Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1997-04-22 to 1997-06-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report date:
1997

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
17th July 1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
July 31st 1992
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Test was performed before the LLNA guideline was available.

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
425-660-0
EC Name:
-
Cas Number:
165101-57-5
Molecular formula:
C14H29NO
IUPAC Name:
3-butyl-2-(heptan-3-yl)-1,3-oxazolidine

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: D Hall Ltd, Burton on Trent, UK
- Age at study initiation: 5-7 weeks old
- Weight at study initiation: 396-471 g
- Housing: groups of five animals in suspended polypropylene cages (six per battery) with open tops, solid floors and stainless steel mesh front panels (providing minimum internal dimensions of 61 x 81 x 25 cm)
- Diet: SQC FD1 (pelleted) diet, ad libitum
- Water: mains water, ad libitum
- Acclimation period: at least seven days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
other: Alembicol D
Concentration / amount:
intradermal induction: three paired injections of 0.1 mL of FCA emulsion (control: FCA emulsion), 2.5 % m/v test substance in Alembicol D (control: only Alembicol D), 2.5 % m/v in FCA emulsion (control: 50% v/v Alembicol D in FCA emulsion)
dermal induction: 0.5 mL of 60 % m/m of the test item in Alembicol D (test animals) or Alembicol D alone (control group)
challenge: left flank: 0.1 mL Alembicol D; right flank: Finn chambers containing 20 % and 10 % m/m Incozol 2 in Alembicol D
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: Alembicol D
Concentration / amount:
intradermal induction: three paired injections of 0.1 mL of FCA emulsion (control: FCA emulsion), 2.5 % m/v test substance in Alembicol D (control: only Alembicol D), 2.5 % m/v in FCA emulsion (control: 50 % v/v Alembicol D in FCA emulsion)
dermal induction: 0.5 mL of 60 % m/m of the test item in Alembicol D (test animals) or Alembicol D alone (control group)
challenge: left flank: 0.1 mL Alembicol D; right flank: Finn chambers containing 20 % and 10 % m/m Incozol 2 in Alembicol D
No. of animals per dose:
test group: 10 animals
control group: 5 animals
Details on study design:
RANGE FINDING TESTS:

First screening test (intradermal injection phase of induction)
The vehicle and six formulations were selected. The dorsa of two guinea pigs were clipped on the day prior to dosing. On Day 1, intradermal injections (0.1 mL per site), incorporating a range of test article concentrations, (from 1 % m/v in Alembicol D up to the maximum practical concentration of 25 % m/v in Alembicol D), were made into the scapular zone of the denuded dorsum. Dermal reactions were individually assessed and recorded approximately 24 and 72 hours later.

Second screening test (topical application phase of induction)
Four formulations, incorporating the test article at a range of concentrations from 40 % m/m up to the maximum practical concentration of the undiluted test article as supplied, were selected. Two guinea pigs were prepared by receiving two 0.1 mL intradermal injections of FCA emulsion into the suprascapular dorsum at least five days prior to application of the test formulation. The dorsum and flanks were clipped and shaved on the day before application of the test formulations. The animals were subject to occluded, topical application of four 20 x 20 mm patches of Whatman No 4 filter paper each saturated with approximately 0.2 mL of one of the test formulations. Occlusion was effected by covering the Whatman patches with successive layers of "Blenderm" adhesive dressing from 3M Co, Loughborough and "Steroban" open-weave, elasticated bandage from Steroplast Ltd, Bredbury. The last layer completely enveloped the torso to ensure the patches remained secure. The dressings and bandages were removed approximately 48 hours after application and the treatment sites were washed with arachis oil. The location of each patch was marked with indelible ink. Treated areas of skin were reshaved approximately 21 hours after removal of the bandages. Dermal reactions were assessed approximately 24 and 96 hours after removal of the patches.

Third screening test (topical application at challenge)
Four formulations, ranging from 20 to 70 % m/m in Alembicol D, were chosen to identify the maximum non-irritant concentration of test article after occluded, topical application to skin. Three guinea pigs were prepared by receiving two 0.1 mL intradermal injections of FCA emulsion into the suprascapular dorsum between 14 and 28 days prior to application of the test formulations. The flanks were clipped on the day before application. The same areas were shaved approximately two hours before treatment. Each animal was subject to occluded topical application of four 12 mm Finn chambers from Biodiagnostics Ltd, Upton-upon-Sevem, each loaded with approximately 0.1 mL of one of the four selected test formulations. The Finn chambers were secured by successive applications of Blenderm and Steroban. The dressings and chambers were removed after 24 hours and the treated areas of skin were washed with arachis oil. The location of each challenge site was marked on the skin using indelible ink. The treated areas of skin were reshaved approximately 21 hours after removal of the chambers. Dermal reactions were assessed approximately 24 and 48 hours after removal of the chambers.

MAIN STUDY
A. INDUCTION EXPOSURE

1. Intradermal injection
The dorsum overlaying the scapulae of each guinea pig was clipped on Day 1 shortly before treatment commenced. The area was confirmed to be free from injury or irritation. On Day 1 three paired intradermal injections (0.1 mL per site) were placed in single rows parallel to and on either side of the dorsal mid-line of each guinea pig such that the anterior and posterior injection sites marked the corners of an approximate 20 x 40 mm area. The middle injection sites were positioned close to the anterior sites. The concentration of test article selected after the first screen was administered as follows:

Site Test group Control group

Anterior FCA emulsion FCA emulsion

Middle Incozol 2, 2.5 % m/v Alembicol D
in Alembicol D

Posterior Incozol 2, 2.5 % m/v 50 % v/v Alembicol D
in FCA emulsion in FCA emulsion

Irritation or other dermal changes at the injection site were recorded by group on Day 2.

2. Topical application
On Day 7 the areas of dorsum denuded for the first phase of induction were clipped. On Day 8 each dorsum was shaved. Approximately two hours later the area of skin including the intradermal injection sites was subject to application of a 25 x 45 mm patch of Whatman No 4 filter paper loaded with approximately 0.5 mL of 60 % m/m Incozol 2 in Alembicol D (test animals) or Alembicol D alone (control group). Occlusion of the treated skin was effected by successive layers of Blenderm and Steroban. The patches and dressings remained in place for 48 hours. The treated areas of skin were washed with arachis oil. Irritation or other dermal changes at the sites of occluded topical application were recorded by group on Day 11.

B. CHALLENGE EXPOSURE

Both flanks of all guinea pigs were clipped on Day 21 and shaved to remove hair stubble on Day 22. At least two hours later the left flank of each animal was subject to application of a 12 mm Finn chamber loaded with approximately 0.1 mL vehicle. Finn chambers containing the formulations selected for challenge, 20 % and 10 % m/m Incozol 2 in Alembicol D were applied to the right flank. The chambers were kept in place by successive layers of Blenderm and Steroban. The chambers and dressings were removed approximately 24 hours after application and the treated areas of skin were washed with arachis oil. Challenge site locations were marked with indelible ink immediately after the washing procedure. Dermal responses to challenge were assessed approximately 24 and 48 hours after removal of the chambers. Responses to challenge were recorded individually.
Challenge controls:
Control animals were treated similarly to test animals, except that the test item was omitted in the induction phase.
Positive control substance(s):
no

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
10 % m/m in Alembicol D
No. with + reactions:
0
Total no. in group:
5
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
20 % m/m in Alembicol D
No. with + reactions:
2
Total no. in group:
5
Clinical observations:
Both animals developed an induration of the test site. Further desquamation and slight erythema were found in one of them..
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
only Alembicol D
No. with + reactions:
0
Total no. in group:
5
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
10 % m/m in Alembicol D
No. with + reactions:
0
Total no. in group:
5
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
20 % m/m in Alembicol D
No. with + reactions:
2
Total no. in group:
5
Clinical observations:
Two animals (same as in first reading) showed induration and slight erythema.
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
only Alembicol D
No. with + reactions:
0
Total no. in group:
5
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
10 % m/m in Alembicol D
No. with + reactions:
3
Total no. in group:
10
Clinical observations:
Two animals showed desquamation. One developed slight erythema.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
20 % m/m in Alembicol D
No. with + reactions:
4
Total no. in group:
10
Clinical observations:
One animal showed desquamation. Two developed an induration and one animal had slight erythema.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
only Alembicol D
No. with + reactions:
2
Total no. in group:
10
Clinical observations:
A desquamation was read in two animals.
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
10 % m/m in Alembicol D
No. with + reactions:
1
Total no. in group:
10
Clinical observations:
A desquamation was developed.
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
20 % m/m in Alembicol D
No. with + reactions:
4
Total no. in group:
10
Clinical observations:
Two animals developed a slight erythema and induration. The other two showed desquamation and one of them a slight erythema.
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
only Alembicol D
No. with + reactions:
1
Total no. in group:
10
Clinical observations:
A desquamation was developed.
Group:
positive control
Remarks on result:
not measured/tested

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the findings in this guinea pig maximisation test with Freund´s complete adjuvants the test substance Incozol 2 is not sensitising to the skin.
Executive summary:

This study was conducted to assess the potential of Incozol 2 to elicit skin sensitisation (delayed contact hypersensitivity) in the guinea pig. The method followed was based on the procedures developed by Magnusson and Kligman and was in compliance with that described in EU Method B6 and OECD TG 406. Ten test and five control animals were used in this study. Based on the results of the preliminary screening studies, the following dose levels were chosen:

Intradermal injection: 2.5% m/v in Alembicol D and/or adjuvant

Topical induction: 60% m/m in Alembicol D

Challenge application: 20 and 10% m/m in Alembicol D

The diluted test substance (60% m/m in vehicle) was applied topically to the intact skin of 10 (test group) respectively 5 (negative control group) guinea pigs after intradermal induction by 2.5% m/v dilution of the test substance (test group) or the vehicle and /or adjuvant emulsion only (control group). Two weeks following the last induction exposure the challenge doses of 10% and 20% m/m in Alembicol D were administered. Challenge with Incozol 2 caused slight erythema, desquamation and induration. The results were read after 24 and 48 hours. Incozol 2 elicited no positive response, indicative of skin sensitisation (delayed contact hypersensitivity) in any of the test animals following the challenge application. Based on these findings Incozol 2 is not sensitising to the skin (Covance, 1997).