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Skin sensitisation

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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:
key study
Study period:
1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented, according to accepted guideline.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report date:
1996

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
year not reported; reliability scoring based on 1992 guideline
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes
Type of study:
Buehler test

Test material

Constituent 1
Chemical structure
Reference substance name:
Butanone
EC Number:
201-159-0
EC Name:
Butanone
Cas Number:
78-93-3
Molecular formula:
C4H8O
IUPAC Name:
butan-2-one
Details on test material:
- Name of test material (as cited in study report): Methyl ethyl ketone
- Physical state: Colorless liquid
- Analytical purity: 99.7%
- Lot/batch No.: F113600445
- Expiration date of the lot/batch: 22 November 1996
- Storage condition of test material: Room temperature

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.
- Age at study initiation: 4 to 6 weeks old
- Weight at study initiation: 343 to 403 g
- Housing: Up to five guinea pigs were accommodated in suspended polypropylene cages with a solid floor relined with a whitewood bedding.
- Diet (e.g. ad libitum): SQC FD1 (pelleted) diet from Special Diets Services Ltd., Witham was freely available to the animals at all times
- Water (e.g. ad libitum): Mains water was provided, ad libitum, via cage mounted water bottles
- Acclimation period: at least 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16 to 22°C
- Humidity (%): 40-80% RH
- Air changes (per hr): at least 10 air changes per hour
- Photoperiod (hrs dark / hrs light): illuminated by flourescent strip-lights for 14 hrs daily.


Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: Alembicol D
Concentration / amount:
1.) Induction phase:
test group: undiluted test substance (0.3 mL)
control: 0.3 mL Alembicol D

2.) Challenge phase:
test group and control: test substance is undiluted and 50% m/m dilution in Alembicol D.
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: Alembicol D
Concentration / amount:
1.) Induction phase:
test group: undiluted test substance (0.3 mL)
control: 0.3 mL Alembicol D

2.) Challenge phase:
test group and control: test substance is undiluted and 50% m/m dilution in Alembicol D.
No. of animals per dose:
20 in treatment group and 10 in control group.
Details on study design:
RANGE FINDING TESTS:
Screening test for induction
Five formulations, incorporating from 20% m/m in Alembicol D up to the maximum practical concentration, undiluted test article, were selected. The dorsum and flanks of two guinea pigs were clipped on the day before application of the test formulations. The same area was shaved shortly prior to treatment. The animals were subject to occluded, topical application of five 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, aluminium foil 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 24 hours after application and 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 2, 24, and 48 hours after removal of the patches and the results were recorded individually.

Screening test for challenge application
Four formulations, 25, 50, and 75% m/m in Alembicol D and undiluted test article, were chosen based on the results of the first screen to identify the maximum non-irritant concentration of test article after occluded, topical application to skin. The flanks of three guinea pigs 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-Severn, 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 six hours and the treated areas of skin were washed with water. 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 24 and 48 hours after removal of the chambers.

No reactions were elicited by the undiluted test article nor by any of the lower applied concentrations.
MAIN STUDY:
Control group: left side of flank induced with vehicle and challenged with vehicle; right side of flank not induced but challenged with test substance (undiluted and 50% mixture in vehicle).
Test group: left side of flank induced with test substance and challenged with vehicle; right side not induced but challenged with test substance (undiluted and 50% mixture in vehicle).

A. INDUCTION EXPOSURE
- No. of exposures: 3
- Exposure period: 24 hours
- Test groups: 20 females were treated with 0.3 mL of undiluted methyl ethyl ketone on left flank.
- Control group: 10 females were treated with 0.3 mL of Alembicol D (vehicle) on left flank.
- Site: Left flank
- Frequency of applications: Days 1, 8, and 15.
- Duration: 3 weeks
- Concentrations: 0.3 mL undiluted methyl ethyl ketone or vehicle.


B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: Animals were challenged at Day 29
- Exposure period: 6 hours
- Test groups: Animals were tested on the left flank with Alembicol D (vehicle) and on the right flank with undiluted methyl ethyl ketone and a 50% m/m dilution in Alembicol D (vehicle); a volume of 0.1 mL was used for challenge.
- Control group: Animals were tested on the left flank with Alembicol D (vehicle) and on the right flank with undiluted methyl ethyl ketone and a 50% m/m dilution in Alembicol D (vehicle); a volume of 0.1 mL was used for challenge.
- Site: Left flank for challenge with Alembicol D (vehicle) and right flank for challenge with methy ethyl ketone (undiluted and 50% dilution).
- Concentrations: undiluted methyl ethyl ketone and 50% m/m dilution in Alembicol D.
- Evaluation (hr after challenge): 24 and 48 hours

Challenge controls:
The control group was challenged with both the vehicle and the test article to determine any unusual response in animals induced with vehicle.
Positive control substance(s):
yes

Study design: in vivo (LLNA)

Vehicle:
not specified
Details on study design:
Not applicable
Statistics:
Not applicable

Results and discussion

Positive control results:
Positive control results with alpha-hexylcinnamaldehyde provided in Appendix 1 (pg 25); however, study report states that regular (six monthly) testing to confirm susceptibility of guinea pigs to 2-mercaptobenzothiazole were performed (pg 8). It is unclear what positive control compound was used in this study.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
other: control
Dose level:
undiluted
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: other: control. Dose level: undiluted. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.
Reading:
2nd reading
Hours after challenge:
48
Group:
other: control
Dose level:
undiluted
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
focal eschar in one animal at anterior site exposed to undiluted methyl ethyl ketone
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: control. Dose level: undiluted. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: focal eschar in one animal at anterior site exposed to undiluted methyl ethyl ketone.
Reading:
1st reading
Hours after challenge:
24
Group:
other: control
Dose level:
50%
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: other: control. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.
Reading:
2nd reading
Hours after challenge:
48
Group:
other: control
Dose level:
50%
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: other: control. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.
Reading:
1st reading
Hours after challenge:
24
Group:
other: control
Dose level:
0 (vehicle)
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: other: control. Dose level: 0 (vehicle). No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.
Reading:
2nd reading
Hours after challenge:
48
Group:
other: control
Dose level:
0 (vehicle)
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: other: control. Dose level: 0 (vehicle). No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
undiluted
No. with + reactions:
2
Total no. in group:
20
Clinical observations:
slight erythema in 1 animal at anterior site exposed to undiluted methyl ethyl ketone (authors judged this response as inconclusive); necrotic focus on dose site in 1 animal at anterior site exposed to undiluted methyl ethyl ketone.
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: undiluted. No with. + reactions: 2.0. Total no. in groups: 20.0. Clinical observations: slight erythema in 1 animal at anterior site exposed to undiluted methyl ethyl ketone (authors judged this response as inconclusive); necrotic focus on dose site in 1 animal at anterior site exposed to undiluted methyl ethyl ketone..
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
undiluted
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
focal eschar (appeared to be scabs over pre-existing spots rather than treatment effects) noted in two animals at anterior site exposed to undiluted methyl ethyl ketone.
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: undiluted. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: focal eschar (appeared to be scabs over pre-existing spots rather than treatment effects) noted in two animals at anterior site exposed to undiluted methyl ethyl ketone..
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
50%
No. with + reactions:
1
Total no. in group:
20
Clinical observations:
slight erythema (barely perceptible) in one animal at the posterior site exposed to methyl ethyl ketone, 50% m/m in Alembicol D (the authors judged this response to be inconclusive).
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 50%. No with. + reactions: 1.0. Total no. in groups: 20.0. Clinical observations: slight erythema (barely perceptible) in one animal at the posterior site exposed to methyl ethyl ketone, 50% m/m in Alembicol D (the authors judged this response to be inconclusive)..
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
50%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: none.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
0 (vehicle)
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 0 (vehicle). No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: none.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
0 (vehicle)
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 0 (vehicle). No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: none.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
Undiluted methyl ethyl ketone elicited a slight erythematous response in one of the test animals following the challenge application. This response was deemed inconclusive. The results for the remaining guinea pigs were negative. Under the conditions of the study, MEK was not considered to be a skin sensitiser in guinea pigs.
Executive summary:

In a dermal sensitisation study (Denton, 1996) 50% m/m in Alembicol D and undiluted methyl ethyl ketone (MEK) were applied to young Dunkin-Hartley Guinea pigs. 20 females in each treatment group and 10 females in the control group were tested using the method of Buehler (test group: induction 100 % epicutaneous, challenge: 50 % and 100 % epicutaneous) according to OECD Guideline 406. Using three guinea pigs, a screening test for challenge application was performed with 25, 50, and 75% MEK m/m in Alembicol D and undiluted test substance before. In the main study 1/20 animals treated with undiluted MEK and 1/20 animal treated with 50 % MEK m/m in Alembicol D elicited a slight erythematous response following the challenge application noted as the 24 h-reading but not at the 48 h-reading. These responses were judged as inconclusive. The results for the remaining guinea pigs were negative at all. Under the conditions of the study, MEK was not considered to be a skin sensitiser in guinea pigs.