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Description of key information

The test substance showed no indication of skin sensitising potential in a GLP compliant OECD 406 study.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
not specified
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
1992
Deviations:
yes
Remarks:
see "Principles of method if other than guideline"
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
1992
Qualifier:
according to
Guideline:
other: Japanese MAFF
Principles of method if other than guideline:
DEVIATIONS: to comply with the Japanese guidelines and requirements, the following deviations were made from the standard protocol:
- Increased group size of 20 animals (10 males and 10 females per group)
- Additional positive control group Mercaptobenzothiazole (MBT)
- Test pattern (cranial to caudal sequence in duplicate)
- Test group: test article/test article + adjuvant/adjuvant
- Negative control: Adjuvant/adjuvant + vehicle/vehicle
- Positive control: MBT/MBT + adjuvant/adjuvant
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The guinea pig maximisation test is the recommended adjuvant test for predictive sensitisation according to the above guidelines
Species:
guinea pig
Strain:
other: Pirbright White (Tif:DHP)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: Young adult animals
- Weight at study initiation: 309 to 422 g
- Housing: Individually in Macrolon cages
- Diet: Pig pellets, ad libitum
- Water: Fresh water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 to 70
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12 / 12

- IN-LIFE DATES: Start: 16 Oct 1995 End: 9 Nov 1995
Route:
intradermal
Vehicle:
other:
Remarks:
peanut oil or physiological saline
Concentration / amount:
1% of the test substance in peanut oil; 1% of the test substance in the adjuvant/physiological saline mixture (w/v); adjuvant/physiological saline mixture 1:1 (v/v) / 0.1 mL
Day(s)/duration:
3 pairs of injections
Route:
epicutaneous, occlusive
Vehicle:
other: vaseline
Concentration / amount:
30 % of the test substance in vehicle (vaseline)
Day(s)/duration:
2 days
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: vaseline
Concentration / amount:
10 % of the test substance / 0.35 mL
Day(s)/duration:
1 day
No. of animals per dose:
10 male and 10 female animals
Details on study design:
INDUCTION EXPOSURE:
A. INTRADERMAL INDUCTION (day 0):
Three pairs of intradermal injections (0.1 mL) were made consecutively on the left and right side of the shaved neck in test group, control group and positive control group animals as follows:
-Test group: 1% of the test substance in peanut oil; 1% of the test substance in the adjuvant/physiological saline mixture (w/v); adjuvant/physiological saline mixture 1:1 (v/v)
- Control group: adjuvant/physiological saline mixture 1:1(v/v); adjuvant/physiological saline mixture 1:1(v/v) + peanut oil (mixture 1:1 v/v); peanut oil
- Positive control group: 0.5% mercaptobenzothiazole in peanut oil; 0.5% mercaptobenzothiazole in adjuvant/physiological saline mixture (w/v); adjuvant/physiological saline mixture 1:1 (v/v)

B. EPIDERMAL INDUCTION APPLICATION (day 8):
- In the test group, a 30% solution of the test substance was applied on a filter paper patch to the neck of the animals (patch 2x4 cm; approx. 0.4 g per patch and held in place under an occlusive dressing for 48 hours).
- The control group was treated with the vehicle (Vaseline) only.
- The positive control group was treated with 50% Mercaptobenzothiazole in Vaseline
- Irritation was assessed on day 10, 1 hour after removal of the dressings.

CHALLENGE EXPOSURE (day 22):
- After 2 weeks without treatment, the test and negative controls were challenged on one flank with a 10% solution of the test substance in Vaseline and on the other flank with the vehicle (Vaseline) alone.
- Animals of the positive control group were challenged with Mercaptobenzothiazole in Vaseline on one flank and with the vehicle on the other. The challenge applications were made using Hilltop chambers containing approximately 0.35 mL per chamber and held in place with occlusive dressing for 24 hours.
- Evaluations: The challenge reactions were assessed 24 and 48 hours after removing the dressings, according to the Draize scale.
Positive control substance(s):
yes
Remarks:
Mercaptobenzothiazole
Positive control results:
Epidermal challenge of 10 male and 10 female guinea pigs with Mercaptobenzothiazole resulted in positive responses in 17 (8 males. 9 females) of 20 guinea pigs after 24 and 48 hours, corresponding to a sensitisation rate of 85%. Scaling erythema reactions were noticed in all animals with positive responses after 48 hours. A very slight erythema reaction in male No. 26 faded after the scoring at 24 hours and was recognised as transient irritation (false positive reaction) based on clinical appearance and duration. Irritant skin reactions were recorded on the vehicle challenge flanks of 2 females after 24 hours and of 3 females after 48 hours.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
10 %w/v
No. with + reactions:
0
Total no. in group:
20
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
10 %w/v
No. with + reactions:
1
Total no. in group:
20
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
10 %w/v
No. with + reactions:
0
Total no. in group:
20
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
10 %w/v
No. with + reactions:
0
Total no. in group:
20
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
10 %w/v
No. with + reactions:
17
Total no. in group:
20
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
10 %w/v
No. with + reactions:
17
Total no. in group:
20

Mortality/body weight

All animals survived till the end of the study.There were no treatment-related effects on body weight during the study.

Induction reactions and duration

After removal of the occlusive dressing on day 10 after epidermal induction, irritation at the application site was seen in all 40 guinea pigs of the test and positive control groups

 

Challenge reactions and duration

Epidermal challenge of 10 male and 10 female guinea pigs with the test substance resulted in positive response in 1 male guinea pig after 48 hours, corresponding to a sensitisation rate of 5%. No irritant skin reactions were recorded among control animals.

Interpretation of results:
GHS criteria not met
Conclusions:
The test substance is graded as a weak sensitiser according to Magnusson and Kligman, which is the lowest possible grade however based on the sensitisation rate of 5%, which is below the threshold of significance (i.e. below 30%), set in Commission Directive 93/21/EEC, classification of the test substance as to its sensitising properties is not required.
Executive summary:

In a dermal sensitisation study with the test substance (98.6% a.i) in Oleum arachidis, young adult male and female Pirbright White (Tif:DHP) guinea pigs (10/sex) were tested using the Magnusson and Kligman guinea pig maximisation test (Magnusson and Kligman, 1980). Two main procedures were involved in the study; (a) the potential induction of an immune response; (b) a challenge of that response. From the findings of a pilot study the concentrations for use in the main study were selected. On the basis of the results, a 1% w/v preparation of the test substance in physiological saline was used for intradermal induction, a 30% w/v preparation in Vaseline was used for the epidermal induction and a 10% w/v preparation in Vaseline was used for the challenge. Epidermal challenge of 10 male and 10 female guinea pigs with the test substance resulted in positive response in 1 male guinea pig after 48 hours, corresponding to a sensitisation rate of 5%. No irritant skin reactions were recorded among control animals. Epidermal challenge test of 10 male and 10 female guinea pigs with Mercaptobenzothiazole (positive control groups) resulted in positive responses in 17 (8 males and 9 females) of the 20 guinea pigs after 24 and 48 hours, corresponding to a sensitisation rate of 85%. The test substance was graded as a weak sensitiser according to the Magnusson and Kligman maximisation scale. Based on the sensitisation rate of 5%, which is below the threshold of significance (i.e. below 30%), set in Commission Directive 93/21/EEC (18th adaptation to technical progress of Council Directive 67/548/EEC), classification of the test substance as to its sensitising properties is not required.

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

Skin sensitisation

In a dermal sensitisation study (Winkler, 1995) with the test substance (98.6% a.i) in Oleum arachidis, young adult male and female Pirbright White (Tif:DHP) guinea pigs (10/sex) were tested using the Magnusson and Kligman guinea pig maximisation test (Magnusson and Kligman, 1980). Two main procedures were involved in the study; (a) the potential induction of an immune response; (b) a challenge of that response. On the basis of the results of a pilot study, a 1% w/v preparation of the test substance in physiological saline was used for intradermal induction, and a 30% w/v preparation in Vaseline was used for the epidermal induction and a 10% w/v preparation in Vaseline was used for the challenge. Epidermal challenge of 10 male and 10 female guinea pigs with the test substance resulted in positive response in 1 male guinea pig after 48 hours, corresponding to a sensitisation rate of 5%. No irritant skin reactions were recorded among control animals. An epidermal challenge test of 10 male and 10 female guinea pigs with Mercaptobenzothiazole (positive control groups) resulted in positive responses in 17 (8 males and 9 females) of the 20 guinea pigs after 24 and 48 hours, corresponding to a sensitisation rate of 85%. The test substance was graded as a weak sensitiser according to the Magnusson and Kligman maximisation scale. Based on the sensitisation rate of 5%, which is below the threshold of significance (i.e. below 30%), set in Commission Directive 93/21/EEC (18th adaptation to technical progress of Council Directive 67/548/EEC), classification of the test substance as to its sensitising properties is not required.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the available data on skin sensitisation, classification for skin sensitisation is not warranted in accordance with the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.