Saccharomyces cerevisiae cell wall, extracted

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

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

Test material information

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 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

Details on mating procedure:

- M/F ratio per cage: one female and one male from the same dose group (1/1)
- Length of cohabitation: 5 days
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy
- After 5 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): Sperm positive females were housed individually.

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

Details on study schedule:

- No F1 parental animals were used in this study. F1 pups were evaluated.
- Age at mating of the mated animals in the study: Mating began after the animals had attained full sexual maturity

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

12 animals per sex per group

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

Parental animals: Observations and examinations:

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

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.

Oestrous cyclicity (parental animals):

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. The number of implantation sites and of corpora lutea were recorded in the females as applicable. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.

Sperm parameters (parental animals):

Parameters examined in P male parental generations:
testis weight, epididymis weight, Special attention was paid to the evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes,all pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1] offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups

GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities; possible cause of death was not determined for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: After 28 Days of exposure period
- Maternal animals: At day 13 post partum

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at PND4 or PND13 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination)


For thyroid hormone analysis, blood samples were taken by cardiac puncture or venepuncture (or decapitation in case of pups) into tubes containing K3-EDTA as anticoagulant as follows:
-from up to two pups per litter on PND4,
-from all dams at termination (PPD14) and up to two pups per litter on PND13,

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.

Reproductive indices:

Parental Males

- Number of pairings
- Number of fertile pairings
- Number of infertile males
- Male mating index
- Male fertility index
- Thyroid hormone (T4) levels

Parental Females

- Oestrus cycle data
- Number of pairings
- Number of pregnant females
- Number of sperm positive, but non-pregnant females
- Number of non-mated females
- Female mating index
- Female fertility index
- Gestation index

Offspring viability indices:

- Number of live births per litter, and number of viable pups per litter on PND 0, 4 and 13
- Survival Index of pups on PND 0, 4 and 13 †

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Each female selected for the treatment showed acceptable cycles (mean cycle length was 4.00 days for each group) before starting the treatment period. There was no effect on test item on the oestrus cycle of females (mean cycle length was in the 3.89-4.03 days for each group after the treatment). No prolonged oestrus or prolonged dioestrus was recorded in any test item treated groups. The frequency of pseudopregnancy in the test item treated groups was similar to control (cases of 1, 0, 3 and 1 in Control, Low dose, Mid dose and High dose groups respectively), this data was in line with the normal, expected range.

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, fertility or gestation. The mating and fertility indices (males and females) were 100% in all groups. There was no non-pregnant female in the study. The gestation index was 100% in all groups.

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 in the test item treated group. The mean duration of mating was 1.92, 2.92, 2.67 and 2.50 days in Control, Low, Mid and High dose groups, respectively. These data are in line with results of earlier studies.

Details on results (P0)

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* (*detailed in section 7.5.1 Repeated dose toxicity: oral).
No test item related effect was detected during neurotoxicity assessment.

Effect levels (P0)

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 (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

There was no test item effect on mortality or survival of the pups. There were no significant differences or effects that could be ascribed to treatment on the pre-natal, post-natal or total mortality values (litter mean and %) in any of the dose groups. Slightly higher number of pups died in the lactation period in the Low dose group, but 6 out of the 9 animals in this group belonged to one dam with difficult delivery (#2511*). There was no dose response, and this fact was considered as animal variability not related to the treatment.

The number of viable pups on PND0, 4 and 13 as well as the survival index of the pups at given time points were comparable to control value in each dose group , thus there was no treatment-related effects on the viability of pups at those time points (the slightly lower number in the Mid dose group (significant at p<0.05) was without dose response, thus considered as animal variability).
The ratio of female pups was slightly higher in the Mid dose group than in the Control group, but as there was no statistical significance and no dose response, this fact was considered as biological variability, not related to the test item.

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. When evaluated per litter basis, the mean litter body weights on PND 0, PND 4 and PND 13 and body weight gain in the relevant periods showed no statistically significant differences compared to controls in the F1 generation. In summary, there were no effects of treatment on pup weights or weight gains.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Based on the external evaluation, no clinical signs or abnormalities were recorded for any pups.

No test item-related macroscopic findings were seen in unscheduled deaths of F1 pups. Three stillborn pups were recorded in the Low dose group. Cannibalization of 2 Control, 3 Low dose, 5 Mid dose and 2 High dose pups was observed at birth or in the lactation period. There were 2, 12, 6 and 1 autolysed pups from the Control, Low, Mid and High dose groups. No special findings were recorded for those pups. The cause of death was not established for any pups.

No test item-related macroscopic findings were recorded in F1 offspring generation euthanized and examined externally at scheduled termination on PND13

Other effects:

no effects observed

Description (incidence and severity):

No test item effect was observed on anogenital distance or nipple retention during the study.
No statistically significant changes in the anogenital distance measured on PND 0 were noted for test item treated male and female pups when compared to control. Slight statistical difference (p<0.05) compared to control was noted in High dose females when all the individual data were used. However, litter mean values showed no statistically significant difference and the range of the individual data were in line with the control range, thus this fact was considered not being treatment related.

No endocrine disruptor effect of test item was observed in the study based on the results of thyroid hormone analysis and thyroid gland weights.
Compared to the relevant control values, there were no statistically significant differences in T4 thyroid hormone concentrations for parental males and PND13 pups. Statistically significant increase (p<0.01) compared to control was detected in the absolute thyroid weight of Low and Mid dose pups, but based on the lack of dose response and as the relative (to body) weights did not reach statistical significance, these differences were considered as animal variability, not being a test item related effect.

Details on results (F1)

There were no test item related effects on the F1 offspring viability, clinical signs, physical or sexual development. No test item related macroscopic finding were recorded for F1 pups at necropsy.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

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 (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

Table 1:Summary of reproductive parameters (males)

Parameters	Dose groups
	Control	Low dose	Mid dose	High dose
Number of treated animals	12	12	12	12
Number of pre-terminal death	0	0	0	0
Number of males used for mating	12	12	12	12
Number of successful mating	12	12	12	12
Number of fertile males	12	12	12	12
Male mating index (%)	100	100	100	100
Male fertility index (%)	100	100	100	100

Table 2:Summary of reproductive parameters (females)

Parameters	Dose groups
	Control	Low dose	Mid dose	High dose
Number of treated animals	12	12	13	12
Number of pre-terminal death	0	0	0	0
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	0	0	0
Number of pregnant females	12	12	12	12
Pregnant females with live born(s)	12	12	12	12
Pregnant females not delivered living pups	0	0	0	0
Female mating index (%)	100	100	100	100
Female fertility index (%)	100	100	100	100
Female gestation index (%)	100	100	100	100

 

Table 3:Summary of the pregnancy evaluation

Parameters	Dose groups
	Control	Low	Mid	High
Number of evaluated females	12	12	12	12
Duration of pregnancy (days)	22.25	22.42	22.25	22.58	NS
Number of implantations, mean	15.42	16.58	15.25	14.92	NS
Number of pups born, mean	15.00	16.08	14.08	14.08	NS
Number of live born pups, mean	14.83	15.58	13.58	14.00	NS
Pre-natal mortality, mean	0.58	1.00	1.67	0.92	NS
Pre-natal mortality (%), mean	4.17	6.21	10.26	5.61	NS
Post-natal mortality, mean	0.17	1.00	0.42	0.17	NS
Post-natal mortality (%), mean	1.01	8.23	2.60	1.11	NS
Total mortality, mean	0.75	2.00	2.08	1.08	NS
Total mortality (%), mean	5.18	12.04	12.65	6.68	NS

Notes: Data (group mean values) were rounded to two decimal places. Pre-natal mortality includes intrauterine mortality and the loss at delivery. Post-natal mortality PND0-13 and total mortality on PND13 are shown in the table (the number of pups culled for blood sampling on PND4 were excluded).

NS: Statistically not significant compared to control

 

Table 4:Summary of survival (offspring)

Parameters	Dose groups
	Control	Low dose	Mid dose	High dose
Number of evaluated litters	12	12	12	12
Number of implantations, mean	15.42	16.58	15.25	14.92	NS
Number of pups born, mean	15.00	16.08	14.08	14.08	NS
Number of live born pups, mean	14.83	15.58	13.58	14.00	NS
Number of living pups on PND13, mean	13.00	12.75	11.33*	12.00	U
Pups culled for blood sampling, mean	1.67	1.83	1.83	1.83	NS
Post-natal mortality on PND0-4, mean	0.08	1.00	0.25	0.17	NS
Post-natal mortality on PND0-4 (%), mean	0.49	8.23	1.62	1.11	NS
Total mortality on PND4, mean	0.67	2.00	1.92	1.08	NS
Total mortality on PND4 (%), mean	4.66	12.04	11.67	6.68	NS
Post-natal mortality on PND0-13, mean	0.17	0.75	0.42	0.17	NS
Post-natal mortality on PND0-13 (%), mean	1.01	7.67	2.60	1.11	NS
Total mortality on PND13, mean	0.75	2.00	2.08	1.08	NS
Total mortality on PND13 (%), mean	5.18	12.04	12.65	6.68	NS
Survival index on PND0	98.61	94.92	96.50	99.44	NS
Sex ratio (%) on PND0	47.70	49.13	55.24	49.33	NS
Survival index on PND4	99.51	92.33	98.38	98.89	NS
Sex ratio (%) on PND4	47.39	52.68	55.62	49.13	NS
Survival index on PND13	99.40	100.00	98.89	100.00	NS
Sex ratio (%) on PND13	46.00	52.51	53.54	48.99	NS

Notes: Data (group mean values) were rounded to two decimal places. Sex ratio means the ratio of females. Culling for blood sampling was made on PND4. Survival index was calculated in comparison with the end of previous period (on PND0 it was compared to the number of pups born, on PND4 it was compared to the number of live born pups, on PND13 it was compared to the number of pups after culling on PND4).

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

U: Mann-Whitney U-test, NS: Statistically not significant compared to control

Table 5:Summary of mortality (offspring)

Parameters	Dose groups
	Control	Low dose	Mid dose	High dose
Number of evaluated litters	12	12	12	12
Number of pups born	180	193	169	169	NS
Number of cannibalized pups	0	0	2	0	NS
Number of autolyzed pups	2	6	4	1	NS
Number of stillborn pups	0	3	0	0	NS
Number of live born pups	178	187	163	168	NS
Number of found dead pups (born alive)	0	3	0	0	NA
Number of living pups on PND0	178	184	163	168	NA
Number of cannibalized pups (PND0-13)	2	3	3	2	NA
Number of autolyzed pups (PND0-13)	0	6	2	0	NA
Number of found dead, intact pups (PND0-13)	0	0	0	0	NA
Total number of pups died (born alive)	2	9*	5	2	CH
Culled for blood sampling on PND4	20	22	22	22	NA
Number of viable pups on PND13	156	153	136	144	NS

Notes: Mortality numbers mean number of pups / number of affected litters. PND0-13 means the lactation period, counted after the delivery was ended.

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

NA: Not applicable, CH: Chi square test, NS: Statistically not significant compared to control

 

Table 6:Bodyweight data (offspring)

Parameters	Dose groups
	Control	Low dose	Mid dose	High dose
Number of evaluated litters	12	12	12	12
Mean litter body weight (PND0), g	6.35	6.56	6.49	6.59	NS
Mean litter body weight (PND4), g	10.43	10.52	10.61	10.66	NS
Mean litter body weight gain (PND0-4), g	4.08	5.80	4.11	4.06	NS
Mean litter body weight (PND13), g	27.05	28.03	29.90	28.65	NS
Mean litter body weight gain (PND4-13), g	16.58	17.52	19.26	17.98	NS
Mean litter body weight gain (PND0-13), g	20.68	21.47	23.39	22.04	NS

Notes: Body weight / body weight gain data (litter mean values) were rounded to two decimal places.

NS: Statistically not significant compared to control

 

Table 7:Anogenital distance

Parameters	Dose groups
	Control	Low dose	Mid dose	High dose
Male pups
Number of evaluated male pups	94	93	73	85	NS
Anogenital distance, litter mean of males (mm)	3.48	3.44	3.49	3.51	NS
Anogenital distance, all male pups (mm)	3.50	3.48	3.47	3.48	NS
Minimum / Maximum value, litter mean (mm)	2.25 / 4.21	1.92 / 3.48	2.39 / 4.69	2.46 / 4.53
Female pups
Number of evaluated female pups	84	91	90	83	NS
Anogenital distance, litter mean of females (mm)	1.72	1.70	1.73	1.79	NS
Anogenital distance, mean of all females (mm)	1.71	1.69	1.73	1.79*	U
Minimum / Maximum value (mm)	1.35 / 2.10	1.17 / 3.08	1.40 / 2.57	1.29 / 2.28

Notes: Data (group mean or litter mean values) were rounded to two decimal places. Data of 12 litters were evaluated in all cases.

NS: Statistically not significant compared to control

 

Table 8: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  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 effect on oestrus cycle of parental females was noted. No test item related changes were noted in the reproductive parameters, gestation, parturition and lactation. There were no test item related effects on the F1 offspring viability, clinical signs, physical or sexual development. No test item related macroscopic finding were recorded for F1 pups at necropsy.
No test item-related macroscopic or microscopic findings were recorded in any of the dose groups at necropsy or during histopathology evaluation. There were no test item effects on organ weights. Under the experimental conditions of this study and based on the results of thyroid, nipple retention, anogenital distance and external reproductive organs analysis, no endocrine signal was highlighted.

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 and also for the F1 generation (pups) based on no adverse effect on reproductive functions and general observations of the treated animals and F1 generation pups.

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, weekly body weight and food consumption, and clinical pathology evaluation. 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 PND 13. The anogenital distance (AGD) and presence of nipples/areolae in all pups were recorded on 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 and offspring. The thyroxine (T4) levels in the adult males and PND 13 pups were also assessed.

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  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 effect on oestrus cycle of parental females was noted. No test item related changes were noted in the reproductive parameters, gestation, parturition and lactation. There were no test item related effects on the F1 offspring viability, clinical signs, physical or sexual development. No test item related macroscopic finding were recorded for F1 pups at necropsy.

No test item-related macroscopic or microscopic findings were recorded in any of the dose groups at necropsy or during histopathology evaluation. There were no test item effects on organ weights. Under the experimental conditions of this study and based on the results of thyroid, nipple retention, anogenital distance and external reproductive organs analysis, no endocrine signal was highlighted.

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 and also for the F1 generation (pups) based on no adverse effect on reproductive functions and general observations of the treated animals and F1 generation pups.