in the control or test item treated groups.
No statistically significant or biologically relevant differences were noted during the assessment of landing foot splay test or grip strength. In case of locomotor activity measurements (SMART), all dose groups of males and females had a normal locomotor activity ; in all cases the initial activity was high, with generally a reduced activity in the 5-minute periods to an approximate plateau by about 20-30 minutes. There was no statistical significance between the test item treated animals and the relevant control groups when evaluating the total travelled distance (period of 0-60 minutes).
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test item related microscopic changes up to dose level of 1000 mg/kg bw/day.
Other changes were seen in control and treated animals without meaningful differences in severity and incidence, and were considered to be incidental or a common background. These included slight bilateral vacuolation of the adrenal gland cortex in one Control male. In the oesophagus, focal / multifocal cellular degeneration of cells necrosis two Control males and one High dose male and focal / multifocal infiltration of inflammatory cells were presented in one Control female. Focal / multifocal infiltration of inflammatory cells were presented in the heart myocardium of two Control males. Unilateral / bilateral renal pelvic dilatation was also recorded for two Control males and one High dose male.
Other effects:
no effects observed
Description (incidence and severity):
No effect of test item was observed in the study based on the results of thyroid hormone analysis and thyroid gland weights.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Overall observations

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

Table 5:Selected body weight parameters of parental animals

Parameters

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Male, Body weight on Day 27 (g)

541.1

535.7

539.8

546.1

NS

difference(%)

-1.0

-0.2

0.9

 

Male, Body weight gain Day 0-27 (g)

61.6

55.8

59.1

65.7

NS

difference(%)

-9.5

-4.1

6.6

 

Female, Body weight on GD20 (g)

436.1

445.5

429.0

430.5

NS

difference(%)

2.2

-1.6

-1.3

 

Female, Body weight on PPD13 (g)

352.7

363.1

362.4

366.8

NS

difference(%)

3.0

2.0

4.0

 

Female, Body weight gain Day 0-PPD13 (g)

75.0

85.6

84.2

88.5

NS

difference(%)

14.1

12.2

18.1

 

Notes: Data (group mean values, n=12) were rounded to one decimal place.

NS: Statistically not significant when compared to control

 

Table 6:Summary of selected FOB and SMART parameters

Parameters

Dose groups

 

 

Control

Low dose

Mid dose

High dose

 

Males

 

Landing foot splay, mm (hind paws)

97.1

98.0

89.9

94.4

NS

HC range: 26-129

difference(%)

1.0

-7.4

-2.7

 

Grip-strength, g (forelimbs)

1961.1

1873.4

1733.3

1788.3

NS

HC range: 1100.0-2332.9

difference(%)

-4.5

-11.6

-8.8

 

Grip-strength, g (hind limbs)

1036.1

1028.9

805.8

950.3

NS

HC range: 483.3-1377.6

difference(%)

-0.7

-22.2

-8.3

 

Total travelled distance (cm)

7206.3

7417.6

7304.1

7754.7

NS

HC range

difference(%)

2.9

1.4

7.6

 

Females

 

Landing foot splay, mm (hind paws)

76.6

72.1

81.3

92.0

NS

HC range: 35-108

difference(%)

-5.9

6.1

20.1

 

Grip-strength, g (forelimbs)

1355.1

1533.9

1494.3

1540.6

NS

HC range: 795.1-1935.1

difference(%)

13.2

10.3

13.7

 

Grip-strength, g (hind limbs)

743.5

846.5

827.7

759.1

NS

HC range: 391.7-1265.4

difference(%)

13.9

11.3

2.1

 

Total travelled distance (cm)

5749.0

6162.6

5071.7

6830.9

 

HC range:

difference(%)

7.2

-11.8

18.8

NS

Notes: Data (group mean values, n=5) are rounded to one digit or to whole number.

Total travelled distance of 0-60 minutes was calculated. HC: Historical control

NS: Statistically not significant when compared to the control.

 

Table 7:Summary ofselected haematology parameters

Parameters

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Males

Relative amount of Eosinophils (%)

1.42

1.68

2.64

2.04

NS

HC range: 0.00-3.90

difference(%)

18.3

85.9

43.7

 

Females

 

Relative amount of Eosinophils (%)

2.64

1.18

1.24

1.02

NS

HC range: 0.30-9.60

difference(%)

-55.3

-53.0

-61.4

 

Notes: Data (group mean values, n=5) were rounded to two decimal places. HC: Historical control.

NS: Statistically not significant compared to control

 

Table 8:Summary of selected clinical chemistry parameters

Parameters

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Males

AST (GOT) activity (U/L)

191.4

124.2

156.2

175.6

NS

HC range: 93-628

difference(%)

-35.1

-18.4

-8.3

 

Females

 

AST (GOT) activity (U/L)

232.6

261.0

307.8

368.8

NS

HC range: 89-533

difference(%)

12.2

32.3

58.6

 

Notes: Data (group mean values, n=5) were rounded to one decimal place. HC: Historical control.

AST (GOT): Aspartate Aminotransferase (GlutamicOxaloaceticTransaminase)

NS: Statistically not significant compared to control

 

Table 9:Summary of selected urinary analysis parameters

Parameters

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Males

Volume (mL)

9.4

9.3

10.6

12.1

NS

HC range: 1.0-51.0

difference(%)

-1.1

12.8

28.7

 

Females

 

Volume (mL)

7.4

12.0

12.8

10.8

NS

HC range: 0.8-32.0

difference(%)

62.2

73.0

45.9

 

Notes: Data (group mean values, n=5) were rounded to one decimal place. HC: Historical control.

NS: Statistically not significant compared to control

 

Table 10:Organ weight data

Organ weight

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Males

 

Terminal body weight, g

516.3

514.3

518.3

522.5

NS

(difference%)

-0.4

0.4

1.2

 

Thyroid(absolute), g

0.0195

0.0188

0.0188

0.0189

NS

(difference%)

-3.8

-3.8

-3.0

 

Thyroid (relative to body), %

0.00590

0.00551

0.00559

0.00557

NS

(difference%)

-6.7

-5.2

-5.6

 

Females

 

Females

Terminal body weight, g

332.3

340.6

335.9

341.2

NS

(difference%)

2.5

1.1

2.7

 

Thyroid(absolute), g

0.0247

0.0282

0.0306

0.0260

NS

(difference%)

14.2

24.0

5.4

 

Thyroid (relative to body), %

0.00476

0.00547

0.00593

0.00497

NS

(difference%)

14.9

24.5

4.4

 

Notes: Data (group mean values, n=12) were rounded to one decimal place. Thyroid and parathyroid weights were measured together. NS: Statistically not significant compared to control

 

Table 11:Selected parameters related to thyroid hormone levels

Parameters

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Parental males

 

Number of evaluated males

12

12

12

12

 

T4 concentration(ng/mL)

42.16

41.78

41.78

43.41

NS

Thyroid gland weights (g)

0.0247

0.0282

0.0306

0.0260

NS

Thyroid gland / body weight (%)

0.0048

0.0055

0.0059

0.0050

NS

PND13 pups

 

Number of evaluated litters

12

12

12

12

 

T4 concentration(ng/mL)

41.33

42.52

40.72

42.25

NS

Thyroid gland weights (g)

0.0053

0.0062**

0.0063**

0.0057

DN

Thyroid gland / body weight (%)

1.965

2.180

2.097

2.025

NS

Notes: Data (group mean values) were rounded to two or four decimal places. Thyroid and parathyroid weights were measured together. Thyroid gland weight for one male and one female pup per litter were determined except of litter #2511 where no male pup survived until PND13. Pups blood were pooled for T4 (thyroxin) determination. Historical control range for T4 was 23.6-61.6 ng/mL (parental males) and 34.3-60.7 ng/mL (PND13 pups).

Statistical significance compared to control: * = p<0.05, ** = p<0.01

DN: Duncan’s Multiple range test; NS: Statistically not significant compared to control

 

 

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions of the study, daily administration of Saccharomyces cerevisiae, lysate test item by oral gavage to Wistar rats at dose levels of 100, 300 or 1000 mg/kg bw/day (Low, Mid and High dose groups, respectively) during the treatment period of this study did not result in test item related mortality, clinical signs, or significant changes in body weight / body weight gain, food consumption, haematology, clinical chemistry or urinalysis parameters. No test item related effect was detected during neurotoxicity assessment. No test item-related macroscopic or microscopic findings were recorded in any of the dose groups at necropsy or during histopathology evaluation. In conclusion, under the conditions of this study, the No Observed Adverse Effect Level (NOAEL) for Saccharomyces cerevisiae, lysate was considered to be 1000 mg/kg bw/day for the female and male parental (adult) generation.
Executive summary:

The purpose of this GLP-compliant Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Rats was to obtain information on the toxicity of Saccharomyces cerevisiae, lysate test item following repeated daily administration by oral gavage to Wistar rats according to OECD TG 422 method.

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 (63 days). Parameters measured during the study included signs of morbidity and mortality twice daily, detailed observation of clinical signs daily or weekly, neurological assessment, weekly body weight and food consumption, and clinical pathology evaluation, including haematology, coagulation, clinical chemistry and urinalysis. Neurological assessment of functional observation battery (including the measurements of the landing foot splay and grip strength) and measurement of locomotor activity was performed during the last week of the treatment. 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 adult males was also assessed.

Under the experimental conditions of the study, with daily administration of Saccharomyces cerevisiae, lysate test item by oral gavage to Wistar rats at dose levels of 100, 300 or 1000 mg/kg bw/day (Low, Mid and High dose groups, respectively) no mortality, clinical signs, or significant changes in body weight / body weight gain, food consumption, haematology, clinical chemistry or urinalysis parameters were observed. No test item related effect was detected during neurotoxicity assessment. No test item-related macroscopic or microscopic findings were recorded in any of the dose groups at necropsy or during histopathology evaluation. In conclusion, under the conditions of this study, the No Observed Adverse Effect Level (NOAEL) for Saccharomyces cerevisiae, lysate was considered to be 1000 mg/kg bw/day for the female and male parental (adult) generation according to CLP criteria.