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

Acute Toxicity: dermal

Administrative data

Endpoint:

acute toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because the physicochemical and toxicological properties suggest no potential for a significant rate of absorption through the skin

Justification for type of information:

As stated in COMMISSION REGULATION (EU) 2016/863, amending Annexes VII and VIII to Regulation (EC) No 1907/2006:
"Recent scientific analysis of available data from in vivo acute toxicity studies have shown that substances that are not toxic via the oral route may be expected with high certainty to be also non-toxic via the dermal route. Therefore, testing those substances via the dermal route does not provide essential information for their safety assessment."

As shown in the acute oral toxicity endpoint, the substance is not toxic via the oral route. In the toxikokinetic assessment it is explained why the substance is not easily absorbed through skin, see cross-refs. below.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

acute 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 and common knowledge

Justification for type of information:

An 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, 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. In case of an acute (instead of sub-acute) duration, this extrapolation can be even considered as a conservative approach. Under these circumstances an 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 acute oral toxicity should be 2’000 mg/kg bw, testing the 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

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 400 mg/kg bw

Interpretation of results:

GHS criteria not met

Conclusions:

An 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.

Cross-reference 2

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

not specified

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 423 (Acute Oral toxicity - Acute Toxic Class Method)

Deviations:

not specified

GLP compliance:

not specified

Test type:

acute toxic class method

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

Remarks:

(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

Doses:

2000 mg/kg bw
4000 mg/kg bw

No. of animals per sex per dose:

5

Control animals:

yes

Details on study design:

Powdered FM was suspended in water (5%, 10%, or 20%) and the suspension shaken vigorously until the contents showed no evidence of test article sedimentation.
Animals were fasted overnight prior to administration of the solution/suspension and for approximately 3 h after dosing.
Rats administered the single doses of powdered FM were observed for grossly observable clinical signs for 6 h after dosing, then once daily for 14 consecutive days. Individual animal body weights were determined on the day of dosing and on days 2, 4, 7, and 14 after dosing. On experimental day 14, all rats were anesthetized (sodium thiopental or ether anesthesia) and exsanguinated, then subjected to detailed necropsy. In the absence of abnormalities observed at necropsy, histopathology evaluations were not conducted.

Statistics:

Quantitative data were analyzed for homogeneity of variance using Bartlett’s test (Sokal and Rohlf 1969a). Homogeneous data were subjected to one-way analysis of variance and differences between dosed and control groups were assessed for statistical significance with Dunnett’s test (Dunnett 1955). Heterogeneous data were subjected to Kruskal-Wallis’ test (Sokal and Rohlf 1969b) and differences between dosed and control groups were assessed with Dunnett’s rank test (Dunnett 1955).

Key result

Sex:

male/female

Dose descriptor:

other: single-dose LOEL

Effect level:

> 4 000 mg/kg bw

Based on:

test mat.

Mortality:

no mortality observed

Clinical signs:

no effects observed

Body weight:

no effects observed

Gross pathology:

no effects observed

Other findings:

A single female dosed with 4000 mg/kg powdered FM had soft stool 4 h after dosing; however, soft stools and perianal fecal smudging were observed sporadically in dosed as well as control rats throughout the 2-week, postdosing observation period.

Interpretation of results:

GHS criteria not met

Conclusions:

Single-dose oral administration of 4000 mg/kg of powdered FM caused no evidence of either systemic or local toxicity in male or female rats (e.g., behavioral observations; clinical signs; food consumption; body weight gains; ophthalmologic examinations; clinical pathology [hematology, clinical chemistry]; urinalysis).
According to the considerations previously listed on the field "justification for type of information", it can be concluded that a single dose of 4000 mg/kg "Bos taurus, milk, pasteurized, homogenized, skimmed, fermented, spray-dried" (EC No. 917-734-0) would likewise not cause either systemic or local toxicity in male or female rats.

Executive summary:

The objective of this study was to assess the toxicological potential of powdered Lactobacillus helveticus–fermented milk (FM). All test articles were administered by oral gavage to male and female Sprague-Dawley rats. Specific goal of the single-dose study was to identify doses that produce evidence of systemic and/or local (i.e., gastrointestinal) toxicity (e.g., lowest-observable effect level [LOEL]). Single doses of powdered FM (2000 or 4000 mg/kg) were administered 14 days prior to study termination. No treatment regimen caused either antemortem (gross observations, body weight, and food consumption parameters) or postmortem (necropsy) evidence of either systemic or local toxicity. Under the conditions of these experiments, the single-dose LOEL of powdered FM was found to be greater than 4000 mg/kg bw.

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 single-dose LOEL of 'Bos taurus, milk, pasteurized, homogenized, skimmed, fermented, spray dried' would also be greater than 4000 mg/kg bw.

Cross-reference 3

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

The substance Bos taurus, milk, pasteurized, homogenized, skimmed, fermented, spray-dried (EC 917-734-0) is a UVCB substance originating from biochemical changes produced in milk by various species of bacteria or yeast, where the main constituents are raw proteins (approx. 34.1% w/w), lactose (approx. 40.3% w/w) and lactic acid (=2.5 - =5% w/w); minor constituents include water (3 – 10% w/w), raw ash (inorganic constituents) (>3 – <10% w/w), raw fat (>0.25 – <1.5% w/w), and other carbohydrates (=2 – =3% w/w). Raw ash contains calcium (=7 – =26% w/w), sodium (=0 - =13% w/w), potassium (=13 - =26% w/w) and magnesium (=0 - =13% w/w). The proteins consist mainly of caseines (80%), serum albumines, beta-lactoglobulines, immunoglobulines and lactalbumines.

After ingestion, raw proteins are generally broken down into amino acids through denaturation of the protein in the acidic environment of the stomach and hydrolysis by proteases in the intestine. They are absorbed from the intestine and distributed systemically across various compartments of the body. Since amino acids are of natural origin and form an essential part of the normal human diet, no further consideration of the toxicokinetics of raw protein is necessary

Likewise, ingested lactose is digested through the enzyme lactase, which is expressed in the brush border membrane of enterocytes in the small intestine, and cleaved into the sugar monomers, glucose and galactose which are taken up in the small intestine (Hampson et. al., 1986). Therefore, it is absorbed and distributed as glucose and galactose. Based on the long history dietary human exposure to lactose, the substance is not acute orally toxic and the evaluation of the toxicokinetics of lactose is not considered necessary. Furthermore, lactose is exempted from REACH registration requirements according to REACH Annex IV (substances included in Annex IV: sufficient information is known about these substances that they are considered to cause minimum risk because of their intrinsic properties) and can thus be considered to have non-critical properties.

Due to the low volatility (lactose: <0.1 Pa at 25°C, lactic acid: 11 Pa at 25°C) the main constituents of the substance are not present in a gaseous state and gas-phase absorption through inhalation is therefore highly unlikely at normal ambient temperatures. The 10th percentile and median size of the particles of the substance (D10 = 75.3 µm and D50 = 127.5 µm, mass based) indicates that the large majority of the particles are too big to be inhaled. If some portion of the substance should reach the respiratory tract, the main constituents could cross the alveolar and capillary membranes by a combination of active and passive uptake mechanisms. Additionally, there is human data on the respiratory effects of occupational exposure to milk powder available which, can be used as an analog to provide evidence for yoghurt powder. Although there were no signs of systemic toxicity, increased risk of nasal symptoms, wheezing, breathlessness and reduced spirometric lung function were observed; based on these findings the substance was classified as a Category 1 respiratory sensitizer.

The physical state (solid) and high molecular weight (only <100 g/mol can be readily taken up dermally) of the main constituents suggest that the substance will not be easily absorbed through skin. Additionally, the high water solubility and low log P values (-0.72 to -3.77) will further decrease the penetration into the lipid rich stratum corneum and therefore dermal absorption of the substances will be limited due to high hydrophilicity.. In a worst-case approach, based on the classification of the substance L (+) Lactic Acid (CAS-No. 79-33-4, EC-No. 201-196-2), Bos Taurus, milk, fermented, spray-dried (EC 917-734-0) was classified as “Skin Irritation Cat. 2” (H315). A damaged skin surface, induced through the substance, may enhance the penetration of the substance.

 

Sources:

Hampson, D., Kidder, DE (1986). Influence of creep feeding and weaning on brush border enzyme activities in the piglet small intestine. Res Vet Sci. 40(1):24-31.

Boiling point lactic acid: Haynes, W.M. (ed.). CRC Handbook of Chemistry and Physics. 95th Edition. CRC Press LLC, Boca Raton: FL 2014-2015, p. 3-336

Vapour pressure Lactic acid: Yaws CL; Handbook Chem Compd Data Process Saf, Houston, TX: Gulf Publishing, p. 33 (1997).

Log P Lactic acid: Hansch, C., Leo, A., D. Hoekman. Exploring QSAR - Hydrophobic, Electronic, and Steric Constants. Washington, DC: American Chemical Society

 

 

Bioaccumulation potential:

no bioaccumulation potential

A toxicokinetic assessment based on the physical properties (see ECHA Guidance on Information Requirements and Chemical Safety Assessment, R7c, version 3.0, June 2017) and toxicity data was performed. NOTE: this is mostly a generic assessment based on general statements in the guidance document that are derived from physical parameters.