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Endpoint:
additional toxicological information
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
15 Mar - 28 Jul 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report date:
1995

Materials and methods

Type of study / information:
This exploratory study was conducted primarily in order to determine the difference in development of corneal opacities between rats and mice after administration of diets containing dietary tyrosine. A secondary objective was to determine whether there was a difference in sensitivity between two strains of rats (Brown Norway and CD).
Test guideline
Qualifier:
no guideline available
Version / remarks:
exploratory/mechanistic study
Principles of method if other than guideline:
Groups of 5 male and 5 female CD rats, Brown Norway rats and CD 1 mice were administered either basal diet or diets supplemented with 2 or 5% tyrosine for 14 days. Animals were weighed weekly, observed for clinical signs daily and subjected to ophthalmological examinations on Days 2, 3, 7, 8 and 14. At study termination, eyes were taken from selected animals for histological examination and plasma taken for free plasma tyrosine analysis of selected groups.
GLP compliance:
yes

Test material

Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Aldrich, France, batch 68160-123
- Purity: 98%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in an air-tight, light-resistant container at approximately 5°C
- Homogeneity and stability of the test substance in the diet were analytically verified. On all occasions results for homogeneity, concentrations and stability were within the acceptable ranges. The phenylalanine and tyrosine content of basal diet was analyzed (total amino acid levels of protein origin)
and were found to be 0.77 ± 0.04% and 0.55 ± 0.03% respectively.

Results and discussion

Any other information on results incl. tables

No deaths occurred during the study. No treatment-related clinical signs were observed in CD rats of either sex at 2%, or in Brown Norway rats or CD 1 mice in either sex at 2 or 5% tyrosine. The observations in the 5% tyrosine male CD rat group were restricted to dark urine (all males during the second week of study and 3/5 females on Day 14) and one male which appeared thin and showed ptosis and piloerection from Days 10 and 13, respectively. In this animal, corneal opacity was severe and visible to the naked eye.

Body weights and food consumption were not affected by treatment.

No treatment-related corneal effects were observed in female groups of rats at 2% or 5% tyrosine. Male CD and Brown Norway rats were not affected at 2% tyrosine. No mice of either sex were affected at any dose. Slight corneal opacities were noted for 3 of 5 male CD rats at 5% tyrosine on Day 2. On the following day, these opacities had progressed to "moderate" and a fourth animal in the group showed a "slight" opacity. By Day 7, the opacities in these four animals had become "severe" and the fifth male had developed a "slight" opacity. By Day 14, two males developed a very severe opacity and showed signs of edema and vascularisation of the cornea. In three animals, congestion of the iris became evident. Only one Brown Norway rat at 5% tyrosine developed any corneal changes; in this animal the corneal opacity was slight and was only observed on Day 14.

Table 1: Individual ophthalmological findings

Group/Dietary tyrosine (%)

Animal No.

Sex

Study Day

2

3

7

8

14

CD rats

0

all

-

-

-

-

-

-

2

all

-

-

-

-

-

-

5

ET3M 1380

M

Sl B

M B

SE B

Se B Im

Se B Im

ET3M 1381

M

Sl U

M B If

Se B Is

Se B Is

VS B Is EV

ET3M 1382

M

Sl B

M B

Se B

Se B Im

VS B Is EV

ET3M 1383

M

-

Sl U

Se B

Se B

Se B

ET3M 1384

M

-

-

Sl B

Sl B

Sl B

all females

F

-

-

-

-

-

Brown Norway rats

0

all

-

-

-

-

-

-

2

all

-

-

-

-

-

-

5

ET6M 1410

M

-

-

-

-

-

ET6M 1411

M

-

-

-

-

-

ET6M 1412

M

-

-

-

-

Sl B

ET6M 1413

M

-

-

-

-

-

ET6M 1414

M

-

-

-

-

-

all females

F

-

-

-

-

-

CD 1 mice

0

all

-

-

-

-

-

-

2

all

-

-

-

-

-

-

5

all

-

-

-

-

-

-

-             no finding

Sl           Slight opacity                B            Bilateral opacity

M          Moderate opacity          If            Iris failed to dilate

Se          Severe opacity              Im         Congestion of iris, mild

VS         Very severe opacity      Is           Congestion of iris, severe

U           Unilateral opacity           EV         Edema and vascularization of cormea

In male CD rats, the 2% and 5% tyrosine diets caused a 3-fold increase in plasma tyrosine to 59 mg/L and a 5-fold increase to 114 mg/L, respectively. Only the 5% group developed corneal opacity. In females the basal levels were lower than males, but the percentage increase in plasma tyrosine was similar in the 5% group for males and females.

In male Brown Norway rats, the basal plasma tyrosine levels were similar to female CD rats, as were the levels with 5% diets (68 mg/L). The levels for the 2% and 5% dietary male groups were about 3 fold and 5 fold the basal level, respectively. One male in the 5% group had a very high plasma tyrosine level (nearly 10 times that of the other four rats in the group), and was the only Brown Norway rat with a corneal lesion.

In CD 1 mice, the basal levels were similar to female CD rats and male Brown Norway rats but with a 5% tyrosine diet, there was no significant increase in plasma tyrosine levels.

Table 2: Mean plasma tyrosine concentrations

Animal strain

Dietary tyrosine (%)

Mean plasma tyrosine (mg/L)

Males

Females

Mean

S.D.

Mean

S.D.

CD rat

0

21

4.2

13

2.0

2

59

4.8

-

-

5

114

39.9

62

29.8

Brown Norway rat

0

12

0.6

-

-

2

32

15.0

-

-

5

68*

13.0

-

-

CD 1 mouse

0

13

1.8

-

-

5

18

7.1

-

-

- not determined                   * excluding one high outlier

At necropsy, no significant gross changes attributable to treatment were observed except for the corneal opacities described below.

Histopathology

No treatment-related changes were seen in female CD rats at any dose level or in males at 2% tyrosine. The two male rats examined at 5% tyrosine presented qualitatively and quantitatively similar lesions. They were characterized by the following changes:

- diffuse corneal epithelial intracytoplasmic vacuolation

- severe interstitial edema of the basal epithelial layer of the cornea

- edematous and/or swollen nuclear changes in the corneal epithelial cells

- inflammatory reaction involving the whole cornea: diffuse polymorphonuclear (PMNs) cell infiltration of the corneal stroma and epithelium, focal infiltration of the corneal epithelium and ciliary processes.

These changes were correlated to the corneal opacities (superficial keratitis) observed for almost two weeks at the ophthalmologic examinations.

No treatment-related changes were seen in male Brown Norway rats at 2% tyrosine. One of the examined eyes from the 5% male group was from the only animal in the group of five to show changes visible at ophthalmoscopy. The histological changes in this eye were characterized by:

- focal accumulation of PMNs in the anterior chamber

- mild diffuse infiltration of the corneal stroma by PMNs lining up at the base of the epithelium

- solitary focus of superficial epithelial desquamation of non-keratinized cells in central portion of cornea.

These changes were correlated with the observation of a slight corneal opacity which was first detected shortly before necropsy.

No treatment-related changes were observed for any group of male mice.

Applicant's summary and conclusion