[No public or meaningful name is available]

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Milk protein hydrolyzate

IUPAC Name:

Milk protein hydrolyzate

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

The animals were about 6 weeks old at the start of the treatment period. The body weights at initiation of treatment ranged from 132 to 169 g (mean 152 g) for males and from 102 to 134 g (mean 118 g) for females. The animals were housed under conventional conditions, in macrolon cages (1 rat/cage) with wood shavings as bedding material and shreds of paper as environmental enrichment. The cages and bedding were changed weekly. During urine collection in week 13, rats were kept individually in stainless-steel metabolism cages. The number of air changes in the animal room was about 10 per h. The temperature in the animal room was between 20 and 24 C; the relative humidity was between 40–70%. Lighting was artificial using fluorescent tubes (12 h light, 12 h dark). Feed (provided as a powder in stainless-steel cans) and drinking water (tap water, provided in polypropylene bottles) were available ad libitum, unless precluded by the collection of blood and urine from fasted rats, and refreshed weekly.

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

Tested material was incorporated into purified, AIN-93G based rodent diet, at the expense of casein, at constant levels of 0, 1.2% (low-dose), 2% (mid-dose) and 4% (high-dose). Food consumption was measured per animal by weighing the feeders at the beginning and end of each week. The intake of tested material per kg body weight was calculated from the nominal dietary levels of tested material and the body weight and food intake results.

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The diets were analyzed by liquid chromatography–mass spectrometry (LC–MS/MS) to verify the concentration and homogeneous distribution of tested material in the diets and to confirm stability under the conditions of the study.

Duration of treatment / exposure:

90 days

No. of animals per sex per dose:

20

Control animals:

yes

Examinations

Observations and examinations performed and frequency:

Each animal was observed daily for clinical signs. The body weight of each animal was recorded once per week, starting at initiation of treatment, and on the day of necropsy. Food consumption was measured per animal by weighing the feeders at the beginning and end of each week.

Neurobehavioural testing was conducted on 10 rats/sex/group (these animals were not the same as those used for blood sampling and urine collection). Detailed clinical observations outside the home cage were performed prior to the first exposure and then once weekly. Functional observational battery (FOB) tests, including assessment of grip strength and sensory reactivity to stimuli of different types, and spontaneous motor activity measurements were performed towards the end of the treatment period.
Routine haematology and clinical chemistry determinations were conducted on 10 rats/sex/group on day 13, day 50 and at termination of the treatment period. The rats were fasted overnight before blood sampling (water was freely available). For the haematological analyses, K2-EDTA was used as anticoagulant. The examination included haemoglobin, packed cell volume, red blood cell count, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, percentage of reticulocytes, total and differential white blood cell counts, thrombocyte count and prothrombin time. The plasma was examined for alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl transferase, sorbitol dehydrogenase, 5’-ND nucleotidase, total bilirubin, bile acids, total protein, albumin, albumin/globulin ratio, glucose, total cholesterol, phospholipids, triglycerides, creatinine, urea, inorganic phosphate, calcium, chloride, potassium and sodium.
Urinalysis was conducted on 10 rats/sex/group (the same as those used for haematology and clinical chemistry). The selected animals were deprived of water for 24 h and of food during the last 16 h of this period. During the last 16 h of deprivation, the rats were kept individually in stainless-steel metabolism cages and urine was collected in glass tubes. The volume and density of the urine were measured to assess the renal concentrating ability. The appearance of the urine was recorded and the samples were analysed semi-quantitatively (test strips) for pH, glucose, occult blood, ketones, protein, bilirubin and urobilinogen. Centrifuged sediment was examined microscopically.
Ophthalmoscopic observations were performed in all rats prior to the start of treatment and in all animals of the control group and the high-dose group in the last week of the treatment period.
At the end of the treatment period (males on days 91–92, females on days 93–94; 10 rats/group/day), after overnight fasting (water was freely available), the animals were sacrificed by exsanguination from the abdominal aorta under CO2/O2 anaesthesia and subsequently thoroughly examined for macroscopic changes. A large number of organs were collected and the following organs were weighed: brain, heart, adrenals, kidneys, liver, spleen, thymus, thyroid, testes, epididymides, ovaries and uterus. The relative organ weights (g/kg body weight) were calculated from the absolute organ weights and the terminal body weight of the rats. All samples of organs were preserved in a neutral aqueous phosphate buffered 4% solution of formaldehyde. Samples of the preserved organs from all rats of the control group and the high-dose group were processed, embedded in paraffin, sectioned at 5 lm, stained with haematoxylin and eosin, and examined by light microscopy.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

There was no treatment-related mortality and the daily clinical observations revealed no
treatment-related effects. Ophthalmoscopy and neurobehavioural examinations revealed no treatment-related changes either. All groups showed normal growth, food consumption and water
consumption

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Clinical biochemistry findings:

effects observed, non-treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Organ weights showed slight increases in the relative weight of the kidneys and the absolute weight of the testes and epididymides in high-dose males. Slight fluctuations in the weights of
the brain, heart and thymus were considered chance findings because there was no dose-related response

Gross pathological findings:

no effects observed

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

> 2 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

ophthalmological examination

organ weights and organ / body weight ratios

serum/plasma biochemistry

urinalysis

water consumption and compound intake

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Executive summary:

No adverse effects were observed for the highest dose used and therefore the NOAEL is >2000 mg/kg BW/ day.