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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:
24 August 1989 - 1 October 1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP study performed according to OECD guideline 406 with minor deviations: no data about purity and no certificate of analysis of the test substance
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
yes
Remarks:
No data about purity/stability and no certificate of analysis of the test substance
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of study:
guinea pig maximisation test

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
417-790-1
EC Name:
-
Cas Number:
78418-01-6
Molecular formula:
C15H20O4
IUPAC Name:
2-Hydroxy-5-(1-oxooctyl)benzoic acid
Constituent 2
Reference substance name:
MEXORYL SAB
IUPAC Name:
MEXORYL SAB
Details on test material:
- Name of test material (as cited in study report): ER 195
- Physical state: white crystalline powder
- Lot/batch No.: DG6
- 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: David Hall Limited, Burton-on-Trent, U.K.
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 326-402 g
- Housing: in groups of up to four in solid-floor polypropylene cages with softwood shavings
- Diet (e.g. ad libitum): Guinea Pig FD1 Diet, Special Diet Services Limited, Witham, U.K., ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-23
- Humidity (%): 60-68%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: from 24 August 1989 to 1 October 1989

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
arachis oil
Concentration / amount:
Intradermal induction:
Test group: three pairs of intradermal injections (0.1 mL)
1) 1:1 mixture of Freund's Complete Adjuvant and distilled water
2) 1% (w/v) dilution of test material in arachis oil
3) 1% (w/v) dilution of test material in a 1:1 preparation of Freund's Complete Adjuvant and arachis oil

Control group: three pairs of intradermal injections (0.1 mL)
1) 1:1 mixture of Freund's Complete Adjuvant and distilled water
2) 1% (w/v) dilution of arachis oil
3) 1% (w/v) dilution of arachis oil in a 1:1 preparation of Freund's Complete Adjuvant and arachis oil

Topical induction: 0.5% (w/w) in arachis oil

Challenge: 2% (w/w) in arachis oil
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
arachis oil
Concentration / amount:
Intradermal induction:
Test group: three pairs of intradermal injections (0.1 mL)
1) 1:1 mixture of Freund's Complete Adjuvant and distilled water
2) 1% (w/v) dilution of test material in arachis oil
3) 1% (w/v) dilution of test material in a 1:1 preparation of Freund's Complete Adjuvant and arachis oil

Control group: three pairs of intradermal injections (0.1 mL)
1) 1:1 mixture of Freund's Complete Adjuvant and distilled water
2) 1% (w/v) dilution of arachis oil
3) 1% (w/v) dilution of arachis oil in a 1:1 preparation of Freund's Complete Adjuvant and arachis oil

Topical induction: 0.5% (w/w) in arachis oil

Challenge: 2% (w/w) in arachis oil
No. of animals per dose:
20 animals treated with test item
10 animals as control
Details on study design:
RANGE FINDING TESTS:
Intradermal irritancy (see table1):
Two animals were intradermally injected 1 and 5% of test item in arachis oil, respectively, and were observed for 7 days. At 5%, necrosis was observed and as well as eschar 7 days after injection.
Topical irritancy (see tables 2 and 3):
Four animals were each exposed to 4 concentrations of the test item by topical application for 48 h and were observed up to 48 h after exposure: two of them were exposed to 50, 25, 10 and 5% and the two others were tested at 2, 1, 0.5 and 0.1%. Exposure at 0.5% caused irritation effects scored 1 in both animals, one hour after exposure, and reversible within 24 h.
Two animals were each exposed to 2, 1, 0.5 and 0.1% of the test item by topical application for 24 h and were observed up to 48 h after exposure: exposure to 2% gave slight irritation effects scored 1, one hour after exposure, and reversible within 24 h.
Thus, concentrations of 1, 0.5 and 2% were chosen for intradermal induction, topical induction and challenge, respectively.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2
- Exposure period: 7 days + 48 h
- Test groups: 20 animals intradermally injected 3 pairs of 0.1 mL injections:
1) 1:1 mixture of Freund's Complete Adjuvant and distilled water
2) 1% (w/v) dilution of test material in arachis oil
3) 1% (w/v) dilution of test material in a 1:1 preparation of Freund's Complete Adjuvant and arachis oil
One week later, topical application of the test article over the injection sites was done with 0.2-0.3 mL of 0.5% (w/w) of test item in arachis oil for 48 h under occlusive conditions.
Site: shoulder region

- Control group: 10 animals intradermally injected 3 pairs of 0.1 mL injections:
1) 1:1 mixture of Freund's Complete Adjuvant and distilled water
2) 1% (w/v) dilution of arachis oil
3) 1% (w/v) dilution of arachis oil in a 1:1 preparation of Freund's Complete Adjuvant and arachis oil
One week later, topical application of 0.2-0.3 mL of arachis oil over the injection sites for 48 h under occlusive conditions.
Site: shoulder region

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 2 weeks after topical induction
- Exposure period: 24 h under occlusive consitions
- Site: right flank for test substance, left flank for arachis oil only, for all animals
- Concentrations: 2% w/w in arachis oil
- Evaluation (hr after challenge): 24 and 48 h
Challenge controls:
Patches of arachis oil applied on the left flanks of all animals
Positive control substance(s):
yes
Remarks:
2,4-dinitrochlorobenzene

Study design: in vivo (LLNA)

Concentration:
Not applicable
No. of animals per dose:
Not applicable
Details on study design:
Not applicable
Statistics:
Not applicable

Results and discussion

Positive control results:
The positive control, challenged at 0.1%, produced 15/19 positive responses, corresponding to 79% of positive responses.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
2 %
No. with + reactions:
14
Total no. in group:
20
Clinical observations:
mild to moderate redness with haemorrhage of the dermal capillaries in 3 animals
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 2 %. No with. + reactions: 14.0. Total no. in groups: 20.0. Clinical observations: mild to moderate redness with haemorrhage of the dermal capillaries in 3 animals.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
2 %
No. with + reactions:
4
Total no. in group:
20
Clinical observations:
Desquamation in positive animals and in 8 other animals
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 2 %. No with. + reactions: 4.0. Total no. in groups: 20.0. Clinical observations: Desquamation in positive animals and in 8 other animals.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
2 %
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 2 %. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
2 %
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 2 %. No with. + reactions: 0.0. Total no. in groups: 10.0.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: Not applicable
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Not applicable

Any other information on results incl. tables

Table 1: Intradermal irritancy during range-finding test

Animal

Time of observation

Concentration of test material (% w/v)

Evidence of necrosis

Evidence of systemic toxicity

A

24 h

1

None

None

48 h

None

None

72 h

None

None

7 days

None

None

B

24 h

5

Necrosis

None

48 h

Necrosis

None

72 h

Necrosis

None

7 days

Eschar

None

Table 2: Topical irritancy during range-finding test after 48-h exposure

Animal

Concentration of test material (% w/v)

Time of observation after dressing removal

1 h

24 h

48 h

C

50

?eOeWeN

?eThN

?eThN

25

?eOeWeN

?eThN

?eThN

10

?eWeN

?eThN

?eThN

5

?eHdWe

?eHdTh

?eHdTh

D

50

?eOeHdWeN

?eThN

?eThN

25

?eOeHdWeN

?eThN

?eThN

10

?eOeHd

?eThHd

?eThHd

5

?eOeHd

?eThHd

?eThHd

E

2

2 Oe

1

1 ThD

1

1 Oe

1

1

0.5

1 Oe

0

0

0.1

1

0

0

F

2

?eOeHdLe

?eStOeBs

?eStOeBs

1

?eHdLeOe

?eStOeBs

?eStOeCr

0.5

1 Oe

0

0

0.1

1

0

0

?e = evaluation of erythema precluded by other adverse reactions

Bs = bleeding due to fissuring

Cr = cracking of the skin

D = desquamation

Hd = haemorrhage of dermal capillaries

Le = loss of skin elasticity

N = necrosis

Od = well-defined oedema

Oe = oedema extending beyond treatment site

R = reaction extending beyond treatment site

Ss = small superficial scattered scabs

St = small areas of black-coloured scabs over test site

Th = thickening of the skin

We = well-defined erythema surrounding treatment site

Table 3: Topical irritancy during range-finding test after 24-h exposure

Animal

Concentration of test material (% w/v)

Time of observation after dressing removal

1 h

24 h

48 h

G

2

1

0

0

1

1

0

0

0.5

0

0

0

0.1

0

0

0

H

2

1

0

0

1

0

0

0

0.5

0

0

0

0.1

0

0

0

Table 4: Skin reactions after challenge in the main study

 

Skin reaction after topical induction

(hours after removal of dressing)

Skin reaction after challenge

(hours after removal of dressing)

1

24

24

48

Test

Vehicle

Test

Vehicle

Test group

1

1

1 R

0

0 D

0

1

0

0

0

0

0

1

0

?eHdOd

0

?eDTh

0

2

?eSs

1

0

0 D

0

1

0

1

0

0 D

0

1

0

1

0

0 D

0

1

0

0

0

0

0

2

1

1

0

0 D

0

1

0

0

0

0

0

2

1

1

0

0 D

0

1

0

0

0

0

0

1

1

0

0

0

0

1

0

0

0

0 D

0

1

0

2

0

1 D

0

2

1

2 Hd

0

1 D

0

1

1

1

0

0 D

0

2

1

?eHdOd

0

1 D

0

2

0

1

0

0

0

1

1

1

0

0

0

1

1

1

0

0

0

Control group

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

?e = evaluation of erythema precluded by other adverse reactions

D = desquamation

Hd = haemorrhage of dermal capillaries

Od = well-defined oedema

Ss = small superficial scattered scabs

Th = thickening of the skin

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information
Conclusions:
Under these test conditions, Mexoryl SAB can be classified as skin sensitiser according to the Annex VI to the Directive 67/548/EEC and in category 1 of the CLP Regulation (EC) N° (1272-2008).
Executive summary:

A dermal sensitisation study using Magnusson and Kligman method on guinea pigs with ER 195 was done according to OECD guideline n°406 and in GLP conditions. 20 test animals were induced intradermally with 1% w/v of test substance in arachis oil with Freund's Complete Adjuvant and 10 additional animals served as controls (vehicle and Freund's Complete Adjuvant). After 1 week, topical application of the test article (or arachis oil alone for controls) over the injection sites was done with 0.5% w/w concentration of the test article in arachis oil for 48 h under occlusive conditions. Two weeks later, animals were challenged using occlusive topical application with 2% w/w test article in arachis oil and placed onto the right flank of all animals for 24 h. The left flank was similarly treated with arachis oil alone and served as control. Skin reactions were scored 24 and 48 h after dressing removal.

14 out of 20 animals had positive reactions 24 h after challenge and 4 animals were positive at 48 h reading whereas no reaction could be observed in controls. As more than 30 % of total tested animals (70%) had positive response, it is concluded that under these test conditions, ER 195 needs to be classified as skin sensitiser according to the Annex VI to the Directive 67/548/EEC and in category 1 of the CLP Regulation (EC) N° (1272-2008).