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

Endpoint summary

Description of key information

- Long history of human exposure to dairy products including yoghurt, without signs of chronic toxicity.

- Chronic 400 mg/kg BW ingestion of Yogurt is considered safe for humans. Therefore, by extrapolation, a sub-acute ingestion of 2400 mg/kgBW yoghurt is considered safe in humans.

- 28-days repeated dose toxicity on rats (Fermented Milk LOEL > 2000 mg/kg BW)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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.

Reason / purpose for cross-reference:

reference to same study

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

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.

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

Route of administration:

oral: gavage

Vehicle:

water

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

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:

10

Control animals:

yes, concurrent vehicle

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

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

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

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

Key result

Critical effects observed:

no

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.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

calculation (if not (Q)SAR)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Calculation based on various sources of information and common knowledge.

Justification for type of information:

A sub-acute exposure of 2’400 mg/kg body weight (bw) of the substance Bos taurus, milk, pasteurized, homogenized, skimmed, fermented, spray-dried (EC 917-734-0) is considered safe in humans. Therefore, animal testing of the oral, sub-acute toxicity of the substance is not necessary.
Reasoning:
The Swiss Food Pyramid proposes a daily consume of 200 g Yoghurt per day (BLV, 2017). Under the assumption of a water content of 86 % of yoghurt, the dry substance of yoghurt and therefore yoghurt powder is 14 % of wet weight of yoghurt (BLV, 2018). This results in a proposed intake of 28 g dry weight of yoghurt powder per day. Applied to an average person (70 kg) results in a daily intake of yoghurt of 400 mg/kg bw/day. This value represents a safe, chronic intake of yoghurt per day without adverse effects.
For the derivation of a chronic Derived No Effect Level (DNEL) for human health, the ECHA provides guidance on the extrapolation from shorter studies to chronic studies (ECHA, 2012). For the extrapolation of a sub-acute study to a chronic study ECHA proposes an assessment factor of 6 to more serious adverse effects with increasing exposure times into account. The inverse conclusion suggests that the extrapolation of a chronic duration to a sub-acute duration with a factor of 6 is also possible. Under these circumstances a sub-acute exposure to yoghurt powder of 2’400 mg/kg bw can be considered safe for humans.
The same value can be assumed for the exposure of animals to yoghurt powder. Since, according to the OECD guidelines the highest tested concentrations in rats for sub-acute oral toxicity should be 1’000 mg/kg bw, testing the sub-acute oral toxicity of yoghurt powder is not considered necessary.
Sources:
Bundesamt für Lebensmittelsicherheit und Veterinärwesen (BLV). Fachinformation Ernährung: Milch- und Milchproduktekonsum in der Schweiz 2014/2015. März 2017.
Bundesamt für Lebensmittelsicherheit und Veterinärwesen. Schweizer Nährwertdatenbank, Lebensmittelsuche für Joghurt. State of knowledge: 04.05.2018.
European Chemicals Agency (ECHA). Guidance on information requirements and chemical safety assessment: Chapter R.8: Characterization of dose [concentration]-response for human health. Version 2.1. November 2012.

Qualifier:

no guideline required

Principles of method if other than guideline:

See Field Justification for type of information

Key result

Dose descriptor:

NOAEL

Effect level:

> 2 400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Conclusions:

An sub-acute exposure of 2’400 mg/kg body weight (bw) of the substance Bos taurus, milk, pasteurized, homogenized, skimmed, fermented, spray-dried (EC 917-734-0) is considered safe in humans.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

2 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Justification for classification or non-classification

There is a long history of human exposure to dairy products including yoghurt, and there have been no reports or signs of toxicity due to chronic yoghurt consumption.

Moreover, based on dietary recomendations chronic ingestion of 400 mg dry weight/kg body weight (bw) per day of yoghurt is considered safe in humans. Therefore, by extrapolation, sub-acute ingestion of 2400 mg/kgBW of yoghurt can be considered safe in humans.

A repeated dose toxicity test on rats, exposed during 28 days to up to 2000 mg/kg BW fermented milk demostrated no toxic effects. The test material used to test the toxicological properties described in the mentioned study 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 oral dose 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 on a weight of evidence analysis to asses the toxicological properties of Bos taurus, milk, pasteurized, homogenized, skimmed, fermented, spray-dried for this endpoint.