Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Remarks:
Test substance is one component of the target (UVCB) substance; it is one of the major components, ranging between 10 - 60 % w/w. Other components show slight differences in terms of position / type of functional groups.
Adequacy of study:
key study
Study period:
From December 20, 1994 to January 20, 1995
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Source study has reliability 1. Details on the read across are available in section 13.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
1992
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
A guinea pig maximisation test was available.

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: Himalayan spotted
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BRL, Biological Research Laboratories ltd.
- Females: nulliparous and non-pregnant
- Age at the beginning of accliamtisation period: 5 -7 weeks
- Weight at beginning of acclimatisation period: control and test group 336- 429 g; pretest 367 - 402 g
- Housing: individually in Macrolon cages (type 3) with autoclaved standard softwood bedding
- Diet: ad libitum
- Water: ad libitum
- Acclimatisation: 1 week for the control and test group under test conditions after health examination; no acclimatisation for the animals of the pretest; only animals without any visual signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 19 - 23 °C
- Humidity: 46 - 60 %
- Air changes per hour: 10 - 15
- Photoperiod: 12 hours light cycle

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
physiological saline
Concentration / amount:
5 %
Day(s)/duration:
day 0
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
intradermal
Vehicle:
other: 1:1 adjuvant / saline mixture
Concentration / amount:
5 %
Day(s)/duration:
day 1
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, semiocclusive
Vehicle:
physiological saline
Concentration / amount:
25 %
Day(s)/duration:
day 8
Adequacy of induction:
other: most qualified conc. to assure an optimum technical application procedure
Challenge
No.:
#1
Route:
epicutaneous, semiocclusive
Vehicle:
physiological saline
Concentration / amount:
15 %
Day(s)/duration:
day 22
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Intracutaneous pretest: 2
Epicutaneous pretest: 4
Main test: 10 in control group; 20 in test group
Details on study design:
RANGE FINDING TEST
Intradermal route
Intradermal injections (0.1 ml/site) were made into the clipped flank of 2 guinea-pigs at concentrations of 5, 3 and 1% of the test article in bi-distilled water.
The resulting dermal reactions were assessed 24 hours later. For intradermal induction application a 5 % test article dilution was selected.

Epidermal applications
Both flanks of each of 4 guinea pigs were clipped and shaved just prior to the application. Thereafter 4 patches of filter paper ( 2 × 2 cm) were saturated with the test article at A = 25 % (this concentration used was found to be the most qualified to assure an optimum technical application procedure), B = 15 %, C = 10 % and D = 5 % of the test article in bi-distilled water and applied to the clipped and shaved flanks. The patches were covered by a strip of aluminum foil and firmly secured by elastic plaster wrapped around the trunk and covered with impervious adhesive tape. This procedure ensured the intensive contact of test article. The dressings were removed after an exposure period of 24 hours. Approximately 21 hours after removing of the dressing the application site was depilated with an approved depilatory cream (VEET cream) to clean the application site from staining produced by test article, so that possible erythema reactions were clearly visible at that time.
The depilatory was placed on the patch sites and surrounding areas, and left on for 3-5 minutes. It was then thoroughly washed off with a stream of warm, running water. The animals were then dried with a disposable towel, and returned to their cages. The reaction sites were assessed 24 and 48 hours after removal of the bandage for erythema and oedema on a numerical basis according to Draize described above.

The allocation of the different test dilutions to the sites (A,B,C,D) on the animals was alternated in order to minimize site to site variation in responsiveness.
For the epidermal induction, test article at 25 % and for the challenge procedure, test article at 15 % was selected.

MAIN STUDY
Intradermal injections - test day 1
An area of dorsal skin from the scapular region (approximately 6 × 8 cm) was clipped free of hair. Three pairs of intradermal injections (0.1 ml/site) were made at the border of a 4 × 6 cm area in the clipped region as follows:
Test Group:
1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline
2) test article, diluted to 5 % with bi-distilled water
3) test article diluted to 5 % by emulsion in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
Control Group:
1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline
2) bi-distilled water
3) 1:1 (w/w) mixture of bi-distilled water in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.

Epidermal applications - test day 8
One week after the injections, the scapular area (approximately 6 × 8 cm) was again clipped and shaved free of hair. A 2 × 4 cm patch of filter paper was saturated with test article (25 % in bi-distilled water) and placed over injection sites of test animals. The patch was covered with aluminum foil and firmly secured by an elastic plaster wrapped around the trunk of the animal and secured with impervious adhesive tape. The dressings were left in place for approximately 48 hours. The epidermal application procedure described ensured intensive contact of test substance.
Guinea-pigs of the control group were treated as described above with bidistilled water only.
Reaction sites were assessed for erythema and oedema 24 and approximately 48 hours after removal of the dressing, using the numerical grading system according to Draize.

Challenge - test day 22
Test and control guinea-pigs were challenged two weeks after the epidermal induction application. Test and control guinea-pigs were treated in the same way.
Hair was clipped and shaved from a 5 × 5 cm area on the left and right flank of each guinea-pig just prior to the application. Two patches ( 2 × 2 cm) of filter paper were saturated with the highest non-irritating concentration of 15 % (left flank) and the vehicle only (bi-distilled water, applied to the right flank) using the same method as for the epidermal application. The dressing were left in place for 24 hours.
22 hours after removing of the dressing the test sites treated with the test article were depilated with an approved depilatory cream (VEET cream). The cream was placed on the patch sites for 3-5 minutes and then washed off with a stream of warm running water. When application sites were clean and any stains from test substance removed, the animals were dried with a disposable paper towel and returned to their cages. 24 and approximately 48 hours after the removal of the dressing, application sites were assessed for erythema and oedema using the numerical scoring system according to Draize.
Challenge controls:
yes, test group treated with 0 % test substance at challenge.
Positive control substance(s):
yes
Remarks:
2-mercaptobenzothiazole

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0 % (vehicle control) and 15 % (test substance control)
No. with + reactions:
0
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0 % (vehicle control) and 15 % (test substance control)
No. with + reactions:
0
Total no. in group:
10
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
0 %
No. with + reactions:
0
Total no. in group:
20
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
0 %
No. with + reactions:
0
Total no. in group:
20
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
15 %
No. with + reactions:
0
Total no. in group:
20
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
15 %
No. with + reactions:
0
Total no. in group:
20

Any other information on results incl. tables

Skin effects after intradermal induction - day 1

Control group

Injection site 1 (1:1 (v/v) mixture of Freund's Complete Adjuvant [FCA] and physiological saline)

The area around the injection site was oedematous and erythematous from day 2 to 4, and became necrotic from day 5 to 7 followed by encrustation and exfoliation of encrustation up to the termination of test.

Injection site 2 (bi-distilled water)

No local symptoms were observed.

Injection site 3 (1:1 (w/w) mixture of bi-distilled water in a 1:1 (v/v) mixture of FCA and physiological saline)

The reactions observed were identical to those obtained at injection site 1 with the mixture of FCA and physiological saline.

As the animals were bandaged with the semi-occlusive dressing no observations of the skin were possible on day 9.

Test group

Injection site 1 (1:1 (v/v) mixture of Freund's Complete Adjuvant [FCA] and physiological saline)

The area around the injection site was oedematous and erythematous from day 2 to 4, and became necrotic from day 5 to 7 followed by encrustation and exfoliation of encrustation up to the termination of test.

Injection site 2 (5 % dilution of test article in bi-distilled water)

The area around the injection site was oedematous and red discoloured from day 2 to 4, and became necrotic from day 5 to 7 followed by encrustation and exfoliation of encrustation up to the termination of test.

Injection site 3 (5 % dilution of test article in a 1:1 (v/v) mixture of FCA and physiological saline)

The reactions observed were identical to those obtained at injection site 2 in the test group.

As the animals were bandaged with the semi-occlusive dressing no observations of the skin were possible on day 9.

Skin effects after epidermal induction - day 8

Control group

No erythematous or oedematous reaction was observed in the animals treated with bi-distilled water only.

Test group

As the test article stained the skin red, it was not possible to determine whether erythema was present. However, no oedema was observed. Red discolouration was noted from day 10 to 25.

Skin effects after challenge - day 22

Control and test group

No positive reactions were observed in the animals either when treated with bidistilled water alone or when treated with the test article at 15 % in bidistilled water.

Red discolouration was noted from test day 23 (after removal of the dressing) to 24 (prior to the depilation).

animal 24 h 48 h
erythema oedema eythema oedema
519 0 0 0 0
520 0 0 0 0
521 0 0 0 0
522 0 0 0 0
523 0 0 0 0
524 0 0 0 0
525 0 0 0 0
526 0 0 0 0
527 0 0 0 0
528 0 0 0 0
529 0 0 0 0
530 0 0 0 0
531 0 0 0 0
532 0 0 0 0
533 0 0 0 0
534 0 0 0 0
535 0 0 0 0
536 0 0 0 0
537 0 0 0 0
538 0 0 0 0

Other observations

Viability / mortality / macroscopic findings: no deaths occurred, thus no necropsies were performed.

Clinical signs, systemic: no symptoms of systemic toxicity were observed in the animals.

Body weights: within the normal range of variability. One animal (no. 541) of the epidermal pretest lost weight during the acclimatization period and one animal (no. 517) of the control group lost weight during the treatment period. The lost of weight is considered to be of incidental nature and treatment unrelated.

Applicant's summary and conclusion

Interpretation of results:
other: not classified within the CLP Regulation (EC 1272/2008)
Conclusions:
No skin reactions were noted in animals of test group, 24 and 48 h after removal of the dressings.
Executive summary:

Method

Test substance was assessed for skin sensitisation according to OECD guideline 406.

In a pre-test, a concentration of 5 % in physiological saline was selected for intradermal induction, as it could be injected and was well tolerated; doses of 5, 10, 15 and 25 % in physiological saline were tested by topical application under semi-occlusive dressing for 24 h. 24 and 48 h after removal of the dressings, reactions were noted at 25 %.

On day 1 of main test, intradermal induction was done by injecting 5 % test substance in physiological saline or 1:1 adjuvant/saline mixture.

On day 8 of main test, induction by epidermal occlusive application was done with test substance at 25 % in bi-distilled water.

On day 22 of main test, challenge was done by epidermal semi-occlusive application of test substance at 15 % in bi-distilled water for 24 h. Observations after challenge were done 24 and 48 h after removal of the dressing.

Results

Challenge reactions were scored 24 and 48 h after removal of the dressings; none of treated and control animals showed skin reactions.