Registration Dossier
Registration Dossier
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EC number: 305-230-8 | CAS number: 94350-12-6
Endpoint summary
Administrative data
Description of key information
No information via the dermal and inhalation routes is available.
A subacute repeated dose toxicity study via the oral route is available for Saccharomyces cerevisiae, lysate (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test). Based on the results of this key study (Hargitai, Klimisch 1, 2018, GLP, OECD TG 422), the No Observed Adverse Effect Level (NOAEL) for the test item Saccharomyces Cerevisiae, lysate was defined to be 1000 mg/kg bw/day for rats, through oral route.
In order to meet the standard information requirements of Regulation (EC) 1907/2006, Annex IX, Column 1, 8.6.2, a reliable subchronic toxicity study should be submitted for Saccharomyces cerevisiae, lysate. No subchronic or chronic repeated dose toxicity study via the oral route is specifically available for Saccharomyces Cerevisiae, lysate substance. However, several reliable studies ranging from subacute to chronic durations are available for either live and dead Saccharomyces Cerevisiae (SC) yeast or various SC-derived substances. Therefore, an overall weight of evidence is used to fulfil the standard information requirements of Regulation (EC) 1907/2006 for this endpoint.
Saccharomyces cerevisiae, lysate is the concentrated, non-extracted, partially soluble digest obtained from yeast biomass produced according to common yeast fermentation process. The lysis of yeast cells results in both soluble and insoluble components (cell walls). Saccharomyces cerevisiae, lysate can therefore reasonably be considered as a combination of the two SC derivative substances Saccharomyces cerevisiae, ext. (SC Yeast extract EC # 283-294-5) and Saccharomyces cerevisiae cell wall, extracted (SC cell walls; EC 949-711-6). Oral (gavage) subchronic studies (GLP compliant) in the rat are available for both SC yeast extract and SC cell walls (Krapivin, Klimisch 2, 2020, GLP, Russian guideline). Both tests were performed as a mandatory requirement for the registration of the substances as feed additives on the territory of the Russian Federation. Female rats were daily treated for 90 days with doses of 0, 1000 and 2000 mg/kg bw/day for Saccharomyces cerevisiae, ext. and doses of 0, 500 and 1000 mg/kg bw/day for Saccharomyces cerevisiae cell wall, extracted. Parameters evaluated were general condition and behaviour, functional state of the central nervous system, manifestation of toxicity symptoms, signs of morbidity and mortality, body weight, blood counts [Haematocrit (%), Haemoglobin (g/l), Erythrocytes (number/L), Leucocytes (number/L), Platelets (number/L), Leukogram], clinical biochemistry [Bilirubin, Aspartate Aminotransferase (AST), Alanine Aminotransferase (ALT), Urea, Creatinine, Total protein, Alkaline phosphatase, Glucose], macroscopic examination of internal organs (liver, lungs, kidneys, heart, spleen, stomach, intestine) and mass ratios internal organs (liver, kidneys, spleen, lungs and heart). Although the method followed was not compliant with the current EU OECD TG 408, the results from the two studies did not reveal significant treatment related toxicological effects up to the highest concentrations tested and the derived subchronic NOAEL for both substances was the maximum dose level recommended by the OECD TG 408 (i.e., 1000 mg/kg bw/day). Taken together the results of the subacute study on the target substance and the results of the subchronic studies on SC yeast extract and SC cell walls, it not expected that Saccharomyces cerevisiae, lysate would show any toxicity upon subchronic repeated exposure.
Other Saccharomyces cerevisiae derivatives were also tested for oral repeated dose toxicity. The yeast hydrolysate from Saccharomyces cerevisiae (Protein hydrolyzates, yeast; EC # 309-709-2) was evaluated for subacute (14 days) toxicity in the rat (male and female) at the limit dose of 1000 mg/kg bw/day (Jung, Klimisch 2, 2010, GLP not specified, OECD TG 407). The results of this study showed that the yeast hydrolysate from SC did not induce toxicity upon a subacute repeated exposure to the limit dose of 1000 mg/kg bw/day in male and female rats and the subacute oral NOAEL was considered to be ≥ 1000 mg/kg bw/day. Yeast hydrolysate from SC (Protein hydrolyzates, yeast; EC # 309-709-2) was also tested for oral subchronic toxicity in the rat (male and female) (Yejin, Klimisch 2, 2021, GLP not specified, OECD TG 408). The substance was administered to rats at a limit dose level of 1000 mg/kg for 90 days. Results of this study showed the SC yeast hydrolysate to be safe and non-toxic following subchronic repeated administration of 1000 mg/kg bw/day.
A dried fermentate preparation of Saccharomyces cerevisiae was orally tested for both subchronic and chronic repeated dose toxicity in the rat (male and female) (Schauss, Klimisch 2, 2012, GLP, OECD TG 408 and 452). The tested doses were 0, 30, 200, and 1500 mg/kg bw/day for the subchronic study and 0, 20, 200, and 800 mg/kg bw/day for the chronic study. Results from both studies showed that the dried fermentate preparation of Saccharomyces cerevisiae was well tolerated up to the highest doses tested of 1500 mg/kg bw/day and 800 mg/kg bw/day and the subchronic and chronic NOAEL were 1500 mg/kg bw/day and 800 mg/kg bw/day respectively.
Repeated dose toxicity of the Saccharomyces cerevisiae yeast itself, as live or dead whole organism, was also assessed via the oral route. The subchronic (60 day-study) toxicity of live S. cerevisiae suspension was assessed in male rats to acquire data on the safety-in-use of the probiotic Saccharomyces cerevisiae strain RC016 (Gonzalez Pereyra, Klimisch 2, 2014, GLP not specified, non-guideline test). The results of this study demonstrated that oral daily administration of the probiotic Saccharomyces cerevisiae strain RC016 in male rats for 60 days did not result in any illness, mortality or any other treatment-related health changes. No negative impact on the animals’ weight gain, Feed Conversion or Feed Efficiency was found and no macroscopic or microscopic differences or lesions was observed, which suggested the strain to be safe to use in vivo.
Subchronic repeated dose toxicity of a fractionated Saccharomyces cerevisiae biomass as a single source of dietary protein was evaluated in male rats (Caballero-Cordoba, Klimisch 2, 2000, GLP not specified, non-guideline test). Animals were fed diets with 15 and 30% protein from the fractionated yeast biomass during 45 and 90 days. The results of this study showed no evidence of toxicity for the whole Saccharomyces cerevisiae yeast biomass after 45 and 90 days of feeding. No mortality, no evidence of toxicity and no effects on body weights or food consumption. Furthermore, no toxicologically relevant effect was noted based on the blood chemistry, urinalysis and organ analysis results.
Furthermore, the Expert Panel for Cosmetic Ingredient Safety recently reviewed the safety of Saccharomyces cerevisiae yeast-derived ingredients (CIR, 2021). Among assessed Saccharomyces cerevisiae (SC) yeast derived substances, a SC beta-glucan extract was tested for oral subchronic repeated dose toxicity [Babicek et al. 2007; GLP, OECD TG 408 (1998)] in male and female rats. Rats (10/sex/group) were given either 2, 33.3, or 100 mg/kg bw/d of the test item in water, via gavage, once a day, for 91 d. A control group was given water only. No mortality, clinical pathology, functional/behavioral, microscopic, or gross observations indicating toxicity were observed. In addition, no negative effects on animal weights or food consumption were noted. No dose-dependent hematological or biochemical toxicities were observed. A no-observed-adverse-effect level (NOAEL) of 100 mg/kg bw/d was established based on the results of this study.
Taking into account all the reliable repeated dose toxicity data available on the target substance Saccharomyces cerevisiae, lysate as well as on the various Saccharomyces cerevisiae derivative substances or the whole SC yeast organism (ranging from subacute to chronic), it can be concluded on a weight of evidence basis that no toxicity upon repeated exposure is expected for the registered substance Saccharomyces cerevisiae, lysate and no additional testing is considered necessary.
In the absence of adverse effects, no STOT-RE classification is required for Saccharomyces cerevisiae, lysate substance according to CLP criteria.
Key value for chemical safety assessment
Repeated dose toxicity: via oral route - systemic effects
Link to relevant study records
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- other: experimental study performed on a SC yeast derivative used in a WoE approach for the target substance
- Adequacy of study:
- weight of evidence
- Study period:
- 2020
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study without detailed documentation
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
- Version / remarks:
- 2018
- Deviations:
- no
- GLP compliance:
- not specified
- Limit test:
- yes
- Specific details on test material used for the study:
- Saccharomyces cerevisiae was cultured according to a previously described method to recover the cells (Jung et al.,2009). To the recovered cells was added 50 mM phosphate buffer (pH 7.0) 10 times the amount of the cells. Then, protease was added to 0.5% of the cells, and hydrolysis was performed at 30°C for 6 h. To inactivate the enzyme, the supernatant obtained by centrifugation at 8 000 g after standing for 10 min in a water bath maintained at 100°C was passed through a 10 kDa Hydrosart membrane (Sartorius Stedim Biotech GmbH, Göttingen, Germany). The peptide in the filtrate was separated, dried, and designated as yeast hydrolysate DNF-10.
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Orient Bio, Seongnam, Korea
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: not specified in the publication
- Weight at study initiationnot specified in the publication
- Fasting period before study: no
- Housing: not specified in the publication
- Diet (e.g. ad libitum): unrestricted access to solid feed (PMI Nutrition International, Richmond, VA, USA), which was sterilized by irradiation.
- Water (e.g. ad libitum): ad libitum access to sterilized tap water, which was filtered using a microfilter and sterilized using an ultraviolet water sterilizer.
- Acclimation period: 1 week (animals were visually inspected once they were obtained, and after 1 week of quarantine and purification, animals considered to be healthy were selected and used in the experiment).
DETAILS OF FOOD AND WATER QUALITY: not specified in the publication
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ±3
- Humidity (%): 50 ± 10
- Air changes (per hr): 10 to 20 times
- Photoperiod (hrs dark / hrs light): 12/12 (lights on from 08:00 to 20:00 h)
IN-LIFE DATES: From: To: not specified in the publication - Route of administration:
- oral: gavage
- Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS: not specified in the publication
DIET PREPARATION
- Rate of preparation of diet (frequency): not applicable
- Mixing appropriate amounts with (Type of food): not applicable
- Storage temperature of food: not applicable
VEHICLE
- Justification for use and choice of vehicle (if other than water): not specified in the publication
- Concentration in vehiclenot specified in the publication
- Amount of vehicle (if gavage): not specified in the publication
- Lot/batch no. (if required): not specified in the publication
- Purity: not specified in the publication - Analytical verification of doses or concentrations:
- not specified
- Duration of treatment / exposure:
- 90 days.
- Frequency of treatment:
- Once daily for 90 days.
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 5/sex/dose group
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: not specified in the publication.
- Rationale for animal assignment (if not random): random
- Fasting period before blood sampling for clinical biochemistry: fasted for 12 h prior to autopsy
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random): not applicable
- Dose range finding studies: not specified in the publication
- Other: none specified in the publication - Positive control:
- None.
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Not specified in the publication
- Time schedule: Not specified in the publication
- Cage side observations: Not specified in the publication
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Not specified in the publication
BODY WEIGHT: Yes
- Time schedule for examinations: Not specified in the publication
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
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified in the publication
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: Not specified in the publication
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes (not a drinking study)
- Time schedule for examinations: Not specified in the publication
OPHTHALMOSCOPIC EXAMINATION: Not specified in the publication
- Time schedule for examinations: Not specified in the publication
- Dose groups that were examined: Not specified in the publication
HAEMATOLOGY: Yes
- Time schedule for collection of blood: animals were fasted for 12 h prior to autopsy and anaesthetised with CO2. The blood sample was collected from the abdominal aorta. The blood samples were obtained using blood collection tubes containing heparin (anticoagulant) for the whole blood sample. Whole blood samples were immediately analysed using the KN-21Nsystem (Sysmex, Seoul, Korea).
- Anaesthetic used for blood collection: Yes (CO2)
- Animals fasted: Yes; 12 h prior to autopsy
- How many animals: all animals
- Parameters examined: red blood cells (RBCs), white blood cells (WBCs), haematocrit (Hct), haemoglobin (Hb), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), and mean corpuscular haemoglobin concentration (MCHC).
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: animals were fasted for 12 h prior to autopsy and anaesthetised with CO2. The blood sample was collected from the abdominal aorta. The blood samples were obtained using blood collection tubes containing ethylenediamine tetra acetic acid (anticoagulant) for the plasma sample. Plasma was centrifuged at 4°C and 3 000 rpm for 15min, separated, stored at −70 °C until analysis, and then subjected to a blood biochemical test. The blood biochemical test was performed using the FUJI DRI-CHEM3500 analyser (Fuji Photo Film Co., Osaka, Japan).
- Animals fasted: Yes; 12 h prior to autopsy
- How many animals: all animals.
- Parameters examined: albumin (ALB), alanine aminotransferase(ALT), aspartate aminotransferase (AST), ammonia(NH3), blood glucose (GLU), blood urea nitrogen (BUN),creatinine (CRE), creatine phosphokinase (CPK), high density lipoprotein (HDL), lactate dehydrogenase (LDH),total bilirubin (BIL), total cholesterol (TCHO), triglyceride(TG), total protein (TP), and uric acid (UA).
PLASMA/SERUM HORMONES/LIPIDS: No
- Time of blood sample collection: not applicable
- Animals fasted: not applicable
- How many animals: not applicable
URINALYSIS: Not specified in the publication
- Time schedule for collection of urine: Not specified in the publication
- Metabolism cages used for collection of urine: Not specified in the publication
- Animals fasted: Not specified in the publication
- Parameters examined: Not specified in the publication
NEUROBEHAVIOURAL EXAMINATION: Not specified in the publication
- Time schedule for examinations: Not specified in the publication
- Dose groups that were examined: Not specified in the publication
- Battery of functions tested: Not specified in the publication
IMMUNOLOGY: Not specified in the publication
- Time schedule for examinations: Not specified in the publication
- How many animals: Not specified in the publication
- Dose groups that were examined: Not specified in the publication
- Parameters examined: Not specified in the publication
OTHER: none specified in the publication - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
The animals’ organs were first visually examined by excising the brain, epididymis, heart, kidney, liver, lung, ovary, spleen, testis, thyroid, and uterus. The extracted organs were treated with saline solution and their weights were measured after removing moisture. The measured organ weight was expressed as relative weight per 100 g body weight.
HISTOPATHOLOGY: Yes - Statistics:
- All experimental results were expressed as mean ± standard error of the mean (SEM) values. The statistical program Statistical Package for the Social Science (SPSS; ver.12.0, SPSS Inc., Chicago, IL, USA) was used for all statistical analyses. The significance between the values was determined using the independent sample t-test.
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- Symptoms such as fur loss, diarrhoea, faeces, decreased motor activity, and oedema were not observed in female and male animals treated with the test item.
- Mortality:
- no mortality observed
- Description (incidence):
- No death occurred after the repeated administration of the test item at 1 000 mg/kg for 90days
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Weight of test item-treated male rats at 6 weeks after oral administration significantly differed from the male controls. Furthermore, the weight of test item-treated female rats differed from the female controls. However, the intergroup difference was not significant. When the test item was repeatedly administered at 1000 mg/kg for 90 days, the weight gain in the test item-treated male rats was significantly different from the male controls (reduced) (Table 1). The weight gain of the test item-treated female rats decreased relative to the female controls; however, the intergroup difference was not significant.
The test item was developed as an anti-obesity material and its weight loss effects have been reported previously (Kim et al., 2004; Jung et al., 2009; Jung etal., 2012). It seems that there was a difference in weight gain due to the anti-obesity effect of the test item. - Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- No specific abnormalities in feed intake was noted.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- no effects observed
- Ophthalmological findings:
- not specified
- Haematological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Changes in haematological parameters are presented in Table 3.
The hematologic parameters among male rats in the experimental group were not significantly different relative to the corresponding parameters in the controls (Table 3). However, the WBC and RBC counts, and Hct level for female rats in the experimental group significantly decreased relative to the corresponding values in the control group (p<0.05). However, the three parameters showed changes within the normal range (WBC count, 3.0∼14.3; RBC count, 6.69∼8.92; Hct level, 36.9∼51.8).
The hematologic parameters were within the normal range and indicated no toxicity of the test compound. - Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Changes in blood biochemical parameters are presented in Table 4.
The ALB level in male rats of the experimental group increased significantly compared to that of male controls (p<0.05). However, the change was within the normal range (ALB level: 3.70∼5.00). The CRE level in female rats of the experimental group significantly decreased compared to the level for female controls (P<0.05). Nevertheless, the change was within the normal range (CRE level: 0.35∼0.79). CRE is mainly produced by a non-enzymatic dehydration reaction from creatine in the muscles and is excreted through the kidneys. It is not reabsorbed; therefore, it is used as an indicator of abnormality in kidney function. An increase in the CRE level within the normal range does not indicate test item toxicity. Another indicator used to evaluate renal function is BUN. It is an indicator of the urea nitrogen level in blood; it is produced when proteins break down in the body. Since it is excreted through the kidneys, the BUN level can indirectly reflect kidney function (Kwon et al., 2003). There was no significant difference in BUN between the experimental and control groups.
Intergroup differences in some blood biochemical parameters were significant. Again, the changes in these parameters were within the normal range and did not indicate that the test compound was toxic - Endocrine findings:
- not examined
- Urinalysis findings:
- not specified
- Behaviour (functional findings):
- not specified
- Immunological findings:
- not specified
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Description (incidence and severity):
- No pathological findings were found in the organs of male animal in the experimental group.
In the male experimental group, brain weight increased significantly compared to male controls. However, the increased brain weight was still within the normal range (brain weight: 0.39∼0.84g/100 g of body weight). Brain weight for female rats in the experimental group did not significantly differ from female controls (Table 2). - Gross pathological findings:
- not specified
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- not specified
- Histopathological findings: neoplastic:
- not examined
- Other effects:
- not specified
- 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:
- Remarks on result:
- other: none
- Key result
- Critical effects observed:
- no
- Conclusions:
- Under the experimental conditions of the study, yeast hydrolysate was found to be safe and non-toxic following repeated dose of 1000 mg/kg/day.
- Executive summary:
This study was performed according to OECD TG 408 and aimed at investigating the subchronic oral toxicity of yeast hydrolysate in Sprague-Dawley rats. The test item was orally administered to female and male rats at a dose of 1000 mg/kg for 90 days. Results of this study showed that the administration of yeast hydrolysate significantly reduced body weight among male rats, which appears attributable to the anti-obesity effect of the compound. In the experimental groups, significant differences in some haematological parameters were considered as non-toxic because the results were within the normal range. Under the experimental conditions of the study, yeast hydrolysate was found to be safe and non-toxic following repeated dose of 1000 mg/kg/day.
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- other: experimental study performed on a SC yeast derivative used in a WoE approach for the target substance
- Adequacy of study:
- weight of evidence
- Study period:
- 2012
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study without detailed documentation
- Remarks:
- in the format of publication (not as detailed as a study report)
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
- Version / remarks:
- 1995 (test performed in 2012); analytical verifiction of doses not specified in the publication
- GLP compliance:
- yes
- Limit test:
- no
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: EpiCor powder from Embria Health Sciences (Ankeny, Iowa).
- Purity, including information on contaminants, isomers, etc.: not applicable; UVCB
RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: not applicable
- Specific activity: not applicable
- Locations of the label: not applicable
- Expiration date of radiochemical substance: not applicable
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: not specified in the publication
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: suspended in 1% methylcellulose containing distilled water not earlier than 30 minutes before administration. Concentration and homogeneity of the test article were checked by gravimetry.
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: not applicable.
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: not specified in the publication
- Reactivity of the test material with the incubation material used (e.g. plastic ware): not specified in the publication
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): none
- Preliminary purification step (if any): none
- Final concentration of a dissolved solid, stock liquid or gel:
- Final preparation of a solid (e.g. stock crystals ground to fine powder using a mortar and pestle): none
FORM AS APPLIED IN THE TEST (if different from that of starting material)
- Specify the relevant form characteristics if different from those in the starting material, such as state of aggregation, shape of particles or particle size distribution: no
INFORMATION ON NANOMATERIALS
- Chemical Composition: not applicable
- Density: not applicable
- Particle size & distribution: not applicable
- Specific surface area: not applicable
- Isoelectric point: not applicable
- Dissolution (rate): not applicable
TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)
- Description of the formulation, e.g. formulated product for foliar application; formulated product soil application; solution in organic solvent for soil application; formulated product seed treatment; solution in organic solvent seed treatment: not applicable
OTHER SPECIFICS
- Other relevant information needed for characterising the tested material, e.g. if radiolabelled, adjustment of pH, osmolality and precipitate in the culture medium to which the test chemical is added: not applicable - Species:
- rat
- Strain:
- Sprague-Dawley
- Details on species / strain selection:
- Specific-pathogen-free (SPF)
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories (Budapest, Hungary)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 4 to 5 weeks of age at arrival
- Weight at study initiation: 101.3 to 124.5 g (females) and 101.4 to 124.9 g (males)
- Fasting period before study: no
- Housing: 2 of the same sex were housed in a cage.
- Diet (e.g. ad libitum): free access to standardized rat and mouse diet S8106-SO11 SM R/M-ZþH (Ssniff Spezialdiäten, Soest, Germany)
- Water (e.g. ad libitum): free access to tap water
- Acclimation period: 5 days
DETAILS OF FOOD AND WATER QUALITY: not specified in the publication
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 15 times
- Photoperiod (hrs dark / hrs light): 12 / 12
IN-LIFE DATES: From: To: not specified in the publication - Route of administration:
- oral: gavage
- Details on route of administration:
- The test item was administred by gavage over 90 consecutive days at doses of 30, 200, and 1500 mg/kg bw. Doses were adjusted for weight on a weekly basis.
- Vehicle:
- other: 1% methylcellulose in distilled water
- Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
EpiCor was suspended in 1% methylcellulose containing distilled water not earlier than 30 minutes before administration (once daily).
DIET PREPARATION
- Rate of preparation of diet (frequency): not applicable
- Mixing appropriate amounts with (Type of food): not applicable
- Storage temperature of food: not applicable
VEHICLE
- Justification for use and choice of vehicle (if other than water): not specified in the publication
- Concentration in vehicle: not specified in the publication
- Amount of vehicle (if gavage): not specified in the publication
- Lot/batch no. (if required): not specified
- Purity: not specified - Analytical verification of doses or concentrations:
- not specified
- Duration of treatment / exposure:
- 90 days.
- Frequency of treatment:
- Daily
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Dose / conc.:
- 30 mg/kg bw/day (nominal)
- Dose / conc.:
- 200 mg/kg bw/day (nominal)
- Dose / conc.:
- 1 500 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 20/sex/dose group
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: the test item being intended to for use as an ingredient in single- and multi-ingredient dietary supplements and as an ingredient in food, dosages were based on proposed human use levels: the oral dose of 500 mg once daily (approximately 7 mg/kg, calculated as 70 kg bw in an adult). The highest dose 1500 mg/kg corresponds to about 200-fold of the 7 mg/kg daily dose if consumed by an adult, in addition to a previous acute toxicity study and dose range finding preliminary experiment. The lower dose of 30.0 mg/kg is still more than 4-fold the adult dose. The intermediate dose is approximately the geometrical mean of the high and the lower dose. The applied volume was adjusted weekly in accordance with the changes in the animals’ body weight.
- Rationale for animal assignment (if not random): not specified in the publication
- Fasting period before blood sampling for clinical biochemistry: yes; overnight
- Rationale for selecting satellite groups: no satellite groups
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random): not applicable
- Dose range finding studies: none
- Other: none - Positive control:
- none
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Not specified
- Time schedule: not spcified
- Cage side observations checked in table [No.?] were included: not specified
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were checked for mortality, general state, external appearance, behavior, and clinical signs twice daily at the beginning and end of the workday on weekdays and once on weekend days until the morning of the 91st day.
Necropsy occurred on the 91st day after a 16-hour fast. Organ weights were recorded and histopathological examinations were performed.
BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed on the first day of treatment (day 1) and weekly thereafter.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption: Food consumption was measured weekly and the daily average food consumption was calculated.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: no
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.
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water consumption was measured once a week for 24 hours, and microbiological testing was performed.
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Direct opthalmological examination was performed once during week 11 of treatment by a veterinary ophthalmologist using a Welch Allyn Pan Optic Ophthalmoscope (Jungingen, Germany).
- Dose groups that were examined: performed on the first 5 males and first 5 females of each dose group prior to the treatment period and all high-dose and control animals.
HAEMATOLOGY: Yes
- Time schedule for collection of blood: After the last treatment, rats were fasted overnight then anesthetized and blood samples were examined for hematological parameters.
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes; overnight.
- How many animals: all animals
- Parameters checked: red blood cells (RBCs), white blood cells (WBCs), hemoglobin (Hb), hematocrit (Hct), platelet count blood (prothrombin).
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: After the last treatment, rats were fasted overnight then anesthetized and blood samples were examined for clinical chemistry parameters
- Animals fasted: Yes; overnight
- How many animals: all animals
- Parameters checked: compliant to version 1995 of OECD TG 408.
PLASMA/SERUM HORMONES/LIPIDS: No
- Time of blood sample collection: not applicable.
- Animals fasted: not applicable.
- How many animals: not applicable.
URINALYSIS: Yes
- Time schedule for collection of urine: Urinalysis was carried out once during week 12 on 10 males and 10 females
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified
- Parameters checked: volume, specific gravity, pH glucose, blood and protein.
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during week 11.
- Dose groups that were examined: all animals.
- Battery of functions tested: Sensory reactivity to auditory, visual, and proprioceptive stimuli, assessment of grip strength, and motor activity was conducted according to routine methods.
IMMUNOLOGY: No
- Time schedule for examinations: not applicable.
- How many animals: not applicable.
- Dose groups that were examined: not applicable.
- Parameters checked in table [No.?] were examined: not applicable.
OTHER: none - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
Necropsy occurred on the 91st day after a 16-hour fast.
HISTOPATHOLOGY: Yes
Organ weights were recorded and histopathological examinations were performed. - Optional endpoint(s):
- Optional endpoints: No
- Other examinations:
- None
- Statistics:
- Statistical evaluations were performed by Statistica Version 5.5 (StatSoft, Edition 99, Tulsa, Oklahoma). Calculations included mean values, standard deviations, comparison of variances, 1-way analysis of variance (ANOVA), Tukey test, and/or Kruskal-Wallis nonparametric 1-way ANOVA.
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- No treatment related changes in general state, external appearance, or behavior were observed in 160 Sprague-Dawley rats, in the 30, 200, and 1500 mg/kg male and female groups. Furless areas without signs of inflammation were noted in 3 rats (1 from the 200 mg/kg males group and 2 from the 1500 mg/kg female group). However, these symptoms have been observed sporadically in other studies and are not considered treatment related.
- Mortality:
- no mortality observed
- Description (incidence):
- No deaths occurred.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- There were no significant differences in body weight or body weight gain among groups treated with EpiCor when compared to the control group at any time point during the experimental period.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- All test item treated male and female groups consumed similar amounts of food compared to the corresponding control groups throughout the study.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- no effects observed
- Description (incidence and severity):
- All test item treated male and female groups consumed similar amounts of water compared to the corresponding control groups throughout the study.
- Ophthalmological findings:
- no effects observed
- Description (incidence and severity):
- No ophthalmologic changes were observed in the control or 1500 mg/kg groups.
- Haematological findings:
- no effects observed
- Description (incidence and severity):
- No compound related effects were observed in red blood cells (RBCs), white blood cells (WBCs), hemoglobin (Hb), hematocrit (Hct), and platelet count values. No alterations in blood coagulation were seen via prothrombin time measurements.
- Clinical biochemistry findings:
- no effects observed
- Description (incidence and severity):
- Clinical chemistry data showed no treatment related toxic alterations at the end of the 90-day lasting treatment. Individual values and group mean values were within the physiological ranges.
Dose-related slight decease of total cholesterol level was measured in the male rats that reached the statistically significant level in the high-dose group only. Total cholesterol levels of the female group were similar to that of the controls. - Endocrine findings:
- not examined
- Urinalysis findings:
- no effects observed
- Description (incidence and severity):
- There were no differences in the volume, specific gravity, or pH of the discharged urine among control and test vehicle treated groups, nor for glucose, blood, or protein.
- Behaviour (functional findings):
- no effects observed
- Description (incidence and severity):
- No treatment related differences in the visual (finger approach) or auditory (startle) reactivity, pain perception (tall pinch), grip strength, or motor activity was observed at examination of male and female rats during week 11 of treatment.
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Description (incidence and severity):
- No differences in the organ weights or organ weights related to body weights were noted.
- Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- Gross necropsy examination revealed no compound-related lesions. The few sporadically developed pathological changes were determined unrelated to treatment. Internal examination showed that subcutaneous tissue, regional lymph nodes, fatty tissue, skeletal muscles, joints, and bone system were normal in all animals.
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- no effects observed
- Description (incidence and severity):
- No treatment related histopathological findings were observed.
- Histopathological findings: neoplastic:
- not examined
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- >= 1 500 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: test material well tolerated up to 1500 mg/kg bw/d dose level
- Key result
- Critical effects observed:
- no
- Conclusions:
- In conclusion, under the conditions of this 90-days study, the test material was well tolerated up to the highest dose tested of 1500 mg/kg bw/d. Based on these results, the no observable adverse effect level (NOAEL) for the Dried Fermentate Preparation of Saccharomyces cerevisiae test item is considered to be 1500 mg/kg bw/d.
- Executive summary:
This GLP-compliant Subchronic Repeated Dose Toxicity Study in the Rat (OECD TG 408, version 1995), was performed to assess the safety of a Dried Fermentate Preparation of Saccharomyces cerevisiae (EpiCor) upon repeated oral intake.
160 SPF Sprague-Dawley rats (80 females and 80 males), were administered the test item by gavage (suspended in 1% methylcellulose in distilled water), once daily over 90 consecutive days at doses of 0, 30, 200, and 1500 mg/kg bw/day. Animals were checked for mortality, general state, external appearance, behavior, and clinical signs twice daily at the beginning and end of the workday on weekdays and once on weekend days until the morning of the 91st day. They were weighed on the first day of treatment (day 1) and weekly thereafter. Food consumption was measured weekly and the daily average food consumption was calculated. Water consumption testing was performed. Direct opthalmological examination was performed on the first 5 males and first 5 females of each dose group prior to the treatment period and all high-dose and control animals once during week 11 of treatment. Sensory reactivity to auditory, visual, and proprioceptive stimuli, assessment of grip strength, and motor activity was conducted on all animals during week 11, according to routine methods. After the last treatment, rats were fasted overnight then anesthetized and blood samples were examined for hematological and clinical chemistry parameters. Urinalysis was carried out once during week 12 on 10 males and 10 females. Necropsy occurred on the 91st day after a 16-hour fast. Organ weights were recorded and histopathological examinations were performed.No deaths occurred and no treatment related changes in general state, external appearance, or behavior were observed in any animal of the study. Furless areas without signs of inflammation were noted in 3 rats (1 from the 200 mg/kg males group and 2 from the 1500 mg/kg female group). However, these symptoms have been observed sporadically in other studies and are not considered treatment related. There were no significant differences in body weight or body weight gain among groups treated with the test item when compared to the control group at any time point during the experimental period. All test item treated male and female groups consumed similar amounts of food and water compared to the corresponding control groups throughout the study. No ophthalmologic changes were observed in the control or 1500 mg/kg groups. No treatment related differences in the visual (finger approach) or auditory(startle) reactivity, pain perception (tall pinch), grip strength, or motor activity was observed at examination of male and female rats during week 11 of treatment. No compound related effects were observed in red blood cells (RBCs), white blood cells (WBCs), hemoglobin (Hb), haematocrit (Hct), and platelet count values. No alterations in blood coagulation were seen via prothrombin time measurements. No clinical chemistry data showed any treatment related toxic alterations at the end of the 90-day lasting treatment. Individual values and group mean values were within the physiological ranges. Dose-related slight decrease of total cholesterol level was measured in the male rats that reached the statistically significant level in the high-dose group only. Total cholesterol levels of the female group were similar to that of the controls. There were no differences in the volume, specific gravity, or pH of the discharged urine among control and test vehicle treated groups, nor for glucose, blood, or protein. Gross necropsy examination revealed no compound-related lesions. The few sporadically developed pathological changes were determined unrelated to treatment. Internal examination showed that subcutaneous tissue, regional lymph nodes, fatty tissue, skeletal muscles, joints, and bone system were normal in all animals. No differences in the organ weights or organ weights related to body weights were noted, and no treatment related histopathological findings were observed.
In conclusion, under the conditions of this 90-days study, the test material was well tolerated up to the highest dose tested of 1500 mg/kg bw/d. Based on these results, the no observable adverse effect level (NOAEL) for the Dried Fermentate Preparation of Saccharomyces cerevisiae test item is considered to be 1500 mg/kg bw/d.
- Endpoint:
- chronic toxicity: oral
- Type of information:
- other: experimental study performed on a SC yeast derivative used in a WoE approach for the target substance
- Adequacy of study:
- weight of evidence
- Study period:
- 2012
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study without detailed documentation
- Remarks:
- in the format of publication (not as detailed as a study report)
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 452 (Chronic Toxicity Studies)
- Version / remarks:
- 1981 (test performed in 2012); analytical verifiction of doses not specified in the publication
- GLP compliance:
- yes
- Limit test:
- no
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: EpiCor powder from Embria Health Sciences (Ankeny, Iowa).
- Purity, including information on contaminants, isomers, etc.: not applicable; UVCB
RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: not applicable
- Specific activity: not applicable
- Locations of the label: not applicable
- Expiration date of radiochemical substance: not applicable
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: not specified in the publication
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: suspended in 1% methylcellulose containing distilled water not earlier than 30 minutes before administration. Concentration and homogeneity of the test article were checked by gravimetry.
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: not applicable.
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: not specified in the publication
- Reactivity of the test material with the incubation material used (e.g. plastic ware): not specified in the publication
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): none
- Preliminary purification step (if any): none
- Final concentration of a dissolved solid, stock liquid or gel:
- Final preparation of a solid (e.g. stock crystals ground to fine powder using a mortar and pestle): none
FORM AS APPLIED IN THE TEST (if different from that of starting material)
- Specify the relevant form characteristics if different from those in the starting material, such as state of aggregation, shape of particles or particle size distribution: no
INFORMATION ON NANOMATERIALS
- Chemical Composition: not applicable
- Density: not applicable
- Particle size & distribution: not applicable
- Specific surface area: not applicable
- Isoelectric point: not applicable
- Dissolution (rate): not applicable
TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)
- Description of the formulation, e.g. formulated product for foliar application; formulated product soil application; solution in organic solvent for soil application; formulated product seed treatment; solution in organic solvent seed treatment: not applicable
OTHER SPECIFICS
- Other relevant information needed for characterising the tested material, e.g. if radiolabelled, adjustment of pH, osmolality and precipitate in the culture medium to which the test chemical is added: not applicable - Species:
- rat
- Strain:
- Sprague-Dawley
- Details on species / strain selection:
- Specific-pathogen-free (SPF)
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories (Budapest, Hungary)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 4 to 5 weeks of age at arrival
- Weight at study initiation: 101.0 to 112.8 g (females) and 126.0 to 149.3 g (males)
- Fasting period before study: no
- Housing: 2 of the same sex were housed in a cage (housed 1 per cage and observed for at least 5 days prior to initiation of treatment).
- Diet (e.g. ad libitum): free access to standardized rat and mouse diet S8106-SO11 SM R/M-ZþH (Ssniff Spezialdiäten, Soest, Germany)
- Water (e.g. ad libitum): free access to tap water
- Acclimation period: 5 days
DETAILS OF FOOD AND WATER QUALITY: not specified in the publication
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 15 times
- Photoperiod (hrs dark / hrs light): 12 / 12
IN-LIFE DATES: From: To: not specified in the publication - Route of administration:
- oral: gavage
- Details on route of administration:
- The test item was administred by gavage over 364 consecutive days (1 year) at doses of 20, 200, and 800 mg/kg bw. The applied volume was adjusted weekly in accordance with the changes in the animals’ body weight.
- Vehicle:
- other: 1% methylcellulose in distilled water
- Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
EpiCor was suspended in 1% methylcellulose containing distilled water not earlier than 30 minutes before administration (once daily).
DIET PREPARATION
- Rate of preparation of diet (frequency): not applicable
- Mixing appropriate amounts with (Type of food): not applicable
- Storage temperature of food: not applicable
VEHICLE
- Justification for use and choice of vehicle (if other than water): not specified in the publication
- Concentration in vehicle: not specified in the publication
- Amount of vehicle (if gavage): not specified in the publication
- Lot/batch no. (if required): not specified
- Purity: not specified - Analytical verification of doses or concentrations:
- not specified
- Duration of treatment / exposure:
- 364 days.
- Frequency of treatment:
- Daily
- Dose / conc.:
- 20 mg/kg bw/day (nominal)
- Dose / conc.:
- 200 mg/kg bw/day (nominal)
- Dose / conc.:
- 800 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 20/sex/dose group
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: Dosages applied in this study were selected on the basis of results obtained in previous acute toxicity and 90-day subchronic studies, where it was well tolerated in daily doses up to 1500 mg/kg for 90 days. The safety factor of 100 to 1 for food additives used in applying animal experimentation data to man was taken into consideration, that is, a food additive for use by man will not be granted a tolerance that will exceed 1/100th of the maximum amount demonstrated to be without harm to experimental animals. The highest dose chosen, 800 mg/kg, corresponds to 100-fold of the approximate 7.1 mg/kg daily dose, for a planned supplement oral dose of 500 mg once daily, if consumed by an adult or 32-fold of the 25 mg/kg daily dose when calculated for a child’s body weight (i.e., 20 kg). The lower dose of 20 mg/kg is still 2.8-fold of the adult’s dose. The intermediate dose is 2.8-fold of the 7.1 mg/kg daily dose if consumed by an adult.
- Rationale for animal assignment (if not random): not specified in the publication
- Fasting period before blood sampling for clinical biochemistry: yes; overnight
- Rationale for selecting satellite groups: no satellite groups
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random): not applicable
- Dose range finding studies: none
- Other: none - Positive control:
- none
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Not specified
- Time schedule: not spcified
- Cage side observations checked in table [No.?] were included: not specified
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were checked for mortality, general state, external appearance, behavior, and clinical signs twice daily at the beginning and end of the workday on weekdays and once on weekend days until the morning of the final day of the study.
Necropsy occurred on day 365 after fasting for 16 hours. Organ weights were recorded and histopathological examinations were performed.
BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed upon arrival to the laboratory, on the day of randomization, on day 1 (first treatment), once weekly during the treatment period, and on the day of necropsy.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption: Food consumption was measured weekly and the daily average food consumption was calculated.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: no
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
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water consumption was measured once a week for 24 hours and microbiologically tested.
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Direct ophthalmologic examination was conducted on all animals prior to the treatment period and on all high-dose and control animals at week 51 by a veterinary ophthalmologist using a Welch Allyn Pan Optic Ophthalmoscope (Jungingen, Germany).
- Dose groups that were examined: on all animals prior to the treatment period and on all high-dose and control animals at week 51.
HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were obtained twice during the study at weeks 14 and 33 from 10 male and 10 female rats per group and 1 day after the last treatment before necropsy. Rats were fasted overnight, anesthetized, and blood samples were examined for hematological parameters.
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes; overnight.
- How many animals: all animals
- Parameters checked: RBC, red blood cells; WBC, white blood cells; Hb, hemoglobin; Hct, hematocrit; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; PLT, platelet; Ly, lymphocytes; Ne, neutrophils; Eo, Eosinophils; Mo, Monocytes; Ba, Basophils; Pt, prothrombin time.
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were obtained twice during the study at weeks 14 and 33 from 10 male and 10 female rats per group and 1 day after the last treatment before necropsy. Rats were fasted overnight, anesthetized, and blood samples were examined for clinical chemistry parameters.
- Animals fasted: Yes; overnight
- How many animals: all animals
- Parameters checked: GPT, glutamic pyruvic transaminase; GGT, gamma-glutamyl transpeptidase; AP, alkaline phosphatase; Cho, total cholesterol; Prot, total protein; Gl, blood glucose; Tg, triglycerides; BUN, blood urea nitrogen; Na, sodium; K, potassium; Cl, chloride; Crea, creatinine; GOT, glutamic oxalacetic transaminase; GLDH, glutamate dehydrogenase; Alb, albumin; Glob, globulin; Ca, total calcium.
PLASMA/SERUM HORMONES/LIPIDS: No
- Time of blood sample collection: not applicable.
- Animals fasted: not applicable.
- How many animals: not applicable.
URINALYSIS: Yes
- Time schedule for collection of urine: Urinalysis was carried out during weeks 13, 32, and 51 on 10 males and 10 females. Because live bacteria were seen in the sediment of 2 rats from the 800 mg/kg group during week 32, sediment analysis was repeated on week 36, and sediments from the 2 rats were sent to an outside laboratory for parasitology and bacteriology examinations.
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: no
- Parameters checked: volume, specific gravity, pH glucose, blood, protein and sediment analysis (parasitology and bacteriology examinations).
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during weeks 15, 27, 40, and 52.
- Dose groups that were examined: all animals.
- Battery of functions tested: Sensory reactivity to auditory, visual, and proprioceptive stimuli, assessment of grip strength, and motor activity were conducted according to routine methods.
IMMUNOLOGY: No
- Time schedule for examinations: not applicable.
- How many animals: not applicable.
- Dose groups that were examined: not applicable.
- Parameters checked in table [No.?] were examined: not applicable.
OTHER: none - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
Necropsy occurred on day 365 after fasting for 16 hours.
HISTOPATHOLOGY: Yes
Organ weights were recorded and histopathological examinations were performed. Any grossly visible or palpable tumor appear, time of onset, location, dimensions, appearance, and progression, were recorded. - Optional endpoint(s):
- Optional endpoints: No
- Other examinations:
- None
- Statistics:
- Statistical evaluations were performed by Statistica Version 5.5 (StatSoft, Edition 99, Tulsa, Oklahoma). Calculations included mean values, standard deviations, comparison of variances, 1-way analysis of variance (ANOVA), Tukey test, and/or Kruskal-Wallis nonparametric 1-way ANOVA.
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- No treatment related changes in general state, external appearance, or behavior were observed in 160 Sprague-Dawley rats, in the 30, 200, and 1500 mg/kg male and female groups. Furless areas without signs of inflammation were noted in 3 rats (1 from the 200 mg/kg males group and 2 from the 1500 mg/kg female group). However, these symptoms have been observed sporadically in other studies and are not considered treatment related.
- Mortality:
- no mortality observed
- Description (incidence):
- No spontaneous deaths occurred in any of the 160 animals during the 1-year study, in any group. One male and one female rat, both from 800 mg/kg dose groups, were found in tumor provoked moribund states (the male had a mass on the skin of the left foreleg, and the female had 3 nodules in the inguinal region) and were consequently sacrificed. One female rat was accidentally over-anaesthetized during scheduled blood sampling at week 33.
- Body weight and weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The development of the animals during the experimental period corresponded appropriately to their species and age; body weight gains in treated and control groups of both genders were similar throughout the study. Statistically significant bodyweight loss at week 39 in the 200 mg/kg male group was attributed to low within-group differences, and statistically significant weight gain at week 44 in the male 20 mg/kg group and at weeks 10, 14, 29, and 52 in the female 800 mg/kg group were considered to be of no biological significance. Prolonged bodyweight losses occurred in 3 rats from different groups; 2 were attributed to pituitary adenomas (200 and 800 mg/kg females) and 1 to diabetes (control male).
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- Food consumption was similar between all treatment groups and control groups.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- effects observed, non-treatment-related
- Description (incidence and severity):
- Statistically significant decreased water consumption occurred in the 800 mg/kg group. The male 800 mg/kg group displayed a statistically significant decrease in water consumption on 24 non-consecutive individual weeks. The female from 800 mg/kg group displayed statistically significant decreased water consumption only during the first 10 weeks of the study. No dose-dependent correlation could be established between decreased water consumption and any findings of clinical or histological relevance related to the test substance.
- Ophthalmological findings:
- no effects observed
- Description (incidence and severity):
- No ophthalmologic changes were observed prior to treatment or at week 51 in the control or the treatment groups, except in the 800 mg/kg group, where 1 male kept its head posture in right rotation from week 35 until the end of the study, determined related to handling during blood sampling, resulting in corneal injury, and 1 male was found with congestion of conjunctival blood vessels noted on the left eye lid, considered incidental.
- Haematological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- On blood examination, no compound-related effects were observed in Hematology and blood coagulation at weeks 14, 33, and at the end of the study. Statistically significant alterations in Hematology were found. Results were considered non-dose-dependent or were within historical control ranges and/or were considered not of clinical significance; the results were unrelated to other findings.
- Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- On blood examination, no compound-related effects were observed in blood chemistry at weeks 14, 33, and at the end of the study. Statistically significant alterations in blood chemistry were found. Results were considered non-dose-dependent or were within historical control ranges and/or were considered not of clinical significance; the results were unrelated to other findings.
- Endocrine findings:
- not examined
- Urinalysis findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- During weeks 13, 32, and 51, no treatment related differences were noted. Several sporadic statistically significant differences were noted in the urine samples of several groups. A low amount of total protein was present at a low incidence in the 20 and 800 mg/kg groups on week 32, and protein discharge was present in all dose groups at week 51. During week 51, the pH values of the 20 and 200 mg/kg females were lower compared to the control group. Low incidence of blood, crystals, and epithelial cells were noted, but were not consistent at any timepoint in the study. Live bacteria were noted in the urinary sediment from 2 symptom-free male rats from the 800mg/kg group in week 32. The sediment analysis was repeated 1 month later, where live bacteria were again noted. These bacteria were identified as non-pathogenic bacteria (Enterobacter spp., Klebsiella spp., and Citrobacter freundii) generally present in the intestinal and urinary tract of rats. As the viable bacteria were facultative pathogens and the animals were free of clinical symptoms, the animals were left in the study. The sediments analyzed in week 51 were clear of viable bacteria.
- Behaviour (functional findings):
- no effects observed
- Description (incidence and severity):
- No test item-related differences in visual (finger approach) or auditory (startle) reactivity, pain perception (tail pinch), grip strength, or motor activity were noted.
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Description (incidence and severity):
- No treatment-related differences in organ weights or in differences in the bodyweight– to–organ weight ratios were found and no treatment-related histopathological changes were seen.
- Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Internal macroscopic examinations found no treatment related pathological changes in subcutaneous tissues, regional lymph nodes, fatty tissues, skeletal muscles, joints, or bones in any animal. Sporadically developed pathological changes were determined to be unrelated to treatment and include 1- and 2-sided testicular atrophy in low incidence, fawn-coloured, and nutmeg-patterned livers in males and females of both control and treatment groups (with higher incidence in the control groups), adrenal gland cystic degeneration in female control and treated groups, age-related thymus atrophy in control and treated groups, occurrence of hydrometria, tumor-like changes in the pituitary and skin in low incidence, and tumor-like tissue changes in mammary glands of 15% to 20% of the 200 and 800 mg/kg female treatment groups. The mammary tumor rates were not significant and fell within the historical control incidences of 6.3%to 32%.
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The histopathological findings are commonly considered incidental and spontaneous in nature. Only benign tumors were found in the animals, which were confirmed histopathologically.
- Histopathological findings: neoplastic:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Only benign tumors were found in the animals, which were confirmed histopathologically. Findings were not statistically significant and included 2 and 3 mammary gland adenomas in the 200 and 800 mg/kg female groups, respectively, a single mammary gland fibroma in each of the same female groups and pituitary adenomas in both treatment and control groups, along with 7 non-dose treatment-related benign skin lesions. While the incidence of pituitary adenomas appeared dose-dependent in nature, the occurrences were not statistically significant, and were within historical control ranges (internal data).
- Other effects:
- no effects observed
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- >= 800 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: based on overall results
- Key result
- Critical effects observed:
- no
- Conclusions:
- In conclusion, under the conditions of this 1-year study and given the overall results, the no observable adverse effect level (NOAEL) for the Dried Fermentate Preparation of Saccharomyces cerevisiae test item is considered to be 800 mg/kg bw/day (highest dose tested).
- Executive summary:
This GLP-compliant Chronic Repeated Dose Toxicity Study in the Rat (OECD TG 452, version 1981), was performed to assess the safety of a Dried Fermentate Preparation of Saccharomyces cerevisiae (EpiCor) upon chronic repeated oral intake.
160 SPF Sprague-Dawley rats (80 females and 80 males), were administered the test item by gavage (suspended in 1% methylcellulose in distilled water), once daily over 364 consecutive days at doses of 0, 20, 200, and 800mg/kg bw/day. Animals were checked for mortality, general state, external appearance, behavior, and clinical signs twice daily at the beginning and end of the workday on weekdays and once on weekend days until the morning of the final day of the study. Should any grossly visible or palpable tumor appear, time of onset, location, dimensions, appearance, and progression, are recorded. Direct ophthalmologic examination was conducted on all animals prior to the treatment period and on all high-dose and control animals at week 51. Sensory reactivity to auditory, visual, and proprioceptive stimuli, assessment of grip strength, and motor activity were conducted on all animals during weeks 15, 27, 40, and 52. Blood samples were obtained twice during the study at weeks 14 and 33 from10 male and 10 female rats per group and 1 day after the last treatment before necropsy. Rats were fasted overnight, anesthetized, and blood samples were examined for haematological and clinical chemistry parameters. Urinalysis was carried out during weeks 13, 32, and 51 on 10 males and 10 females. Because live bacteria were seen in the sediment of 2 rats from the 800 mg/kg group during week 32, sediment analysis was repeated on week 36, and sediments from the 2 rats were sent to an outside laboratory for parasitology and bacteriology examinations. Necropsy occurred on day 365 after fasting for 16 hours. Organ weights were recorded and histopathological examinations were performed. No spontaneous deaths occurred in any of the 160 animals during the 1-year study, in any group. One male and one female rat, both from 800 mg/kg dose groups, were found in tumor provoked moribund states (the male had a mass on the skin of the left foreleg, and the female had 3 nodules in the inguinal region) and were consequently sacrificed. One female rat was accidentally over-anaesthetized during scheduled blood sampling at week 33. On gross examination, males and females from both control and treatment groups displayed varying degrees of insignificant fur loss without visible changes of epidermis or signs of injury or inflammation. Two female rats (200 and 800 mg/kg group) displayed moderate ataxy resulting from pituitary adenomas. An ulcerated area on the right hindleg of 1 female rat in the 20 mg/kg was histopathologically described as an inflamed lipoma. The development of the animals during the experimental period corresponded appropriately to their species and age; body weight gains in treated and control groups of both genders were similar throughout the study. Statistically significant bodyweight loss at week 39 in the 200 mg/kg male group was attributed to low within-group differences, and statistically significant weight gain at week 44 in the male 20 mg/kg group and at weeks 10, 14, 29, and 52 in the female 800 mg/kg group were considered to be of no biological significance. Prolonged bodyweight losses occurred in 3 rats from different groups; 2 were attributed to pituitary adenomas (200 and 800 mg/kg females) and 1 to diabetes (control male). Food consumption was similar between all treatment groups and control groups. Statistically significant decreased water consumption occurred in the 800 mg/kg group. The male 800 mg/kg group displayed a statistically significant decrease in water consumption on 24 non-consecutive individual weeks. The female from 800 mg/kg group displayed statistically significant decreased water consumption only during the first 10 weeks of the study. No dose-dependent correlation could be established between decreased water consumption and any findings of clinical or histological relevance related to the test substance. No ophthalmologic changes were observed prior to treatment or at week 51 in the control or the treatment groups, except in the 800 mg/kg group, where 1 male kept its head posture in right rotation from week 35 until the end of the study, determined related to handling during blood sampling, resulting in corneal injury, and 1 male was found with congestion of conjunctival blood vessels noted on the left eye lid, considered incidental. Otherwise, no compound-related differences in visual (finger approach) or auditory (startle) reactivity, pain perception (tail pinch), grip strength, or motor activity were noted. On blood examination, no compound-related effects were observed in Hematology, blood coagulation, or blood chemistry at weeks 14, 33, and at the end of the study. Statistically significant alterations in Hematology and blood chemistry were found. Results were considered non-dose-dependent or were within historical control ranges and/or were considered not of clinical significance; the results were unrelated to other findings. On urinalysis during weeks 13, 32, and 51, no treatment related differences were noted. Several sporadic statistically significant differences were noted in the urine samples of several groups. A low amount of total protein was present at a low incidence in the 20 and 800 mg/kg groups on week 32, and protein discharge was present in all dose groups at week 51. During week 51, the pH values of the 20 and 200 mg/kg females were lower compared to the control group. Low incidence of blood, crystals, and epithelial cells were noted, but were not consistent at any timepoint in the study. Live bacteria were noted in the urinary sediment from 2 symptom-free male rats from the 800mg/kg group in week 32. The sediment analysis was repeated 1 month later, where live bacteria were again noted. These bacteria were identified as non-pathogenic bacteria (Enterobacter spp., Klebsiella spp., and Citrobacter freundii) generally present in the intestinal and urinary tract of rats. As the viable bacteria were facultative pathogens and the animals were free of clinical symptoms, the animals were left in the study. The sediments analyzed in week 51 were clear of viable bacteria. Internal macroscopic examinations found no treatment related pathological changes in subcutaneous tissues, regional lymph nodes, fatty tissues, skeletal muscles, joints, or bones in any animal. Sporadically developed pathological changes were determined to be unrelated to treatment and include 1- and 2-sided testicular atrophy in low incidence, fawn-coloured, and nutmeg-patterned livers in males and females of both control and treatment groups (with higher incidence in the control groups), adrenal gland cystic degeneration in female control and treated groups, age-related thymus atrophy in control and treated groups, occurrence of hydrometria, tumor-like changes in the pituitary and skin in low incidence, and tumor-like tissue changes in mammary glands of 15% to 20% of the 200 and 800 mg/kg female treatment groups. The mammary tumor rates were not significant and fell within the historical control incidences of 6.3%to 32%. The histopathological findings are commonly considered incidental and spontaneous in nature. Only benign tumors were found in the animals, which were confirmed histopathologically. Findings were not statistically significant and included 2 and 3 mammary gland adenomas in the 200 and 800 mg/kg female groups, respectively, a single mammary gland fibroma in each of the same female groups and pituitary adenomas in both treatment and control groups, along with 7 non-dose treatment-related benign skin lesions. While the incidence of pituitary adenomas appeared dose-dependent in nature, the occurrences were not statistically significant, and were within historical control ranges (internal data). No treatment-related differences in organ weights or in differences in the bodyweight– to–organ weight ratios were found and no treatment-related histopathological changes were seen.
In conclusion, under the conditions of this 1-year study and given the overall results, the no observable adverse effect level (NOAEL) for the Dried Fermentate Preparation of Saccharomyces cerevisiae test item is considered to be 800 mg/kg bw/day (highest dose tested).
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- other: experimental study performed on a SC yeast derivative used in a WoE approach for the target substance
- Adequacy of study:
- weight of evidence
- Study period:
- From July 2020 to November 2020
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- test procedure in accordance with national standard methods with acceptable restrictions
- Qualifier:
- according to guideline
- Guideline:
- other: Russian Guidelines for conducting preclinical studies of medicinal products. Part One / Edited by A.N. Mironov. -M.: Griff & K, 2012. -536 с
- Qualifier:
- according to guideline
- Guideline:
- other: Russian Federal Law No. 61-FZ of 12.04.2010. "On Circulation of Medicines". (ed. from 04.06.2018)
- Qualifier:
- according to guideline
- Guideline:
- other: Russian Order of the Ministry of Agriculture of 6 March 2018 No. 101
- Principles of method if other than guideline:
- The study is a subchronic oral toxicity test (gavage) in outbred rats performed with the aim of introducing the feed additive into clinical veterinary practice.
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- rat
- Strain:
- not specified
- Remarks:
- white outbred rats
- Details on species / strain selection:
- General toxicity studies are preferably performed on rodents, as they are a standard test system for evaluating the general toxic properties of feed additives.
- Sex:
- female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Branch "Andreevka" of the Federal State Budgetary Institution "Scientific Centre for Biomedical Technologies" of the Russian Academy of Medical Sciences (Moscow, Russia)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8-9 weeks
- Weight at study initiation: 173-195 g (body weight of the animals at the start of the experiment was in the range ±20% of the mean)
- Fasting period before study: no
- Housing: rats were kept in polypropylene cages covered with steel lattice covers with feeding cavity, 10 animals in each. The size of the cages was 50×35×20 (L×W×H, cm). Wood shavings for laboratory animals were used as bedding.
- Diet (e.g. ad libitum): fed with full-fat extruded compound feed for laboratory animals (rats, mice, hamsters)
- Water (e.g. ad libitum): ad libitum; water from standard rodent drinkers with prepared drinking water.
- Acclimation period: Immediately after arrival at the research centre, the animals were placed in the quarantine and adaptation room for 7 days. During this period, the animals were inspected daily.
DETAILS OF FOOD AND WATER QUALITY: not specified in the study report
ENVIRONMENTAL CONDITIONS
Temperature and humidity were monitored daily using a combined thermohygrometer
- Temperature (°C): 20-26
- Humidity (%): 30-70
- Air changes (per hr): not specified in the study report
- Photoperiod (hrs dark / hrs light): not specified in the study report
IN-LIFE DATES: From: To: not specified in the study report - Route of administration:
- oral: gavage
- Details on route of administration:
- Oral route was chosen as the test item is a feed additive and the test was conducted in the framework of mandatory studies required for the registration of feed additives on the territory of the Russian Federation according to the Order of the Ministry of Agriculture of the Russian Federation N 48 « About approval of rules of the state registration of medicines for animals and feed additives » dated 01.04.2005.*
Suspension of the test item was administered to rats by gavage to rats using a stainless steel atraumatic probe in a fixed volume of 1.0 ml per 100 g body weight. - Vehicle:
- water
- Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
Before administration, the test item was suspended in water in the ratios required to obtain the doses required for the study (1/5 and 1/10 of the maximum soluble amount of the test item in 100 ml of water).
The suspensions were prepared according to the following scheme:
-->20 g of test item + 87.4 ml water until a 100 ml solution is obtained (2000 mg/kg dose is 1/5 of the maximum soluble amount of test item in 100 ml of water);
--> 10 g feed additive + 94 ml water until a 100 ml solution is obtained (1000 mg/kg dose is 1/10 of the maximum soluble amount of test item in 100 ml water);
The test item was weighed on an laboratory scale with an accuracy of 0.1 g.
Animals in the control group were given water at a rate 1 ml per 100 g body weight.
DIET PREPARATION
- Rate of preparation of diet (frequency): not applicable
- Mixing appropriate amounts with (Type of food): not applicable
- Storage temperature of food: not applicable
VEHICLE
- Justification for use and choice of vehicle (if other than water): vehicle is water
- Concentration in vehicle: suspensions were prepared according to the following scheme:
-->20 g of test item + 87.4 ml water until a 100 ml solution is obtained (2000 mg/kg dose is 1/5 of the maximum soluble amount of test item in 100 ml of water);
--> 10 g feed additive + 94 ml water until a 100 ml solution is obtained (1000 mg/kg dose is 1/10 of the maximum soluble amount of test item in 100 ml water);
- Amount of vehicle (if gavage): 1 ml per 100 g body weight
- Lot/batch no. (if required): not applicable
- Purity: not applicable - Analytical verification of doses or concentrations:
- not specified
- Duration of treatment / exposure:
- 90 days.
- Frequency of treatment:
- Daily
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- Dose / conc.:
- 2 000 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 30 female rats; 10 females/dose group (2 test dose groups and one vehicle control group)
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: not specified in the study report
- Rationale for animal assignment (if not random): random; rats were allocated to groups randomly. As body weight was taken as the criterion, so that the individual weight did not deviate from the average by more than 20%.
- Fasting period before blood sampling for clinical biochemistry: yes; prior to the terminal procedures, rats were deprived of food for 16-18 hours while maintaining access to water, then weighed.
- Rationale for selecting satellite groups: additional specific satellite groups (control and top dose groups) were not used but the following manipulations were performed on days 91 and 100 of the study with half of the rats from each test and control groups (5 animals were used for each period):
--> blood samples were taken for haematology and clinical chemistry analysis;
--> animals were autopsied with macroscopic examination of internal organs;
--> mass ratios of the internal organs of the animals were determined.
- Post-exposure recovery period in satellite groups: 10 days
- Section schedule rationale (if not random): not specified
- Dose range finding studies: none
- Other: none - Positive control:
- None
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Not specified
- Time schedule: Not specified
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
During the whole experimental period of the study, the animals were observed for their general condition and behaviour, reaction to stimuli (sound, light), manifestation of toxicity symptoms, possible death
BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of animals in experimental and control groups was monitored on the day of the experiment (before introduction of the feed additive), as well as on days 1, 14, 28, 42, 56, 70, 84 and 90 (i.e., at 2-week intervals ).
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): not applicable
FOOD EFFICIENCY: No
- 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
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): not applicable
OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations: not applicable
- Dose groups that were examined: not applicable
HAEMATOLOGY: Yes
- Time schedule for collection of blood: On the day following the end of test item administration (day 91), half of the animals from control and test group 1 (2000 mg/kg bw/day) (n=5) and 4 animals for test group 2 (1000 mg/kg bw/day) (due to death of one rat from this group not test-item related) (into tubes with anticoagulant EDTA-K3) to determine hematological parameters. On the 10th day after completion of test item administration, the other half of the animals (n=5) were euthanized and blood samples were taken to assess the degree of reversibility of possible effects after repeated administration of the test item.
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes; animals were deprived of food for 16-18 hours while maintaining access to water, then weighed.
- How many animals: 5/dose/study period
- The following parameters were examined: basic rat peripheral blood parameters were determined on an haematology analyser (Haematocrit (%), Haemoglobin (g/l), Erythrocytes (number/L), Leucocytes (number/L), Platelets (number/L), Leukogram).
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On the day following the end of test item administration (day 91), half of the animals from control and test group 1 (2000 mg/kg bw/day) (n=5) and 4 animals for test group 2 (1000 mg/kg bw/day) (due to death of one rat from this group not test-item related) were euthanized and blood samples were taken (into tubes with coagulation activator) to determine biochemical parameters. On the 10th day after completion of test item administration (day 100), the other half of the animals (n=5) were euthanized and blood samples were taken to assess the degree of reversibility of possible effects after repeated administration of the test item.
- Animals fasted: Yes; animals were deprived of food for 16-18 hours while maintaining access to water, then weighed.
- How many animals: 5/dose/study period
- Parameters examined: Samples after coagulation were centrifuged at 3,000 rpm for 5 minutes to obtain serum. Biochemical blood values were determined on an A25 analyser [Bilirubin, Aspartate Aminotransferase (AST), Alanine Aminotransferase (ALT), Urea, Creatinine, Total protein, Alkaline phosphatase, Glucose].
PLASMA/SERUM HORMONES/LIPIDS: No
- Time of blood sample collection: not applicable
- Animals fasted: not applicable
- How many animals: not applicable
URINALYSIS: No
- Time schedule for collection of urine: not applicable
- Metabolism cages used for collection of urine: not applicable
- Animals fasted: not applicable
- Parameters checked in table [No.?] were examined: not applicable
NEUROBEHAVIOURAL EXAMINATION: Yes; the functional state of the CNS was assessed visually by observation of motor activity and response to external stimuli (reaction to stimuli (sound, light))
- Time schedule for examinations: During the whole experimental period of the study
- Dose groups that were examined: all
- Battery of functions tested: motor activity and response to external stimuli (reaction to stimuli (sound, light))
IMMUNOLOGY: No
- Time schedule for examinations: not applicable
- How many animals: not applicable
- Dose groups that were examined: not applicable
- Parameters checked in table [No.?] were examined: not applicable
OTHER: None. - Sacrifice and pathology:
- SACRIFICE: On the 1st and 10th days after completion of test item administration (91 and 100 days of the experiment), an autopsy of the experimental animals was performed after blood sampling.
GROSS PATHOLOGY: Yes
The following organs and tissues were subjected to macroscopic examination: skin with subcutaneous fatty tissue, liver, lungs, kidneys, heart, spleen, stomach, large and small intestine.
The body weight of the rats was used for subsequent calculation of the mass ratios of the following organs: liver, kidneys, spleen, lungs and heart.
The relative weight of each organ was calculated using the formula:
S = (m / M) ×100 g
where S is the relative mass of the organ,
m - mass of the organ (g)
M is the body weight of the animal (g).
HISTOPATHOLOGY: No - Optional endpoint(s):
- Optional endpoints: No.
- Other examinations:
- None.
- Statistics:
- The experimental data was statistically processed using Microsoft Excel PC software.
Mean values and the width of the confidence interval (P=0.95) were calculated for all data. To determine the validity of intergroup differences, statistical processing was performed in two stages: first, hypothesis testing for equality of variance of the control sample and each of the test samples was performed (Fisher test, 0.05 probability threshold), then hypothesis testing for equality of mean values of the samples was performed (Student's test, Cramer-Welch approximation, 0.05 probability threshold). - Clinical signs:
- no effects observed
- Description (incidence and severity):
- No signs of toxicity were observed in the animals of the test groups. The general condition of the rats remained satisfactory. No changes in behaviour were noted. Convulsions were not observed; coordination of movements was not impaired; skeletal muscle tone was normal; reaction to tactile, pain, sound and light stimuli was adequate; integrity and elasticity of the skin were preserved, hyperemia was absent; coloration of visible mucous membranes corresponded to normal; frequency and depth of respiratory movements were unchanged; feces were dark brown, of dense consistency, characteristic oval-oblong shape with a specific odor.
- Mortality:
- no mortality observed
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Body weight changes results are presented in Table 1.
On day 1, rats weight in the two test groups did not statistically differ from the control group, which shows homogeneity of the formed animal groups: 184.2 ± 5.1 g (2000 mg/kg bw/day test group) and 183.6 ± 4.5 g (1000 mg/kg bw/day test group) versus 182.1 ± 4.5 g (vehicle control group).
On day 79 of test item administration, rat No. 5 of test group 2 (1000 mg/kg bw/day test group) died. The death of the animal was not due to the test item treatment. Therefore, the results of weighing from day 84 are presented only for 9 animals for this group, and this was taken into account for statistical calculations made on days 91 and 100 of the experiment.
On day 14, there was a statistically significant difference in the weight of the rats from the 1000 mg/kg bw/day test group compared to the control group: 214.8 ± 7.2 g versus 211.5 ± 6.4 g, respectively. On day 28, in contrast, there was a statistically significant difference in the weight of the rats from the 2000 mg/kg bw/day test group compared to the control group: 232.1± 8.6 g versus 227.1 ± 7.1 g respectively. On days 42 and 56 this difference was retained (245.1 ± 9.5 g in the test group versus 240.1 ± 6.9 g in the control and 255.4 ± 11.4 g versus 250.8 ± 7 69g respectively). By day 70, the differences in weight had levelled out.
A slight increase in animals weight throughout the study was noted.
Analysis of body weight gain data (Table 2), showed no significant difference in the percentages of weight gain between animals of the test groups and the control group. - Food efficiency:
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- The results of haematological analyses performed on days 91 and 100 of the experiment are summarised in Tables 5 and 6.
On the first day after completion of test item administration, there was a statistically significant (t-test, p<0.05) change in blood parameters for the 2000 mg/kg bw/day test group:
- Lower haematocrit concentration than in the control group (42.14 ± 3.69% vs. 44.33 ± 2.75%)
- Concentration of segmented neutrophils was higher than in controls (16.2 ± 6.65% vs. 11.2 ± 5.98%)
- Concentration of eosinophils was higher than in the control group: 3.0 ± 3.17% versus 1.40 ± 1.88%;
- Lymphocyte levels were reduced compared to controls: 80.40 ± 7.27% and 86.8 0± 6.17%, respectively.
In the second test group (1000 mg/kg), significant differences were found only in the leucocyte count: 8.29 ± 2.33% compared to 13.59 ± 3.11% in controls.
Statistical analysis of the results of the haematological examinations on the tenth day after the end of test item administration showed a statistically significant change (t-test, p<0.05) in blood values for the 2000 mg/kg bw/day test group:
- As with the 91 day group, the haematocrit level of the 100 day group was lower than that of the control animals: 42.14 ± 3.69% compared to 44.33 ± 2.75%
- An increase in the level of segmented neutrophils (19.60± 9.06% vs. 11.8 0± 6.65%), which was also observed immediately after the end of the experiment
- Decrease in monocyte concentration: 2.40 ± 1.42 and 4.2 0 ± 1.84% in controls
- Decrease in white blood cell concentration: 74.80 ±11.53% vs. 82.40 ± 8.36%, which is consistent with the results of the primary analysis.
No significant differences were found in the 1000 mg/kg bw/day test group. - Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The results of clinical biochemistry analyses carried out on days 91 and 100 of the experiment are summarised in Tables 7 and 8.
Statistical processing of the results on the first day after the end of test item administartion showed significant differences from animals of the control group for the 2000 mg/kg bw/day test group:
- ALT levels were decreased (80.60 U/l ± 18.22 compared to 102.60 U/l ± 21.67 in controls). However, a decrease in the activity of this enzyme is not clinically significant.
- Lower alkaline phosphatase levels than in the control group (102.80 ± 26.44 U/l versus ± 122.60 ± 20.34 U/l).
In the Clinical biochemistry evaluation performed on the tenth day after completion of test item administration, the following parameters were found to differ significantly from those of the control animals for the 2000 mg/kg bw/day test group:
- statistically significant differences in the total bilirubin level (2.44 ± 0.84 µmol/l compared to 3.08 ± 0.34 µmol/l in the control group) and creatinine level: 77.8 0± 5.15 µmol/l compared to 81.20 ± 4.60 µmol/l in the control group.
In the 1000 mg/kg bw/day test group, the following differences with the control were found:
- Lower alanine aminotransferase level: 55.20 ± 2.69 U/l compared to 61.6 0± 10.07 U/l
- Reduced creatinine level as in the 2000 mg/kg bw/day test group: 76.0 ± 5.82 µmol/l
- Total protein levels were also lower than in controls: 74.0 ± 2.48 g/l versus 77.6 0± 4.53 g/l;
- The number of alkaline phosphatase molecules is lower than that of the control: 64.20 ± 23.48 U/l compared to 83.6 0± 40.14 U/l;
- Glucose levels were higher than in the control group: 5.82 ± 0.76 mmol/l versus 5.18 ± 0.70 mmol/l, while at the same time being higher than in the 2000 mg/kg bw/day test group (4.88 ± 0.75 mmol/l), which suggests that this difference is due to the individual characteristics of the animals.
The Statistically significant differences in the blood chemistry for test groups rats are ambiguous and cannot be of considered of biological significance. - Endocrine findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- no effects observed
- Description (incidence and severity):
- No changes in behaviour were noted. Coordination of movements was not impaired; skeletal muscle tone was normal; reaction to tactile, pain, sound and light stimuli was adequate.
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The day after completion of test item administration, a statistically significant difference in liver mass ratio was observed in animals of the 1000 mg/kg bw/day test group compared to the control group animals (results are shown in Table 3).
10 days after the end of test item administration, there was a statistically significant difference in the kidney heart ratio in the rats given test item at 1000 mg/kg bw/day (0.34 ± 0.03) compared to the control group animals (0.35 ± 0.03) (results are shown in Table 4).
As there is no time- or dose-dependent effect in these differences, they are not considered to be treatment related, but due to the individual characteristics of the animals. - Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- The macroscopic examination of organs and tissues showed no differences between the groups.
No abnormal discharge from natural orifices was detected on external inspection of the rats. The coat was lustrous, without alopecia, and the teeth were preserved.
Visible mucous membranes were pale pink and shiny. No deformity or swelling of the limbs was detected.
The development of the external genitalia corresponded to the physiological norm.
The thoracic and abdominal cavities were free of effusion. The position of the internal organs of the thoracic and abdominal cavities were anatomically correct. The parietal and visceral sheets of the pleura and peritoneum were thin, shiny and smooth.
Macroscopic examination of the liver, lungs, kidneys, heart, stomach, intestines, spleen and skin showed the following:
Liver: No changes in the structure of the organ were found in the test groups animals. The shape and size of the liver were normal. The surface of the liver was smooth, uniformly dark red in colour, the capsule thin and transparent. The liver tissue on the section was full-blooded andmoderately dense.
Lungs: In animals of the test groups, the structure of the organ was normal. The lumen of the trachea and large bronchi was unchanged, the mucous membrane was shiny, smooth, pale in colour. The lungs were airy, without thickening to the touch, pale pink in colour.
Kidneys: There were no changes in animals of all test groups. The size and shape of the kidneys were unchanged. The surface of the kidneys was brownish, smooth, and the capsule was thin, transparent and easily removable. Cortical and medullary substance was clearly distinguishable on the section of the organ.
Heart: In the test groups animals, the structure of the organ was normal. The size and shape of the heart of the control rats did not differ visually from those of the experimental animals. The right and left ventricles contained insignificant amounts of dark liquid blood. The valves of the heart were thin, shiny and smooth. The heart muscle had a uniform cherry-brownish colour and moderately dense consistency on section.
Spleen: In the rats of the test groups, the spleen was of normal shape, dark cherry colour, of moderately dense consistency. The surface of the organ was smooth, capsule was thin. Small grayish follicles could be seen on a section on the dark red background of the spleen.
Stomach: The structure of the stomach was normal in all animals. The thickness of the stomach wall was unchanged.There was no hyperemia. After opening the organ along the small curvature, an accumulation of contents of mushy consistency was found in the pyloric part, greenish-brown in colour.
Intestine: The mesentery and omentum of all test groups animals were not altered, the vessels were not bloody, the mesenteric lymph nodes were not enlarged. The intestinal mucosa was unchanged. The rectum contained unformed brownish-black faeces.
Skin: No changes were detected in the skin surface, or in the subcutaneous fatty tissue of the rats of the test groups.
No differences in the macroscopic structure of the organs of the experimental group rats compared to the control group animals were observed during the autopsy on day 100 of the experiement. - Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- not examined
- Histopathological findings: neoplastic:
- not examined
- Other effects:
- not examined
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 1 000 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- other: changes in haematological parameters at the 2000 mg/kg bw/day dose level.
- Key result
- Critical effects observed:
- no
- Conclusions:
- Under the experimental conditions of the study, with daily administration of Saccharomyces cerevisiae, ext. test item by oral gavage to female white rats at dose levels of 1000 or 2000 mg/kg bw/day, no mortality, clinical signs, or significant changes in body weight gain were observed. No test item-related macroscopic finding was recorded in any of the dose groups at necropsy. Effects observed on organ mass ratios were not time- or dose-dependent and were therfore not considered to be treatment related. The changes in Clinical biochemistry parameters for test groups rats were ambiguous and cannot be of considered of biological significance. Only the observed dose-dependent effects on blood counts were considered test item related. In conclusion, under the conditions of this study, the No Observed Adverse Effect Level (NOAEL) for Saccharomyces cerevisiae, ext. is considered to be 1000 mg/kg bw/day for the female rat based on effects on cell counts.
- Executive summary:
This GLP-compliant Subchronic Repeated Dose Toxicity Study in the Rat was performed as a mandatory test for the registration of a Saccharomyces cerevisiae, ext. substance as a feed additive on the territory of the Russian Federation according to the Order of the Ministry of Agriculture of the Russian Federation N 48.
20 female white outbred rats (strain not specified) were daily treated for 90 days by gavage with Saccharomyces cerevisiae, ext. Doses tested were 1000 and 2000 mg/kg bw/day (10 rats/dose level). 10 animals treated in the same conditions with water served as vehicle control group. During the whole period of exposure, animals were observed for their general condition and behaviour, functional state of the central nervous system [assessed visually by observation of motor activity and response to external stimuli (sound, light)], manifestation of toxicity symptoms, signs of morbidity and mortality. The body weight of animals in test and control groups was monitored the day before first administration of the test item, as well as on days 1, 14, 28, 42, 56, 70, 84 and 90. Prior to the terminal procedures, the animals were deprived of food for 16-18 hours while maintaining access to water. On the day following the end of test item administration (day 91) and the 10th day (day 100), half of the animals from control and 2000 mg/kg bw/day test group (n=5) and 4 animals for 1000 mg/kg bw/day (due to death of one rat from this group not test-item related), were used to take blood samples for blood counts (Haematocrit (%), Haemoglobin (g/l), Erythrocytes (number/L), Leucocytes (number/L), Platelets (number/L), Leukogram) and clinical biochemistry analysis [Bilirubin, Aspartate Aminotransferase (AST), Alanine Aminotransferase (ALT), Urea, Creatinine, Total protein, Alkaline phosphatase, Glucose]. Animals were then autopsied with macroscopic examination of internal organs (liver, lungs, kidneys, heart, spleen, stomach, intestine). Mass ratios of the internal organs of the animals were also determined (liver, kidneys, spleen, lungs and heart).
Female rats of both test groups showed no signs of toxicity during the 90 days of the experiment. A slight increase in animals’ weight throughout the study was noted, but there was no significant difference in the percentage of weight gain for rats of the test groups compared to the control group. On the first day after completion of test item administration, a statistically significant difference in liver mass ratio was observed in animals of the 1000 mg/kg bw/day test group compared to the control group animals. On the tenth day, there was a statistically significant difference in the kidney heart ratio in the rats given test item at 1000 mg/kg bw/day (0.34 ± 0.03) compared to the control group animals. As there is no time- or dose-dependent effect in these differences, they are not considered to be treatment related, but due to the individual characteristics of the animals. No differences in the macroscopic structure of the organs of rats of the test groups compared to their control counterparts were observed during the autopsy on days 91 and 100. The macroscopic examination of all the assessed organs and tissues showed no differences between the test and the control groups (liver, lungs, kidneys, heart, spleen, stomach, intestine and skin). The changes in Clinical biochemistry parameters for test groups rats were ambiguous and cannot be of considered of biological significance. On the first day after completion of test item administration, there was a statistically significant change in the following blood parameters for the 2000 mg/kg bw/day test group: lower haematocrit concentration; higher segmented neutrophils and eosinophils, lower lymphocytes. In the 1000 mg/kg bw/day test group, only a lower leucocyte count was found. On the tenth day after completion of test item administration, there was a statistically significant change in the following blood parameters for the 2000 mg/kg bw/day test group: lower haematocrit concentration; increase in segmented neutrophils, decrease in monocytes and leucocytes. No significant differences were found in the 1000 mg/kg bw/day test group.
Under the experimental conditions of the study, with daily administration of Saccharomyces cerevisiae, ext. test item by oral gavage to female white rats at dose levels of 1000 or 2000 mg/kg bw/day, no mortality, clinical signs, or significant changes in body weight gain were observed. No test item-related macroscopic finding was recorded in any of the dose groups at necropsy. Effects observed on organ mass ratios were not time- or dose-dependent and were therefore not considered to be treatment related. The changes in Clinical biochemistry parameters for test groups rats were ambiguous and cannot be of considered of biological significance. Only the observed dose-dependent effects on blood counts were considered test item related. In conclusion, under the conditions of this study, the No Observed Adverse Effect Level (NOAEL) for Saccharomyces cerevisiae, ext. is considered to be 1000 mg/kg bw/day for the female rat based on effects on cell counts.- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- other: experimental study performed on a SC yeast derivative used in a WoE approach for the target substance
- Adequacy of study:
- weight of evidence
- Study period:
- From August 2020 to November 2020
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- test procedure in accordance with national standard methods with acceptable restrictions
- Qualifier:
- according to guideline
- Guideline:
- other: Russian Guidelines for conducting preclinical studies of medicinal products. Part One / Edited by A.N. Mironov. -M.: Griff & K, 2012. -536 с
- Qualifier:
- according to guideline
- Guideline:
- other: Russian Federal Law No. 61-FZ of 12.04.2010. "On Circulation of Medicines". (ed. from 04.06.2018)
- Qualifier:
- according to guideline
- Guideline:
- other: Russian Order of the Ministry of Agriculture of 6 March 2018 No. 101
- Principles of method if other than guideline:
- The study is a subchronic oral toxicity test (gavage) in outbred rats performed with the aim of introducing the feed additive into clinical veterinary practice.
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- rat
- Strain:
- not specified
- Remarks:
- white outbred rats
- Details on species / strain selection:
- General toxicity studies are preferably performed on rodents, as they are a standard test system for evaluating the general toxic properties of drugs.
- Sex:
- female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Branch "Andreevka" of the Federal State Budgetary Institution "Scientific Centre for Biomedical Technologies" of the Russian Academy of Medical Sciences (Moscow, Russia)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 10-11 weeks
- Weight at study initiation: 173-199 g (body weight of the animals at the start of the experiment was in the range ±20% of the mean)
- Fasting period before study: no
- Housing: rats were kept in polypropylene cages covered with steel lattice covers with feeding cavity, 10 animals in each. The size of the cages was 50×35×20 (L×W×H, cm). Wood shavings for laboratory animals were used as bedding.
- Diet (e.g. ad libitum): fed with full-fat extruded compound feed for laboratory animals (rats, mice, hamsters)
- Water (e.g. ad libitum): ad libitum; water from standard rodent drinkers with prepared drinking water.
- Acclimation period: Immediately after arrival at the research centre, the animals were placed in the quarantine and adaptation room for 7 days. During this period, the animals were inspected daily.
DETAILS OF FOOD AND WATER QUALITY: not specified in the study report
ENVIRONMENTAL CONDITIONS
Temperature and humidity were monitored daily using a combined thermohygrometer
- Temperature (°C): 20-26
- Humidity (%): 30-70
- Air changes (per hr): not specified in the study report
- Photoperiod (hrs dark / hrs light): not specified in the study report
IN-LIFE DATES: From: To: not specified in the study report - Route of administration:
- oral: gavage
- Details on route of administration:
- Oral route was chosen as the test item is a feed additive and the test was conducted in the framework of mandatory studies required for the registration of feed additives on the territory of the Russian Federation according to the Order of the Ministry of Agriculture of the Russian Federation N 48 « About approval of rules of the state registration of medicines for animals and feed additives » dated 01.04.2005.*
Suspension of the test item was administered to rats by gavage to rats using a stainless steel atraumatic probe in a fixed volume of 1.0 ml per 100 g body weight. - Vehicle:
- water
- Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
Before administration, the test item was suspended in water in the ratios required to obtain the doses required for the study (1/5 and 1/10 of the maximum soluble amount of the test item in 100 ml of water).
The suspensions were prepared according to the following scheme:
-->10 g of test item + 91,6 ml water (1000 mg/kg dose is 1/5 of the maximum soluble amount of test item in 100 ml of water);
--> 5 g feed additive + 96 ml water (500 mg/kg dose is 1/10 of the maximum soluble amount of test item in 100 ml water);
The test item was weighed on an laboratory scale with an accuracy of 0.1 g.
Animals in the control group were given water at a rate 1 ml per 100 g body weight.
DIET PREPARATION
- Rate of preparation of diet (frequency): not applicable
- Mixing appropriate amounts with (Type of food): not applicable
- Storage temperature of food: not applicable
VEHICLE
- Justification for use and choice of vehicle (if other than water): vehicle is water
- Concentration in vehicle: suspensions were prepared according to the following scheme:
-->10 g of test item + 91,6 ml water (1000 mg/kg dose is 1/5 of the maximum soluble amount of test item in 100 ml of water);
--> 5 g feed additive + 96 ml water (500 mg/kg dose is 1/10 of the maximum soluble amount of test item in 100 ml water);
- Amount of vehicle (if gavage): 1 ml per 100 g body weight
- Lot/batch no. (if required): not applicable
- Purity: not applicable - Analytical verification of doses or concentrations:
- not specified
- Duration of treatment / exposure:
- 100 days.
- Frequency of treatment:
- Daily
- Dose / conc.:
- 500 mg/kg bw/day (nominal)
- Dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 30 female rats; 10 females/dose group (2 test dose groups and one vehicle control group)
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: not specified in the study report
- Rationale for animal assignment (if not random): random; rats were allocated to groups randomly. As body weight was taken as the criterion, so that the individual weight did not deviate from the average by more than 20%.
- Fasting period before blood sampling for clinical biochemistry: yes; prior to the terminal procedures, rats were deprived of food for 16-18 hours while maintaining access to water, then weighed.
- Rationale for selecting satellite groups: additional specific satellite groups (control and top dose groups) were not used but the following manipulations were performed on days 101 and 110 of the study with half of the rats from each test and control groups (5 animals were used for each period):
--> blood samples were taken for general clinical and biochemical analysis;
--> animals were autopsied with macroscopic examination of internal organs;
--> mass ratios of the internal organs of the animals were determined.
- Post-exposure recovery period in satellite groups: 10 days
- Section schedule rationale (if not random): not specified
- Dose range finding studies: none
- Other: none - Positive control:
- None
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Not specified
- Time schedule: Not specified
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
During the whole experimental period of the study, the animals were observed for their general condition and behaviour, reaction to stimuli (sound, light), manifestation of toxicity symptoms, possible death
BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of animals in experimental and control groups was monitored on the day of the experiment (before introduction of the feed additive), as well as on days 1, 14, 28, 42, 56, 70, 84 and 100 (every 2 weeks).
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): not applicable
FOOD EFFICIENCY: No
- 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
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): not applicable
OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations: not applicable
- Dose groups that were examined: not applicable
HAEMATOLOGY: Yes
- Time schedule for collection of blood: On the day following the end of test item administration (day 101), half of the animals from each group (n=5) were euthanized and blood samples were taken (into tubes with anticoagulant EDTA-K3) to determine hematological parameters. On the 10th day after completion of test item administration, the other half of the animals (n=5) were euthanized and blood samples were taken to assess the degree of reversibility of possible effects after repeated administration of the test item.
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes; animals were deprived of food for 16-18 hours while maintaining access to water, then weighed.
- How many animals: 5/dose/study period
- The following parameters were examined: basic rat peripheral blood parameters were determined on an haematology analyser (Haematocrit (%), Haemoglobin (g/l), Erythrocytes (number/L), Leucocytes (number/L); Leukogram).
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On the day following the end of test item administration (day 101), half of the animals from each group (n=5) were euthanized and blood samples were taken (into tubes with coagulation activator) to determine biochemical parameters. On the 10th day after completion of test item administration, the other half of the animals (n=5) were euthanized and blood samples were taken to assess the degree of reversibility of possible effects after repeated administration of the test item.
- Animals fasted: Yes; animals were deprived of food for 16-18 hours while maintaining access to water, then weighed.
- How many animals: 5/dose/study period
- Parameters examined: Samples after coagulation were centrifuged at 3,000 rpm for 5 minutes to obtain serum. Biochemical blood values were determined on an A25 analyser [Bilirubin, Aspartate Aminotransferase (AST), Alanine Aminotransferase (ALT), Urea, Creatinine, Total protein, Alkaline phosphatase, Glucose].
PLASMA/SERUM HORMONES/LIPIDS: No
- Time of blood sample collection: not applicable
- Animals fasted: not applicable
- How many animals: not applicable
URINALYSIS: No
- Time schedule for collection of urine: not applicable
- Metabolism cages used for collection of urine: not applicable
- Animals fasted: not applicable
- Parameters checked in table [No.?] were examined: not applicable
NEUROBEHAVIOURAL EXAMINATION: Yes; the functional state of the CNS was assessed visually by observation of motor activity and response to external stimuli (reaction to stimuli (sound, light))
- Time schedule for examinations: During the whole experimental period of the study
- Dose groups that were examined: all
- Battery of functions tested: motor activity and response to external stimuli (reaction to stimuli (sound, light))
IMMUNOLOGY: No
- Time schedule for examinations: not applicable
- How many animals: not applicable
- Dose groups that were examined: not applicable
- Parameters checked in table [No.?] were examined: not applicable
OTHER: None. - Sacrifice and pathology:
- SACRIFICE: On the 1st and 10th days after completion of test item administration (101 and 110 days of the experiment), an autopsy of the experimental animals was performed after blood sampling.
GROSS PATHOLOGY: Yes
The following organs and tissues were subjected to macroscopic examination: skin with subcutaneous fatty tissue, liver, lungs, kidneys, heart, spleen, stomach, large and small intestine.
The body weight of the rats was used for subsequent calculation of the mass ratios of the following organs: liver, kidneys, spleen, lungs and heart.
The relative weight of each organ was calculated using the formula:
S = (m / M) ×100 g
where S is the relative mass of the organ,
m - mass of the organ (g)
M is the body weight of the animal (g).
HISTOPATHOLOGY: No - Optional endpoint(s):
- Optional endpoints: No.
- Other examinations:
- None.
- Statistics:
- The experimental data was statistically processed using Microsoft Excel PC software.
Mean values and the width of the confidence interval (P=0.95) were calculated for all data. To determine the validity of intergroup differences, statistical processing was performed in two stages: first, hypothesis testing for equality of variance of the control sample and each of the test samples was performed (Fisher test, 0.05 probability threshold), then hypothesis testing for equality of mean values of the samples was performed (Student's test, Cramer-Welch approximation, 0.05 probability threshold). - Clinical signs:
- no effects observed
- Description (incidence and severity):
- No signs of toxicity were observed in the animals of the test groups. The general condition of the rats remained satisfactory. Appetite and thirst were not altered, seizures were not observed.
Integrity and elasticity of the skin was preserved; there was no hyperemia; the coloration of visible mucous membranes was normal; the frequency and depth of breathing movements were unchanged; the feces were dark brown, of dense consistency, characteristic oval-long form with a specific odor. - Mortality:
- no mortality observed
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Body weight changes results are presented in Table 1.
On day 1, rats weight in the two test groups did not statistically differ from the control group, which shows homogeneity of the formed animal groups: 183.3 ± 6.22 g (1000 mg/kg bw/day test group) and 182.8 ± 4.48 g (500 mg/kg bw/day test group) versus 182.1 ± 4.34 g (vehicle control group).
On day 14, a statistically significant difference in the weight of the rats from the 1000 mg/kg bw/day test group compared to the control group: 217.5 ± 11.4 g versus 209.4 ± 4.96 g, respectively. On day 28, there was also a statistically significant difference in the weight of the rats from the 1000 mg/kg bw/day test group compared to the control group: 234.0 ± 10.52 versus 224.1 ± 6.6 g respectively.
On day 42, there was a statistically significant difference in the weight of the rats from the 500 mg/kg bw/day test group compared to the control group: 239.3 ± 10.14 g versus 247.5 ± 8.71 g, respectively.
On day 56, there was a statistically significant difference in the weight of the rats from 1000 mg/kg bw/day test group compared to the control group: 253.6 ± 13.54 g compared to 248.4 ± 8.86 g respectively.
On day 84, a statistically significant difference in the weight of rats from 500 mg/kg bw/day test group compared to the control group was observed: 269.9 ± 10.03 g versus 264.1 ± 12.79 g, respectively.
Significant differences between the body weight of the control group rats and the animals of the experimental groups were characterized by an increase in the weight of the experimental individuals at the indicated time intervals. This situation was probably due to the individual characteristics of the experimental rats.
Analysis of body weight gain data (Table 2), showed no significant difference in the percentages of weight gain between animals of the test groups and the control group. - Food efficiency:
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The results of haematological analyses performed on days 101 and 110 of the experiment are summarised in Tables 5 and 6.
On the first day after completion of test item administration, there was a statistically significant change in blood values:
- a decrease in white blood cell count in the first (1000 mg/kg bw/day) and second (500 mg/kg bw/day) test groups compared to that of control group rats (15.72 ± 7.67 */109/l, 10.85 ± 2.56 */109/l compared to 20.51 ± 6.12 */109/l);
- a decrease in the number of eosinophils in the first (1000 mg/kg bw/day) and second (500 mg/kg bw/day) test groups compared to the control group (1.0 ± 2.15%, 1.4 ± 0.68% vs. 3.2 ± 2.69%);
- a decrease in the number of monocytes in the first (1000 mg/kg bw/day) and second (500 mg/kg bw/day) test groups compared the control group (4.0 ± 2.63%, 3.6 ± 1.42% vs. 5.6 ± 1.11%);
- increased lymphocytes count in the first (1000 mg/kg bw/day) test group compared to the control group (80.6 ± 4.17% vs. 74.4 ± 9.4%).
Statistical analysis of the results of the haematological examinations on the tenth day after the end of test item administration showed a statistically significant change in blood values:
- increased haematocrit level in the second experimental group compared to the control group (45.16 ± 2.04% vs. 43.75 ± 1.94%);
- increased haemoglobin in the second test group (500 mg/kg bw/day) compared to the control group (151.6 ± 7.98 g/l versus 145.8 ± g/l9.31);
- a decrease in white blood cell count in the first (1000 mg/kg bw/day) and second (500 mg/kg bw/day) test groups compared to the control group (9.54 ± 3.8 */109/l and 11.64 ± 4.28 */109/l compared to 15.9 ± 4.43*/109/l).
- a decrease in the number of eosinophils in the first a(1000 mg/kg bw/day) and second (500 mg/kg bw/day) test groups compared to the control group (0.4 ± 1.11% and 0.6 ± 1.11% compared to 1.4 ± 1.42%).
- a decrease in white blood cell count in the first (1000 mg/kg bw/day) and second (500 mg/kg bw/day) test groups compared to the control group (9.54 ± 3.8 */109/l and 11.64 ± 4.28 */109/l compared to 15.9 ± 4.43*/ 109/l).
- increased number of monocytes in the first (1000 mg/kg bw/day) and second (500 mg/kg bw/day) test groups compared to rats of the control group (1.0 ± 1.76% and 0.8 ± 1.04% compared to 0.2 ± 0.56%).
However, the blood values varied within the physiological range for the species, which had no effect on the general health of the animals.
On the 10th day after completion of test item administration, there were differences between the test and control groups in terms of hematocrit and haemoglobin levels. These indicators were not dose-dependent and were of random nature, hence not considered of toxicological relevance. - Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The results of clinical biochemistry analyses carried out on days 101 and 110 of the experiment are summarised in Tables 7 and 8.
Statistical processing of the results on the first day after the end of test item administartion showed significant differences from animals of the control group:
- decrease in total bilirubin in the second (500 mg/kg bw/day) test group compared to control group (1.96 ± 0.34 µmol/l versus 2.18 ± 0.38 µmol/l, respectively);
- decrease in direct bilirubin concentration in the second (500 mg/kg bw/day) test group compared to the control group (0.36 ± 0.19 μmol/l versus 0.84 ± 1.09 μmol/l);
- lower total protein levels in the both test groups compared to control: 73.8 ± 1.62 g/l and 73.8 ± 1.62 g/l versus 75.8 ± 3.66 g/l;
- increased alkaline phosphatase levels in the second (500 mg/kg bw/day) test group compared to the control group: 133.0 ± 40.54 U/L versus 110.8 ± 33.71 U/L.
Results summarised in Table 7 showed that the significant differences seen in some of the values in the test groups compared to the control were not dose-dependent and were of random nature and therefore not considered to be of toxicological significance.
In the Clinical biochemistry evaluation performed on the tenth day after completion of test item administration, the following parameters were found to differ significantly from those of the control animals:
- reduction in total bilirubin in bothe test groups compared to control: 2.28 ± 0.44 µmol/l and 1.88 ± 0.24 µmol/l versus 2.92 ± 0.42 µmol/l;
- reduction in direct bilirubin in the second (500 mg/kg bw/day) test group compared to the control group: 0.14 ± 0.07 μmol/l versus 0.26 ± 0.07 μmol/l;
- decrease in ALT in the second (500 mg/kg bw/day) test group compared to the control group: 65.0 ± 14.48 U/L versus 75.0 ± 11.88 U/L;
- reduction in urea levels in the first (1000 mg/kg bw/day) and second (500 mg/kg bw/day) test group compared to the control group: 5.8 ± 0.64mmol/l and 6.5 ± 1.09mmol/l versus 7.24 ± 0.47mmol/l;
- a decrease in creatinine levels in the first (1000 mg/kg bw/day) test group compared to control: 66.2 ± 2.69 μmol/l compared to 71.6 ± 5.24 μmol/l;
- a decrease in the Alkaline phosphatase concentration in the second (500 mg/kg bw/day) test group compared to the control: 78.4 ± 24.93 U/l compared to 96.0 ± 32.86 U/l;
- increased glucose levels in the second (500 mg/kg bw/day) test group compared to control: 6.66 ± 0.59 mmol/l compared to 6.06 ± 0.59 mmol/l.
Results summarised in Table 8 shows that the statistically significant differences of some values in the test groups compared to the control were not dose-dependent. The absence of a dose-dependent effect may indicate that these changes depended on the individual characteristics of each experimental animal and had a random character. Hence, it can be argued that they have no biological signifiance.
For other parameters (urea, creatinine, total bilirubin), significant variations observed between test and control groups were scattered and not observed in analyses performed at the first day after the end of test item administration. These did not show dose dependence and were consequently considered not to have a biological significance. - Endocrine findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- no effects observed
- Description (incidence and severity):
- No changes in behaviour were noted. Coordination of movements was not impaired; skeletal muscle tone was normal; reaction to tactile, pain, sound and light stimuli was adequate.
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The day after completion of test item administration, no statistically significant differences in organ mass ratios were observed in animals of the test groups compared to the control group animals (results are shown in Table 3).
10 days after the end of test item administration, there was a statistically significant difference in the values of kidney mass ratios in the rats given test item at 1000 mg/kg bw/day (0.63 ± 0.05) compared to the control group animals (0.55 ± 0.06) (results are shown in Table 4). - Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- The macroscopic examination of organs and tissues showed no differences between the groups.
No abnormal discharge from natural orifices was detected on external inspection of the rats. The coat was lustrous, without alopecia, and the teeth were preserved.
Visible mucous membranes were pale pink and shiny. No deformity or swelling of the limbs was detected.
The development of the external genitalia corresponded to the physiological norm.
The thoracic and abdominal cavities were free of effusion. The position of the internal organs of the thoracic and abdominal cavities were anatomically correct. The parietal and visceral sheets of the pleura and peritoneum were thin, shiny and smooth.
Liver: No changes in the structure of the organ were found in the test groups animals. The shape and size of the liver were normal. The surface of the liver was smooth, uniformly dark red in colour, the capsule thin and transparent. The liver tissue on the section was full-blooded andmoderately dense.
Lungs: In animals of the test groups, the structure of the organ was normal. The lumen of the trachea and large bronchi was unchanged, the mucous membrane was shiny, smooth, pale in colour. The lungs were airy, without thickening to the touch, pale pink in colour.
Kidneys: There were no changes in animals of all test groups. The size and shape of the kidneys were unchanged. The surface of the kidneys was brownish, smooth, and the capsule was thin, transparent and easily removable. Cortical and medullary substance was clearly distinguishable on the section of the organ.
Heart: In the test groups animals, the structure of the organ was normal. The size and shape of the heart of the control rats did not differ visually from those of the experimental animals. The right and left ventricles contained insignificant amounts of dark liquid blood. The valves of the heart were thin, shiny and smooth. The heart muscle had a uniform cherry-brownish colour and moderately dense consistency on section.
Spleen: In the rats of the test groups, the spleen was of normal shape, dark cherry colour, of moderately dense consistency. The surface of the organ was smooth, capsule was thin. Small grayish follicles could be seen on a section on the dark red background of the spleen.
Stomach: The structure of the stomach was normal in all animals. The thickness of the stomach wall was unchanged.There was no hyperemia. After opening the organ along the small curvature, an accumulation of contents of mushy consistency was found in the pyloric part, light brown in colour.
Intestine: The mesentery and omentum of all test groups animals were not altered, the vessels were not bloody, the mesenteric lymph nodes were not enlarged. The intestinal mucosa was unchanged. The rectum contained unformed brownish-black faeces.
Skin: No changes were detected in the skin surface, or in the subcutaneous fatty tissue of the rats of the test groups.
No differences in the macroscopic structure of the organs of the experimental group rats compared to the control group animals were observed during the autopsy on day 110 of the experiement. - Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- not examined
- Histopathological findings: neoplastic:
- not examined
- Other effects:
- not examined
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- >= 1 000 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- other: no signs of general toxicity, no macroscopic changes in assessed organs and no significant changes in haematological and biochemical parameters evaluated.
- Key result
- Critical effects observed:
- no
- Conclusions:
- Under the experimental conditions of the study, with daily administration of Saccharomyces cerevisiae cell wall, extracted test item by oral gavage to white female rats at dose levels of 500 or 1000 mg/kg bw/day, no mortality, clinical signs, or significant changes in body weight gain or organ mass ratios. The changes in haematology, clinical chemistry parameters observed during the study were of no clinical significance, as the values were within the physiological ranges for the species. No test item-related macroscopic finding was recorded in any of the dose groups at necropsy. In conclusion, under the conditions of this study, the No Observed Adverse Effect Level (NOAEL) for Saccharomyces cerevisiae cell wall, extracted is considered to be 1000 mg/kg bw/day for the female rat.
- Executive summary:
This GLP-compliant Subchronic Repeated Dose Toxicity Study in the Rat was performed as a mandatory test for the registration of a Saccharomyces cerevisiae cell wall, extracted substance as a feed additive on the territory of the Russian Federation according to the Order of the Ministry of Agriculture of the Russian Federation N 48.
20 female white outbred rats (strain not specified) were daily treated for 100 days by gavage with Saccharomyces cerevisiae cell wall, extracted. Doses tested were 500 and 1000 mg/kg bw/day (10 rats/dose level). 10 animals treated in the same conditions with water served as vehicle control group. During the whole period of exposure, animals were observed for their general condition and behaviour, functional state of the central nervous system [assessed visually by observation of motor activity and response to external stimuli (sound, light)], manifestation of toxicity symptoms, signs of morbidity and mortality. The body weight of animals in test and control groups was monitored the day before first administration of the test item, as well as on days 1, 14, 28, 42, 56, 70, 84 and 100. Prior to the terminal procedures, the animals were deprived of food for 16-18 hours while maintaining access to water. On the day following the end of test item administration (day 101) and the 10th day (day 110), half of the rats from each test and control group (i.e., 5 animals each) were used to take blood samples for blood counts (Haematocrit (%), Haemoglobin (g/l), Erythrocytes (number/L), Leucocytes (number/L); Leukogram) and clinical biochemistry analysis [Bilirubin, Aspartate Aminotransferase (AST), Alanine Aminotransferase (ALT), Urea, Creatinine, Total protein, Alkaline phosphatase, Glucose]. Animals were then autopsied with macroscopic examination of internal organs (liver, lungs, kidneys, heart, spleen, stomach, intestine). Mass ratios of the internal organs of the animals were also determined (liver, kidneys, spleen, lungs and heart).
Female rats of both test groups showed no signs of toxicity during the 100 days of the experiment. There was no significant difference in the percentage of weight gain for rats of the test groups compared to the control group. On the first day after completion of test item administration, no statistically significant differences in organ mass ratios of the test groups compared with the control were found. On the tenth day, a slight statistically significant difference in the mass ratios of the kidneys of the 1000 mg/kg bw/day group compared with the control group was observed. No differences in the macroscopic structure of the organs of rats of the test groups compared to their control counterparts were observed during the autopsy on days 101 and 110. The changes in haematological and Clinical biochemistry parameters were of no clinical significance, as the values were within the physiological range for the species. The macroscopic examination of all the assessed organs and tissues showed no differences between the test and the control groups (liver, lungs, kidneys, heart, spleen, stomach, intestine and skin).
Under the experimental conditions of the study, with daily administration of Saccharomyces cerevisiae cell wall, extracted test item by oral gavage to white female rats at dose levels of 500 or 1000 mg/kg bw/day, no mortality, clinical signs, or significant changes in body weight gain or organ mass ratios. The changes in haematology, clinical chemistry parameters observed during the study were of no clinical significance, as the values were within the physiological ranges for the species. No test item-related macroscopic finding was recorded in any of the dose groups at necropsy. In conclusion, under the conditions of this study, the No Observed Adverse Effect Level (NOAEL) for Saccharomyces cerevisiae cell wall, extracted is considered to be 1000 mg/kg bw/day for the female rat.- Endpoint:
- short-term repeated dose toxicity: oral
- Type of information:
- other: experimental study performed on a SC yeast derivative used in a WoE approach for the target substance
- Adequacy of study:
- weight of evidence
- Study period:
- 2010
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study without detailed documentation
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
- Version / remarks:
- 1995 (14 day study)
- GLP compliance:
- not specified
- Limit test:
- yes
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Saccharomyces cerevisiae IFO 2346 was incubated in medium containing 2% molasses, 0.6% (NH4)2SO4, 0.1% MgSO4·7H2O,0.2% KH2PO4, 0.03% K2HPO4, and 0.1% NaCl for 3 days at 30°C. After incubation, the culture was centrifuged at 10,000 ×g for 20 min. The cells were suspended in 20 mM phosphate buffer (pH 7.0) and hydrolyzed with 1,000 units of bromelain at 30°C for 4 h. The hydrolysate was subsequently centrifuged at10,000 × g for 20 min. The supernatant was lyophilized to obtain the yeast hydrolysate (Notress).
- Purity, including information on contaminants, isomers, etc.: not applicable; UVCB
RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: not applicable
- Specific activity: not applicable
- Locations of the label: not applicable
- Expiration date of radiochemical substance: not applicable
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: not specified in the publication
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: not specified in the publication
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: not specified in the publication
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: not specified in the publication
- Reactivity of the test material with the incubation material used (e.g. plastic ware): not specified in the publication
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): none
- Preliminary purification step (if any): none
- Final concentration of a dissolved solid, stock liquid or gel: none
- Final preparation of a solid (e.g. stock crystals ground to fine powder using a mortar and pestle): none
FORM AS APPLIED IN THE TEST (if different from that of starting material)
- Specify the relevant form characteristics if different from those in the starting material, such as state of aggregation, shape of particles or particle size distribution: not applicable
INFORMATION ON NANOMATERIALS
- Chemical Composition: not applicable
- Density: not applicable
- Particle size & distribution: not applicable
- Specific surface area: not applicable
- Isoelectric point: not applicable
- Dissolution (rate): not applicable
TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)
- Description of the formulation, e.g. formulated product for foliar application; formulated product soil application; solution in organic solvent for soil application; formulated product seed treatment; solution in organic solvent seed treatment: not applicable
OTHER SPECIFICS
- Other relevant information needed for characterising the tested material, e.g. if radiolabelled, adjustment of pH, osmolality and precipitate in the culture medium to which the test chemical is added: not applicable - Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Nara biotech (Seoul,Korea)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 weeks of age
- Weight at study initiation: not specified in the publication
- Fasting period before study: not specified in the publication
- Housing: individually housed in plastic cages with grated stainless-steel floors.
- Diet (e.g. ad libitum): ad libitum; commercial diet (Samyang Co., Seoul, Korea) containing the following (g/kg of diet): moisture, 80; protein, 230; fat, 35; fiber,50; carbohydrate, 600; and water.
- Water (e.g. ad libitum): ad libitum
- Acclimation period: not specified in the publication
DETAILS OF FOOD AND WATER QUALITY: not specified in the publication
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24± 1
- Humidity (%): 60%
- Air changes (per hr): not specified in the publication
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: To: not specified in the publication - Route of administration:
- oral: gavage
- Details on route of administration:
- Not specified in the publication
- Vehicle:
- water
- Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
- not specified in the publication
DIET PREPARATION
- not applicable:
VEHICLE
- Justification for use and choice of vehicle (if other than water): water
- Concentration in vehicle: not specified in the publication
- Amount of vehicle (if gavage): not specified in the publication
- Lot/batch no. (if required): not applicable
- Purity: not applicable - Analytical verification of doses or concentrations:
- not specified
- Duration of treatment / exposure:
- 14 days. The yeast hydrolysate was administered to treated group (1st group) at the dose of 1000 mg/kg body weight for 14 days, whereas an equal volume of water vehicle was given to control group (2nd group). In order to access reversibility and delayed occurrence of toxic effects, satellite group was given the yeast hydrolysate (3rd group) at the dose of 1000 mg/kg body weight or an equal volume of water vehicle (4th group) for 14 days and kept for other 14 days after treatment
- Frequency of treatment:
- Daily
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 5/sex/dose group
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: based on the results of the oral acute toxicity study showing no effect at 5000 mg/kg bw
- Rationale for animal assignment (if not random): random
- Fasting period before blood sampling for clinical biochemistry: yes; for 16–18 h.
- Rationale for selecting satellite groups: not specified in the publication
- Post-exposure recovery period in satellite groups: 14 days
- Section schedule rationale (if not random): random
- Dose range finding studies: not performed
- Other: none - Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Not specified
- Time schedule: not specified
- Cage side observations checked in table [No.?] were included: not specified
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: During the period of administration, the animals were observed daily to detect signs of toxicity. Daily visual observations were made and recorded systematically and included changes in the skin and fur, eyes and mucous membranes, and also respiratory, circulatory, autonomic and central nervous system as well as somatomotor
activity and behavioral pattern.
BODY WEIGHT: Yes / No / Not specified
- Time schedule for examinations:
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 / No / Not specified
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / Not specified
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: Yes / No / Not specified
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / Not specified
- Time schedule for examinations:
OPHTHALMOSCOPIC EXAMINATION: Yes / No / Not specified
- Time schedule for examinations:
- Dose groups that were examined:
HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the study. Blood samples were collected from a common carotid into heparinized and dry non-heparinized centrifuge tubes. The heparinized blood was used for hematological study. Hematological analysis was performed using an automatic hematological analyzer KN-21N (SysmexCo., Kobe, Seoul, Korea).
- Anaesthetic used for blood collection: Yes (ethyl ether)
- Animals fasted: Yes; for 16–18 h
- How many animals: all rats
- Parameters examined: red blood cell (RBC) count, white blood cell (WBC) count, hematocrit (Hct), hemoglobin (Hgb), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC).
CLINICAL CHEMISTRY: Yes. Blood samples were collected from a common carotid into heparinized and dry non-heparinized centrifuge tubes. The serum separated from the non-heparinized blood was assayed for biochemical analysis.Blood was centrifuged at 1,500 × g for 10 min to obtain serum.
- Time schedule for collection of blood: at the end of the study.
- Animals fasted: Yes; for 16–18 h
- How many animals: all rats
- Parameters examined: glucose, blood urea nitrogen (BUN), creatinine, total protein, albumin, total bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT).
PLASMA/SERUM HORMONES/LIPIDS: No
- Time of blood sample collection: not applicable
- Animals fasted: not applicable
- How many animals: not applicable
URINALYSIS: No
- Time schedule for collection of urine: not applicable
- Metabolism cages used for collection of urine: not applicable
- Animals fasted: not applicable
- Parameters checked in table [No.?] were examined: not applicable
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During the period of administration, the animals were observed daily for somatomotor activity and behavioral pattern.
- Dose groups that were examined: all animals
- Battery of functions tested: somatomotor activity and behavioral pattern
IMMUNOLOGY: No
- Time schedule for examinations: not applicable
- How many animals: not applicable
- Dose groups that were examined: not applicable
- Parameters checked in table [No.?] were examined: not applicable
OTHER: none - Sacrifice and pathology:
- SACRIFICE
- At the end of the period, all rats were fasted for 16–18 h, then anesthetized with ethyl ether and sacrificed, then anesthetized with ethyl ether and sacrificed.
GROSS PATHOLOGY: Yes
- After blood collection rats were sacrificed for tissue studies. Rats were perfused with saline solution followed by 10% buffered formalin solution for 10 min and the organs such as liver, kidney, spleen, lung, heart and sex organs were removed, blotted free of blood and weighed immediately on an electronic balance for subsequent analysis. Organ weights were expressed in relative terms (g/100 g of body weight).
HISTOPATHOLOGY: Yes
- After fixation in 10% phosphate buffered formalin, liver and kidney were processed in routine manner, embedded in paraffin, and sectioned. Then to perform light microscopic evaluation, the liver was stained with hematoxylin and eosin (H & E), and kidney was stained with periodic acid schiff (PAS). - Statistics:
- All statistical analyses were performed using the Statistical Package for Social Sciences (SPSS) version 12.0 (SPSS Inc.,IL, USA). The differences between control and treated groups were evaluated by Student’s t-test. The p values less than 0.05were considered significant. All data were reported as means ±standard deviations (SD).
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- No toxicity signs, such as piloerection, alterations in locomotor activity or diarrhea, were recorded during the 14 consecutive days of yeast hydrolysate treatment via the oral route at doses of 1000 mg/kg.
- Mortality:
- no mortality observed
- Description (incidence):
- No animal died during the study.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- The yeast hydrolysate containing the 30-10 kDa molecular weight peptides (YGF) induced significant increases in body weight in relation to the control group in the male rats (p<0.05). This increase in body weight, which was higher than that of the control group, may be due to appetite stimulation by the yeast hydrolysate, leading to increased food consumption and observed body weight (results are presented in Table 1).
For the female rats, no statistically significant differences were recorded for body weight gain, although the body weight gain of the female rats treated with yeast hydrolysate was slightly lower compared to that of the control (results are presented in Table 1). - Description (incidence and severity):
- No significant differences in daily food intake between the control and treated rats was observed (results are presented in Table 1).
- Food efficiency:
- not specified
- Water consumption and compound intake (if drinking water study):
- no effects observed
- Description (incidence and severity):
- No significant differences in daily water intake between the control and treated rats was observed (results are presented in Table 1).
- Ophthalmological findings:
- not specified
- Haematological findings:
- no effects observed
- Description (incidence and severity):
- The hematological profiles of the treated and control groups are presented in Table 2. No statistically significant differences were recorded in any of the hematological parameters analyzed.
- Clinical biochemistry findings:
- no effects observed
- Description (incidence and severity):
- The biochemical profiles of the treated and control groups are presented in Table 3.
The BUN test is a measurement of the amount of nitrogen in the blood in the form of urea, and BUN values are affected by dietary protein (Sarwar et al., 1999). The BUN values of the control rats were slightly lower than the normal range of BUN values for SD rats, and the BUN values of the rats treated with yeast hydrolysate, which is a rich source of protein and amino acids, were increased to normal range. - Endocrine findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- no effects observed
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Description (incidence and severity):
- Generally, the reduction of internal organ weight is a simple and sensitive index of toxicity after exposure to a toxic substance(Teo et al., 2003). The relative internal organ weights of the rats were not altered by the yeast hydrolysate (results are presented in Table 4).
- Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- The macroscopic analysis of the treated animals did not show significant changes in color and texture when compared to the control group in both the male and female rats.
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- no effects observed
- Description (incidence and severity):
- The microscopical findings did not suggest histological alterations in the liver and kidneys. Gross examination of the internal organs of all rats revealed no detectable abnormalities. The yeast hydrolysate did not induce any damage to the internal organs as examined by blood parameters.
- Histopathological findings: neoplastic:
- not examined
- 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:
- Remarks on result:
- other: none
- Key result
- Critical effects observed:
- no
- Conclusions:
- In conclusion, the results of this study showed that the yeast hydrolysate from Saccharomyces cerevisiae did not induce toxicity upon a subacute repeated exposure to the limit dose of 1000 mg/kg bw/day in male and female rats. The subacute oral NOAEL was therefore considered to be ≥ 1000 mg/kg bw/day.
- Executive summary:
The yeast hydrolysate from Saccharomyces cerevisiae was evaluated for subacute toxicity on female and male Sprague-Dawley (SD) rats. The hydrolysate was administered orally at a dose of 1000 mg/kg/day for a period of 14 days. The satellite group was treated with the hydrolysate at the same dose and the same period and kept for another 14 days after treatment. There were no significant differences in organ weights between control and treated group of both sexes. Hematological analysis and blood chemistry revealed no toxicity effects of S. cerevisiae hydrolysate. Pathologically, neither gross abnormalities nor histopathological changes were observed.
In conclusion, the results of this study showed that the yeast hydrolysate from Saccharomyces cerevisiae did not induce toxicity upon a subacute repeated exposure to the limit dose of 1000 mg/kg bw/day in male and female rats. The subacute oral NOAEL was therefore considered to be ≥ 1000 mg/kg bw/day.- Endpoint:
- short-term repeated dose toxicity: oral
- Remarks:
- 7-days (dose range finding stury)
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 10 November 2017 to 09 November 2020
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Qualifier:
- no guideline available
- Guideline:
- other: Dose range finding prior to an OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
- Principles of method if other than guideline:
- - Principle of test:
Rats (Wistar, 4 males and females) were orally dosed (gavage) with the test substance (500, 1000 and 2000 mg/kg bw/d) during 7 consecutive days.
- Short description of test conditions:
Animals were housed in cages (2 animals of the same sex) and daily dosed.
- Parameters analysed / observed:
Animals were subjected to mortality checking, clinical observation, body weight and fod consumption recording. Blood samples were taken before sacrifice. Gross necropsy was performed at termination in each animal, and selected tissues/ogans were weighed. - GLP compliance:
- no
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Batch number of test material: AC17F00560
- Expiration date of the lbatch: 28 February 2019 - 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
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 16 weeks
- Weight at study initiation: Males (482g-541g), Females (290g-309g)
- Fasting period before study: No
- Housing: In Type II and/or III polycarbonate cages (2 animals of the same sex per cage), with LIGNOCEL® ¾ S certified wooden chips bedding and ARBOCEL® nest building material for nesting.
- Diet: ssniff® SM R/M "Autoclavable complete diet for rats and mice, ad libitum.
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: 60 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.4 – 22.1 °C
- Humidity (%): 28-69%
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod: 12 hours/12 hours
IN-LIFE DATES: From: 13 November 2017 To: 20 November 2017. - Route of administration:
- oral: gavage
- Details on route of administration:
- Animals were treated in each group daily for 7 consecutive days
- Vehicle:
- water
- Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared daily (fresh prior to administration to animals) at appropriate concentrations . 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 for 30 minutes by a magnetic stirrer to make a homogenous formulation and was kept mixed until the end of treatment. The formulation preparation was coordinated in such a way that the difference between the end of formulation and start of treatment was as minimal as practicable on each day.
VEHICLE
- Amount of vehicle (if gavage): 10 mL/kg (constant dose volume)
- Lot/batch no. (if required): 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 98.4% and 100.8% of their nominal concentrations (within the +/- 10% limit). No test item was detected in the control samples. 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:
- 7 consecutive days
- Frequency of treatment:
- Once daily
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Dose / conc.:
- 500 mg/kg bw/day (nominal)
- Dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- Dose / conc.:
- 2 000 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 4 animals/sex/dose
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale:
Based on available information (LD50>2000 mg/kg bw for male and female rats in an OECD 423 study.
- Rationale for animal assignment (if not random): Random
- Fasting period before blood sampling for clinical biochemistry: Overnight
- Rationale for selecting satellite groups: Not applicable
- Post-exposure recovery period in satellite groups: Not applicable - 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: Twice daily (before/after treatment)
BODY WEIGHT: Yes
- Time schedule for examinations: at the start (Day 0), then on Days 3, 6 and 7 (fasted, prior to necropsy).
FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: at the start (Day 0) and then on Days 3, and 6.
FOOD EFFICIENCY: No
WATER CONSUMPTION: No
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: Yes
- Time schedule for collection of blood: At study termination (day 7)
- Anaesthetic used for blood collection: Yes (pentobarbital)
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table [No.1] were examined.
CLINICAL CHEMISTRY: Yes / No / Not specified
- Time schedule for collection of blood: At study termination (day 7)
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table [No.2] were examined. - Sacrifice and pathology:
- GROSS PATHOLOGY and ORGAN WEIGHT: Yes (see table 3)
HISTOPATHOLOGY: No - Statistics:
- The normality and heterogeneity of variance between groups was checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests showed no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test was carried out. If the obtained result was positive, Dunnett’s (Multiple Range) test was used to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01 as appropriate. This parametric analysis was the better option when the normality and heterogeneity assumptions implicit in the tests were adequate.
If either of the Shapiro-Wilk or Levene tests showed significance on the data, then the ANOVA type approach was not valid, and a non-parametric analysis was required. 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; identifying differences of <0.05 or <0.01 as appropriate. For non-continuous data, the Cochran-Armitage test for trend was applied and the Chi-squared test was used for statistical differences relative to control. - Clinical signs:
- no effects observed
- Description (incidence and severity):
- No clinical signs were observed during the study.
- Mortality:
- no mortality observed
- Description (incidence):
- There was no mortality during the study.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- No test item effect was seen in the body weight and body weight gain values of male and female animals up to the high dose (2000 mg/kg bw/d) (Table 4).
- Food consumption and compound intake (if feeding study):
- effects observed, non-treatment-related
- Description (incidence and severity):
- No test item related adverse effect was observed on food consumption (Table 5).
In case of females, lower mean food consumption values were recorded compared to control animals, the difference was statistically significant (p<0.01) in all test item treated groups. However, the observed values were within the historical control range and the small sample size might affect the statistical analysis. Thus, no test item related adverse effect was concluded on the food consumption in females. - Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No test item-related effects were seen on the examined haematology parameters (Table 6).
When compared to the control, there were no differences in males and females that could be considered toxicologically significant in the test item treated groups.
Platelet count of Mid dose females was significantly lower (by approx. -13%, p<0.05) than control. A similar trend was also seen in High dose females, but there the difference (although it was higher, approx. 29%) was statistically not significant. As all the observed values were within the historical control range and close to the control level of male animals, these values were considered as being animal variability, not a test item related effect.
There were no other statistically significant values recorded in any test item treated groups compared to the control. - Clinical biochemistry findings:
- no effects observed
- Description (incidence and severity):
- There were no effects on clinical chemistry parameters that could be ascribe to the test item administration. There were no statistically significant values recorded in any of the dose groups compared to the control.
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were no treatment-related statistically significant differences among groups in the weights of other organs measured when compared to controls (Table 7).
The brain-related liver weight was significantly increased (by 10%, at p<0.05) in females of the High dose group (2000 mg/kg bw/day) when compared to control data (as summarized in Table 6). The observed values are within the historical control range, and no similar trend was seen in males. These changes were considered not to be an effect of the treatment with the test item although no histopathology was performed to confirm the lack of adversity.
There were no other statistically significant differences among groups in the weights of organs measured when compared to controls. - Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- There was no evidence of the macroscopic changes related to the administration of the test item up to dose level of 2000 mg/kg bw.
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- not examined
- Histopathological findings: neoplastic:
- not examined
- Other effects:
- not examined
- Key result
- Dose descriptor:
- NOEL
- Effect level:
- 2 000 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Conclusions:
- In conclusion, based on this short 7-day Dose Range Finding (DRF) study, the1000 mg/kg bw/day dose level was considered to be a suitable High dose level for the OECD No. 422 study.
- Executive summary:
In a range finding study, Wistar rats (4 males/females per group) were given Saccharomyces cerevisiae, lysate, by oral gavage at three dose levels (500, 1000 and 2000 ùg/kg bw/d) for 7 days in order to determine the dose levels for a subsequent OECD No. 422 study.
Mortality checking and clinical observations were performed twice daily. Body weight and food consumption were measured for all animals on Days 0, 3 and 6, terminal body weight (fasted) was also recorded at scheduled necropsy on Day 7. Following the daily repeated dose administration for 7 days, blood samples were collected for clinical pathology at necropsy from the surviving animals. Gross necropsy was performed at termination in each animal. Selected tissues / organs were weighed and preserved in fixative.
Results:
All the dose formulations were homogenous.
There was no mortality during the study.
No clinical signs were recorded for any animals of the study.
No test item effect was observed on body weight and body weight gain.
There was no test item related adverse effect on food consumption.
No test item-related effects were seen on the examined haematology and clinical chemistry parameters.
There were no treatment-related statistically significant differences among groups in the weights of other organs measured when compared to controls.
No test item related macroscopic findings were recorded for any animals of the study during necropsy.
In conclusion, based on this short 7-day Dose Range Finding (DRF) study, the 1000 mg/kg bw/day dose level was considered to be a suitable High dose level for the OECD No. 422 study.
In a range finding study, Wistar rats (4 males/females per group) were given Saccharomyces cerevisiae, lysate, by oral gavage at three dose levels (500, 1000 and 2000 mg/kg bw/d) for 7 days in order to determine the dose levels for a subsequent OECD No. 422 study.
Mortality checking and clinical observations were performed twice daily. Body weight and food consumption were measured for all animals on Days 0, 3 and 6, terminal body weight (fasted) was also recorded at scheduled necropsy on Day 7. Following the daily repeated dose administration for 7 days, blood samples were collected for clinical pathology at necropsy from the surviving animals. Gross necropsy was performed at termination in each animal. Selected tissues / organs were weighed and preserved in fixative.
Results:
All the dose formulations were homogenous.
There was no mortality during the study.
No clinical signs were recorded for any animals of the study.
No test item effect was observed on body weight and body weight gain.
There was no test item related adverse effect on food consumption.
No test item-related effects were seen on the examined haematology and clinical chemistry parameters.
There were no treatment-related statistically significant differences among groups in the weights of other organs measured when compared to controls.
No test item related macroscopic findings were recorded for any animals of the study during necropsy.
In conclusion, based on this short 7-day Dose Range Finding (DRF) study, the 1000 mg/kg bw/day dose level was considered to be a suitable High dose level for the OECD No. 422 study.
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- other: experimental study performed on a SC yeast used in a WoE approach for the target substance
- Adequacy of study:
- weight of evidence
- Study period:
- 2014
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Principles of method if other than guideline:
- The objective of this 60-day subchronic oral (gavage) toxicity study in rats was to acquire data on the safety-in-use of the probiotic S. cerevisiae RC016. The bone marrow micronuclei assay and the comet assay were used to evaluate genotoxicity and cytotoxicity of dietary yeast administered to Wistar rats. Also, internal organs were macroscopically and microscopically examined to assess potential impairment in S. cerevisiae RC016- treated and control rats.
- GLP compliance:
- not specified
- Limit test:
- yes
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Saccharomyces cerevisiae RC016 was isolated from pig intestine in a previous study (Armando et al. 2011). The strain is currently deposited in the culture collection of the Universidad Nacional de Rio Cuarto collection centre, located in Rio Cuarto, Cordoba, Argentina.
- Purity, including information on contaminants, isomers, etc.: identified by molecular techniques through DNA extraction and 18S rRNA and 28S rRNA amplification and analysis, comparing sequences with the basic local alignment search tool (BLAST) within the NCBI database as described by Armando et al. (2011).
RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: not applicable
- Specific activity: not applicable
- Locations of the label: not applicable
- Expiration date of radiochemical substance: not applicable
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: live material
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: not applicable
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: not applicable
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: not applicable
- Reactivity of the test material with the incubation material used (e.g. plastic ware): not applicable
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): no
- Preliminary purification step (if any): no
- Final concentration of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid (e.g. stock crystals ground to fine powder using a mortar and pestle): not applicable
FORM AS APPLIED IN THE TEST (if different from that of starting material)
SC cells were resuspended in sterile phosphate buffer saline (PBS) solution and the concentration was adjusted to 109 cells/ml. Concentration of cell suspensions was determined using an haemocytometer. Cell viability was confirmed by standard plate count method using YPD agar.
INFORMATION ON NANOMATERIALS
- Chemical Composition: not applicable
- Density: not applicable
- Particle size & distribution: not applicable
- Specific surface area: not applicable
- Isoelectric point: not applicable
- Dissolution (rate): not applicable
TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)
- Description of the formulation, e.g. formulated product for foliar application; formulated product soil application; solution in organic solvent for soil application; formulated product seed treatment; solution in organic solvent seed treatment: not applicable
OTHER SPECIFICS
- Other relevant information needed for characterising the tested material, e.g. if radiolabelled, adjustment of pH, osmolality and precipitate in the culture medium to which the test chemical is added: none - Species:
- rat
- Strain:
- Wistar
- Details on species / strain selection:
- Inbred
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: not specified in the publication
- Females (if applicable) nulliparous and non-pregnant: not applicable (only males tested)
- Age at study initiation: 8 weeks old
- Weight at study initiation: 173 ± 15 g
- Fasting period before study: no
- Housing: housed in stainless steel metabolic cages (three animals per cage). Animals were housed in the animal facility centre of the National University of Rio Cuarto in accordance with international sanitary and ethical guidelines.
- Diet (e.g. ad libitum): Animals were fed 90 g feed per cage per day (estimating a daily intake of 30 g of per animal, as recommended by the feed manufacturer and the animal house’s personnel).
- Water (e.g. ad libitum): ad libitum
- Acclimation period: not specified in the publication
DETAILS OF FOOD AND WATER QUALITY:
- not specified in the publication for water
- A commercial basal mice/rat diet (GEPSA Feeds; Grupo Pilar S.A., Buenos Aires, Argentina) was used. Centesimal composition of this feed was >24%protein, <7% fibre, 1–1.2% calcium, >6% ether extract,0.5–0.9% phosphorus, <8% total minerals and <13%moisture. The commercial feed was analyzed for Afs (aflatoxins) content by HPLC (Trucksess et al. 1994). Extraction and clean-up procedure was performed using Mycosep® Afla-Pat 228 columns (Romer Labs, Tulln, Austria), following methodology provided by the manufacturer. The basal feed did not show detectable levels of AFs.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): not specified in the publication (animals kept in an environmentally controlled room)
- Humidity (%): not specified in the publication (animals kept in an environmentally controlled room)
- Air changes (per hr): not specified in the publication (animals kept in an environmentally controlled room)
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: To: not specified in the publication - Route of administration:
- oral: gavage
- Details on route of administration:
- Disposable syringes without needle were used. Oral doses of 0.2 ml of a S. cerevisiae suspension (10E9 CFU/ml) were administered daily to test group (yeast) and 0.2 ml of saline solution (0.9% NaCl) was administered to control group (uncontaminated feed control).
- Vehicle:
- other: saline solution (0.9% NaCl)
- Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
- The inoculum of S. cerevisiae RC016 was prepared daily from a 30°C 24 h culture in yeast-peptone-dextrose (YPD) broth and harvested by centrifugation (20 min, 700 g). The cells were resuspended in sterile phosphate buffer saline (PBS) solution and the concentration was adjusted to 10E9 cells/ml. Concentration of cell suspensions was determined using an haemocytometer. Cell viability was confirmed by standard plate count method using YPD agar. Animals were administered a daily dose of 10E8 viable cells or colony-forming units (CFU), as 0.2 ml of the 10E9 CFU/ml S. cerevisiae RC016 suspension in PBS was the maximum volume the animals would take orally. The concentration of the S. cerevisiae suspension was adjusted to the volume that was more easily incorporated by the rats (0.2 ml).
DIET PREPARATION
- A commercial basal mice/rat diet (GEPSA Feeds; Grupo Pilar S.A., Buenos Aires, Argentina) was used as basal feed for formulating control diet. The control diet was prepared by mixing 2-52 kg of finely ground commercial basal feed with 60 g agar dissolved in 2.5 l water. The mixture was homogenized manually for 20 min in a big plastic container, and 30 g pieces were moulded manually. After solidification, feed was stored at -20°C until use.
- Analytical verification of doses or concentrations:
- no
- Remarks:
- The inoculum of S. cerevisiae RC016 was prepared daily and the concentration was adjusted to 109 cells/ml.
- Duration of treatment / exposure:
- 60 days
- Frequency of treatment:
- Oral doses of 0.2 ml of a S. cerevisiae suspension (109 CFU/ml) were administered daily (every day at the same time, 10:00 AM).
- Dose / conc.:
- 0 other: CFU/day
- Dose / conc.:
- 1 000 000 000 other: CFU/day
- No. of animals per sex per dose:
- 6 male rats/dose group
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: not specified in the publication
- Rationale for animal assignment (if not random): not specified in the publication
- Fasting period before blood sampling for clinical biochemistry: not specified in the publication
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random):not applicable
- Dose range finding studies: not performed
- Other: none - Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Not specified
- Time schedule: Not specified
- Cage side observations checked in table [No.?] were included: Not specified
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The general health status of rats was evaluated by recording any changes in behaviour, activity, posture, fur quality, feed and water intake, possible illness and deaths.
BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed three times a week. Total weight gain (TWG) was calculated as the difference between the weight of animals in the beginning and at the end of the experiment. Progressive weight gain (PWG) was calculated considering the PWG of animals (weighed three times a week) during the experiment. Each of these growth parameters was measured individually (per animal), per cage and per experimental group and statistically analyzed.
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: Feed intake was recorded.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Estimated daily feed intake (DI) was 30 g per animal. Ninety grams was administered in each cage per day.
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: Feed efficiency (FE) was calculated as TWG/DI and feed conversion (FC) as DI/TWG. Each of these growth parameters was measured individually (per animal), per cage and per experimental group and statistically analyzed.
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: water intake, was evaluated.
OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations: not applicable
- Dose groups that were examined: not applicable
HAEMATOLOGY:
- Time schedule for collection of blood: Blood samples were also collected and reserved to further analyses.
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Not specified
- How many animals: Not specified
- Parameters checked in table [No.?] were examined: Not specified
CLINICAL CHEMISTRY:
- Time schedule for collection of blood: Blood samples were also collected and reserved to further analyses.
- Animals fasted: Not specified
- How many animals: Not specified
- Parameters checked in table [No.?] were examined: Not specified
PLASMA/SERUM HORMONES/LIPIDS: No
- Time of blood sample collection: not applicable
- Animals fasted: not applicable
- How many animals: not applicable
URINALYSIS: No
- Time schedule for collection of urine: not applicable
- Metabolism cages used for collection of urine: not applicable
- Animals fasted: not applicable
- Parameters checked in table [No.?] were examined: not applicable
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The general health status of rats was evaluated by recording any changes in behaviour.
- Dose groups that were examined: not specified
- Battery of functions tested: not specified
IMMUNOLOGY: No
- Time schedule for examinations: not applicable
- How many animals: not applicable
- Dose groups that were examined: not applicable
- Parameters checked in table [No.?] were examined: not applicable
OTHER:
- Faeces, liver, lungs, intestines and testicle samples were also collected and reserved to further analyses.
- Bone marrow samples were collected for the micronuclei assay. - Sacrifice and pathology:
- SACRIFICE
- After 60 days, animals were sacrificed by decapitation.
GROSS PATHOLOGY: Yes
- Macroscopic characteristics (weight, size and colour) of the organs collected during necropsy (liver, lungs, kidney and intestine) were registered.
HISTOPATHOLOGY: Yes
- Tissue slices were fixed in 4% neutral buffered formalin (pH 7.4) for paraffin routine processing. These samples were cut at 4 µm thickness and stained with haematoxylin/eosin (H&E) stain and Masson’s Trichrome stain to demonstrate the presence of connective tissue. Stained sections were examined by light microscopy. Photomicrographs were taken with a Zeiss Axiostar plus microscope using an Electronic Eye piece camera and a Canon Power Shot G5 camera (Canon Inc.). - Statistics:
- For weight parameters, data were analyzed by the GLMM using INFOSTAT (ver. 2012) software, considering treatments as fixed effects and each animal, cage and weight registration as random effects. First order continuous autoregressive correlations were considered, and the model was corrected to obtain the homogeneity of the variance. The variation between means of each treatment was analyzed by the LSD test (P ≤ 0.05). The general health status of rats was evaluated by recording any changes in behaviour, activity, posture, fur quality, feed and water intake, possible illness and mortality.
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- Rats from both groups appeared healthy, inquisitive and active throughout the experiment. The oral dose of S. cerevisiae RC016, did not cause illness.
- Mortality:
- no mortality observed
- Description (incidence):
- The oral dose of S. cerevisiae RC016, did not cause mortality.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- Results showed that the administration of the oral dose of S. cerevisiae RC016 did not cause any adverse effects on weight gain (TWG and PWG) during the experiment (results are presented in Table 1).
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- No significant difference in feed intake was observed.
- Food efficiency:
- no effects observed
- Description (incidence and severity):
- Results showed that the administration of the oral dose of S. cerevisiae RC016 did not cause any adverse effects on FC or FE during the experiment (results are presented in Table 1).
- Water consumption and compound intake (if drinking water study):
- no effects observed
- Description (incidence and severity):
- No significant difference in water intake was observed.
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Endocrine findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- no effects observed
- Description (incidence and severity):
- The oral dose of S. cerevisiae RC016, did not cause significant changes in the general behaviour of animals.
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Description (incidence and severity):
- Gross examination of the internal organs of all rats revealed no detectable abnormalities, indicating that the probiotic yeast did not induce any organ damage.
- Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- Gross examination of the internal organs of all rats revealed no detectable abnormalities, indicating that the probiotic yeast did not induce any organ damage. Macroscopic analyses of the different organs of S. cerevisiae RC016-treated rats did not show significant changes in colour and texture when compared with the control group.
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- no effects observed
- Description (incidence and severity):
- Histological examinations of liver, lungs, kidneys and intestine of the S. cerevisiae RC016-treated and control rats revealed that there were no differences between S. cerevisiae RC016-treated and control rats.
- Histopathological findings: neoplastic:
- not examined
- Other effects:
- no effects observed
- Description (incidence and severity):
- The effect of S. cerevisiae on genotoxicity in rat erythrocytes and lymphocytes was evaluated through the bone marrow rodent micronuclei assay and the comet assayThe tested S. cerevisiae strain did not cause genotoxicity or cytotoxicity in vivo.
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- >= 1 000 000 000 other: CFU/day
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- other:
- Remarks on result:
- other: none
- Key result
- Critical effects observed:
- no
- Conclusions:
- In conclusion, under the conditions of this study, oral daily administration of the probiotic Saccharomyces cerevisiae RC016 in male rats for 60 days did not result in any illness, mortality or any other health changes related to the intake of this strain. Also, the application of a daily oral dose of S. cerevisiae RC016 did not cause any negative impact on the animals’ weight gain, FC or FE. The examination of internal organs of S. cerevisiae RC016-treated and control rats revealed no macroscopic or microscopic differences or lesions associated to the use of the probiotic. This suggests the strain is safe to use in vivo.
- Executive summary:
The objective of this 60-day subchronic oral (gavage) toxicity study in male rats was to acquire data on the safety-in-use of the probiotic Saccharomyces cerevisiae RC016.
Male rats (n=6) were administered daily oral doses of 0.2 ml of a S. cerevisiae suspension (109 CFU/ml) for 60 days. Animals of the uncontaminated feed control were given 0.2 ml of saline solution (0.9% NaCl) under the same conditions as the test group.
Animals were weighed three times a week. The general health status of rats was evaluated by recording any changes in behaviour, activity, posture, fur quality, feed and water intake, possible illness and deaths. Total weight gain (TWG) was calculated as the difference between the weight of animals in the beginning and at the end of the experiment. Progressive weight gain (PWG) was calculated considering the PWG of animals (weighed three times a week) during the experiment. Feed efficiency (FE) was calculated as TWG/DI and feed conversion (FC) as DI/TWG. Each of these growth parameters was measured individually (per animal), per cage and per experimental group and statistically analyzed. After 60 days, animals were sacrificed by decapitation. Blood, faeces, liver, lungs, intestines and testicle samples were also collected and reserved to further analyses.
Results showed that rats from both test and control groups appeared healthy, inquisitive and active throughout the experiment. The oral dose of S. cerevisiae RC016, did not cause mortality, illness or significant changes in the general behaviour of animals. No significant difference in feed and water intake or fur quality between groups were observed. The administration of the oral dose of S. cerevisiae RC016 did not cause any adverse effects on weight gain (TWG and PWG), FC or FE during the experiment. Gross examination of the internal organs of all rats revealed no detectable abnormalities, indicating that the probiotic yeast did not induce any organ damage. Macroscopic analyses of the different organs of S. cerevisiae RC016-treated rats did not show significant changes in colour and texture when compared with the control group. Histological examinations of liver, lungs, kidneys and intestine of the S. cerevisiae RC016-treated and control rats revealed that there were no differences between S. cerevisiae RC016-treated and control rats.
In conclusion, under the conditions of this study, oral daily administration of the probiotic Saccharomyces cerevisiae RC016 in male rats for 60 days did not result in any illness, mortality or any other health changes related to the intake of this strain. Also, the application of a daily oral dose of S. cerevisiae RC016 did not cause any negative impact on the animals’ weight gain, FC or FE. The examination of internal organs of S. cerevisiae RC016-treated and control rats revealed no macroscopic or microscopic differences or lesions associated to the use of the probiotic. This suggests the strain is safe to use in vivo.- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- other: peer reviewed data
- Adequacy of study:
- weight of evidence
- Study period:
- 2007
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- data from handbook or collection of data
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
- Version / remarks:
- 1998
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Route of administration:
- oral: gavage
- Duration of treatment / exposure:
- 91 days
- Frequency of treatment:
- Daily
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Dose / conc.:
- 2 mg/kg bw/day (nominal)
- Dose / conc.:
- 33.3 mg/kg bw/day (nominal)
- Dose / conc.:
- 100 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 10/sex/dose
- Control animals:
- yes, concurrent vehicle
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- >= 100 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other:
- Remarks on result:
- other: none
- Key result
- Critical effects observed:
- no
- Conclusions:
- A no-observed-adverse-effect level (NOAEL) of 100 mg/kg bw/d was established for a beta-glucan extract derived from Saccharomyces cerevisiae orally administred to male and female rats for 91 days.
- Executive summary:
Specific pathogen free (SPF) Fischer-344 rats (10/sex/group) were given either 2, 33.3, or 100 mg/kg bw/d of a beta-glucan extract derived from Saccharomyces cerevisiae, in water, via gavage, once a day, for 91 d (Babicek et al. 2007). A control group was given water only. No mortality, clinical pathology, functional/behavioral, microscopic, or gross observations indicating toxicity were observed. In addition, no negative effects on animal weights or food consumption were noted. No dose-dependent hematological or biochemical toxicities were observed. A no-observed-adverse-effect level (NOAEL) of 100 mg/kg bw/d was established.
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- other: experimental study performed on a SC yeast used in a WoE approach for the target substance
- Adequacy of study:
- weight of evidence
- Study period:
- 2000
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Principles of method if other than guideline:
- - Principle of test: subchronic toxicity of a fractionated yeast biomass as a single source of dietary protein was evaluated. The study was not conducted according to a standardised method and only limited parameters from the OECD TG 408 were examined. The group used as control is not a true control (vehicle concurrent or plain diet) as animals of the zero-time group (control) were killed prior to the beginning of the experiment.
- Parameters analysed / observed: Subchronic toxicity studies providing information on organ lesions and cumulative toxic effects and liver and kidneys being among the organs most susceptible to damage by toxic substances from the diet, this study focused on those two organs. Lesions in these organs can be measured by anatomical, histological and biochemical alterations. Biochemical alterations in the organs can be monitored by blood determinations such as transaminase (TGO and TGP) activities, blood urea and creatinine, and urine creatinine, urea and uric acid. Liver lesions with hepatocyte destruction normally result in an appreciable increase in TGO and TGP activities in blood serum. On the other hand, renal damage or failure is detected by increased blood urea, creatinine and uric acid. - GLP compliance:
- not specified
- Limit test:
- no
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: provided by a company in Campinas, SaÄo Paulo, Brazil which uses brewer's yeast biomass for the production of yeast extract.
- Purity, including information on contaminants, isomers, etc.: UVCB
RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: not applicable
- Specific activity: not applicable
- Locations of the label: not applicable
- Expiration date of radiochemical substance: not applicable
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: not specified in the publication
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: not applicable
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: not applicable
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: not applicable
- Reactivity of the test material with the incubation material used (e.g. plastic ware): not applicable
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): Prior to incorporation into food, brewer's yeast must undergo a debittering process by washing with alkaline solution and then with distilled water. For the rupturing of yeast cell walls, the procedure recommended by Hedenskog and Mogren (1973) was used. A cell suspension (40% w/v) was forced through a Dynomill at 2400 rev/min at a flow rate of 4.8l/h. Glass spheres of 0.6mm diameter filled 70% of the mill chamber during operation. The efficiency of cell rupture was evaluated by centrifuging aliquots of the mill effluent (10000 xg, 30 min) and determining nitrogen by a semimicro Kjeldahl method in the supernatant. Over 95% of the cell walls were estimated to be ruptured by this procedure. The ruptured cell walls were then submitted to dehydrattion by spray drying.
- Preliminary purification step (if any): none
- Final concentration of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid (e.g. stock crystals ground to fine powder using a mortar and pestle): not applicable
FORM AS APPLIED IN THE TEST (if different from that of starting material)
- Specify the relevant form characteristics if different from those in the starting material, such as state of aggregation, shape of particles or particle size distribution: not applicable
INFORMATION ON NANOMATERIALS
- Chemical Composition: not applicable
- Density: not applicable
- Particle size & distribution: not applicable
- Specific surface area: not applicable
- Isoelectric point: not applicable
- Dissolution (rate): not applicable
TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)
- Description of the formulation, e.g. formulated product for foliar application; formulated product soil application; solution in organic solvent for soil application; formulated product seed treatment; solution in organic solvent seed treatment: not applicable
OTHER SPECIFICS
- Other relevant information needed for characterising the tested material, e.g. if radiolabelled, adjustment of pH, osmolality and precipitate in the culture medium to which the test chemical is added: Chemical characterisation was performed on the ruptured yeast biomass. - Species:
- rat
- Strain:
- Wistar
- Details on species / strain selection:
- SPF
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: not specified in the publication
- Females (if applicable) nulliparous and non-pregnant: not applicable (only males tested)
- Age at study initiation: not specified in the publication
- Weight at study initiation: average 54.78 ± 4.2g
- Fasting period before study: not specified in the publication
- Housing: not specified in the publication
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: not specified in the publication
DETAILS OF FOOD AND WATER QUALITY: not specified in the publication
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): not specified in the publication
- Air changes (per hr): not specified in the publication
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: To: not specified in the publication - Route of administration:
- oral: feed
- Details on route of administration:
- Test item was administred in diet.
- Vehicle:
- not specified
- Details on oral exposure:
- DIET PREPARATION
Animals were fed diets with 15 and 30% protein from fractionated yeast biomass.
- Analytical verification of doses or concentrations:
- no
- Duration of treatment / exposure:
- 90 days
- Frequency of treatment:
- Daily via diet.
- Dose / conc.:
- 15 other: % of protein in test item
- Dose / conc.:
- 30 other: % of protein in test item
- No. of animals per sex per dose:
- 10 males/dose
- Control animals:
- other:
- Details on study design:
- - Dose selection rationale: not specified in the publication.
- Rationale for animal assignment (if not random): rabdom
- Fasting period before blood sampling for clinical biochemistry: not specified in the publication.
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random): not applicable
- Dose range finding studies: not performed.
- Other: none - Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Not specified
- Time schedule: Not specified
- Cage side observations checked in table [No.?] were included: Not specified
DETAILED CLINICAL OBSERVATIONS: Not specified
- Time schedule: Not specified
BODY WEIGHT: Yes
- Time schedule for examinations: body weight gain was recorded weekly.
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; diet consumption was recorded weekly.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified
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: Diet consumption and body weight gain were used for the calculation of diet efficiency ratio (DER).
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations: not applicable
OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations: not applicable
- Dose groups that were examined: not applicable
HAEMATOLOGY: No
- Time schedule for collection of blood: not applicable
- Anaesthetic used for blood collection: not applicable
- Animals fasted: not applicable
- How many animals: not applicable
- Parameters checked in table [No.?] were examined: not applicable
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected after 45 and 90 days of feeding. Blood was collected from the rats by cardiac puncture following ether anaesthesia, kept in a water bath at 37°C for 15min and centrifuged (1514 x g, 10 min, 4°C) in an RC5C Sorvall centrifuge to separate the blood serum.
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Not specified
- How many animals: all animals
- Parameters examined: TGO and TGP transaminase (aminotransferases) activities, creatinine, urea and uric acid contents. Creatinine determination was done by the method of Lima et al (1985) by the reaction of creatinine with alkaline picrate in buffered medium at 510 nm. Prior to colorimetric reaction the blood serum was deproteinated with picric acid. Glutamic-oxalacetic (TGO) and glutamic-pyruvic (TGP) transaminase activities were determined by measuring the quantity of oxalate or pyruvate produced under standardised conditions of pH and temperature. The oxalate is unstable and is transformed into pyruvate. The pyruvate reacts under alkaline conditions with 2,4-dinitrophenylhydrazine(2,4-DNFH), resulting in a coloured product which is quantitated at 505 nm. Uric acid determination was based on the blue colour developed by the reductive action of uric acid on phosphotungstic reagent under alkaline pH in the presence of allantoin and CO2. The tungsten blue formed absorbs at 700 nm. Urea was determined by a combination of the action of urease to liberate ammonia, which then reacts with sodium formate in the presence of hypochlorite to form a chromophore absorbing at 600 nm.
PLASMA/SERUM HORMONES/LIPIDS: No
- Time of blood sample collection: not applicable
- Animals fasted: not applicable
- How many animals: not applicable
URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected after 45 and 90 days.
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified
- Parameters examined: creatinine, urea and uric acid. Creatinine, urea and uric acid were also quantitated in rat urine by the same procedures utilised for blood serum.
NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations: not applicable
- Dose groups that were examined: not applicable
- Battery of functions tested: not applicable
IMMUNOLOGY: No
- Time schedule for examinations: not applicable
- How many animals: not applicable
- Dose groups that were examined: not applicable
- Parameters checked in table [No.?] were examined: not applicable
OTHER: none. - Sacrifice and pathology:
- SACRIFICE
The experimental animals (10 rats per group) were killed after 45 days (five rats) and 90 days (remaining five rats) for organ and blood collection.
GROSS PATHOLOGY: Yes; limited to liver and kidneys.
HISTOPATHOLOGY: No - Statistics:
- Results of biological and chemical assays were submitted to analysis of variance using the SANEST program version 2.1 suggested by Zonta and Machado. Differences among means were tested by Tukey's test with 95% confidence limit (p<0.05).
- Clinical signs:
- not specified
- Mortality:
- no mortality observed
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- Body weight gain for the treated groups of rats is presented in Table 2.
Body weight gain was statistically the same whether the diet contained 15 or 30% protein.
No difference in growth is noticed between 15 and 30% protein groups. - Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- Diet and protein consumption for the treated groups of rats are presented in Table 2.
Food consumption was similar for the 15 and 30% groups. - Food efficiency:
- not specified
- Description (incidence and severity):
- For both groups, ratio was higher after 45 days than after 90 days.
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- The results of determinations done on the blood serum are presented in Table 3.
Variations were detected as a function of time, but not for different protein concentrations, except for uric acid in which variations occurred also with different protein concentrations. A drop of TGO was registered from day 45 to day 90. There was a decrease in activity in the serum of rats day 45 to day 90. An increase was measured in the rats from day 45 to day 90, and these changes were all statistically significant (p<0.05). Regarding urea, an increase from day 45 to day 90 was noted. The 30 % diet promoted a significantly higher serum uric acid concentration. At day 90 a drop in serum uric acid was registered for the 30 % group and remained constant for the other group. - Endocrine findings:
- not examined
- Description (incidence and severity):
- The data for urine creatinine, urea and uric acid are shown in Table 4.
Creatinine did not differ (p<0.05) for both groups at day 45 of feeding. There was a significant drop in Urea for the rats fed the 15% diet. Urine uric acid increased with increase in protein concentration measured at both days 45 and 90 of feeding. Both treatment groups showed an increase in uric acid in rat urine at day 90 (p<0.05). - Behaviour (functional findings):
- not specified
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Gross pathological findings:
- not specified
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- not examined
- Histopathological findings: neoplastic:
- not examined
- Other effects:
- effects observed, treatment-related
- Description (incidence and severity):
- Same determinations as for blood serum were also done on the liver and kidneys of rats at both days 45 and 90 of feeding.
Table 5 presents data on fresh liver weight (g), liver total fat and liver total protein concentrations and liver/body weight ratio, expressed as a percentage.
The ratio seemed to increase with the content of dietary protein. Liver total fat (g per 100g) was slightly higher at day 45 and increased significantly at day 90 for the rats fed the YBM diets.
Kidney fresh weight (g) and kidney total fat and protein concentrations (% dry basis) are shown in Table 6. No statistical differences were observed in lipid content in the kidneys of rats fed 15 or 30% protein in the diets, and no difference, within a treatment, at days 45 and 90 of feeding.
Kidney total protein concentration did not change much among groups. The diet YBM 15% (day 90), the highest value, did not differ statistically from YBM 30% after both 45 and 90 days of the experiment. The diet YBM 15% (day 45), the lowest value, differed statistically from YBM 15% (day 90). - Details on results:
- Composition of the ruptured yeast biomass is presented in Table 1. The yeast biomass proteins presented a well-balanced amino acid profile.
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- >= 30 other: % of protein in test item
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other:
- Remarks on result:
- other: none
- Key result
- Critical effects observed:
- no
- Conclusions:
- When tested for subchronic toxicity at 15 and 30% protein concentration, no evidence of toxicity was found for the whole Saccharomyces cerevisiae yeast biomass after 45 and 90 days of feeding.
- Executive summary:
Th subchronic toxicity of a fractionated yeast biomass as a single source of dietary protein was evaluated in the rat. Saccharomyces cerevisiae yeast was prepared by alkaline treatment for debittering, cell wall rupture and dehydration by spray drying. Male rats (10/group) were fed diets with 15 and 30% protein from the fractionated yeast biomass during 90 days. A group was called the zero-time group (control) and these animals were killed prior to the beginning of the experiment. The experimental animals (10 rats per group) were killed after 45 days (five rats) and 90 days (remaining five rats) for organ (liver and kidneys), blood (TGO and TGP transaminase (aminotransferases) activities, creatinine, urea and uric acid contents) and urine collection (creatinine, urea and uric acid).
No mortality and no evidence of toxicity was found during the study. Body weight gain was statistically the same whether the diet contained 15 or 30% protein. No difference in growth is noticed between 15 and 30% protein groups. Food consumption was similar for the 15 and 30% groups. No toxicologically relevant effect was noted based on the blood chemistry, urinalysis and organ analysis results.
In conclusion, when tested for subchronic toxicity at 15 and 30% protein concentration, no evidence of toxicity was found for the whole Saccharomyces cerevisiae yeast biomass after 45 and 90 days of feeding.- 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
- 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
- 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 - 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 - 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
- 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 - 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. - 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.
- 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
- Critical effects observed:
- no
- 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.
Referenceopen allclose all
Table 1. Body weight gain and daily intake of Sprague-Dawley rats treated orally with the test item
| Control group | Test group |
Female |
|
|
Body weight gain (g) | 122.67±7.13ns | 106.76±5.16 |
Food intake (g/d) | 20.46±0.15ns | 20.64±0.50 |
Water intake (mg/d) | 31.57±1.01ns | 28.55±1.73 |
Male |
|
|
Body weight gain (g) | 337.09±18.04 | 284.07±12.91* |
Food intake (g/d) | 29.27±0.47ns | 27.71±0.16 |
Water intake (mg/d) | 38.34±1.69ns | 32.26±2.37 |
Values are mean ± SEM for each group.
Significant differences at *P <0.05 by Student’s t-test.
nsNot significant.
Table 2. Organ weights of Sprague-Dawley rats treated orally with the test item
Organ | Control group | Test group |
Female |
|
|
Liver | 2.57±0.05 | 2.78±0.07* |
Kidney | 0.60±0.02 | 0.67±0.02* |
Spleen | 0.15±0.01ns | 0.16±0.01 |
Heart | 0.31±0.01ns | 0.33±0.04 |
Lung | 0.46±0.02ns | 0.53±0.03 |
Brain | 0.60±0.02ns | 0.62±0.02 |
Thyroid | 0.16±0.01ns | 0.17±0.01 |
Ovary | 0.04±0.00ns | 0.04±0.00 |
Uterus | 0.18±0.03ns | 0.22±0.02 |
Male |
|
|
Liver | 2.64±0.06ns | 2.78±0.13 |
Kidney | 0.68±0.03ns | 0.69±0.02 |
Spleen | 0.15±0.01ns | 0.15±0.01 |
Heart | 0.32±0.01ns | 0.31±0.01 |
Lung | 0.37±0.01ns | 0.41±0.02 |
Brain | 0.35±0.01 | 0.39±0.01** |
Thyroid | 0.14±0.02ns | 0.14±0.01 |
Testis | 0.63±0.02ns | 0.65±0.04 |
Epididymis | 0.14±0.01ns | 0.16±0.01 |
Values are mean ± SEM for each group.
Significant differences at *P <0.05 and **P <0.01, respectively by Student’s t-test.
nsNot significant.
Table 3. Haematological parameters of Sprague-Dawley rats treated orally with the test item
Haematological parameters | Control group | Test group |
Female |
|
|
WBC (103/L) | 6.87±0.42 | 5.55±0.19* |
RBC (106/L) | 8.52±0.16 | 7.99±0.12* |
Hb (g/L) | 15.74±0.27ns | 15.17±0.14 |
Hct (%) | 53.97±1.02 | 50.96±0.60* |
MCV (fL) | 63.37±0.34ns | 63.82±0.53 |
MCH (pg) | 18.52±0.33ns | 19.01±0.18 |
MCHC (g/dL) | 29.23±0.45ns | 29.77±0.09 |
Male |
|
|
WBC (103/L) | 9.58±0.85ns | 12.17±1.19 |
RBC (106/L) | 9.32±0.09ns | 9.37±0.22 |
Hb (g/L) | 16.43±0.26ns | 16.68±0.40 |
Hct (%) | 58.06±0.76ns | 58.41±1.53 |
MCV (fL) | 62.31±0.47ns | 62.31±0.55 |
MCH (pg) | 17.66±0.25ns | 17.80±0.18 |
MCHC (g/dL) | 28.32±0.24ns | 28.58±0.20 |
Values are mean ± SEM for each group.
Significant differences at *P <0.05 by Student’s t-test.
nsNot significant.
Table 4. Blood biochemical parameters of Sprague Dawley rats treated orally with the test item
Blood biological parameters | Control group | Test group |
Female |
|
|
ALB (g/dL) | 5.27±0.09ns | 5.54±0.12 |
ALT (U/L) | 33.00±4.80ns | 35.22±4.44 |
AST (U/L) | 86.11±15.82ns | 80.22±14.83 |
BIL (mg/dL) | 0.42±0.02ns | 0.36±0.03 |
BUN (mg/dL) | 17.03±0.82ns | 17.38±0.72 |
CPK (U/L) | 63.00±2.45ns | 54.89±2.99 |
CRE (mg/dL) | 0.46±0.03 | 0.35±0.02** |
GLU (mg/dL) | 211.22±15.13ns | 225.89±10.36 |
HDL (mg/dL) | 61.00±3.47ns | 58.00±3.00 |
LDH (U/L) | 175.00±8.85ns | 157.22±19.69 |
NH3 (g/dL) | 355.44±28.35ns | 342.00±25.59 |
TCHO (mg/dL) | 143.78±8.70ns | 146.44±5.44 |
TP (g/dL) | 7.38±0.14ns | 7.56±0.14 |
TG (mg/dL) | 82.11±11.79ns | 66.11±7.89 |
UA (mg/dL) | 4.53±0.29ns | 4.70±0.24 |
Male |
|
|
ALB (g/dL) | 4.28±0.08 | 4.53±0.06* |
ALT (U/L) | 29.78±1.41ns | 33.78±1.09 |
AST (U/L) | 63.67±4.64ns | 77.78±5.08 |
BIL (mg/dL) | 0.26±0.20ns | 0.20±0.02 |
BUN (mg/dL) | 19.90±1.42ns | 17.61±1.12 |
CPK (U/L) | 82.33±20.35ns | 139.33±17.89 |
CRE (mg/dL) | 0.36±0.02ns | 0.32±0.01 |
GLU (mg/dL) | 374.67±25.03ns | 327.22±23.66 |
HDL (mg/dL) | 75.44±3.59ns | 78.78±2.80 |
LDH (U/L) | 183.86±38.47ns | 195.60±12.39 |
NH3 (g/dL) | 381.67±23.50ns | 335.89±35.58 |
TCHO (mg/dL) | 104.89±6.20ns | 105.33±6.74 |
TP (g/dL) | 6.43±0.09ns | 6.33±0.53 |
TG (mg/dL) | 55.33±5.07ns | 54.11±3.39 |
UA (mg/dL) | 6.90±0.53ns | 6.20±0.63 |
Values are mean ± SEM for each group.
Significant differences at *P <0.05 and **P <0.01, respectively by Student’s t-test.
Table 1. Summary of Hematology Findings
Group |
| Dose (mg/kg/d) |
RBC (1012/L) |
WBC (109/L) |
Hb (g/L) |
Hct (%) |
MCV (fL) |
MCH (pg) |
MCHC (g/L) |
PLT (109/L) |
Ly (%) |
Ne (%) |
Eo (%) |
Mo (%) |
Ba (%) |
Pt (min) |
Week 14 | Male | 0 | 8.18 ± 0.50 | 12.4 ± 2.9 | 153 ± 9.0 | 45.9 ± 2.60 | 56.2 ± 2.02 | 18.8 ± 0.84 | 334 ± 7.77 | 1155 ± 148 | 86.6 ± 1.5 | 11.2 ± 1.6 | 0.95 ± 0.60 | 1.30 ± 0.63 | 0.0 ± 0.0 |
|
|
| 20 | 8.24 ± 0.53 | 13.2 ± 1.9 | 157 ± 11.2 | 46.5 ± 2.58 | 56.5 ± 1.36 | 19.0 ± 0.57 | 337 ± 10.33 | 1098 ± 142 | 85.1 ± 2.32 | 12.3 ± 2.4 | 1.25 ± 0.63 | 1.35 ± 0.41 | 0.0 ± 0.0 |
|
|
| 200 | 8.35 ± 0.26 | 13.9 ± 2.7 | 158 ± 5.8 | 46.5 ± 1.54 | 55.7 ± 1.24 | 19.0 ± 0.47 | 340 ± 6.49 | 1116 ± 178 | 84.8 ± 2.18 | 12.5 ± 2.1 | 1.20 ± 0.54 | 1.50 ± 0.58 | 0.0 ± 0.0 |
|
|
| 800 | 8.09 ± 0.22 | 16.5 ± 2.2b | 151 ± 6.4 | 45.1 ± 1.68 | 55.7 ± 1.70 | 18.6 ± 0.63 | 334 ± 4.14 | 1162 ± 136 | 86.0 ± 1.97 | 11.5 ± 1.8 | 1.50 ± 0.91 | 1.15 ± 0.34 | 0.0 ± 0.0 |
|
| Female | 0 | 7.26 ± 0.28 | 8.9 ± 2.2 | 140 ± 7.6 | 41.9 ± 1.96 | 57.7 ± 1.20 | 19.2 ± 0.61 | 333 ± 8.46 | 1078 ± 113 | 86.2 ± 2.21 | 11.6 ± 1.8 | 0.95 ± 0.37 | 1.35 ± 0.82 | 0.0 ± 0.0 |
|
|
| 20 | 7.49 ± 0.22 | 8.5 ± 1.7 | 148 ± 6.8 | 44.1 ± 1.95 | 58.8 ± 1.72 | 19.7 ± 0.57 | 335 ± 4.89 | 1141 ± 85 | 83.6 ± 2.65 | 13.7 ± 2.2 | 1.25 ± 0.49 | 1.50 ± 0.53 | 0.0 ± 0.0 |
|
|
| 200 | 7.26 ± 0.38 | 9.3 ± 1.6 | 144 ± 9.3 | 42.6 ± 2.54 | 58.7 ± 1.16 | 19.8 ± 0.40 | 337 ± 4.06 | 1112 ± 118 | 83.9 ± 2.49 | 13.7 ± 2.1 | 1.30 ± 0.71 | 1.20 ± 0.59 | 0.0 ± 0.0 |
|
|
| 800 | 7.46 ± 0.34 | 7.9 ± 0.6 | 144 ± 7.2 | 42.9 ± 2.30 | 57.4 ± 1.11 | 19.3 ± 0.48 | 335 ± 3.98 | 1094 ± 135 | 84.6 ± 1.5 | 13.4 ± 1.7 | 1.10 ± 0.39 | 0.95 ± 0.28 | 0.0 ± 0.0 |
|
Week 33 | Male | 0 | 8.42 ± 0.43 | 12.3 ± 1.6 | 155 ± 8.2 | 44.5 ± 2.37 | 52.9 ± 2.41 | 18.4 ± 1.01 | 348 ± 11 | 1185 ± 88 | 84.9 ± 1.96 | 13.1 ± 1.6 | 0.65 ± 0.41 | 1.40 ± 0.57 | 0.0 ± 0.0 |
|
|
| 20 | 8.36 ± 0.45 | 12.6 ± 1.7 | 159 ± 7.1 | 45.5 ± 2.35 | 54.4 ± 1.27 | 19.1 ± 0.72 | 350 ± 10.98 | 1131 ± 162 | 84.7 ± 2.73 | 13.0 ± 2.7 | 0.95 ± 0.55 | 1.45 ± 0.60 | 0.0 ± 0.0 |
|
|
| 200 | 8.77 ± 0.29 | 11.4 ± 1.5 | 160 ± 4.5 | 46.0 ± 1.64 | 52.4 ± 1.52 | 18.3 ± 0.72 | 349 ± 9.95 | 1104 ± 183 | 82.4 ± 3.05 | 15.3 ± 2.5 | 1.00 ± 0.91 | 1.35 ± 0.47 | 0.0 ± 0.0 |
|
|
| 800 | 8.73 ± 0.32 | 12.7 ± 1.5 | 156 ± 6.2 | 46.3 ± 2.17 | 53.1 ± 2.01 | 17.9 ± 0.92 | 337 ± 9.57 | 1149 ± 212 | 82.4 ± 2.40 | 15.0 ± 2.0 | 1.10 ± 0.88 | 1.60 ± 0.52 | 0.0 ± 0.0 |
|
| Female | 0 | 7.45 ± 0.34 | 8.3 ± 2.0 | 145 ± 5.1 | 41.7 ± 1.75 | 55.9 ± 0.84 | 19.5 ± 0.61 | 350 ± 8.87 | 1089 ± 102 | 84.3 ± 3.59 | 13.3 ± 3.4 | 0.90 ± 0.61 | 1.55 ± 0.44 | 0.0 ± 0.0 |
|
|
| 20 | 7.43 ± 0.35 | 8.8 ± 1.8 | 146 ± 8.2 | 42.1 ± 2.42 | 56.7 ± 1.77 | 19.6 ± 0.58 | 347 ± 6.90 | 1021 ± 114 | 83.3 ± 2.64 | 14.4 ± 2.3 | 1.18 ± 0.40 | 1.14 ± 0.50 | 0.0 ± 0.0 |
|
|
| 200 | 7.25 ± 0.52 | 7.8 ± 1.7 | 144 ± 10.4 | 41.5 ± 2.89 | 57.3 ± 1.12 | 19.9 ± 0.91 | 347 ± 13.30 | 1045 ± 135 | 81.4 ± 2.70 | 16.0 ± 2.5 | 1.05 ± 0.55 | 1.55 ± 0.44 | 0.0 ± 0.0 |
|
|
| 800 | 7.36 ± 0.23 | 8.7 ± 2.0 | 143 ± 7.1 | 41.5 ± 1.68 | 56.3 ± 0.83 | 19.4 ± 0.55 | 345 ± 8.60 | 976 ± 195 | 80.7 ± 3.72 | 17.0 ± 3.4b | 1.20 ± 0.67 | 1.15 ± 0.47 | 0.0 ± 0.0 |
|
Day 365 | Male | 0 | 8.34 ± 0.41 | 12.7 ± 2.5 | 155 ± 9.8 | 45.9 ± 2.41 | 55.1 ± 2.05 | 18.6 ± 1.14 | 338 ± 17.8 | 1193 ± 153 | 80.8 ± 2.16 | 16.4 ± 2.0 | 1.03 ± 0.44 | 1.85 ± 0.78 | 0.0 ± 0.0 | 18.3 ± 2.5 |
|
| 20 | 8.16 ± 1.08 | 14.9 ± 9.3 | 155 ± 17.1 | 45.6 ± 5.24 | 56.1 ± 3.20 | 19.1 ± 1.05 | 340 ± 12.2 | 1197 ± 187 | 81.2 ± 3.59 | 15.6 ± 3.5 | 1.18 ± 0.61 | 2.10 ± 0.62 | 0.0 ± 0.0 | 16.9 ± 2.0 |
|
| 200 | 8.48 ± 0.55 | 12.3 ± 2.7 | 160 ± 10.3 | 46.6 ± 3.24 | 55.0 ± 1.99 | 18.9 ± 0.85 | 343 ± 10.3 | 1235 ± 237 | 80.3 ± 3.47 | 16.6 ± 3.4 | 1.15 ± 0.59 | 1.95 ± 0.63 | 0.0 ± 0.0 | 17.8 ± 1.5 |
|
| 800 | 8.35 ± 0.24 | 12.1 ± 2.5 | 157 ± 8.2 | 45.8 ± 2.08 | 54.9 ± 2.55 | 18.8 ± 1.00 | 343 ± 12.1 | 1200 ± 154 | 80.2 ± 2.14 | 17.0 ± 2.0 | 1.11 ± 0.43 | 1.63 ± 0.74 | 0.0 ± 0.0 | 18.1 ± 3.4 |
| Female | 0 | 7.07 ± 0.61 | 8.0 ± 1.8 | 146 ± 10.9 | 42.0 ± 2.80 | 59.5 ± 2.33 | 20.7 ± 0.47 | 348 ± 9.26 | 919 ± 218 | 79.8 ± 3.68 | 17.6 ± 3.4 | 1.05 ± 0.32 | 1.63 ± 0.53 | 0.0 ± 0.0 | 15.1 ± 2.2 |
|
| 20 | 7.04 ± 0.75 | 8.9 ± 3.5 | 147 ± 12.6 | 42.3 ± 3.57 | 60.4 ± 3.67 | 21.0 ± 1.12 | 348 ± 8.90 | 970 ± 147 | 79.8 ± 3.45 | 17.3 ± 3.3 | 1.28 ± 0.53 | 1.68 ± 0.57 | 0.0 ± 0.0 | 15.5 ± 1.3 |
|
| 200 | 7.22 ± 0.49 | 8.8 ± 3.5 | 150 ± 9.9 | 43.0 ± 2.79 | 59.6 ± 2.54 | 20.8 ± 0.81 | 349 ± 8.66 | 1028 ± 132 | 78.0 ± 2.50 | 18.7 ± 2.3 | 1.16 ± 0.50 | 2.16 ± 0.69b | 0.0 ± 0.0 | 15.5 ± 1.4 |
|
| 800 | 7.34 ± 0.50 | 8.5 ± 1.7 | 150 ± 10.3 | 43.2 ± 2.63 | 59.0 ± 1.57 | 20.4 ± 0.52 | 346 ± 7.47 | 951 ± 146 | 78.4 ± 3.16 | 18.2 ± 3.1 | 1.24 ± 0.54 | 2.11 ± 0.66 | 0.0 ± 0.0 | 15.6 ± 1.9 |
Abbreviations: RBC, red blood cells; WBC, white blood cells; Hb, hemoglobin; Hct, hematocrit; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; PLT, platelet; Ly, lymphocytes; Ne, neutrophils; Eo, Eosinophils; Mo, Monocytes; Ba, Basophils; Pt, prothrombin time.
a Data represent the mean and the standard deviation
b P < 0.05.
Table 2. Summary of Chemistry Findings
Group |
| Dos (mg/ kg/d) |
GPT (U/L) |
GGT (U/L) |
AP (U/L) |
Cho (mmol/L) |
Prot (g/L) |
Gl (mmol/L) |
Tg (mmol/L) |
BUN (mmol/L) |
Na (mmol/L) |
K (mmol/L) |
Cl (mmol/L) |
Crea (mmol/L) |
GOT (U/L) |
GLDH (U/L) |
Alb (g/L) |
Glob (mmol/L) |
Ca (mmol/L) |
Week 14 | Male | 0 | 40.1 ± 3.23 | 2.2 ± 1.83 | 194 ± 64.8 | 2.12 ± 0.36 | 76.2 ± 4.29 | 4.53 ± 0.60 | 1.57 ± 0.50 | 6.79 ± 0.46 | 159 ± 5.55 | 6.00 ± 0.29 | 96.8 ± 4.16 | 73.2 ± 7.21 |
|
|
|
|
|
|
| 20 | 42.8 ± 6.01 | 2.2 ± 1.73 | 171 ± 42.2 | 1.89 ± 0.29 | 75.7 ± 4.31 | 4.49 ± 0.71 | 1.35 ± 0.48 | 7.01 ± 1.67 | 158 ± 4.11 | 5.69 ± 0.30 | 100.7 ± 2.66b | 74.6 ± 8.71 |
|
|
|
|
|
|
| 200 | 41.5 ± 5.74 | 2.9 ± 2.06 | 182 ± 37.3 | 1.93 ± 0.27 | 73.6 ± 2.85 | 4.48 ± 1.17 | 1.36 ± 0.50 | 7.19 ± 0.55 | 159 ± 5.34 | 6.06 ± 0.57 | 99.4 ± 2.05 | 82.3 ± 11.90 |
|
|
|
|
|
|
| 800 | 46.9 ± 3.68 | 2.9 ± 1.83 | 211 ± 49.6 | 1.85 ± 0.18 | 76.4 ± 3.40 | 4.70 ± 0.82 | 1.42 ± 0.47 | 6.73 ± 1.20 | 156 ± 2.68 | 5.81 ± 0.31 | 97.5 ± 2.02 | 84.6 ± 13.66 |
|
|
|
|
|
| Female | 0 | 31.9 ± 4.1 | 0.7 ± 0.7 | 91.2 ± 15.7 | 2.19 ± 0.39 | 82.6 ± 4.14 | 4.33 ± 0.64 | 0.94 ± 0.63 | 5.19 ± 0.77 | 156 ± 3.9 | 4.58 ± 0.39 | 95.3 ± 2.64 | 80.8 ± 11.07 |
|
|
|
|
|
|
| 20 | 30.0 ± 3.1 | 1.0 ± 0.7 | 85.5 ± 23.1 | 2.48 ± 0.32 | 82.3 ± 3.40 | 4.26 ± 0.41 | 0.79 ± 0.31 | 4.84 ± 0.67 | 154 ± 3.2 | 4.68 ± 0.44 | 98.5 ± 2.16b | 84.9 ± 9.80 |
|
|
|
|
|
|
| 200 | 33.6 ± 4.9 | 1.4 ± 1.0 | 99.2 ± 22.5 | 2.64 ± 0.42 | 81.9 ± 4.15 | 4.95 ± 0.92 | 0.86 ± 0.43 | 5.33 ± 1.22 | 149 ± 3.3c | 4.57 ± 0.36 | 97.7 ± 3.70 | 93.8 ± 13.11 |
|
|
|
|
|
|
| 800 | 31.5 ± 3.6 | 2.1 ± 0.5c | 83.9 ± 18.6 | 2.43 ± 0.35 | 84.1 ± 4.16 | 4.63 ± 0.75 | 0.99 ± 0.49 | 5.06 ± 0.83 | 152 ± 2.2b | 4.58 ± 0.36 | 90.0 ± 1.51c | 83.1 ± 10.79 |
|
|
|
|
|
Week 33 | Male | 0 | 42.9 ± 7.27 | 3.0 ± 2.21 | 145 ± 49.9 | 2.68 ± 0.49 | 78.5 ± 2.83 | 6.63 ± 0.48 | 2.08 ± 0.81 | 6.18 ± 0.56 | 157 ± 2.47 | 5.99 ± 0.52 | 104.0 ± 5.67 | 78.7 ± 7.40 |
|
|
|
|
|
|
| 20 | 41.7 ± 9.52 | 3.3 ± 1.79 | 128 ± 27.6 | 2.57 ± 0.48 | 80.7 ± 5.01 | 6.54 ± 0.50 | 1.73 ± 0.86 | 6.38 ± 0.89 | 157 ± 2.88 | 5.99 ± 0.56 | 102.1 ± 5.00 | 76.8 ± 7.93 |
|
|
|
|
|
|
| 200 | 39.2 ± 10.72 | 3.6 ± 2.29 | 122 ± 25.1 | 2.44 ± 0.43 | 79.9 ± 2.53 | 6.76 ± 0.88 | 1.76 ± 0.71 | 6.53 ± 0.67 | 153 ± 1.24 | 6.07 ± 0.50 | 100.2 ± 2.42 | 73.4 ± 5.63 |
|
|
|
|
|
|
| 800 | 40.5 ± 6.67 | 3.9 ± 3.58 | 142 ± 48.1 | 2.29 ± 0.40 | 82.8 ± 3.55 | 7.08 ± 0.83 | 192 ± 0.63 | 6.28 ± 0.64 | 155 ± 3.01b | 5.97 ± 0.55 | 100.6 ± 4.49 | 75.9 ± 5.42 |
|
|
|
|
|
| Female | 0 | 44.1 ± 11.8 | 1.6 ± 1.54 | 48.9 ± 11.4 | 2.91 ± 0.82 | 88.7 ± 8.27 | 6.20 ± 0.40 | 1.98 ± 0.78 | 5.99 ± 0.95 | 145 ± 1.51 | 4.40 ± 0.26 | 94.0 ± 9.75 | 74.0 ± 5.16 |
|
|
|
|
|
|
| 20 | 47.7 ± 15.9 | 0.9 ± 1.21 | 53.3 ± 19.5 | 3.37 ± 0.51 | 89.5 ± 2.54 | 6.12 ± 0.44 | 2.10 ± 1.01 | 5.36 ± 0.65 | 145 ± 1.23 | 4.28 ± 0.39 | 93.2 ± 2.45 | 78.7 ± 9.02 |
|
|
|
|
|
|
| 200 | 48.3 ± 16.4 | 1.1 ± 0.66 | 55.5 ± 12.6 | 3.37 ± 0.88 | 86.4 ± 5.39 | 6.38 ± 0.57 | 2.46 ± 1.23 | 5.83 ± 0.77 | 150 ± 1.94c | 4.34 ± 0.20 | 95.5 ± 4.27 | 79.0 ± 8.65 |
|
|
|
|
|
|
| 800 | 38.7 ± 12.2 | 1.2 ± 0.89 | 57.1 ± 10.6 | 2.75 ± 0.60 | 86.2 ± 3.78 | 6.30 ± 0.43 | 1.91 ± 1.12 | 5.71 ± 0.46 | 147 ± 1.77b | 4.35 ± 0.41 | 96.6 ± 2.70 | 76.6 ± 5.24 |
|
|
|
|
|
Day 365 | Male | 0 | 42.3 ± 18.7 | 3.2 ± 1.94 | 163.5 ± 56.4 | 2.94 ± 0.80 | 68.8 ± 3.76 | 7.05 ± 2.48 | 2.00 ± 0.81 | 6.10 ± 2.38 | 146 ± 4.56 | 4.99 ± 0.47 | 99.5 ± 2.76 | 79.2 ± 7.39 | 150.2 ± 37.1 | 6.9 ± 2.3 | 42.4 ± 2.31 | 26.4 ± 3.21 | 2.52 ± 0.09 |
|
| 20 | 41.4 ± 20.1 | 2.3 ± 1.19 | 137.9 ± 25.0 | 2.83 ± 1.03 | 68.3 ± 4.50 | 6.51 ± 0.75 | 1.85 ± 0.67 | 5.86 ± 0.94 | 146 ± 1.64 | 4.97 ± 0.41 | 99.9 ± 3.39 | 80.5 ± 7.51 | 140.7 ± 29.1 | 5.5 ± 2.2 | 41.5 ± 1.87 | 26.9 ± 3.28 | 2.52 ± 0.09 |
|
| 200 | 53.6 ± 46.9 | 1.8 ± 0.99 | 139.5 ± 28.1 | 2.67 ± 0.52 | 68.7 ± 3.19 | 6.85 ± 0.75 | 1.52 ± 0.71 | 5.41 ± 0.61 | 146 ± 1.59 | 4.81 ± 0.28 | 101.5 ± 1.91 | 79.9 ± 6.43 | 145.7 ± 34.1 | 5.6 ± 2.8 | 40.6 ± 1.87b | 28.1 ± 2.65 | 2.50 ± 0.07 |
|
| 800 | 36.2 ± 15.8 | 1.1 ± 0.61c | 146.8 ± 34.6 | 2.41 ± 0.70 | 69.3 ± 2.40 | 6.82 ± 0.70 | 1.54 ± 0.42 | 5.25 ± 0.87 | 147 ± 1.50 | 4.80 ± 0.35 | 100.8 ± 4.07 | 74.0 ± 6.12 | 124.2 ± 34.0 | 4.4 ± 1.5c | 39.8 ± 1.84c | 29.5 ± 1.76c | 2.43 ± 0.07c |
| Female | 0 | 37.1 ± 9.37 | 1.0 ± 0.76 | 52.9 ± 8.77 | 3.20 ± 0.76 | 77.8 ± 4.84 | 5.2 ± 0.54 | 2.44 ± 0.90 | 5.23 ± 0.50 | 144 ± 1.89 | 3.77 ± 0.46 | 97.6 ± 3.65 | 82.3 ± 7.39 | 116.1 ± 22.3 | 6.7 ± 6.03 | 48.6 ± 4.24 | 29.2 ± 2.52 | 2.58 ± 0.13 |
|
| 20 | 37.2 ± 10.1 | 0.7 ± 0.52 | 50.4 ± 16.8 | 3.26 ± 0.81 | 77.7 ± 5.33 | 5.4 ± 0.53 | 2.12 ± 1.15 | 5.34 ± 0.68 | 144 ± 1.81 | 3.91 ± 0.37 | 100.2 ± 3.29b | 87.8 ± 13.45 | 129.5 ± 42.9 | 8.7 ± 5.02 | 48.5 ± 4.64 | 29.2 ± 2.28 | 2.66 ± 0.12 |
|
| 200 | 48.4 ± 22.1 | 1.2 ± 0.76 | 50.1 ± 13.5 | 3.24 ± 0.92 | 78.8 ± 4.03 | 5.6 ± 0.59 | 1.69 ± 0.90 | 5.50 ± 1.07 | 144 ± 1.46 | 3.91 ± 0.43 | 98.3 ± 2.40 | 85.4 ± 8.81 | 144.9 ± 42.7 | 8.9 ± 7.15 | 46.7 ± 1.91 | 32.1 ± 2.89b | 2.48 ± 0.19 |
|
| 800 | 38.5 ± 14.8 | 1.1 ± 0.87 | 50.0 ± 14.3 | 3.07 ± 0.71 | 76.7 ± 3.23 | 5.5 ± 0.64 | 2.27 ± 1.23 | 5.43 ± 0.68 | 143 ± 1.76 | 3.83 ± 0.36 | 100.9 ± 9.03b | 85.4 ± 19.73 | 122.5 ± 36.1 | 7.8 ± 6.78 | 46.1 ± 6.13 | 30.6 ± 5.52 | 2.60 ± 0.16 |
Abbreviations: GPT, glutamic pyruvic transaminase; GGT, gamma-glutamyl transpeptidase; AP, alkaline phosphatase; Cho, total cholesterol; Prot, total protein; Gl, blood glucose; Tg, triglycerides; BUN, blood urea nitrogen; Na, sodium; K, potassium; Cl, chloride; Crea, creatinine; GOT, glutamic oxalacetic transaminase; GLDH, glutamate dehydrogenase; Alb, albumin; Glob, globulin; Ca, total calcium.
a Data represents the mean and the standard deviation
b P < .05.
c P < .01.
Table 3. Summary of Gross Pathological Findings
|
| Males | Females | ||||||
|
| Control MC 1% solution
| EpiCor | Control MC 1% solution
| EpiCor | ||||
Groups (n= 20 per groups) | 20 (mg/kg/d)
| 200 (mg/kg/d)
| 800 (mg/kg/d)
| 20 (mg/kg/d)
| 200 (mg/kg/d)
| 800 (mg/kg/d)
| |||
Organ | Lesion | Observed alterations/number of investigate animals | |||||||
Nutrition | Slight | 0 | 1 | 0 | 1 | 0 | 2 | 1 | 1 |
| Moderate | 1 | 1 | 3 | 0 | 7 | 6 | 9 | 7 |
| Well | 10 | 12 | 9 | 14 | 7 | 5 | 3 | 6 |
| Overweight | 8 | 6 | 8 | 5 | 6 | 7 | 7 | 6 |
Liver | Fawn-colored | 12 | 13 | 11 | 13 | 5 | 7 | 8 | 6 |
| Nutmeg-pattern | 8 | 7 | 8 | 10 | 3 | 1 | 3 | 4 |
| Focal | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 |
Skin | Furless areas | 1 | 2 | 2 | 1 | 1 | 4 | 2 | 0 |
| Inflammation | 0 | 1 | 0 | 1 | 0 | 1 | 0 | 0 |
| Tumor-like tissue changes | 0 | 1 | 1 | 2 | 0 | 2 | 1 | 0 |
Mammary gland | Retention of secretum | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 2 |
| Tumor-like tissue changes | 0 | 0 | 0 | 0 | 0 | 0 | 3 | 4 |
Adrenal gland | Cystic degeneration | 0 | 0 | 0 | 0 | 1 | 4 | 2 | 3 |
| Tumor-like tissue changes | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 |
Pituitary | Cystic degeneration | 0 | 0 | 0 | 0 | 0 | 2 | 0 | 0 |
| Tumor-like enlargement | 0 | 0 | 1 | 0 | 1 | 2 | 1 | 3 |
Thymus | Atrophy | 1 | 1 | 0 | 1 | 0 | 3 | 0 | 1 |
Testes | Atrophy | 1 | 1 | 3 | 1 | n/a | n/a | n/a | n/a |
Epididymis | Atrophy | 0 | 1 | 0 | 0 | n/a | n/a | n/a | n/a |
Uterus | Dilation | n/a | n/a | n/a | n/a | 6 | 3 | 4 | 3 |
Abbreviations: n/a, data not available; MC, methylcellulose.
Table 4. Summary of Histopathological Findings
|
| Males | Females | ||
Groups (n= 20 per groups) | Control MC 1% solution
| EpiCor 800 (mg/kg/d)
| Control MC 1% solution
| EpiCor 800 (mg/kg/d)
| |
Organ | Lesion | Observed alterations/number of investigate animals | |||
Liver | Pathological fatty infiltrationa | 17 | 20 | 18 | 10 |
Kidney | Focal lymphohistocytic infiltration*b | 11 | 15 | 2 | 0 |
| Tubular dilatation | 10 | 8 | 4 | 1 |
| Fibrosis | 1 | 0 | 0 | 0 |
| Cyst (1 side) | 0 | 0 | 0 | 1 |
| Mineral deposits | 0 | 0 | 2 | 1 |
Adrenal | Cystic degenerationc | 1 | 0 | 11 | 12 |
Lung | Alveolar emphysemad | 9 | 3 | 2 | 1 |
| Hemorrhage (focal) | 0 | 1 | 1 | 1 |
| Foam cells | 1 | 1 | 0 | 0 |
| Focal catarrhal inflammation | 0 | 1 | 0 | 0 |
Pituitary | Ademoma | 1 | 0 | 1 | 4 |
Skin | Atheroma | 0 | 1 | 0 | 0 |
| Adenoma | 0 | 0 | 0 | 0 |
| Fibroma | 0 | 0 | 0 | 0 |
| Myxoma | 0 | 1 | 0 | 0 |
| Papilloma | 0 | 0 | 0 | 0 |
| Lipoma | 0 | 0 | 0 | 0 |
| Inflammation (subacute) | 0 | 1 | 0 | 0 |
Conjunctiva | Edema | 0 | 1 | 0 | 0 |
Ovary | Cyst | n/a | n/a | 0 | 1 |
Uterus | Fibroma | n/a | n/a | 0 | 1 |
Testes | Decreased spermatogenesis | 1 | 1 | n/a | n/a |
| Edema | 1 | 1 | n/a | n/a |
Epididymis | Lack of spermatocytes | 1 | 1 | n/a | n/a |
Seminal vesicle | Involution | 1 | 0 | n/a | n/a |
Prostate | Involution | 1 | 0 | n/a | n/a |
Mammary gland | Adenoma | 0 | 0 | 0 | 3 |
| Fibroadenoma | 0 | 0 | 0 | 1 |
| Retention of secretum | 0 | 0 | 0 | 2 |
Heart | Focal fibrosis | 2 | 2 | 0 | 0 |
Pancreas | Focal atrophye | 8 | 10 | 0 | 0 |
Spleen | Hyperplasia | 0 | 1 | 0 | 0 |
a Considered a consequence of overnight fasting.
b An age-related spontaneous nephropathy characteristic of male rats.
c Occurs spontaneously in predominantly aging female rats.
d Considered related to extermination.
e Most common spontaneous degenerative change in aged rats.
Table 1 – Body weight change (g)
No. of rats | Control group | Test group 1 (2000mg/kg) | Test group 2 (1000mg/kg) |
Day 1 | |||
1 | 190 | 195 | 176 |
2 | 180 | 178 | 189 |
3 | 193 | 187 | 181 |
4 | 186 | 185 | 182 |
5 | 181 | 174 | 183 |
6 | 173 | 186 | 185 |
7 | 185 | 184 | 193 |
8 | 177 | 173 | 174 |
9 | 177 | 191 | 181 |
10 | 179 | 189 | 192 |
Mean ± SD | 182.1 ± 4.5 | 184.2 ± 5.1 | 183.6 ± 4.5 |
Day 14 | |||
1 | 219 | 221 | 206 |
2 | 216 | 208 | 214 |
3 | 220 | 222 | 211 |
4 | 220 | 209 | 219 |
5 | 218 | 210 | 212 |
6 | 205 | 211 | 217 |
7 | 214 | 227 | 235 |
8 | 194 | 191 | 202 |
9 | 204 | 227 | 206 |
10 | 205 | 213 | 226 |
Mean ± SD | 211.5 ± 6.4 | 213.9 ± 7.8 | 214.8 ± 7.2* |
Day 28 | |||
1 | 241 | 235 | 220 |
2 | 233 | 226 | 230 |
3 | 233 | 244 | 219 |
4 | 227 | 223 | 229 |
5 | 226 | 225 | 224 |
6 | 223 | 226 | 232 |
7 | 236 | 247 | 240 |
8 | 208 | 210 | 221 |
9 | 229 | 245 | 231 |
10 | 215 | 240 | 233 |
Mean ± SD | 227.1 ± 7.1 | 232.1 ±8.6 * | 227.9 ± 4.8 |
Day 42 | |||
1 | 243 | 249 | 235 |
2 | 246 | 242 | 246 |
3 | 248 | 247 | 236 |
4 | 241 | 239 | 238 |
5 | 238 | 240 | 246 |
6 | 238 | 243 | 236 |
7 | 251 | 257 | 255 |
8 | 216 | 215 | 240 |
9 | 244 | 263 | 246 |
10 | 236 | 256 | 245 |
Mean ± SD | 240.1 ± 6.9 | 245.1 ± 9.5* | 242.3 ± 4.6 |
Day 56 | |||
1 | 254 | 264 | 238 |
2 | 250 | 254 | 252 |
3 | 266 | 255 | 237 |
4 | 249 | 253 | 258 |
5 | 252 | 248 | 257 |
6 | 245 | 246 | 250 |
7 | 266 | 266 | 252 |
8 | 228 | 220 | 250 |
9 | 250 | 272 | 258 |
10 | 248 | 276 | 259 |
Mean ± SD | 250.8 ± 7.7 | 255.4 ± 11.4* | 251.1 ± 5.7 |
Day 70 | |||
1 | 259 | 276 | 238 |
2 | 267 | 258 | 263 |
3 | 272 | 268 | 258 |
4 | 257 | 254 | 267 |
5 | 256 | 259 | 268 |
6 | 258 | 255 | 252 |
7 | 265 | 279 | 258 |
8 | 232 | 223 | 270 |
9 | 258 | 273 | 265 |
10 | 259 | 267 | 266 |
Mean ± SD | 258.3 ± 7.6 | 261.2 ± 11.5 | 260.5 ± 6.9 |
Day 84 | |||
1 | 268 | 265 | 246 |
2 | 275 | 265 | 265 |
3 | 274 | 254 | 262 |
4 | 263 | 264 | 273 |
5 | 268 | 268 | - |
6 | 263 | 254 | 277 |
7 | 276 | 285 | 271 |
8 | 238 | 224 | 281 |
9 | 263 | 290 | 270 |
10 | 270 | 272 | 268 |
Mean ± SD | 265.8 ± 7.8 | 264.1 ± 13.1 | 268.11 ± 7.8 |
Day 90 | |||
1 | 270 | 269 | 244 |
2 | 275 | 265 | 266 |
3 | 284 | 280 | 260 |
4 | 266 | 266 | 279 |
5 | 269 | 269 | - |
6 | 265 | 255 | 280 |
7 | 280 | 288 | 274 |
8 | 240 | 228 | 286 |
9 | 265 | 295 | 275 |
10 | 272 | 276 | 273 |
Mean ± SD | 268.6 ± 8.5 | 269.1 ± 13.3 | 270.78 ± 9.7 |
* Statistically significant differences (Student's t-test, p<0.05)
Table 2 - Body weight gain (%)
No. of rats | Group | ||
Control group | Test group 1 (2000mg/kg) | Test group 2 (1000mg/kg) | |
1 | 142.11 | 137.95 | 138.64 |
2 | 15278 | 148.88 | 140.74 |
3 | 147.15 | 149.73 | 143.65 |
4 | 143.01 | 143.78 | 153.30 |
5 | 148.62 | 154.60 | - |
6 | 153.18 | 137.10 | 151.35 |
7 | 151.35 | 156.52 | 141.97 |
8 | 135.59 | 131.79 | 164.37 |
9 | 149.72 | 154.45 | 15193 |
10 | 151.96 | 146.03 | 142.19 |
Mean ± SD | 147.55 ± 4.08 | 146.08 ± 5.98 | 147.57 ± 6.38 |
Table 3 - Organ mass ratios on the first day (day 91) after completion of test item administration (g/100 g)
Organ | Control group; n=5 | Dose, mg/kg | |
Test group 1 (2000mg/kg); n=5 | Test group 2 (1000mg/kg); n=4 | ||
Liver | 3.35 ± 0.27 | 3.24 ± 0.25 | 2,91 ± 0.55* |
Kidneys | 0.56 ± 0.06 | 0.58 ± 0.04 | 0,54 ± 0.06 |
Spleen | 0.26 ± 0.05 | 0.27 ± 0.10 | 0,25 ± 0.07 |
Lungs | 0.61 ± 0.27 | 0.72 ± 0.14 | 0,72 ± 0.13 |
Heart | 0.34 ± 0.06 | 0.35 ± 0.07 | 0,35 ± 0.05 |
* Statistically significant differences (t-test, p<0.05)
Table 4 - Organ mass ratios at the tenth day (day 100) after completion of test item administration (g/100 g)
Organ | Control group; n=5 | Dose, mg/kg | |
Test group 1 (2000mg/kg); n=5 | Test group 2 (1000mg/kg); n=4 | ||
Liver | 2.48 ± 0.07 | 2.43 ± 0.12 | 2.58 ± 0.09 |
Kidneys | 0.55 ± 0.05 | 0.59 ± 0.05 | 0. 58 ± 0.04 |
Spleen | 0.25 ± 0.04 | 0.27 ± 0.04 | 0.28 ± 0.05 |
Lungs | 0. 81 ± 0.20 | 0.71 ± 0.06 | 0.98 ± 0.18 |
Heart | 0.35 ± 0.03 | 0.35 ± 0.03 | 0.34 ± 0.03* |
* Statistically significant differences (t-test, p<0.05)
Table 5 - Blood counts (haematological findings) on the first day (day 91) after completion of test item administration
Indicator | Control group; n=5 | Doses, mg/kg | |
Test group 1 (2000mg/kg); n=5 | Test group 2 (1000mg/kg); n=4 | ||
Haematocrit, % | 44.33 ± 2.75 | 42.14 ± 3.69* | 41.34 ± 10.60 |
Haemoglobin, g/l | 146.80 ± 11.19 | 140.40 ± 16.11 | 135.75 ± 38.02 |
Erythrocytes, 1012/l | 7.63 ± 0.53 | 7.34 ± 0.81 | 7.17 ± 1.82 |
Leucocytes, 109/l | 13.59 ± 3.11 | 13.49 ± 6.78 | 8.29 ± 2.33* |
Platelets, 109/l | 748.60 ± 131.85 | 680.0 ± 194.97 | 678.50 ± 204.40 |
Leukogram | |||
Stab neutrophils, % | 0.0 ± 0.0 | 0.0 ± 0.0 | 0.25 ± 0.80 |
Segmentonuclear neutrophils, % | 11.20 ± 5.98 | 16.20 ± 6.65* | 14.25 ± 9.75 |
Eosinophils, % | 1.40 ± 1.88 | 3.0 ± 3.17* | 0.50 ± 0.92 |
Monocytes, % | 0.60 ± 1.67 | 0.40 ± 0.68 | 0.75 ± 0.80 |
Basophils, % | 0.0 ± 0.0 | 0.0 ± 0.0 | 0.0 ± 0.0 |
Lymphocytes, % | 86.80 ± 6.17 | 80.40 ± 7.27* | 84.25 ± 9.75 |
* Statistically significant differences (Student's t-test, p<0.05)
Table 6 - Blood counts (haematological findings) at the tenth day (day 100) after completion of test item administration
Indicator | Control group; n=5 | Doses, mg/kg | |
Test group 1 (2000mg/kg); n=5 | Test group 2 (1000mg/kg); n=4 | ||
Haematocrit, % | 46.54 ± 2.1 | 45.37 ± 1.59* | 46.05 ± 2.53 |
Haemoglobin, g/l | 153.60 ± 6.18 | 154.20 ± 5.3 | 155.80 ± 8.48 |
Erythrocytes, 10/l12 | 8.02 ± 0.57 | 7.87 ± 0.35 | 8.09 ± 0.68 |
Leucocytes, 10/l9 | 16.29 ± 1.98 | 15.65 ± 4.68 | 18.51 ± 6.93 |
Platelets, 109/l | 831.40 ± 102.36 | 838.0 ± 75.82 | 813.60 ± 197.07 |
Leukogram | |||
Stab neutrophils, % | 0.40 ± 0.68 | 0.80 ± 1.62 | 0.60 ± 0.68 |
Segmentonuclear neutrophils, % | 11.80 ± 6.65 | 19.6 ± 9.06* | 15.0 ± 9.78 |
Eosinophils, % | 1.20 ± 1.04 | 2.40 ± 3.24 | 1.40 ± 1.67 |
Monocytes, % | 4.20 ± 1.84 | 2.40 ± 1.42* | 3.20 ± 2.39 |
Basophils, % | 0.0 ± 0.0 | 0.0 ± 0.0 | 0.0 ± 0.0 |
Lymphocytes, % | 82.40 ± 8.36 | 74.80 ± 11.53* | 79.80 ± 8.48 |
* Statistically significant differences (t-test, p<0.05)
Table 7 – Clinical biochemistry results on the first day (day 91) after completion of test item administration, n=5
Indicator | Control group; n=5 | Doses, mg/kg | |
Test group 1 (2000mg/kg); n=5 | Test group 2 (1000mg/kg); n=4 | ||
Bilirubin total, μmol/l | 1.82 ± 0.69 | 1.56 ± 0.45 | 2.08 ± 0.98 |
Direct bilirubin, μmol/l | 0.16 ± 0.07 | 0.14 ± 0.07 | 0.20 ± 0.13 |
Aspartate Aminotransferase (AST), U/L | 316.80 ± 358.4 | 310.60 ± 134.39 | 349.75 ± 254.22 |
Alanine Aminotransferase (ALT), U/L | 102.60 ± 21.67 | 80.60 ± 18.22* | 92.50 ± 15.18 |
Urea, mmol/l | 6.86 ± 0.63 | 6.56 ± 1.34 | 6.33 ± 1.93 |
Creatinine, μmol/l | 74.60 ± 4.78 | 75.0 ± 7.4 | 77.0 ± 18.09 |
Total protein, g/l | 76.80 ± 4.84 | 77.80 ± 2.04 | 79.50 ± 3.79 |
Alkaline phosphatase, U/L | 122.60 ± 20.34 | 102.80 ± 26.44* | 165.0 ± 128.42 |
Glucose, mmol/l | 6.64 ± 0.79 | 7.04 ± 0.57 | 6.53 ± 0.15 |
* Statistically significant differences (t-test, p<0.05)
Table 8 - Clinical biochemistry results at the tenth day (day 100) after completion of test item administration, n=5
Indicator | Control group | Doses, mg/kg | |
Test group 1 (2000mg/kg); n=5 | Test group 2 (1000mg/kg); n=4 | ||
Total Bilirubin, μmol/l | 3.08 ± 0.34 | 2.44 ± 0.84* | 2.78 ± 0.89 |
Direct bilirubin, μmol/l | 0.68 ± 0.33 | 0.52 ± 0.34 | 0.56 ± 0.40 |
Aspartate Aminotransferase (AST), U/L | 147.60 ± 40.23 | 147.0 ± 29.94 | 129.80 ± 29.75 |
Alanine Aminotransferase (ALT), U/L | 61.60 ± 10.07 | 63.60 ± 23.6 | 55.20 ± 2.69* |
Urea, mmol/l | 6.40 ± 1.52 | 6.32 ± 1.38 | 6.52 ± 0.84 |
Creatinine, µmol/l | 81.20 ± 4.6 | 77.80 ± 5.15* | 76.0 ± 5.82* |
Total protein, g/l | 77.60 ± 4.53 | 77.0 ± 1.76 | 74.0 ± 2.48* |
Alkaline phosphatase, U/L | 83.60 ± 40.14 | 74.20 ± 26.81 | 64.20 ± 23.48* |
Glucose, mmol/l | 5.18 ± 0.7 | 4.88 ± 0.75 | 5.82 ± 0.76* |
* Statistically significant differences (t-test, p<0.05)
Table 1 – Body weight change (g)
No. of rats | Control group | Test group 1 (1000mg/kg) | Test group 2 (500mg/kg) |
Day 1 | |||
1 | 189 | 199 | 173 |
2 | 180 | 184 | 190 |
3 | 193 | 186 | 180 |
4 | 186 | 173 | 177 |
5 | 181 | 175 | 186 |
6 | 173 | 173 | 181 |
7 | 185 | 188 | 191 |
8 | 177 | 178 | 176 |
9 | 178 | 184 | 186 |
10 | 179 | 193 | 188 |
Mean ± SD | 182.1 ± 4.34 | 183.3 ± 6.22 | 182.8 ± 4.48 |
Day 14 | |||
1 | 213 | 250 | 197 |
2 | 206 | 217 | 216 |
3 | 208 | 236 | 203 |
4 | 211 | 202 | 197 |
5 | 215 | 203 | 216 |
6 | 203 | 201 | 209 |
7 | 209 | 220 | 234 |
8 | 219 | 207 | 203 |
9 | 195 | 215 | 225 |
10 | 215 | 224 | 217 |
Mean ± SD | 209.4 ± 4.96
| 217.5 ± 11.4*
| 211.7 ± 8.7
|
Day 28 | |||
1 | 227 | 260 | 221 |
2 | 222 | 227 | 233 |
3 | 215 | 255 | 210 |
4 | 231 | 222 | 237 |
5 | 241 | 227 | 213 |
6 | 219 | 218 | 218 |
7 | 217 | 244 | 246 |
8 | 228 | 226 | 217 |
9 | 210 | 222 | 245 |
10 | 231 | 239 | 236 |
Mean ± SD | 224.1 ± 6.6
| 234.0 ± 10.52*
| 227.6 ± 9.54
|
Day 42 | |||
1 | 246 | 267 | 233 |
2 | 253 | 242 | 241 |
3 | 249 | 280 | 216 |
4 | 234 | 235 | 254 |
5 | 271 | 234 | 226 |
6 | 236 | 223 | 227 |
7 | 249 | 259 | 257 |
8 | 260 | 231 | 238 |
9 | 231 | 239 | 258 |
10 | 246 | 248 | 243 |
Mean ± SD | 247.5 ± 8.71
| 245.8 ± 12.74
| 239.3 ± 10.14*
|
Day 56 | |||
1 | 255 | 274 | 250 |
2 | 253 | 247 | 242 |
3 | 249 | 293 | 230 |
4 | 234 | 247 | 266 |
5 | 271 | 245 | 238 |
6 | 236 | 230 | 237 |
7 | 249 | 262 | 265 |
8 | 260 | 238 | 248 |
9 | 231 | 240 | 267 |
10 | 246 | 260 | 251 |
Mean ± SD | 248.4 ± 8.86
| 253.6 ± 13.54*
| 249.4 ± 9.38
|
Day 70 | |||
1 | 264 | 273 | 253 |
2 | 268 | 252 | 259 |
3 | 259 | 302 | 236 |
4 | 245 | 254 | 274 |
5 | 291 | 240 | 250 |
6 | 250 | 224 | 242 |
7 | 256 | 266 | 273 |
8 | 261 | 208 | 252 |
9 | 231 | 253 | 253 |
10 | 253 | 269 | 260 |
Mean ± SD | 257.8 ± 11.25
| 254.1 ± 18.85
| 255.2 ± 8.59
|
Day 84 | |||
1 | 262 | 281 | 265 |
2 | 276 | 260 | 263 |
3 | 266 | 320 | 255 |
4 | 243 | 282 | 282 |
5 | 304 | 255 | 260 |
6 | 248 | 241 | 255 |
7 | 263 | 276 | 290 |
8 | 274 | 243 | 261 |
9 | 247 | 261 | 292 |
10 | 258 | 277 | 276 |
Mean ± SD | 264.1 ± 12.79
| 269.6 ± 16.56
| 269.9 ± 10.03*
|
Day 100 | |||
1 | 274 | 277 | 263 |
2 | 286 | 262 | 265 |
3 | 272 | 316 | 249 |
4 | 255 | 278 | 282 |
5 | 309 | 252 | 261 |
6 | 246 | 242 | 265 |
7 | 270 | 272 | 283 |
8 | 286 | 248 | 261 |
9 | 246 | 269 | 310 |
10 | 266 | 279 | 271 |
Mean ± SD | 271.0 ± 13.98
| 269.5 ± 15.02
| 271.0 ± 12.17
|
* Statistically significant differences (t-test, p<0.05)
Table 2 - Body weight gain (%)
No. of rats | Group | ||
Control group | Test group 1 (1000mg/kg) | Test group 2 (500mg/kg) | |
1 | 144,97 | 139,20 | 152,02 |
2 | 158,89 | 142,39 | 139,47 |
3 | 140,93 | 169,89 | 138,33 |
4 | 137,10 | 160,69 | 159,32 |
5 | 170,72 | 144,00 | 140,32 |
6 | 142,20 | 139,88 | 146,41 |
7 | 145,95 | 144,68 | 148,17 |
8 | 161,58 | 139,33 | 148,30 |
9 | 138,20 | 146,20 | 166,67 |
10 | 148,60 | 144,56 | 144,15 |
Mean ± SD | 148.91 ± 7.99
| 147.08 ± 7.25
| 148.27 ± 6.5
|
Table 3 - Organ mass ratios on the first day (day 101) after completion of test item administration (g/100 g), n=5
Organ | Control group | Dose, mg/kg | |
Test group 1 (1000 mg/kg) | Test group 2 (500 mg/kg) | ||
Liver | 3,27 ± 0,18 | 3,19 ± 0,36 | 3,27 ± 0,2 |
Kidneys | 0,56 ± 0,05 | 0,59 ± 0,08 | 0,58 ± 0,06 |
Spleen | 0,27 ± 0,04 | 0,26 ± 0,05 | 0,24 ± 0,02 |
Lungs | 0,7 ± 0,12 | 0,69 ± 0,14 | 0,68 ± 0,07 |
heart | 0,34 ± 0,04 | 0,34 ± 0,04 | 0,34 ± 0,01 |
Table 4 - Organ mass ratios at the tenth day (day 110) after completion of test item administration (g/100 g), n=5
Organ | Control group | Dose, mg/kg | |
Test group 1 (1000 mg/kg) | Test group 2 (500 mg/kg) | ||
Liver | 2,66 ± 0,1 | 2,76 ± 0,28 | 2,74 ± 0,2 |
Kidneys | 0,55 ± 0,06 | 0,63 ± 0,05* | 0,58 ± 0,05 |
Spleen | 0,23 ± 0,05 | 0,27 ± 0,03 | 0,25 ± 0,04 |
Lungs | 0,84 ± 0,32 | 0,84 ± 0,49 | 0,66 ± 0,11 |
heart | 0,37 ± 0,06 | 0,35 ± 0,04 | 0,38 ± 0,06 |
* Statistically significant differences (t-test, p<0.05)
Table 5 - Blood counts (haematological findings) on the first day (day 101) after completion of test item administration, n=5
Indicator | Control group | Doses, mg/kg | |
Test group 1 (1000 mg/kg) | Test group 2 (500 mg/kg) | ||
Haematocrit, % | 43.57 ± 1.04 | 43.86 ± 2.07 | 43.45 ± 3.09 |
Haemoglobin, g/l | 146.8 ± 5.15 | 148.2 ± 6.71 | 147.0 ± 11.34 |
Erythrocytes, 1012/l | 7.5 ± 0.33 | 7.61 ± 0.6 | 7.69 ± 0.49 |
Leucocytes, 109/l | 20.51 ± 6.12 | 15.72 ± 7.67* | 10.85 ± 2.56* |
Leukogram | |||
Stab neutrophils, % | 0.2 ± 0.56 | 0.0 ± 0.0 | 0.4 ± 1.11 |
Segmentonuclear neutrophils, % | 16.6 ± 7.73 | 14.4 ± 7.38 | 18.4 ± 10.15 |
Eosinophils, % | 3.2 ± 2.69 | 1.0 ± 2.15* | 1.4 ± 0.68* |
Monocytes, % | 5.6 ± 1.11 | 4.0 ± 2.63* | 3.6 ± 1.42* |
Lymphocytes, % | 74.4 ± 9.4 | 80.6 ± 4.17* | 76.2 ± 10.59 |
* Statistically significant differences (t-test, p<0.05)
Table 6 - Blood counts (haematological findings) at the tenth day (day 110) after completion of test item administration, n=5
Indicator | Control group | Doses, mg/kg | |
Test group 1 (1000 mg/kg) | Test group 2 (500 mg/kg) | ||
Haematocrit, % | 43.75 ± 1.94 | 44.07 ± 2.75 | 45.16 ± 2.04* |
Haemoglobin, g/l | 145.8 ± 9.31 | 147.6 ± 13.21 | 151.6 ± 7.98* |
Erythrocytes, 10/l12 | 7.75 ± 0.37 | 7.87 ± 0.54 | 7.95 ± 0.37 |
Leucocytes, 10/l9 | 15.9 ± 4.43 | 9.54 ± 3.8* | 11.64 ± 4.28* |
Leukogram | |||
Stab neutrophils, % | 0.0 ± 0.0 | 0.0 ± 0.0 | 0.0 ± 0.0 |
Segmentonuclear neutrophils, % | 10.6 ± 5.86 | 10.6 ± 2.86 | 10.6 ± 5.01 |
Eosinophils, % | 1.4 ± 1.42 | 0.4 ± 1.11* | 0.6 ± 1.11* |
Monocytes, % | 0.2 ± 0.56 | 1.0 ± 1.76* | 0.8 ± 1.04* |
Lymphocytes, % | 87.8 ± 6.88 | 88.0 ± 4.12 | 88.0 ± 6.21 |
* Statistically significant differences (t-test, p<0.05)
Table 7 – Clinical biochemistry results on the first day (day 101) after completion of test item administration, n=5
Indicator | Control group | Doses, mg/kg | |
Test group 1 (1000 mg/kg) | Test group 2 (500 mg/kg) | ||
Bilirubin total, μmol/l | 2.18 ± 0.38 | 2.18 ± 0.49 | 1.96 ± 0.34* |
Direct bilirubin, μmol/l | 0.84 ± 1.09 | 0.42 ± 0.16 | 0.36 ± 0.19* |
Aspartate Aminotransferase (AST), U/L | 148.2 ± 63.95 | 159.6 ± 39.64 | 131.0 ± 26.79 |
Alanine Aminotransferase (ALT), U/L | 72.4 ± 8.31 | 77.0 ± 12.78 | 76.2 ± 30.27 |
Urea, mmol/l | 7.04 ± 0.91 | 6.88 ± 1.32 | 6.82 ± 1.22 |
Creatinine, μmol/l | 69.6 ± 5.52 | 69.8 ± 4.34 | 70.4 ± 7.88 |
Total protein, g/l | 75.8 ± 3.66 | 73.8 ± 1.62* | 73.8 ± 2.22* |
Alkaline phosphatase, U/L | 110.8 ± 33.71 | 122.8 ± 39.12 | 133.0 ± 40.54* |
Glucose, mmol/l | 8.06 ± 1.66 | 7.5 ± 0.95 | 7.5 ± 0.88 |
* Statistically significant differences (t-test, p<0.05)
Table 8 - Clinical biochemistry results at the tenth day (day 110) after completion of test item administration, n=5
Indicator | Control group | Doses, mg/kg | |
Test group 1 (1000 mg/kg) | Test group 2 (500 mg/kg) | ||
Total Bilirubin, μmol/l | 2.92 ± 0.42
| 2.28 ± 0.44*
| 1.88 ± 0.24*
|
Direct bilirubin, μmol/l | 0.26 ± 0.07
| 0.26 ± 0.07
| 0.14 ± 0.07*
|
Aspartate Aminotransferase (AST), U/L | 162.6 ± 73.36
| 169.6 ± 122.26
| 162.4 ± 29.35
|
Alanine Aminotransferase (ALT), U/L | 75.0 ± 11.88
| 72.0 ± 15.08
| 65.0 ± 14.48*
|
Urea, mmol/l | 7.24 ± 0.47
| 5.8 ± 0.64*
| 6.5 ± 1.09*
|
Creatinine, µmol/l | 71.6 ± 5.24
| 66.2 ± 2.69*
| 73.2 ± 8.06
|
Total protein, g/l | 76.6 ± 4.78
| 74.8 ± 2.83
| 77.0 ± 2.32
|
Alkaline phosphatase, U/L | 96.0 ± 32.86
| 99.0 ± 30.32
| 78.4 ± 24.93*
|
Glucose, mmol/l | 6.06 ± 0.59
| 6.14 ± 0.64
| 6.66 ± 0.59*
|
* Statistically significant differences (t-test, p<0.05)
Table 1. Body weight gain and daily intake of SD rats treated orally with yeast hydrolysate (Notress)
| Group for subacute toxicityb | Satellite group for subacute toxicityc | ||
Control | Yeast hydrolysate | Control | Yeast hydrolysate | |
Female |
|
|
|
|
Body weight gain (g) | 44.83 ± 2.86 | 50.20 ±15.27 | 74.30 ± 6.98 | 88.70 ± 17.67 |
Food intake (g/day) | 17.25 ± 3.06 | 17.15 ± 3.85 | 18.13 ± 4.48 | 18.15 ± 3.79 |
Water intake (ml/day) | 41.18 ± 4.05 | 42.25 ± 2.21 | 43.32 ± 4.27 | 44.45 ± 3.98 |
Male |
|
|
|
|
Body weight gain (g) | 76.47 ± 8.11 | 90.33* ± 6.47 | 146.53 ± 9.55 | 159.07 ± 21.33 |
Food intake (g/day) | 20.85 ± 4.14 | 20.25 ± 4.05 | 23.98 ± 4.45 | 23.78 ± 3.33 |
Water intake (ml/day) | 43.22 ± 3.24 | 44.47 ± 5.02 | 48.26 ± 4.29 | 46.62 ± 4.10 |
Values are means ± SD for 5 rats/group. bThe group for subacute toxicity was given water vehicle or yeast hydrolysate at 1000 mg/kg daily for 14 days. cSatellite group for subacute toxicity was given water vehicle or yeast hydrolysate at 1000 mg/kg daily for 14 days followed by no treatment for 14 days. The differences between the control and treated groups were evaluated by Student’s t-test.
* Statistically significant (p<0.05)
Table 2. Hematological parameters of SD rats treated orally with yeast hydrolysate (Notress)
Hematological parameters | Group for subacute toxicitya | Satellite group for subacute toxicityb | ||
Control | Yeast hydrolysate | Control | Yeast hydrolysate | |
Female |
|
|
|
|
RBC (×106/µl) | 7.33 ± 0.52 | 7.48 ± 0.92 | 7.47 ± 0.30 | 7.87 ± 0.24 |
WBC (×103/µl) | 8.57 ± 1.55 | 8.08 ± 1.58 | 8.00 ± 1.34 | 7.11 ± 0.90 |
Hct (%) | 43.77 ± 3.75 | 43.27 ± 3.87 | 44.73 ± 2.74 | 39.80 ± 1.13 |
Hgb (g/dl) | 13.51 ± 1.52 | 14.42 ± 2.74 | 14.50 ± 0.87 | 13.60 ± 0.26 |
MCV (fl) | 56.77 ± 1.45 | 59.82 ± 3.75 | 59.83 ± 1.37 | 58.17 ± 1.74 |
MCH (pg) | 18.34 ± 0.58 | 17.79 ±1.22 | 19.33 ± 0.50 | 20.00 ± 0.53 |
MCHC (g/dl) | 31.12 ± 1.23 | 33.09 ± 1.22 | 32.37 ± 0.21 | 33.97 ± 0.68 |
Platelets (×103/µl) | 845.26 ± 54.42 | 856.41 ± 53.39 | 728.00 ± 41.52 | 821.45 ± 51.11 |
Male |
|
|
|
|
RBC (×106/µl) | 7.06 ± 0.78 | 7.62 ± 1.09 | 7.96 ± 0.38 | 7.91 ± 0.21 |
WBC (×103/µl) | 11.21 ± 1.02 | 11.92 ± 1.20 | 10.47 ± 0.31 | 10.87 ± 1.41 |
Hct (%) | 45.15 ± 3.35 | 47.12 ± 2.48 | 47.25 ± 2.35 | 45.55 ± 0.95 |
Hgb (g/dl) | 13.99 ± 0.98 | 13.94 ± 1.28 | 14.97 ± 0.21 | 14.80 ± 0.10 |
MCV (fl) | 57.78 ± 1.24 | 57.77 ± 2.25 | 58.93 ± 1.11 | 58.47 ± 0.55 |
MCH (pg) | 17.49 ± 0.52 | 17.59 ± 1.36 | 18.50 ± 0.52 | 18.70 ± 0.44 |
MCHC (g/dl) | 31.07 ± 1.01 | 31.24 ± 1.13 | 31.27 ± 0.21 | 32.30 ± 0.66 |
Platelets (×103/µl) | 721.42 ± 58.87 | 742.49 ± 66.03 | 692.67 ± 60.34 | 707.89 ± 55.47 |
Values are means ± SD for 5 rats/group. aThe group for subacute toxicity was given water vehicle or yeast hydrolysate at 1,000 mg/kg daily for 14 days. bThe satellite group for subacute toxicity was given water vehicle or yeast hydrolysate at 1,000 mg/kg daily for 14 days followed by no treatment for 14 days. RBC; red blood cell, WBC; white blood cell, Hct; hematocrit, Hgb; hemoglobin, MCV; mean corpuscular volume, MCH; mean corpuscular hemoglobin, MCHC; mean corpuscular hemoglobin concentration. The differences between the control and treated groups were evaluated by Student’s t-test.
Table 3. Blood biochemical parameters of SD rats treated orally with yeast hydrolysate (Notress)
Blood biochemical parameters | Group for subacute toxicitya | Satellite group for subacute toxicityb | ||
Control | Yeast hydrolysate | Control | Yeast hydrolysate | |
Female |
|
|
|
|
Glucose (mg/dl) | 89.67 ± 8.02 | 91.50 ± 1.50 | 114.00 ± 9.70 | 112.33 ± 7.02 |
BUN (mg/dl) | 8.57 ± 0.50 | 12.57 ± 1.31** | 13.40 ± 1.40 | 15.47 ± 0.55 |
Creatinine (mg/dl) | 0.30 ± 0.01 | 0.33 ± 0.06 | 0.27 ± 0.05 | 0.27 ± 0.06 |
Total protein (g/dl) | 5.27 ± 0.06 | 6.10 ± 0.10 | 5.81 ± 0.13 | 6.20 ± 0.26 |
Albumin (g/dl) | 3.53 ± 0.12 | 3.93 ± 0.06 | 3.79 ± 0.15 | 4.00 ± 0.10 |
Total bilirubin (mg/dl) | 0.47 ± 0.06 | 0.57 ± 0.12 | 0.58 ± 0.08 | 0.77 ± 0.15 |
AST (U/l) | 85.33 ± 5.08 | 94.33 ± 8.39 | 90.00 ± 6.97 | 94.67 ± 1.53 |
ALT (U/l) | 27.67 ± 4.16 | 28.33 ± 3.51 | 29.33 ± 4.18 | 30.00 ± 2.65 |
Male |
|
|
|
|
Glucose (mg/dl) | 102.00 ± 8.89 | 105.00 ± 13.45 | 118.67 ± 4.04 | 111.33 ± 11.02 |
BUN (mg/dl) | 7.03 ± 0.32 | 11.50 ± 0.70** | 13.00 ± 0.56 | 12.87 ± 1.23 |
Creatinine (mg/dl) | 0.30 ± 0.01 | 0.33 ± 0.06 | 0.30 ± 0.01 | 0.23 ± 0.06 |
Total protein (g/dl) | 5.40 ± 0.20 | 5.77 ± 0.06 | 5.60 ± 0.32 | 5.80 ± 0.10 |
Albumin (g/dl) | 3.50 ± 0.01 | 3.80 ± 0.10 | 3.53 ± 0.06 | 3.73 ± 0.06 |
Total bilirubin (mg/dl) | 0.47 ± 0.06 | 0.47 ± 0.07 | 0.47 ± 0.06 | 0.53 ± 0.06 |
AST (U/l) | 95.00 ± 5.01 | 104.67 ±6.43 | 96.33 ± 8.13 | 101.33 ± 7.08 |
ALT (U/l) | 37.33 ± 2.16 | 34.00 ± 1.73 | 41.67 ± 2.58 | 42.33 ± 3.21 |
Values are means ± SD for 5 rats/group. aThe group for subacute toxicity was given water vehicle or yeast hydrolysate at 1,000 mg/kg daily for 14 days. bThe satellite group for subacute toxicity was given water vehicle or yeast hydrolysate at 1,000 mg/kg daily for 14 days followed by no treatment for 14 days. BUN; lood urea nitrogen, AST; aspartate aminotransferase, ALT; alanine aminotransferase. **Significantly different from control, p<0.01. The differences between the control and treated groups were evaluated by Student’s t-test.
Table 4. Relative organ weights of SD rats treated orally with yeast hydrolysate (Notress)
Relative organ weight (g/100 g of body weight) | Group for subacute toxicityb | Satellite group for subacute toxicityc | ||
Control | Yeast hydrolysate | Control | Yeast hydrolysate | |
Female |
|
|
|
|
Liver | 3.77 ± 0.16 | 3.31 ± 0.31 | 3.72 ± 0.20 | 3.12 ± 0.39 |
Kidney | 0.89 ± 0.05 | 0.85 ±0.09 | 0.84 ± 0.11 | 0.82 ± 0.09 |
Spleen | 0.26 ± 0.03 | 0.26 ± 0.03 | 0.23 ± 0.02 | 0.20 ± 0.04 |
Lung | 0.48 ± 0.03 | 0.45 ± 0.05 | 0.42 ± 0.04 | 0.44 ± 0.05 |
Heart | 0.34 ± 0.01 | 0.39 ± 0.03 | 0.30 ± 0.01 | 0.35 ± 0.05 |
Ovary | 0.06 ± 0.01 | 0.07 ± 0.05 | 0.05 ± 0.01 | 0.05 ± 0.01 |
Male |
|
|
|
|
Liver | 3.80 ± 0.13 | 3.21 ± 0.13 | 3.37 ± 0.10 | 3.23 ± 0.09 |
Kidney | 0.87 ± 0.07 | 0.87 ± 0.09 | 0.82 ± 0.01 | 0.79 ± 0.05 |
Spleen | 0.24 ± 0.01 | 0.26 ± 0.03 | 0.20 ± 0.01 | 0.24 ± 0.02 |
Lung | 0.41 ± 0.02 | 0.45 ± 0.05 | 0.36 ± 0.04 | 0.38 ± 0.05 |
Heart | 0.34 ± 0.01 | 0.39 ± 0.04 | 0.36 ± 0.01 | 0.32 ± 0.04 |
Testis | 0.71 ± 0.07 | 0.73 ± 0.09 | 0.68 ± 0.05 | 0.72 ± 0.06 |
Values are means ± SD for 5 rats/group. aThe group for acute toxicity was given water vehicle or yeast hydolysate at 5,000 mg/kg once followed by no treatment for 14 days. bThe group for subacute toxicity was given water vehicle or yeast hydrolysate at 1,000 mg/kg daily for 14 days. cSatellite group for subacute toxicity was given water vehicle or yeast hydrolysate at 1,000 mg/kg daily for 14 days followed by no treatment for 14 days. The differences between the control and treated groups were evaluated by Student’s t-test.
Table 1. Effect of Saccharomyces cerevisiae RC016 on total weight gain (TWG), progressive weight gain (PWG), feed conversion (FC) and feed efficiency (FE) in rats administered S. cerevisiae RC016 and Uncontaminated feed control rats
| Production parameters (mean ± SE) | |||
Test group | TWG (g) | PWG (g) | FC index | FE index |
Uncontaminated feed control | 79.17 ± 7.31 | 3.22 1.42 | 0.41 0.04 | 2.64 0.21 |
S. cerevisiae RC016 | 83.33 ± 7.31 | 3.58 1.28 | 0.37 0.03 | 2.78 0.23 |
SE, standard error.
TWG: difference between initial and final weight of animals
PWG: weight changes calculated through the experiment, weighing animals three times a week
FC: TWG/ daily feed intake
FE: daily feed intake/TWG
Table 1. Percentage compositions (dry basis) of yeast biomass (YBM)
Component | YBM |
Protein (% N_5.5) | 47.19 |
RNA | 7.04 |
Ash | 8.55 |
Soluble fibre | 9.65 |
Insoluble fibre | 2.57 |
Total lipids | 3.53 |
Total carbohydrate | 21.52 |
Table 2. Diet consumption (DC), protein consumption (PC) and body weight gain (BWG) of rats fed diets containing 15 or 30% protein from yeast biomass (YBM 15, 30)
Protein source | DCa | PCa | BWGa |
YBM 15 | 1254.76 ± 60.55 | 193.86 ± 9.36 | 259.67 ± 19.04 |
YBM 30 | 1229.50 ± 52.55 | 359.14* ± 15.35 | 255.05 ± 23.31 |
a Values given in grams for the 90day period.
*statistically different results (p<0.05) among treatments.
Table 3. Blood serum levels of transaminases, creatinine, urea and uric acid of rats fed diets containing 15 or 30% protein from yeast biomass (YBM 15, 30) after 45 and 90 days of feeding
| TGO (UI-1) | TGP (UI-1) | Creatinine (mg dl-1) | Urea (mg dl-1) | Uric acid (mg dl-1) | |||||
Protein source | 45d | 90d | 45d | 90d | 45d | 90d | 45d | 90d | 45d | 90d |
Control | 67.58 ± 10.64 | 67.58 ± 10.64 | 18.34 ± 1.83 | 18.34 ± 1.83 | 0.90 ± 0.05 | 0.90 ± 0.05 | 12.48 ± 1.62 | 12.48 ± 1.62 | 1.45 ± 0.58 | 1.45 ±0.58 |
YBM 15 | 71.76 ± 3.26 | 52.76 ** ± 4.93 | 15.91 ± 0.98 | 11.28** ± 2.33 | 1.40 ± 0.13 | 2.15** ± 0.09 | 13.95 ± 2.66 | 14.73** ± 4.12 | 3.12± 0.55 | 3.35 ± 0.52 |
YBM 30 | 71.39 ± 2.15 | 49.36 ** ± 6.94 | 18.10 ± 1.27 | 9.50** ± 3.26 | 1.35 ± 0.11 | 2.06** ± 0.13 | 14.43 ± 1.51 | 17.23** ± 3.69 | 5.36 * ± 1.89 | 2.84** ± 0.18 |
* Statistically different results (p<0.05) among treatments; ** statistically different results (p<0.05) within treatment as a function of feeding time; TGO, glutamic-oxalacetic transaminase; TGP, glutamic-pyruvic transaminase.
Table 4. Urinary levels of creatinine, urea and uric acid of rats fed diets containing 15 or 30% protein from yeast biomass (YBM 15,30) after 45 and 90 days of feeding
| Creatinine (gl-1) | Urea (mg per 24h) | Uric acid (mg per 24h) | |||
Protein source | 45d | 90d | 45d | 90d | 45d | 90d |
YBM 15 | 1.16 ± 0.11 | 1.09 ± 0.47 | 18.04 ± 3.69 | 11.65** ±2.46 | 52.07 ± 13.891 | 106.89** ± 2.41 |
YBM 30 | 1.26 ± 0.04 | 1.29 ± 0.50 | 18.86 ± 2.29 | 27.88* ± 1.37 | 82.32* ± 9.46 | 114.58 **± 5.63 |
* Statistically different results (p<0.05) among treatments; ** statistically different results (p<0.05) within treatment as a function of feeding time
Table 5. Liver weight (g fresh tissue) and total liver protein and fat concentrations (% dry basis) of rats fed 15 or 30% dietary protein from yeast biomass (YBM 15, 30) after 45 and 90 days of feeding
| Liver weight (g) | Liver/body weight ratio (%) | Liver total protein (%) | Liver total fat (%) | ||||
Protein source | 45d | 90d | 45d | 90d | 45d | 90d | 45d | 90d |
YBM 15 | 10.24 ± 1.19 | 9.86 ±1.79 | 3.96 ± 0.35 | 3.17 ± 0.48 | 25.93 ± 0.15 | 22.94 ± 1.01 | 7.08 ± 0.19 | 11.05 ± 0.75
|
YBM 30 | 10.23 ± 0.97 | 10.18 ± 0.98 | 4.17 ± 0.15 | 3.34 ± 0.38 | 28.19 ± 0.62 | 26.46 ± 0.44 | 7.11 ± 0.45 | 9.02 ± 0.21 |
* Statistically different results (p<0.05) among treatments
Table 6. Kidney weight (g fresh tissue) and total kidney protein and fat concentrations (% dry basis) of rats fed 15 or 30% dietary protein from yeast biomass (YBM 15, 30) after 45 and 90 days of feeding
| Kidney weight (g) | Kidney/body weight ratio (%) | Kidney total protein (%) | Kidney total fat (%) | ||||
Protein source | 45d | 90d | 45d | 90d | 45d | 90d | 45d | 90d |
YBM 15 | 1.74 ± 0.13 | 1.83 ± 0.14 | 0.68 ± 0.05 | 0.59 ± 0.03 | 18.86 ± 0.06 | 19.14* ± 0.07 | 5.75 ± 0.46 | 5.37 ± 0.42 |
YBM 30 | 1.72 ± 0.16 | 1.89 ± 0.26 | 0.70 ± 0.03 | 0.62 ± 0.06 | 16.91 ± 0.02 | 17.94 ± 0.04 | 5.91 ± 0.72 | 5.84 ± 0.25
|
* Statistically different results (p<0.05) among treatments
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
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 1 000 mg/kg bw/day
- Study duration:
- subacute
- Species:
- rat
- Quality of whole database:
- Reliability 1 and 2 studies relevant for assessment.
Repeated dose toxicity: inhalation - systemic effects
Endpoint conclusion
- Endpoint conclusion:
- no study available
Repeated dose toxicity: inhalation - local effects
Endpoint conclusion
- Endpoint conclusion:
- no study available
Repeated dose toxicity: dermal - systemic effects
Endpoint conclusion
- Endpoint conclusion:
- no study available
Repeated dose toxicity: dermal - local effects
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Justification for classification or non-classification
Based on all the reliable repeated dose toxicity data available on the target substance Saccharomyces cerevisiae, lysate (key study, Hargitai, Klimisch 1, 2018, GLP, OECD TG 422, subacute NOAEL ≥ 1000 mg/kg bw/day), as well as on the various Saccharomyces cerevisiae derivative substances or the whole Saccharomyces cerevisiae yeast organism (ranging from subacute to chronic), it is concluded on a weight of evidence basis that no toxicity upon repeated exposure is expected for the registered substance Saccharomyces cerevisiae, lysate. In the absence of adverse effects, no STOT-RE classification is required for Saccharomyces cerevisiae, lysate substance according to CLP criteria.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
