Bos taurus, milk, pasteurized, homogenized, skimmed, fermented, spray-dried

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Not specifed

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non-GLP, near guideline, published in peer reviewed literature, adequate for assessment

Justification for type of information:

The test material used to test the toxicological properties described in the study used for this RSS is not exactly the same as Bos taurus, milk, pasteurized, homogenized, skimmed, fermented, spray-dried. However, the process used to prepare ‘Powdered Lactobacillus helveticus-fermented milk’ (FM) is qualitatively similar to that used in the production of ‘Bos taurus, milk, pasteurized, homogenized, skimmed, fermented, spray dried’ and is based on the same natural material (cow milk) and biological production process (bacterial fermentation). The components that are additionally removed from FM, lactic acid and casein, occur naturally in milk and milk that has been naturally exposed to microorganisms and are not expected to have important toxicological effects regarding acute oral toxicity, repeated dose oral toxicity, reproduction, or genotoxicity at the concentrations present in fermented milk products. Therefore, the toxicological properties reported on this study can be used in combination with the long historical safe consumption of yogurt and yogurt-derived products by humans in a weight of evidence analysis to assess the toxicological properties of 'Bos taurus, milk, pasteurized, homogenized, skimmed, fermented, spray dried' for this endpoint.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

publication

Title:

Studies of the Toxicological Potential of Tripeptides: III. Single- and/or Repeated-Dose Toxicity of Tripeptides-Containing Lactobacillus helveticus–Fermented Milk Powder and Casein Hydrolysate in Rats

Author:

Maeno M, Nakamura Y, Mennear JH, Bernard BK

Year:

2005

Bibliographic source:

International Journal of Toxicology, 24(Suppl. 4):13–23

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

not specified

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

powdered L. helveticus–fermented milk

IUPAC Name:

powdered L. helveticus–fermented milk

Test material form:

solid

Details on test material:

Reconstituted skim milk (9%, w/w) was pasteurized and fermented with L. helveticus CM4 at 37°C for 22 h. Casein was removed by centrifugation and lactic acid was eliminated from the supernatant by electrodialysis. The residual supernatant was converted to fermented milk whey powder by using maltodextrin as a bulking agent and spray drying.

Specific details on test material used for the study:

Reconstituted skim milk (9%, w/w) was pasteurized and fermented with L. helveticus CM4 at 37°C for 22 h. Casein was removed by centrifugation and lactic acid was eliminated from the supernatant by electrodialysis. The residual supernatant was converted to fermented milk whey powder by using maltodextrin as a bulking agent and spray drying.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

(Crj:CD(SD), SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
- Age at study initiation: 6 to 7 weeks old
- Weight at study initiation: 140 to 201 g (males) and 112 to 164 g (females)
- Housing: individually in metal cages
- Diet: ad libitum pelleted CRF; Oriental Yeast Co., Ltd, or Rodent diet 5002; PMI Inc.
- Water: tap water ad libitum
- Acclimation period: 1 week

DETAILS OF FOOD AND WATER QUALITY:
Drinking water and food were routinely analyzed for contaminants. Analyses revealed no evidence of contamination that either compromised or influenced the outcome of the studies.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12h/12h

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: grossly observable clinical signs
- Time schedule: Daily at the time of dosing and 2 to 4 h later

BODY WEIGHT: Yes
- Time schedule for examinations: on dosing days 1, 8, 15, 22, and 28, as well as on the day of necropsy (after an overnight fast).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal was determined twice weekly throughout the study

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: During week 4
- Dose groups that were examined: all. Half of the animals in each group received a mydriatic to facilitate examination of the intermediate optic media and fundus.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 28
- Anaesthetic used for blood collection: Yes, ethyl ether
- Animals fasted: Yes, overnight
- Animals euthanized by exsanguination

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 28
- Animals fasted: Yes, overnight

URINALYSIS: Yes
- Time schedule for collection of urine: during week 4
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Not specified
- Parameters checked: sediment and color, pH, protein, glucose, ketone bodies, urobilinogen, bilirubin, and occult blood measured using a test paper method. Twentyfour-hour samples were used for determination of volume, specific gravity and electrolyte excretion

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, major organs were weighed

HISTOPATHOLOGY: Yes. Approximately 35 organs and tissues from each animal were fixed
in 10% formalin. Organs from all control and high-dose group animals were embedded, sectioned, stained with hematoxylineosin, and examined microscopically
Because of the presence of eosinophilic bodies in the proximal tubular epithelium of kidneys
from two high-dose males, kidneys from mid-dose males were also processed and examined microscopically

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

increases (3.8% to 5.2%) in hematocrit and hemoglobin concentrations in the low- and mid-dose male groups. There was no indication of similar changes in the highdose rats of either sex. Lack of evidence of a dose-response relationship indicates that these small changes are not related to
powdered FM administration.

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Some male rats exhibited proteinuria and ketonuria, but these small changes were observed in both control and treated animals. The presence of white blood cells and epithelial cells in the urinary sediment was similar in dosed and control rats. Urinary volumes and excretion of electrolytes
were similar in all groups of rats.

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

1 male rat from highest dose had a swollen right lateral lobe and the quadrate lobe of the liver had adhesions to the diaphragm.
1 male rat from highest dose had grossly observable white spots on the surface of a kidney.

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Effect levels

1

Dose descriptor:

other: maximally tolerated dose

Effect level:

> 2 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

2

Key result

Dose descriptor:

LOEL

Effect level:

> 2 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Administration of powdered fermented milk (FM) for 28 consecutive days produced no signs of toxicity and resulted in the conclusion that the LOEL must be greater than 2000 mg/kg BW/day
According to the considerations previously listed on the field "justification for type of information", it can be concluded that "Bos taurus, milk, pasteurized, homogenized, skimmed, fermented, spray-dried" (EC No. 917-734-0) would likewise have a LOEL greater that 200 mg/kg BW day for rats, after 28 days consecutive oral exposure.

Executive summary:

Powdered Lactobacillus helveticus–fermented milk (FM) was administered by oral gavage Sprague- Dawley rats. 0, 500, 1000, or 2000 mg/kg body weight [BW]/day was administered by gastric gavage to male and female rats for 28 consecutive days.

Antemortem evaluative parameters included gross observations, ophthalmic examinations, and clinical pathology (clinical chemistry, hematology, and urinalysis). Post mortem parameters included necropsy, determination of organ weights, and

microscopic examination of major organs. There was neither inlife nor postmortem evidence that powdered FM administration

caused physiological or toxicological changes.

There was no evidence to support establishment of either the LOEL or MTD; both being greater than 2000 mg/kg/day for up to 28 consecutive days.

The process used to prepare ‘Powdered Lactobacillus helveticus-fermented milk’ (FM) is qualitatively similar to that used in the production of ‘Bos taurus, milk, pasteurized, homogenized, skimmed, fermented, spray dried’ and is based on the same natural material (cow milk) and biological production process (bacterial fermentation).The components that are additionally removed from FM, lactic acid and casein, occur naturally in milk and milk that has been naturally exposed to microorganisms and are not expected to have important toxicological effects regarding acute oral toxicity, repeated oral dose toxicity,

reproduction, or genotoxicity at the concentrations present in fermented milk products.

Therefore, taking into account the previous considerations, it is safe to assume that the LOEL for repeated exposure of rats during 28 consecutive days to 'Bos taurus, milk, pasteurized, homogenized, skimmed, fermented, spray dried' would also be greater than 2000 mg/kg bw day.