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Key value for chemical safety assessment

Skin sensitisation

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Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January 23 to February 16, 1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: guideline test with GLP
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
Forty female SPF albino guinea pigs of the Dunkin Hartley strain were obtained from the Møllegaard Breeding Centre ApS, Ejby, DK-4623 Lille Skensved. At start of the experiment the guinea pigs were 6 to 7 weeks old and weighed between 291 and 381 g.
The guinea pigs were housed in opaque PPL (Type IV) cages, two or three to a cage. The bedding used was pinewood sawdust "Spanvall Special White" from Spanvall A/S, Jorløse, DK-4490 Jerslev. The guinea pigs had free access to a pelleted diet, "3113 Altromin", from Chr. Petersen A/S, DK-4100 Ringsted, and vitamin C enriched tap water. Regular analyses for relevant possible water contaminants are performed. The room temperature was set at 21 ±2 °C and the relative humidity at 55 ±15%, Air change 6 times an hour and light on from 6 to 18 h. Analysis certificate of bedding is presented. Declaration and analysis of diet mixture are given in Appendices.
Route:
intradermal and epicutaneous
Vehicle:
other: groundnut oil for intradermal induction, Ethanol 96% : diethyl phthalate (DEP) 1:1 for dermal induction and challenge
Concentration / amount:
On the basis of the preliminary investigations, the following dose levels were selected: A concentration of 2.5% (w/w) was selected for the intradermal induction. A 50% (w/w) test concentration was chosen for the dermal induction and for the challenge application a 25% (w/w) test concentration was selected.
Route:
epicutaneous, occlusive
Vehicle:
other: groundnut oil for intradermal induction, Ethanol 96% : diethyl phthalate (DEP) 1:1 for dermal induction and challenge
Concentration / amount:
On the basis of the preliminary investigations, the following dose levels were selected: A concentration of 2.5% (w/w) was selected for the intradermal induction. A 50% (w/w) test concentration was chosen for the dermal induction and for the challenge application a 25% (w/w) test concentration was selected.
No. of animals per dose:
20
Details on study design:
Groups and numbers
The guinea pigs were randomly allocated into two groups and earmarked with a punched number. To secure that each animal could easily be identified the guinea pig with the highest number in a cage was marked on the forehead with a red India ink pen and the guinea pig with the second highest number was marked green.
The groups and numbers were as follows:
Control group: 20
test group: 20
PRELIMINARY INVESTIGATIONS
The intradermal irritancy of the test article was investigated in order to find a slight to moderate irritant test concentration for the intradermal induction. The topical irritancy of the test article was investigated in order to find a slight to moderate irritant concentration for the dermal induction.The topical irritancy of the test article was investigated in order to find the highest non-irritant concentration for the challenge application.
The procedure was in two parts: induction and challenge
Induction test group:
intradermal injection: An area of dorsal skin 4 x 6 cm in the scapular region was clipped free of hair with an electric clipper. Three pairs of intradermal injections were given simultaneously into this area. Each dose was 0.05 ml. The vehicle was groundnut oil. Preparations for injection were as follows:
1. Freund's complete adjuvant diluted 1:1 with water for injection.
2. 2.5% (w/w) test article.
3. 5.0% (w/w) test article and Freund’s complete adjuvant in the ratio 1:1.
Topical Application
Six days after the injections, the same 4 x 6 cm scapular area was clipped free of hair. To provoke a mild inflammatory reaction 10% sodium lauryl sulphate in petrolatum was massaged into the skin with a glass rod. 24 hours later 0,4 ml of 50% (w/w) test article in ethanol 96%/DEP (1:1) was spread over a 3 x 5 cm patch of Whatman No. 3 MM paper. The patch was placed on the skin and covered with 7 cm impermeable adhesive tape (Blendenn 5 cm width). This was firmly secured with an adhesive bandage (Scanpor Tape 5 cm width) wound round the trunk. The dressing was left in place for 48 hours.
Induction control group
intradermal injection:
An area of dorsal skin 4 x 6 cm in the scapular region was clipped free of hair with an electric clipper. Three pairs of intradermal injections were given simultaneously into this area. Each dose was 0.05 ml. The vehicle was groundnut oil, Ph. Nord. Preparation for injections were as follows:
1. Freund's complete adjuvant diluted 1:1 with water for injection.
2. vehicle.
3. vehicle and Freund’s complete adjuvant in the ratio 1:1.
Topical Application:
Six days after the injections, the same 4 x 6 cm scapular area was clipped free of hair. To provoke a mild inflammatory reaction 10% sodium lauryl sulphate in petrolatum was massaged into the skin with a glass rod* 24 hours later 0,4 ml ethanol 96%/DEP (1:1) was spread over a 3 x 5 cm patch of Whatman No. 3 MM paper. The patch was placed on the skin and covered with 7 cm impermeable adhesive tape (Blenderm 5 cm width). This was firmly secured with an adhesive bandage (Scanpor Tape 5 cm width) wound round the trunk. The dressing was left in place for 48 hours.
challenge:
The guinea pigs were challenged topically three weeks after the intradermal induction. The vehicle was ethanol 96% and diethyl phthalate (DEP) in the ratio 1:1 (w/w).
Hair was removed with an electric clipper from a 5 x 5 cm area from both flanks of the guinea pigs in the test group and the control group. About 0.1 ml of 25% (w/w) test article was applied to a 2 x 2cm Whatman No. 3 MM paper and placed on the left flank of the guinea pigs in the test group and on the right flank of the guinea pigs in the control group. In a similar way 0.1 ml vehicle was applied on the right flank of the guinea pigs in the test group. The patches were covered with 4 cm Blenderm (5 cm width) and secured with Scanpor Tape (7.5 cm width) wound round the trunk. After 24 hours of exposure the patches and tape were removed.
Reading Challenge Reactions
Each challenge site was read 24, 48 and 72 hours after removal of the patch. At least three hours before the 24-hour reading the sites were clipped and shaved facilitating the evaluation.


Positive control substance(s):
not specified
Positive control results:
no data
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
25%(w/w) test article
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
6/20 animals showed grade 1 reaction on the vehicle site, one of these also a grade 1 reaction on the test site.
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 25%(w/w) test article. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: 6/20 animals showed grade 1 reaction on the vehicle site, one of these also a grade 1 reaction on the test site..
Reading:
1st reading
Hours after challenge:
24
Group:
other: vehicle control
Dose level:
0
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
5/20 animals showed grade 1 reaction to the vehicle
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: other: vehicle control. Dose level: 0. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: 5/20 animals showed grade 1 reaction to the vehicle.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
25%(w/w) test article
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
2/20 animals showed grade 1 reaction on the vehicle site, one of these also a grade 1 reaction on the test site.
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 25%(w/w) test article. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: 2/20 animals showed grade 1 reaction on the vehicle site, one of these also a grade 1 reaction on the test site..
Reading:
2nd reading
Hours after challenge:
48
Group:
other: vehicle control
Dose level:
0
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
1/20 animals showed grade 1 reaction to the vehicle
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: vehicle control. Dose level: 0. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: 1/20 animals showed grade 1 reaction to the vehicle.
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
test group
Dose level:
25%(w/w) test article
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
no positive reaction was observed
Remarks on result:
other: Reading: other: 3rd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 25%(w/w) test article. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: no positive reaction was observed.
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
other: vehicle control
Dose level:
0
No. with + reactions:
0
Total no. in group:
29
Clinical observations:
no positive reaction was observed
Remarks on result:
other: Reading: other: 3rd reading. . Hours after challenge: 72.0. Group: other: vehicle control. Dose level: 0. No with. + reactions: 0.0. Total no. in groups: 29.0. Clinical observations: no positive reaction was observed.

Health and body weight

The guinea pigs made normal body weight changes over the duration of the study and exhibited normal appearance and behaviour.
Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the experimental conditions described in the authorized report the test article did not produce any evidence of delayed contact hypersensitivity in guinea pigs.
Executive summary:

This study was conducted to investigate the allergenic potential of test article according to OECD guideline No. 406. The procedure was in two parts: induction and challenge. On the basis of the preliminary investigations, the following dose levels were selected: A concentration of 2.5% (w/w) was selected for the intradermal induction. A 50% (w/w) test concentration was chosen for the dermal induction and for the challenge application a 25% (w/w) test concentration was selected. The guinea pigs were challenged topically three weeks after the intradermal induction. Each challenge site was read 24, 48 and 72 hours after removal of the patch. At least three hours before the 24-hour reading the sites were clipped and shaved facilitating the evaluation. No skin responses evaluated as positive were observed.

In test and control groups sporadic grade 1 reactions to the vehicle were observed.

The guinea pigs made normal body weight changes over the duration of the study and exhibited normal appearance and behaviour.

Therefore, under the experimental condition, test article failed to cause allergenic response to guinea pigs.

 

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The sensitisation study was performed to investigate the allergenic potential of test article. The procedure was in two parts: induction and challenge. On the basis of the preliminary investigations, the following dose levels were selected: A concentration of 2.5% (w/w) was selected for the intradermal induction. A 50% (w/w) test concentration was chosen for the dermal induction and for the challenge application a 25% (w/w) test concentration was selected. The guinea pigs were challenged topically three weeks after the intradermal induction. No skin responses evaluated as positive were observed. In test and control groups sporadic grade 1 reactions to the vehicle were observed.The guinea pigs made normal body weight changes over the duration of the study and exhibited normal appearance and behaviour.

Therefore, under the experimental condition, test article did not cause allergenic response to guinea pigs.

Migrated from Short description of key information:
Test article was not considered to be a sensitizer to guinea pig based on the available data.

Justification for selection of skin sensitisation endpoint:
one study according to OECD guideline and GLP was available

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

In a guideline-compliant primary skin sensitisation study in guinea pigs, 2-ethylhexyl salicylate showed no skin sensitising properties. Therefore, 2-ethylhexyl salicylate does not have to be classified as a skin sensitizer according to the criteria laid down in the EU Dangerous Substances Directive (67/548/EEC) and in the EU Classification Labelling and Packaging Regulation (1272/2008/EC).

For respiratory sensitisation no data is available.