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Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation test
Species:
guinea pig
Strain:
other: BOR: DHPW (SPF)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Winkelmann, Borchen, Germany
- Age at study initiation: approximately 4-7 weeks
- Weight at study initiation: mean 321 g
- Housing: in groups
- Diet and water: ad libitum
- Acclimation period: at least 7 days
Route:
intradermal and epicutaneous
Vehicle:
polyethylene glycol
Concentration / amount:
test substance formulated in polyethylene glycol 400 (PEG 400)
intradermal induction: 5% test item
epicutaneous induction: 50% test item
1. challenge: 50% test item
2. challenge: 25% and 12% test item
Route:
epicutaneous, semiocclusive
Vehicle:
polyethylene glycol
Concentration / amount:
test substance formulated in polyethylene glycol 400 (PEG 400)
intradermal induction: 5% test item
epicutaneous induction: 50% test item
1. challenge: 50% test item
2. challenge: 25% and 12% test item
No. of animals per dose:
in test group: 20
in control group: 10
Details on study design:
RANGE FINDING TESTS:
The dose finding for the intradermal induction was performed with 1 animal and the following test substance concentrations: 0%, 1%, 2.5%, 5% (injection volume 0.1 ml). Thereafter the concentration for the main test was set 5%.
The dose finding for the epicutaneous induction was performed with 4 animals and the following test substance concentrations: 6%, 12%, 25%, 50% (application volume 0.5 ml). No skin reddening or erythema was observed. In the pre-testing no concentration 100% was tested by mistake, therefore the concentration for the main test was set 50%.
The dose finding for the challenge was performed with 5 animals and the following test substance concentrations: 6%, 12%, 25%, 50% (application volume 0.5 ml). No skin reddening or erythema was observed. In the pre-testing no concentration 100% was tested by mistake, therefore the concentration for the main test was set 50% for the first and 25% and 12% for the second challenge.

MAIN STUDY
A. INDUCTION EXPOSURE
Day 0 - intradermal injections: One day prior to intradermal injections the skin areas of the test and control group animals were shorn. Three pairs of injections of 0.1 ml were given.
Injection 1: 1:1 mixture FCA/saline solution
Injection 2: test substance 5% in PEG 400
Injection 3: test substance 5% in PEG 400 in a 1:1 mixture with FCA
For control group animals the test substance was replaced by the same amount of PEG 400.

Day 7 - epicutaneous induction: One day prior to epicutaneous induction the skin areas of the test and control group animals were shorn and coated with a 10% formulation of sodium lauryl sulfate in paraffin oil.
Patches loaded with 0.5 ml 50% test item were applied on the skin of the test group animals, covered with aluminium foil and fixed with an adhesive tape. After an exposure duration of 48 h, remaining test substance was removed with saline solution. For control group animals the amount of test item was replaced by PEG 400.

B. CHALLENGE EXPOSURE
1. and 2. challenge were conducted 3 and 4 weeks after the intradermal induction, respectively. One day prior to challenge the skin areas of the test and control group animals were shorn.
1. Challenge: Patches loaded with 0.5ml 50% test item in PEG 400 were applied on the skin of test and challenge control group, covered with aluminium foil and fixed with an adhesive tape for an exposure duration of 24 hours.
2. Challenge: Patches loaded with 0.5 ml 25% or 12% test item in PEG 400 were applied on the skin of test and challenge control group, covered with aluminium foil and fixed with an adhesive tape for an exposure duration of 24 hours.
After challenge exposure the remaining test substance was removed with saline solution.
Treated skin ares were evaluated 48 and 72 hours after the beginning of challenge testing.
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
50%
No. with + reactions:
17
Total no. in group:
20
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50%. No with. + reactions: 17.0. Total no. in groups: 20.0.
Reading:
2nd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
50%
No. with + reactions:
9
Total no. in group:
20
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 50%. No with. + reactions: 9.0. Total no. in groups: 20.0.
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
50%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
50%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: negative control. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
other: rechallenge, 1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
25%
No. with + reactions:
10
Total no. in group:
20
Remarks on result:
other: Reading: other: rechallenge, 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 25%. No with. + reactions: 10.0. Total no. in groups: 20.0.
Reading:
other: rechallenge, 2nd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
25%
No. with + reactions:
4
Total no. in group:
20
Remarks on result:
other: Reading: other: rechallenge, 2nd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 25%. No with. + reactions: 4.0. Total no. in groups: 20.0.
Reading:
other: rechallenge, 1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
25%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: other: rechallenge, 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
other: rechallenge, 2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
25%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: other: rechallenge, 2nd reading. . Hours after challenge: 72.0. Group: negative control. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
other: rechallenge, 1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
12%
No. with + reactions:
7
Total no. in group:
20
Remarks on result:
other: Reading: other: rechallenge, 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 12%. No with. + reactions: 7.0. Total no. in groups: 20.0.
Reading:
other: rechallenge, 2nd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
12%
No. with + reactions:
4
Total no. in group:
20
Remarks on result:
other: Reading: other: rechallenge, 2nd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 12%. No with. + reactions: 4.0. Total no. in groups: 20.0.
Reading:
other: rechallenge, 1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
12%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: other: rechallenge, 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 12%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
other: rechallenge, 2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
12%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: other: rechallenge, 2nd reading. . Hours after challenge: 72.0. Group: negative control. Dose level: 12%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Study was performed with Aspartic acid, N,N'-[methylenebis(2-methyl-4,1-cyclohexanediyl)]bis-, 1,1',4,4'-tetraethyl ester which is a structural analogue to Aspartic acid, N,N'-(methylenedi-4,1-cyclohexanediyl)bis-, 1,1',4,4'-tetraethyl ester. Both substances are diethyl esters of aspartic acid linked to a dicyclohexylmethyldiamine moiety. The difference between these two substances is merely the presence of two methyl groups connected to the cyclohexane rings. This structural analogy was confirmed by the Member State responsible for the notification of both substances under the NONS regulation. The Member State decided that test results obtained for one substance can be transferred to the other substance and that testing of both substances is usually not required. This decision is in accordance with the grouping of substances and read-across approach in Annex XI, 1.5 of the REACH Regulation.

Further remarks on results:

Signs of irritation during induction: After the second induction two animals showed an open wound at the application area on day 9. On day 10 the application area of six animals became encrusted. These encrustations stayed up to day 16.

No systemic intolerance reactions occurred during treatment.

Executive summary:

A guinea pig maximisation test according to Magnusson and Kligman (OECD TG 406) was performed. Aspartic acid, N,N'-[methylenebis(2-methyl-4,1-cyclohexanediyl)]bis-,1,1',4,4'-tetraethyl ester, 5 % in polyethylene glycol (PEG) and Freund’s Complete Adjuvant was injected intracutaneously on day 0 in 20 Guinea pigs. For topical induction on day 7 50 % test item was applied for 48 hours. Dermal challenge was conducted with 50 % test item on day 21 (1st challenge) and with 25% and 12% test item on day 28 (rechallenge), each application for 24 hours, semiocclusive.

After challenge 17/20 (85%) animals of the test group exhibited skin reactions to the 50% test substance formulation, and after rechallenge 10/20 (50%) and 7/20 (35%) to the 25% and 12% test substance formulation, respectively. No animal showed skin effects in the challenge control group. Thus, in this study aspartic acid, N,N'-[methylenebis(2-methyl-4,1-cyclohexanediyl)]bis-,1,1',4,4'-tetraethyl ester was considered to be a skin sensitizer.

The undiluted test substance (100%) was not tested during the dose finding study by mistake and therefore also not in the main study. However, results of the main study indicate, that evidence exist for a primary irritating potency of the 50% test item formulation, furthermore the unchanged substance was tested as slightly to moderately irritating in an earlier conducted skin irritation test (OECD TG 404) and therefore, and also because of the clear positive result no further testing is required.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

The structural analogue aspartic acid, N,N'-[methylenebis(2-methyl-4,1-cyclohexanediyl)]bis-,1,1',4,4'-tetraethyl ester showed skin sensitisation potential in a guinea pig maximisation test according to Magnusson and Kligman (OECD TG 406). After an intracutaneous induction (test substance 5% in polyethylene glycol (PEG) and Freund’s Complete Adjuvant) on day 0 and a topical induction on day 7 (test substance 50% in PEG, 48 hours) a first challenge was performed on day 21 (exposure 24 hours, vehicle PEG, semiocclusive) with 50% test substance and a rechallenge on day 28 (exposure 24 hours, vehicle PEG, semiocclusive) with 25% and 12% test substance.

After challenge 17/20 (85%) animals of the test group showed skin reactions to the 50% test substance formulation, and after rechallenge 10/20 (50%) and 7/20 (35%) to the 25% and 12% test substance formulation, respectively. No animal showed skin effects in the challenge control group. Thus, a sensitisation potential is concluded for the substance.

The study investigating skin sensitisation was performed with aspartic acid, N,N’-[methylenebis(2-methyl-4,1-cyclohexanediyl)]bis-, 1,1',4,4'-tetraethyl ester which is a structural analogue to aspartic acid, N,N’-(methylenedi-4,1-cyclohexanediyl)bis-, 1,1',4,4'-tetraethyl ester. Both substances are diethyl esters of aspartic acid linked to a dicyclohexylmethyldiamine moiety. The difference between these two substances is merely the presence of two methyl groups connected to the cyclohexane rings. This structural analogy was confirmed by the Member State responsible for the notification of both substances under the NONS regulation. The Member State decided that test results obtained for one substance can be transferred to the other substance and that testing of both substances is usually not required. This decision is in accordance with the grouping of substances and read-across approach in Annex XI, 1.5 of the REACH Regulation.


Justification for selection of skin sensitisation endpoint:
Only one study available.

Justification for classification or non-classification

According to EU-Directive 67/548/EEC, Annex I, the test substance requires R43: May cause sensitisation by skin contact

According to Regulation (EC) No 1272/2008, Annex VI, the test substance requires classification as Skin Sens.1; H317: May cause an allergic skin reaction.

Regulation (EU) No 286/2011, amending Regulation (EC) No 1272/2008, states that, where data are sufficient, a refined evaluation allows the allocation of skin sensitisers into sub-categories (1A: strong sensitisers; 1B: other skin sensitiser). In this case, for assessing sub-categorization, one guinea pig maximation test is available, which reveals 85% positive responses at first reading of 1st challenge (48 h after start of challenge application) with the challenge concentration of 50%. The intradermal induction dose was 5%. Applying the criteria of Regulation (EU) No. 286/2011 leads to sub-categorization 1B.

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