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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
15 Jul - 15 Oct 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Inspectorate for Health Protection, Commodities and Veterinary Public Health; Ministry of Health, Welfare and Sport; The Netherlands.
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Sirional E. Coli (LYS + THR), (Liquid by product from the production of lysine and threonine by E. Coli K12)
- Physical state: Dark brown viscous liquid
- Analytical purity: Not relevant (complex mixture used as feed material)
- Lot/batch No.: Not given (only one batch was produced; CP trial 13/14 November 2001)
- Expiration date of the lot/batch: 12 April 2004
- Storage condition of test material: 2-10 "C, in a dry place
- Composition
dry matter: 61.14%, water: 38.86%, total nitrogen: 6.22%, crude proteins: 38.87%, ammoniacal nitrogen: 4.18%
pH (solution 10%) 4.4, ashes: 2.78%, K: 0.17%, Na: 0.87%, Ca: n.d., Mg: n.d., Cl: 0.375%, SO4: 14.23%, P: 0.12%

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Weight at study initiation: 174.8-215.8 g (males), 126.7-156.9 g (females)
- Age at study initiation: approx. 7 weeks
- Fasting period before study: no
- Diet (ad libitum): AIN-93G control diet from 4 days before study initiation on
- Water: ad libitum
- Acclimation period: the animals were delivered approximately 2 weeks before the start of the study

IN-LIFE DATES: 03 July 2002 - 15 Oct 2002

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: plain diet
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Integrity of the test diets
In the first batch of test diets the following analyses were performed:
- dry matter (TNO SOP DASlLNCl069);
- crude protein (TNO SOPS LNCIlO1 en LNC102);
- crude fat (TNO SOP LNCl139);
- crude fibre (TNO SOP DASlLNCIO11);
- ash (TNO SOP DAS/LNCl131);
- Ca, P, Na, K (TNO SOP DAMlLSPl057);
- Cl (TNO SOP LNCl216).
Duration of treatment / exposure:
13 weeks of dosing
Frequency of treatment:
daily ad libitum
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 2, 6 and 15%
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0, 1000, 3000 and 8000 mg/kg bw /day
Basis:
other: mean test substance intakes (approx.) in males
Remarks:
Doses / Concentrations:
0, 1200, 3300, 9600 mg/kg bw /day
Basis:
other: mean test substance intakes (approx.) in females
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
other: additional ammonium (derived from (NH4)2S04); approx. 0.6%) control group
Positive control:
0.6% ammonium (derived from (NH4)2S04)

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: At initiation of treatment and once weekly thereafter

FOOD CONSUMPTION: Yes
- Time schedule for examinations: 3- or 4-day periods, measured by cage

FOOD EFFICIENCY:
- The intake of test substance per kg body weight per day was calculated from the nominal dietary concentration of the test substance, the food consumption and the mean body weight in the period for which the intake of test substance is calculated.

WATER CONSUMPTION: Yes
- Time schedule for examinations: 4-day periods in weeks 1,6 and 12

FAECES COLLECTION: Yes
- Time schedule for examinations: In weeks 2, 7 and 13

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to the first exposure and then once weekly up to and including week 11. S
- Dose groups that were examined: All animals
- Battery of functions tested: spontaneous motor activity measurements and Functional Observational Battery (FOB) tests were performed towards the end of the study (week 12/13).

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations / Dose groups that were examined: Prior to the start of treatment in all animals and towards the end of the treatment period in all surviving animals of the control groups and the high-dose group.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At necropsy at the end of treatment
- Anaesthetic used for blood collection: CO2/O2
- Animals fasted: Yes
- How many animals: all animals
- Parameters examined: haemoglobin, packed cell volume, red blood cell count, reticulocytes, total white blood cell count, differential white blood cell count, prothrombin time, thrombocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At necropsy at the end of treatment
- Animals fasted: Yes
- How many animals: all animals
- Parameters examined: alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, gamma glutamyl transferase, total protein, albumin ratio albumin to globulin, urea, creatinine, fasting glucose, bilirubin (total), cholesterol (total), triglycerides, phospholipids, Ca, Na, K, Cl, inorganic phosphate

RENAL CONCENTRATION TEST:
- Time schedule for collection of blood: Shortly before the end of the treatment (days 87-88)

URINALYSIS: Yes
- Time schedule for collection of urine: Shortly before the end of the treatment (days 87-88)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: appearance, glucose, pH, occult blood, ketones, protein, bilirubin, urobilinogen, microscopy of the sediment
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

All animals were subjected to a complete gross necropsy. In week 14, after overnight fasting, the surviving animals were killed. A thorough autopsy was also performed on the animal that had to be killed because it was moribund. At final necropsy, the following organs were weighed (paired organs together) as soon as possible after dissection to avoid drying: Adrenals, ovaries, brain, spleen, epididymides, testes, heart, thymus, kidneys, thyroid (with parathyroids), liver and uterus.

Samples of the following tissues and organs of all animals were preserved in a neutral aqueous phosphate-buffered 4 per cent solution of formaldehyde:
Adrenals, parathyroid, aorta, * parotid salivary glands, axillary lymph nodes pituitary, brain (brain stem, cerebrum, cerebellum), prostate, caecum, rectum, colon * seminal vesicles with coagulating glands, epididymides, skeletal muscle (thigh),* exorbital lachrymal glands, skin (flank), eyes, small intestine (duodenum, ileum, jejunum), femur with joint spinal cord (at three levels), GALT (gut associated lymphoid tissue including Peyer's patches), sternum with bone marrow, stomach (glandular and non-glandular), heart, sublingual salivary glands, kidneys, submaxillary salivary glands, liver, testes, lungs, thymus, mammary gland (females), thyroid, * mandibular (cervical) lymph nodes, trachea/bronchi, mesenteric lymph nodes, urinary bladder, nerve-peripheral (sciatic), uterus (with cervix), oesophagus, * vagina, ovaries, pancreas, all gross lesions.

* The tissues marked with an asterisk were preserved but not processed for histopathological examination, since histopathological examination was not considered necessary on the basis of the results of gross observations.
Histopathological examination was performed on all tissues and organs listed above - except those marked with an asterisk - of all animals of the control groups (groups A and B) and of the high-dose group (group E), and of the animal that was killed in extremis. In addition, the lungs, liver, kidneys and gross lesions were examined microscopically in all rats of the intermediate dose-groups.
Other examinations:
ORGAN WEIGHTS: adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus, thyroid (with parathyroids), uterus
SPERM ANALYSIS AND ESTRUS CYCLE
Statistics:
The statistical procedures used in the evaluation of data were as follows:
- body weight: one-way analysis of covariance (covariate: body weight on day 0) followed by
Dunnett's multiple comparison tests;
- red blood cell and clotting potential variables, total white blood cell counts, absolute differential
white blood cell counts, clinical chemistry values, volume and density of the urine, organ
weights: one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison
tests;
- food and water consumption and food efficiency: one-way analysis of variance (ANOVA) followed
by L.S.D. tests;
- reticulocytes, relative differential white blood cell counts, urinary parameters except for volume
and density: Kruskal-Wallis non-parametric ANOVA followed by Mann-Whitney U-tests;
- histopathological changes: Fisher's exact probability test;
- neurobehavioural observations: parameters assessed during functional observations were
measured on different measurement scales (e.g., continuous, rank, categorical). Functional
observational battery and motor activity results: one-way analysis of variance (Anova) followed
by Dunnett's multiple comparison tests (e.g. continuous and motor activity data),
Kruskal-Wallis non-parametric Anova followed by multiple comparison tests (rank order), or
Pearson chi-square test (categorical data);
- Sperm parameters were evaluated by ANOVA followed by Dunnett's multiple comparison tests
or Kruskal-Wallis non-parametric ANOVA followed by Mann-Whitney U-tests;
TNO report V46 1 9/04
July 2003
- Estrus cyclicity was evaluated by Fisher's exact probability test or ANOVA followed by
Dunnett's multiple comparison tests.
All tests were two-sided. Probability values of pC0.05 were considered significant.

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS AND MORTALITY: No effects

BODY WEIGHT AND WEIGHT GAIN:
A slight reduction in growth and slightly reduced food consumption (during the first week of the study) were observed in the high-dose group, which were probably related to the high level of ammonium nitrogen in the test substance.

FOOD CONSUMPTION AND COMPOUND INTAKE:
The overall intake of Sirional E. Coli (LYS + THR) for the low-, mid-, and high-dose groups was approximately 1000, 3000 and 8000 mg/kg bw/d in males and 1200, 3300 and 9600 mg/kg bw/d in females, respectively. The intake of ammonium for the ammonium-control group was approximately 320 and 380 mg/kg bw/d in males and females, respectively.

WATER CONSUMPTION:
Water consumption was generally increased in a dose-related manner in males and females.

NEUROBEHAVIOUR: No effects

OPHTHALMOSCOPIC EXAMINATION: No effects

HAEMATOLOGY: No effects

CLINICAL CHEMISTRY:
Clinical chemistry parameters were generally similar among the groups. However, albumin was decreased in females of the ammonium-control group and of the high-dose group and the albumin/globulin ratio was decreased in males of the ammonium-control group and in females of the high-dose group. These changes were probably related to the high level of ammonium nitrogen in the test substance. The decrease in chloride in females of the high-dose group was considered treatment-related.

URINALYSIS:
Urinary density was increased in males of the ammonium-control group. Urinary volume and density were similar among the treated groups. Urinary pH was lower and urinary crystals were increased in males of the ammonium-control group and of the high dose group. These changes were considered related to the high level of ammonium nitrogen in the test substance.

ORGAN WEIGHTS:
Relative kidney weights were increased in the ammonium-control group (males) and in the high-dose group (males and females). Absolute brain weights were increased in females of the ammonium-control group and of the low- and high-dose groups. Relative brain weights were increased in the ammonium-control group (males) and in the high-dose group (males and females). Relative liver weights were increased in males and females of the high-dose group. Relative testes weights were increased in the ammonium-control group and in the mid-dose group. Absolute and relative uterus weights were increased in the low-dose group. All changes were considered either chance findings or related to the ammonium- level in the test substance, except for the change in relative liver weights, which was considered treatment-related.

GROSS PATHOLOGY: Macroscopic examination at autopsy did not reveal treatment-related gross changes. All lesions observed were about equally distributed amongst the controls and the different test groups, or they occurred in one or a few animals only.

HISTOPATHOLOGY: Microscopic examination revealed a statistically significant decrease in the incidence of microvesicular hepatocellular vacuolation in males of the high-dose group only. This histopathological lesion is not considered to be related to treatment, since a large variation in incidence of this condition is not uncommon in animals of this strain and age. All other lesions were about equally distributed between the controls and the different test groups, or they occurred in a single animal only.

FERTILITY PARAMETERS: No effects of the test substances on estrous cyclicity, epididymal sperm motility, epididymal sperm count, daily sperm production and sperm morphology were observed.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
3 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: equivalent to 6% in the diet. No obvious signs of toxicity.
Dose descriptor:
NOAEL
Effect level:
3 300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: equivalent to 6% in the diet. No obvious signs of toxicity.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion