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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:
13 March 1989 to 25 September 1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report date:
1989

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The in vivo guinea pig maximisation test was already available (performed in 1989) and assessed reliable to evaluate the skin sensitization potential and the classification determination.

Test material

Constituent 1
Chemical structure
Reference substance name:
Cyclopentanone, 2-[(2R)-2-[(1R)-4-methyl-3-cyclohexen-1-yl]propyl]-, (2S)-rel-
Molecular formula:
C14H24O1
IUPAC Name:
Cyclopentanone, 2-[(2R)-2-[(1R)-4-methyl-3-cyclohexen-1-yl]propyl]-, (2S)-rel-
Constituent 2
Chemical structure
Reference substance name:
Cyclopentanone, 2-[(2S)-2-[(1R)-4-methyl-3-cyclohexen-1-yl]propyl]-, (2R)-rel-
Molecular formula:
C15H24O
IUPAC Name:
Cyclopentanone, 2-[(2S)-2-[(1R)-4-methyl-3-cyclohexen-1-yl]propyl]-, (2R)-rel-
Constituent 3
Chemical structure
Reference substance name:
Cyclopentanone, 2-[(2S)-2-[(1R)-4-methyl-3-cyclohexen-1-yl]propyl]-, (2S)-rel-
Molecular formula:
C15H24O
IUPAC Name:
Cyclopentanone, 2-[(2S)-2-[(1R)-4-methyl-3-cyclohexen-1-yl]propyl]-, (2S)-rel-
Constituent 4
Chemical structure
Reference substance name:
Cyclopentanone, 2-[(2R)-2-[(1R)-4-methyl-3-cyclohexen-1-yl]propyl]-, (2R)-rel-
Molecular formula:
C15H24O
IUPAC Name:
Cyclopentanone, 2-[(2R)-2-[(1R)-4-methyl-3-cyclohexen-1-yl]propyl]-, (2R)-rel-
Test material form:
liquid
Specific details on test material used for the study:
Substance name (as stated in the report): NECTARYL - LRG 1371
Identification for the study: 07683 D9 001
Description: colourless slightly viscous liquid
Batch: AS1241001

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male/female
Details on test animals and environmental conditions:
- Supplier: Elevage Lebeau (78950 Gambais - France)
- Age: young adults
- Weight at the start of treatment : preliminary studies 394 g to 497 g and main study: 365 g to 470 g
- Number and sex at preliminary studies : 6 males, 6 non-pregnant females
- Number and sex at main study: control group: 10 males, 10 non-pregnant females ; treated group : 10 males, 10 non-pregnant females. Two supplementary guinea-pigs (1 male+ 1 female) will also be treated to replace any non­ treatment related deaths.
- Cages: housing by sex, in groups of 5 or 6 (or 2 for the studies), in polystyrene cages with perforated flooring dimensions 560 x 355 x 315 mm).
- Temperature: 17.5 to 24.5°c
- Humidity: 23 to 78 % R.H.
- Lighting: artificial, 12 hours out of 24 (photoperiod = 7h30 - 19h30)

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
paraffin oil
Remarks:
sterile codex liquid paraffin
Concentration / amount:
10% (w/w) / 2 x 0.1 ml injection
Day(s)/duration:
Day 1
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
intradermal
Vehicle:
physiological saline
Concentration / amount:
50% (v/v) in an isotonic injectable solution / 2 x 0.1 ml injection
Day(s)/duration:
Day 1
Adequacy of induction:
other: Magnusson and Kligman protocol
Route:
intradermal
Vehicle:
paraffin oil
Remarks:
sterile codex liquid paraffin
Concentration / amount:
mixture 50/50 (V/V) : test substance in a 10% (w/w) solution in sterile codex liquid paraffin + Freund's complete adjuvant at 50% (v/v) in an isotonic injectable solution / 2 x 0.1 ml injection
Day(s)/duration:
Day 1
Adequacy of induction:
other: Magnusson and Kligman protocol
Route:
epicutaneous, occlusive
Vehicle:
paraffin oil
Remarks:
sterile codex liquid paraffin
Concentration / amount:
0.5mL of 10% (W/W) suspension of sodium lauryl sulphate in Codex paraffin
Day(s)/duration:
Day 8 / 48 hours
Adequacy of induction:
other: Magnusson and Kligman protocol
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
0.5 ml of the test substance as supplied (undiluted)
Day(s)/duration:
Day 9 / 48 hours
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
Challengeopen allclose all
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Remarks:
as supplied (no dilution)
Concentration / amount:
0.5 ml on the left flank
Day(s)/duration:
24 hours
Adequacy of challenge:
highest non-irritant concentration
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
paraffin oil
Remarks:
sterile codex liquid paraffin
Concentration / amount:
0.5 ml on the right flank
Day(s)/duration:
24 hours
No. of animals per dose:
Induction preliminary studies:
- Intradermal route: one treated group of 2 clipped males and 2 clipped females
- Topical route: a new treated group of 2 clipped and shaved males and 2 clipped and shaved females
"Challenge" application preliminary study:
- Topical route: a new treated group of 2 clipped and shaved males and 2 clipped and shaved females
MAIN STUDY
- group of 10 (+ 1) males, 10 (+ 1) females
Challenge controls:
The intradermal injections and the topical occlusive application for 48 hours were carried out under the same conditions as in the treated group, sterile Codex liquid paraffin replacing the test substance
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:
test chemical
Dose level:
0.5 ml of the test substance as supplied
No. with + reactions:
0
Total no. in group:
40
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
0.5 ml of the test substance
No. with + reactions:
0
Total no. in group:
40
Reading:
other: all readings
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
40
Reading:
other: all readings
Group:
positive control
Dose level:
not performed
No. with + reactions:
0
Total no. in group:
0
Remarks on result:
not measured/tested

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Taking into account the macroscopic results obtained under the experimental conditions employed, the test substance did not provoke any reaction of cutaneous sensitization in the 20 guinea-pigs of the treated group. No cutaneous abnormality was noted in the 20 guinea-pigs of the control group.
Executive summary:

Evaluation of the delayed cutaneous hypersensitivity of the test material was performed in the albino Dunkin-Hartley guinea pig. Forty male and female animals were divided into a test group (10 males and 10 females) and a control group (10 males and 10 females). Animals weighed 465 g to 470 g at the start of treatment. Animals were housed by sex in groups of 5 in polystyrene cages with perforated flooring. Animal room temperature was 17.5 to 24.5 C; relative humidity was 23 to 78%; artificial lighting was 12 hours light/dark. Animals received a complete pelleted guinea pig maintenance diet and water (softened and filtered 15 um) mains drinking water ad libitum. Animals were acclimated for 19 days before the start of treatment. Before test material administration the back and flanks of each animal were clipped free of hair. Induction was by the intradermal route and consisted of 3 series of 2 x 0.1 ml injections (2 cm x 4 cm clipped area): Treated group - Freund's complete adjuvant at 50% (v/v) in an isotonic injectiable solution (0.9% NaCl); test material in a 10% (w/w) solution in sterile Codex liquid paraffin; mixture 50/50 (v/v): test material in a 20% (w/w) solution in sterile Codex liquid paraffin plus Freund's complete adjuvant at 50% (v/v) in an isotonic injectable solution, i.e. a final 10% solution of the sample controlled. The control group received the same injections only without the test material. On day 8, sodium lauryl sulphate was applied under the following conditions to induce a local irritation in the treated and control groups as application of the test material as supplied during the preliminary study was non-irritant. 0.5 ml of a 10% (w/w) suspension of sodium lauryl sulphate in Codex paraffin was applied to the clipped and shaved skin of each guinea pig. On Day 9 the topical induction of the test material was conducted under an occlusive patch. Application area was the clipped and shaved area of 8 cm2 corresponding to that used for the intradermal injections. Topical induction: 0.5 ml of test material as supplied or Codex liquid paraffin (vehicle for control group) was applied to the skin of each guinea pig under an occlusive patch composed of Whatman 2 cm x 4 cm filter paper held in contact with the skin by means of a 3 cm x 5 cm impermeable and hypoallergenic bandage (Blenderm). The patch was secured by a material adhesive tape 4 cm wide which was applied on an absorbent gauze compress covering the entire clipped area. The patches remained in place for 48 hours. A rest period of 11 days was provided. No treatment was administered. After the rest period a challenge application was administered on Day 22 of the study. The application area was the left lateral abdominal region (test material) and right lateral abdominal region (vehicle) to an area of 4 cm2, clipped, shaved and previously untreated. 0.5 ml of the test material as supplied (Maximum Non-Irritant Concentration) was applied to the left flank; 0.5 ml of Codex liquid paraffin (vehicle) was applied to the right flank. Application was to the skin under an occlusive dressing composed of 2 cm x 2 cm filter paper (Whatman) held in contact with the skin wih an adhesive hypoallergenic patch made up of an occlusive disc 28 mm in diameter and kept in contact with the skin by means of an adhesive and microporous ring 10 mm wide. Fixation of this patch was reinforced with a material adhesive band 4 cm wide, which was applied on an absorbent gauze compress covering the entire clipped surface. Patches remained in place for 24 hours. Evaluation of the macroscopic cutaneous reactions (erythema and oedema) were made at 24 and 48 hours after removal of dressings according to the following scale: 0 = no reaction; 1 = slight erythema (hardly visible); 2 = moderate erythema (easily visible); 3 = severe erythema with oedema. Any other abnormality was also noted: vesicles, thickening, dryness of the skin etc. A positive reaction was when one of the following reactions was observed: focal reaction, vesicular effect, erythematous or oedematous lesions interpreted as a difference equal to or greater than 2 in comparison with the vehicle application site (right flank) and with the control group guinea pigs (vehicle plus test material). A negative reaction was a score = to 0 or a score of "1" equal to that noted at the vehicle application site (right flank) and to that of the control group guinea pigs (vehicle plus test material).

Histopathological examinations of the skin were not conducted as no macroscopic reactions were observed in the guinea pigs examined. The sensitivity of the method was evaluated separately using several different substances well known for their sensitizing potential. Positive sensitization results proved the sensitivity of the method.

One guinea pig in the test group was found dead. No abnormality was noted when necropsying. It was replaced by a guinea pig in the complementary group which was treated in the same way.

Under the conditions of the study, the test material did not produce any skin sensitization reactions in the guinea pigs tested.

NECTARYL was considered to be not skin sensitizer according to this test and it does not meet the criteria to be classified according to the CLP Regulation 1272/2008/EC.

 

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