Registration Dossier

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:
39 Days
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Reliable without restrictions; study conducted according to Annex of Directive 92/69/EEC (OJ No. L383A, 29.12.92), Part B, Method B.6. Skin sensitisation and GLPs.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The study was conducted before the LLNA was an approved method

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
-Test material (as cited in report): Nesterol
-Chemical name (as cited in report):2,2,4-Trimethyl-1 ,3-pentanediol-isobutyrate
-Purity: 99.5%
-Expiration date: September 28, 1995
-Physical description: Colorless to light yellow liquid
-Storage: Room temperature

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
Test animals:
-Source: D. Hall, Newchurch, Staffordshire, England
-Sex: male
-Age at receipt: approximately 4-5 weeks
-Acclimation period: 12 days
-Weight at study initiation: 311-343g
-Housing: Grouped housed in groups of five in suspended metal cages with wire mesh floors.
-Diet: vitamin C enriched guinea-pig diet FD2, ad libitum. Hay was given weekly.
-Water: local municipality, ad libitum
-Method of animal identification: ear tattoo

Environmental Conditions:
-Temperature: approximately 21°C
-Humidity: 30-70%
-Photoperiod: 12 hour light/dark cycle
-Air exchanges: approximately 15 air exchanges per hour

In-Life Study Dates:
-Study Initiation Date: March 8, 1995
-Experimental Start Date: April 4, 1995
-Experimental Completion Date: May 13, 1995

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
other: acetone and acetone in Alembicol D
Concentration / amount:
Preliminary study:
Induction intradermal injection: 0.1, 0.25, 0.5, 1.0, 2.5, 5.0, 7.5, and 10% Nesterol (v/v) in 5% acetone in Alembicol D
Induction topical application: 30, 50, 70 and 100% Nesterol
Topical challenge: as supplied and 50% (v/v) in acetone

Main Study:
Induction intradermal injections:
The following three injectables were prepared:
-Freund's complete adjuvant diluted with an equal volume of water for irrigation
-Nesterol 10% (v/v) in 5% acetone in Alembicol D
-Nesterol 10% (v/v) in a 50:50 mixture of Freund's complete adjuvant and 5% acetone in Alembicol D
Induction topical application: Nesterol neat (as supplied)
Topical challenge: Nesterol as supplied and 50% (v/v) in acetone
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: acetone and acetone in Alembicol D
Concentration / amount:
Preliminary study:
Induction intradermal injection: 0.1, 0.25, 0.5, 1.0, 2.5, 5.0, 7.5, and 10% Nesterol (v/v) in 5% acetone in Alembicol D
Induction topical application: 30, 50, 70 and 100% Nesterol
Topical challenge: as supplied and 50% (v/v) in acetone

Main Study:
Induction intradermal injections:
The following three injectables were prepared:
-Freund's complete adjuvant diluted with an equal volume of water for irrigation
-Nesterol 10% (v/v) in 5% acetone in Alembicol D
-Nesterol 10% (v/v) in a 50:50 mixture of Freund's complete adjuvant and 5% acetone in Alembicol D
Induction topical application: Nesterol neat (as supplied)
Topical challenge: Nesterol as supplied and 50% (v/v) in acetone
No. of animals per dose:
Preliminary study: 8 animals
Main study: 5 controls and 10 Nesterol-treated animals
Details on study design:
Induction:
-Induction intradermal injections - test animals
A 40 x 60 mm area of dorsal skin on the scapular region of the guinea pig was clipped free of hair with electric clippers. Three pairs of intradermal injections: 1) Freund's complete adjuvant diluted with an equal volume of water for irrigation, 2) Nesterol, 10% (v/v) in 5% acetone in Alembicol D, and 3) Nesterol, 10% (v/v) in a 50:50 mixture of Freund's complete adjuvant and 5% acetone in Alembicol D were made into a 20 x 40 mm area within the clipped area.

-Induction topical application - test animals
Six days after the intradermal injections, the same 40 x 60 mm interscapular area was clipped and shaved free of hair and the site was pre-treated with 0.5 ml per site of 10% (w/w) sodium lauryl sulphate in petrolatum. Twenty-four hours later a 20 x 40 mm patch of Whatman No. 3 paper was saturated with approximately 0.4 ml of Nesterol (neat), placed on the skin of the test animals, covered by a length of impermeable plastic adhesive tape (50 mm width Blenderm), secured by elastic adhesive bandage (50 mm width Elastoplast) wound round the torso of the animal and fixed with an impervious plastic adhesive tape. The dressing was left in place for 48 hours.

-Induction - control animals
During the induction phase, the control animals were treated similarly to the test animals except that the test substance was omitted from the intradermal injections and topical application.

Challenge:
-Challenge - control and test animals
The test animals were challenged topically two weeks after the topical induction using Nesterol (neat) and 50% (v/v) in acetone. Hair was removed by clipping followed by shaving from an area on the left flank of each guinea-pig. A 20 x 20 mm patch of Whatman NO.3 paper was saturated with approximately, 0.2 ml of Nesterol (neat) and applied to an anterior site on the flank. Nesterol 50% (v/v) in acetone was applied in a similar manner to the posterior site. The patches were sealed in the same manner as the induction topical applications. The challenge sites were evaluated 24, 48 and 72 hours after removal of the patches.

Observations:
-Clinical observations:
All animals were observed daily for signs of toxicity.

-Body weights:
The body weight of each guinea pig on the main study was recorded on the say of study start (day of intradermal injections) and on the last day observations were made of dermal responses to the challenge applications.

-Dermal responses:
Erythema and eschar formation:
0-No erythema
1-Slight erythema
2-Well-defined erythema
3-Moderate erythema
4-Severe erythema (beet redness) to slight eschar formation (injuries in depth)

Edema formation:
0-No edema
1-Slight edema
2-Well-defined edema (edges of area well-defined by definite raising)
3-Moderate edema (raised approximately 1 millimeter)
4-Severe edema (raised more than 1 millimeter and extending
beyond the area of exposure)

Interpretation of Results:
Dermal reactions in the test animals elicited by the challenge application were compared with the control animals. A test animal was considered to show positive evidence of delayed contact hypersensitivity if the
observed dermal reaction at challenge was more marked and/or persistent than the maximum reaction seen in the controls.

If the dermal reaction seen in a test animal at challenge was slightly more marked and/or persistent than (but not clearly distinguishable from) the maximum reaction seen in controls, the result for that test animal was classified as inconclusive.

A test animal was considered to show no evidence of delayed contact hypersensitivity if the dermal reaction resulting from the challenge application was the same as, or less marked and/or persistent than the maximum reaction seen in the control group.
Challenge controls:
The control animals were challenged topically two weeks after the topical induction using Nesterol (neat) and 50% (v/v) in acetone. Hair was removed by clipping followed by shaving from an area on the left flank of each guinea-pig. A 20 x 20 mm patch of Whatman NO.3 paper was saturated with approximately, 0.2 ml of Nesterol (neat) and applied to an anterior site on the flank. Nesterol 50% (v/v) in acetone was applied in a similar manner to the posterior site. The patches were sealed in the same manner as the induction topical applications. The challenge sites were evaluated 24, 48 and 72 hours after removal of the patches.
Positive control substance(s):
yes
Remarks:
The sensitivity of the guinea pig strain was periodically checked at the testing facility (not on this particular study)with hexyl cinnamic aldehyde.

Results and discussion

Positive control results:
Clinical observations:
No signs of toxicity were observed in the study.

Body weights:
All animals gained weights during the study.

Induction:
Intradermal injections:
Necrosis was recorded at injection sites receiving Freund's Complete Adjuvant in test and control animals. Slight irritation was seen in test animals at injection sites receiving Nesterol 10% (v/v) in 5% acetone in
Alembicol D and slight irritation was observed in control animals receiving 5 % acetone in Alembicol D alone.

Topical application:
Slight erythema was observed in test animals following topical application with Nesterol (neat) and slight erythema was seen in the controls

Challenge:
No dermal reactions seen in any of the test or control animals.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
All animals
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No erythema or edema was noted in any animals 24 hours after the challenge dermal application of the test substance.
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: All animals. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No erythema or edema was noted in any animals 24 hours after the challenge dermal application of the test substance..
Reading:
1st reading
Hours after challenge:
24
Group:
other: control
Dose level:
All animals
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
No erythema or edema was noted in any animals 24 hours after the challenge dermal application of the test substance.
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: other: control. Dose level: All animals. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: No erythema or edema was noted in any animals 24 hours after the challenge dermal application of the test substance..
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
All animals
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No erythema or edema was noted in any animals 48 hours after the challenge dermal application of the test substance.
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: All animals. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No erythema or edema was noted in any animals 48 hours after the challenge dermal application of the test substance..
Reading:
2nd reading
Hours after challenge:
48
Group:
other: control
Dose level:
All animals
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
No erythema or edema was noted in any animals 48 hours after the challenge dermal application of the test substance.
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: control. Dose level: All animals. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: No erythema or edema was noted in any animals 48 hours after the challenge dermal application of the test substance..
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
test group
Dose level:
All animals
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No erythema or edema was noted in any animals 72 hours after the challenge dermal application of the test substance.
Remarks on result:
other: see Remark
Remarks:
Reading: other: 3rd reading. . Hours after challenge: 72.0. Group: test group. Dose level: All animals. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No erythema or edema was noted in any animals 72 hours after the challenge dermal application of the test substance..
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
other: control
Dose level:
All animals
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
No erythema or edema was noted in any animals 72 hours after the challenge dermal application of the test substance.
Remarks on result:
other: see Remark
Remarks:
Reading: other: 3rd reading. . Hours after challenge: 72.0. Group: other: control. Dose level: All animals. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: No erythema or edema was noted in any animals 72 hours after the challenge dermal application of the test substance..
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
all animals
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
not determinable
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
all animals
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
none
Remarks on result:
not determinable

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
Nesterol did cause irritation (necrosis at injection sites receiving Freund's Complete Adjuvant in test and control animals, slight irritation at injection sites receiving Nesterol 10% (v/v) in 5% acetone in Alembicol D and in control animals receiving 5 % acetone in Alembicol D alone) but did not sensitize any animal.

Under the conditions of the present study, Nesterol is not classified under GHS.
Executive summary:

In a guinea pig maximization test, 10 guinea pigs/sex were intradermally injected with the following: Freund's complete adjuvant diluted with an equal volume of water for irrigation, Nesterol 10% (v/v) in 5% acetone in Alembicol D, and Nesterol 10% (v/v) in a 50:50 mixture of Freund's complete adjuvant and 5% acetone in Alembicol D. Six days after intradermal injections, the animals were exposed topically to a 10% solution of sodium lauryl sulfate. The following day 0.4ml Nesterol (neat) was topically applied and in contact with the skin for 24 hours. Two weeks after the induction period, the animals were challenged with 0.4 ml of either Nesterol (neat) or a 50:50 solution of Nesterol in acetone. Nesterol remained in place for 24 hours under occlusion at which time the patches were removed. The animals were observed for dermal responses at 24, 48, and 72 hours after application of the challenge patch. Irritation, necrosis at injection sites receiving Freund's Complete Adjuvant in test and control animals, slight irritation at injection sites receiving Nesterol 10% (v/v) in 5% acetone in Alembicol D and in control animals receiving 5 % acetone in Alembicol D alone, was observed during the induction phase. No dermal irritation or sensitization was observed after the challenge phase of the study. Under the conditions of the study, Nesterol did not indicate a potential for dermal sensitization.