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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27.11.2002-20.12.2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed according to the OECD guidelines and according to GLP principles.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
In vivo skin sensitisation studies that were carried out or initiated before 11 October 2016, and that meet the requirements set out in Article 13(3), first subparagraph, and Article 13(4) REACH shall be considered appropriate to address this standard information requirement.

Test material

Constituent 1
Chemical structure
Reference substance name:
Lysine hydrochloride
EC Number:
211-519-9
EC Name:
Lysine hydrochloride
Cas Number:
657-27-2
Molecular formula:
C6H14N2O2.ClH
IUPAC Name:
lysine hydrochloride
Details on test material:
- Name of test material (as cited in study report): L-Lysine HCL crystals
- Substance type: white to pale yellow crystals
- Physical state: solid
- Analytical purity: 99%
- Lot/batch No.: CP trial 13-14 November 2001
- Expiration date of the lot/batch: 1 November 2004
- Stability under test conditions: not specified
- Storage condition of test material: Ambient temperature, storage under dry conditions

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Germany
- Age at study initiation: ca 4 weeks
- Weight at study initiation: no data
- Housing: in a mobile battery, containing 6 cages; maximal 10 animals per cage
- Diet (e.g. ad libitum): standard laboratory diet ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 (+/-) 3C
- Humidity (%): 30-70%. Upper limit incidentally up to 98% , because of meteorological circumstances and/or wet cleaning of the animal room.
- Air changes (per hr): ca 10 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark cycle

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
physiological saline
Concentration / amount:
Preliminary experiment: 1%, 3%, 10% and 30% (intradermal treament); 10% and 30% (topical treatment)
Selected concentrations for main test:
-Intradermal induction: 3% in physiological saline; Topical induction: 30% in physiological saline (pretreatment of test site with 10% SLS); Topical chalenge: 30% in physiological saline.
Challengeopen allclose all
Route:
epicutaneous, semiocclusive
Vehicle:
physiological saline
Concentration / amount:
Preliminary experiment: 1%, 3%, 10% and 30% (intradermal treament); 10% and 30% (topical treatment)
Selected concentrations for main test:
-Intradermal induction: 3% in physiological saline; Topical induction: 30% in physiological saline (pretreatment of test site with 10% SLS); Topical chalenge: 30% in physiological saline.
No. of animals per dose:
For preliminary irritation experiment: 2
For main test: 10 treated animals and 5 control animals.
Details on study design:
RANGE FINDING TESTS: The irritation response to intradermal injection of various test concentrations of the test substance was examined in 2 animals. Amounts of 0.1 mL of the selected concentrations were applied by intradermal injection to the flank, which had been clipped free of hair. About 24 hours later the animals were observed for signs of irritation.
The irritation response to topical application of various test concentrations of the test substance was also examined in 2 animals. Patches loaded with the test substance were placed on the clipped skin of the flanks and covered with semi-occlusive dressing. The dressing was left in place for 24 hours and about 24 and 48 hours later the animals were observed for signs of irritation.

MAIN STUDY
A. INDUCTION EXPOSURE
a. Intradermal induction injection
An area of about 24 cm2 of dorsal skin in the scapular region was clipped free of hair. Pairs of intradermal injections (0.1 mL) were made simultaneously in the clipped area as follows:
Test animals
- Two injections with Freund’s Complete Adjuvant (FCA)/physiological saline (1:1).
- Two injections with the selected test concentration (3%).
- Two injections with the selected test concentration (3%) in FCA/vehicle (1:1).
Control animals
- Two injections with FCA/physiological saline (1:1).
- Two injections with the vehicle.
- Two injections with FCA/vehicle (1:1).
- Skin readings were made at ca 24 hours after the treatment.
b. Topical induction application
Five days after the intradermal injections, the clipped dorsal skin of the test and control animals was treated with 10% sodium lauryl sulfate in vaseline (open application) to enhance skin sensitivity. One the next day, patches loaded with the selected concentration of the test substance (30%) were placed over the site of the intradermal injections and covered with semi-occlusive dressing. The dressing was left in place for 48 hours. The controls were similarly treated with a patch loaded with the vehicle.

B. CHALLENGE EXPOSURE
Fourteen days after the topical induction, patches loaded with the selected concentration of the test substance (30%) were placed on the clipped dorsal skin of the test and control animals. The dressing was left in place for 24 hours and about 24 and 48 hours later the animals were observed for signs of irritation.

OTHER:
Challenge controls:
see above
Positive control substance(s):
yes
Remarks:
Formaldehyde (37%), 3 control animals and 5 treated animals; Intradermal inductions with 0.3%, topical induction application with 20%, challenge with 10% and 3% test dilution; test performed about 8 months earlier.

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
3%
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: 3%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
none
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: none.
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
3%
No. with + reactions:
2
Total no. in group:
5
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: 3%. No with. + reactions: 2.0. Total no. in groups: 5.0.
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
10%
No. with + reactions:
4
Total no. in group:
5
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: 10%. No with. + reactions: 4.0. Total no. in groups: 5.0.
Reading:
1st reading
Hours after challenge:
24
Group:
other: negative control for test with positive control
No. with + reactions:
0
Total no. in group:
3
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: other: negative control for test with positive control. No with. + reactions: 0.0. Total no. in groups: 3.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
3%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 3%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
none
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: none.
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
10%
No. with + reactions:
4
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: positive control. Dose level: 10%. No with. + reactions: 4.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
3%
No. with + reactions:
2
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: positive control. Dose level: 3%. No with. + reactions: 2.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
other: negative control for test with positive control
No. with + reactions:
0
Total no. in group:
3
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: negative control for test with positive control. No with. + reactions: 0.0. Total no. in groups: 3.0.

Any other information on results incl. tables

PRELIMINARY TEST

Following intradermal treatment, the 3% concentration caused moderate erythema in both animals, wheras the next higher concentration (10%) also caused necrosis, besides moderate eryhtmea. Therefore the 3% concentration was considered to be appropriate for intradermal treatment.

Following topical treatment, the 30% concentration did not cause any skin reactions. A concentration higher than 30% was considered unsuitable for topical application. Therefore the 30% concentration was considered to be acceptable for topical treatment. Since the 30% concentration was not irritating, the induction site was pretreated with a 10% sodium lauryl sulfate solution in vaseline.

MAIN TEST

The intradermal induction injections caused the following skin reactions:

Test animals

FCA/physiological saline (1:1); moderate erythema.

selected test concentration (3%): slight erythema.

selected test concentration (3%) in FCA/vehicle (1:1): moderate erythema.

Control animals

FCA/physiological saline (1:1): moderate erythema.

Vehicle: no skin reactions.

FCA/vehicle (1:1): moderate erythema

 The topical pretreatment with 10% sodium lauryl sulfate caused distinct erythema in test and control animals. The topical application with the vehicle alone caused slight or moderate erythema in controls. After the 48 -h topical application of the selected test concentration, slight or moderate erythema was observed in the test animals.

 At 24 and 48 hours after the topical challenge application, none of the controls or test animals showed any skin reaction.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
In a test according to OECD 406 (Maximization test) on L-Lysine HCl, none of the treated animals showed any skin reaction at 24 and 48 hours after the topical challenge application. The test substance is not a sensitizer.