L-alanine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

1993-04-23 to 1993-06-24

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study, used for read-across

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Test material

Test material information

Constituent 1

Reference substance name:

L-tert-Leucine

IUPAC Name:

L-tert-Leucine

Constituent 2

Reference substance name:

20859-02-3

EC Number:

606-659-6

Cas Number:

20859-02-3

IUPAC Name:

20859-02-3

Constituent 3

Reference substance name:

(s)-2-Amino-3.3-dimethylbutyric acid

IUPAC Name:

(s)-2-Amino-3.3-dimethylbutyric acid

In vivo test system

Test animals

Species:

guinea pig

Strain:

other: Pirbright White

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Winkelmann Versuchstierzuchten GmbH & Co. KG, Borchen, Germany
- Age at study initiation: males: 9 weeks; females: 9 weeks
- Weight at study initiation: males: 431 - 486 g; females: 387 - 440 g
- Caging: Macrolon cages type IV
- Diet (e.g. ad libitum): ad libitum, Standard Diet ssniff G, "Special diet for guinea pigs", supplied by ssniff Spezialdiäten GmbH, Soest, Germany
- Water (e.g. ad libitum): water was provided using a time switched drinking water system
- Acclimation period: The animals were kept 6 days under test conditions before application of the test substance

ENVIRONMENTAL CONDITIONS
- according to guideline

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

other: Physiological saline solution (0.9%)

Induction 2

Concentration / amount:

Preliminary test: 1.) irritant concentration, intracutaneous: 1%; 2.) irritant concentration, epidermal: 50%; 3.) non-irritant concentration, epidermal: 10%.
Selected concentration of the test substance in the vehicle in main study:intracutaneous application: 1 % ; after 1 week, epidermal application: 50% ; 2 weeks after that (challenge), epidermal application: 10% .

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

other: Physiological saline solution (0.9%)

Challenge 2

Concentration / amount:

Preliminary test: 1.) irritant concentration, intracutaneous: 1%; 2.) irritant concentration, epidermal: 50%; 3.) non-irritant concentration, epidermal: 10%.
Selected concentration of the test substance in the vehicle in main study:intracutaneous application: 1 % ; after 1 week, epidermal application: 50% ; 2 weeks after that (challenge), epidermal application: 10% .

No. of animals per dose:

Number of groups: 2 control groups, 1 treatment group

Animals per group:
Control group 1: 6 (Nos. 1- 3 m, 4- 6 f); Control group 2: 6 (Nos. 7- 9 m, 10-12 f); Treatment group: 10 (Nos. 13-17 m, 18-22 f)

Details on study design:

RANGE FINDING TESTS:
In preliminary tests, the maximum non-irritating concentration for the intracutaneous or the slight to moderate irritating concentrations for the epidermal application during the induction phase as well as a non-irritating concentration for the epidermal application during the challenge phase were determined.

MAIN STUDY
A. INDUCTION EXPOSURE

1.) Intracutaneous application:

- Day of application: Day 1
- Immunostimulation: Freunds Gomplete Adjuvant (FGA), batch No. 3231, supplied by Difco Laboratories, Detroit, Michigan, USA
- Vehicle: Physiological saline solution (0.9%), batch No. CM 3590 Al, supplied by Fresenius AG, Bad Homburg, Germany
- Concentration of the test substance in the vehicle: 1%
- Application Volume: 0.1 ml
- Application Site: Scapular area
- Number of Applications: Total 6; 2 x FGA/physiol. saline solution (1:1); 2 x test substance solution; 2 x test substance solution/FCA (1:1)
- Animals of the control group: Identical treatment, however, the vehicle was used instead of the test substance solution.

2.) Epidermal application:

- Day of application: Day 8
- Route of application: Occlusive patch
- Duration of application: 48 h
- Patch material: Filter paper with plastic foil backing (3 x 4 cm)
- Vehicle: Physiological saline solution (0.9%), batch No. CM 3590 Al, supplied by Fresenius AG, Bad Homburg, Germany
- Concentration of the test substance in the vehicle: 50% (slightly to moderately irritant concentration)
- Amount applied: ca. 1 g
- Application Site: Scapular area
- Number of Applications: 1
- Animals of the control group: Identical treatment, however, the vehicle was used instead of the test substance solution.


B. CHALLENGE EXPOSURE

- Day of application: Day 22
- Route of application: Epidermal (occlusive patch)
- Duration of application: 24 hours
- Patch material: Leukotest(R) supplied by Beiersdorf AG, Hamburg, Germany
- Vehicle: Physiological saline solution (0.9%), batch No. CM 3590 Al, supplied by Fresenius AG, Bad Homburg, Germany
- Concentration of the test substance in the vehicle: 10%
- Application volume: 0.2 ml
- Application sites: Right flank (vehicle); Left flank (test substance)
- Number of applications: 2
- Assessment of the reactions at the application sites: Days 24 and 25 (24 and 48 hours after the end of exposure)
- Animals of the control group 1: Identical to treatment of animals of the test substance group. The control animals Nos. 1-3 and 4-6 were used.
- Repetition of the challenge: As the results of the first challenge were unequivocal, no second challenge was performed. The scheduled control group animals Nos. 7-9 and 10-12 were left untreated.

Challenge controls:

Induction exposure of the control animals:
Identical treatment like test animals, however, the vehicle was used instead of the test substance solution.
Challenge exposure:
Animals of the control group 1: Identical to treatment of animals of the test substance group. The control animals Nos. 1-3 and 4-6 were used.

Positive control substance(s):

yes

Remarks:

1,4- phenylenediamine x 2 HCl

Results and discussion

Positive control results:

Proof of the sensitivity of the Guinea Pig Strain:
Last examination: April 1993, test substance: 1,4-phenylenediamine x 2 HCl, Study No. 895140,
result: all animals showed signs of sensitization.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

10%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 10%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.

Results 2

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

10%

No. with + reactions:

0

Total no. in group:

6

Clinical observations:

none

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 10%. No with. + reactions: 0.0. Total no. in groups: 6.0. Clinical observations: none.

Results 3

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

10%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 10%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.

Results 4

Reading:

1st reading

Hours after challenge:

48

Group:

negative control

Dose level:

10%

No. with + reactions:

0

Total no. in group:

6

Clinical observations:

none

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 10%. No with. + reactions: 0.0. Total no. in groups: 6.0. Clinical observations: none.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Migrated information

Conclusions:

L-Tert-Leucine was applied to the skin of guinea pigs to determine its sensitizing properties by the maximization test method.
Following epidermal challenge, none of the animals showed changes at the exposed skin areas.
None of the animals of the control group showed changes at the exposed skin areas.
L-Tert-Leucine has no sensitizing properties on the skin of the guinea pig.