Registration Dossier

Administrative data

Endpoint:
neurotoxicity: sub-chronic oral
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Inconsistent result

Data source

Reference
Reference Type:
publication
Title:
Dietary beta-alanine results in taurine depletion and cerebellar damage in adult cats.
Author:
Lu P.; Xu W., Sturman J.A.
Year:
1996
Bibliographic source:
Journal of Neuroscience Research 43:112-119

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Female domestic cats were fed a completely defined, taurine-free purified diet alone or containing 0.05% taurine for at least 2 years before being assigned to this study. 5 cats in each group were caged individually and fed the same diets and provided with 5% beta-alanine in the drinking water for 20 weeks.
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): beta-alanine
- Analytical purity: no data

Test animals

Species:
cat
Strain:
not specified
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: domestic cats raised in the colony of the Institute for Basic Research in Developmental, NY, USA
- Housing: cats were housed individually
- Diet (e.g. ad libitum): animals were fed a completely defined, taurine-free purified diet (Bioserve, Frenchtown, NJ, USA) alone or containing 0.05% taurine for at least 2 years before being assigned to this study.

Administration / exposure

Route of administration:
oral: drinking water
Vehicle:
water
Details on exposure:
No further data
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
20 weeks
Frequency of treatment:
during the entire study period
Doses / concentrations
Remarks:
Doses / Concentrations:
5% of beta-alanine in drinking water
Basis:
nominal in water
No. of animals per sex per dose:
5 (female cats)
Control animals:
yes, concurrent no treatment

Examinations

Observations and clinical examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

OTHER: From each cat the brain was removed and the right half immersed in cold fixative (1% paraformaldehyde and 2.5% gluteraldehyde in 0.1 M Sorensen´s phosphate buffer, pH 7.4) for several days prior to processing for routine histology and immunohistochemistry.
The left halves of brain were rapidly dissected and frozen on dry ice prior to extraction for determination of taurine and beta-alanine concentrations. Tissues were homogenized in 10 vols 20% trifluoroacetic acid (TFA) and centrifuged at 20000 for 30 min.
The clear supernatants were passed through a 0.45 µm µStar filter and stored at -70 °C until used for taurine and beta-alanine measurements. Analysis was accomplished by derivatizing with phenylisothiocynate and the taurine and beta-alanine derivatives separated by reverse-phase high performance liquid chromatography (HPLC).

Neurobehavioural examinations performed and frequency:
No
Sacrifice and (histo)pathology:
- Time point of sacrifice: at the end of the 20-week exposure period
- Number of animals sacrificed: 5 per experimental group
- Tissues evaluated: brain.
- Type of staining: immunohistochemistry and histology staining. Sections were deparaffined and hydrated by warming at 60 °C for 65 minutes in a histoclear bath, followed by graded ethanol washes. Staining with the antisera (1: 2000 dilution) was carried out at 4°C overnight and visualized using the peroxidise-conjugated avidin/DAB method (Dako Corporation, Carpintera, CA, USA). Control sections were included in every batch, those in which the antisera were preadsorbed with the immunogen prior to processing, and others replacing the primary antiserum with preimmune serum at similar dilutions. Control slides showed no visible staining after processing, whereas slides prepared with the antisera showed brown reaction products. Counterstaining, when employed, was with hematoxylin. Other histological stains employed were cresyl violet, toluidine blue, hematoxylin and eosin, and Luxol fast blue. Photomicrographs were taken on a Zeiss Axiophot using Kodak 40 color reversal film and Kodak Panatomic X film.
- Methodology of preparation of sections: Tissue was embedded in paraffin and serial 6 µm coronal sections cut and mounted on glass slides.
- Thickness: 6 µm
- Embedding media: paraffin
- Number of animals evaluated from each sex and treatment group: 5

Positive control:
No
Statistics:
Standard deviations and significance of difference between groups were calculated using the Student´s t test using a standard computer program (STATA, Computer Resource Center, Santa Monica, CA)

Results and discussion

Results of examinations

Clinical signs:
not examined
Mortality:
not examined
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Clinical biochemistry findings:
not examined
Behaviour (functional findings):
not examined
Gross pathological findings:
not examined
Neuropathological findings:
effects observed, treatment-related
Details on results:
During the 20-week period of study, each cat consumed approximately 500 g of beta-alanine. The concentrations of taurine in the three regions of the brain investigated in this study, cerebellum, occipital lobe, and hippocampus, were significantly reduced in both taurine-supplemented and taurine-deprived cats. A major difference was the accumulation of small amounts of beta-alanine in these regions of taurine-supplemented cats compared with large amounts accumulated in taurine-deprived cats.
Microscopic examination of the cerebellum of cats treated with beta-alanine showed a reduced number of granule cells compared to the cerebellum of cats treated only with water and many of those remaining were pyknotic. There was also a clear reduction in the number of Purkinje cells and many of those still remaining were dead or dying. The cerebellar white matter contained numerous long and swollen fibers which resemble the Rosenthal fibers described in several human disease states of the cerebellum, generally accompanied by reactive or neoplastic astrocytosis. As with Rosenthal fibers, these fibers in the cerebellar white matter also stained on the periphery with an antibody to ubiquitin. Such fibers are not seen in taurine-supplemented or taurine-deprived cats not treated with beta-alanine. There was also prominent gliosis in the cerebellum of cats treated with beta-alanine, shown by GFAP staining, compared to cats treated only with water. Morphological changes in beta-alanine-treated cats were greater in taurine-deprived than in taurine-supplemented cats. This gliosis was much more severe than has been noted in taurine-deprived kittens.
Immunohistochemical staining of cerebellum of cats treated with beta-alanine with an antibody to beta-alanine showed prominent localization in Purkinje cells and their dendrites.
Golgi II cells also contained beta-alanine. Some granule cells contained beta-alanine, especially in taurine-deprived cats treated with beta-alanine while others were devoid. None of the granule cells with pyknotic appearance were stained by antibody with beta-alanine (or taurine).
Cerebellum of cats treated only with water was negative to this antibody. Using an antibody to taurine, this compound appears to have been virtually eliminated from cerebellar Purkinje cell and granular cells of cats treated with beta-alanine and concentrated in Golgi II and glial cells of the hippocampus, and in the visual cortex, suggesting that beta-alanine accumulation with neurons is global. Beta-alanine and taurine have been shown repeatedly to be transported by a single carrier in neuronal cells, so it is not surprising that the prolonged treatment with beta-alanine in this study resulted in displacement of taurine from many neural components.
The authors concluded that although dietary treatment with beta-alanine results in great reductions in the concentration of taurine in cerebellum (and other brain regions) of taurine–supplemented cats treated with beta-alanine, yet taurine-deprived cats do not show the unusual pathology described here. Taurine has an influence on many cellular processes, including calcium concentrations which, in turn, regulated extra- and intracellular glutamate.
Beta-alanine may lack this ability and allow more gluatemate to penetrate the cells and thus cause cell death.
However, the quality of histochemical and immuno-histochemical stainings as presented in the publication is low (contrast quality and magnification scales of the shown cerebellar slides differ between treated and untreated groups). This makes it difficult to interpret the observed effects. Moreover, GFAP-stainings showed also gliosis in the animal group, which was taurine-deprived (0% taurine), but not treated with beta-alanine.
Therefore, the conclusion of the authors, that beta-alanine itself caused the pathologic changes in cerebellum could be wrong.
Taurine is an essential component of cat diets. In contrast to other species including humans, dogs, horses, cattle and rodents, cats cannot synthesize taurine from cysteine due to the lack of the required decarboxylating enzymes. It is well known, that insufficient dietary taurine -supplementation in feline food leads to central retinal atrophy and dilated cardiomyopathy in cats. Beta-alanine is a taurine analogue, which uses the same cellular transport system (carrier). Thus, prolonged exposure to beta-alanine will increase the serum levels of beta-alanine and finally lead to a displacement of intracellular taurine.
In conclusion, the observed neuropathological changes in feline cerebellum could be due to the taurine-deficiency, which was induced through dietary beta-alanine, rather than through beta-alanine treatment itself.




Effect levels

Dose descriptor:
NOAEL
Remarks on result:
not determinable
Remarks:
no NOAEL identified

Applicant's summary and conclusion