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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:
From 25.03.1985 until 10.05.1985; study report 12.06.1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1985
Report date:
1985

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
The test was carried out according to the maximization test of Magnusson and Kligman (J, invest. Dermatol, 52, 268-276, 1969), recommended in the OECD guidelines 1981 and in the EEC directive 79/831.
Principles of method if other than guideline:
Test procedure

Induction, intradermal application:

Two intradermal injections (0,1 ml per injection) were made into the neck of the guinea pigs with a mixture of adjuvant and saline, with the test compound
in sesame oil and with the test compound in the adjuvant saline mixture. Induction, epidermal application: One week later the tet item was incorporated in vaseline and applied on a filterpaper patch to the neck of the animals (occlusive administration for 48 hours),

Challenge:
Two weeks after the epidermal induction application the animals were tested on the flank with 10% of the test compound in vaseline and the vehicle alone (24 h occlusive application) , Twenty four hours after removing the dressings the challenge reactions were graded according the Draize scoring scale. The application sites were chemically depilated 3 hours before examination (Veet, ~5 minutes). A second evaluation is made 48 hours after removing the dressings.
The concentrations of the test compound for induction and challenge periods were determined on separate animals. A control group was treated with adjuvant and the vehicle during the induction period. During the challenge period the group was treated with the vehicle as well as with the test compound to control the maximal subirritant concentration of the test compound in adjuvant treated animals.
GLP compliance:
not specified
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The study was conducted in 1985 before a guideline decribing the LLNA came into force.

Test material

Constituent 1
Chemical structure
Reference substance name:
(benzothiazol-2-ylthio)succinic acid
EC Number:
401-450-4
EC Name:
(benzothiazol-2-ylthio)succinic acid
Cas Number:
95154-01-1
Molecular formula:
C11H9NO4S2
IUPAC Name:
2-(1,3-benzothiazol-2-ylsulfanyl)butanedioic acid
Test material form:
solid
Details on test material:
(benzothiazol-2-ylthio)succinic acid, Batch: Z 334

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: Pirbright White strain (Tif:DHP)
Sex:
male/female
Details on test animals and environmental conditions:
The guinea pig is the animal of choice for sensitization studies.
Animal strain: Guinea pigs of the Pirbright White strain (Tif:DHP), bred on our premises were used.


- Animals received: 18.03.1985
- Source: bred on own premises
- Age at study initiation: ~10 weeks
- Weight at study initiation: 291 and 420 g (It was impossible to select all animals within the weight range proposed in the protocol from the animals received for the test series)
- Housing: housed individually in Macrolon cages (type 3)


Diet (e.g. ad libitum): ad libitum standard guinea pigs pellets - NAFAG No, 846, Gossau SG - , supplemented with fresh carrots
- Water (e.g. ad libitum): ad libitum fresh water
- Acclimation period: 7 days
- Indication of any skin lesions: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 2
- Humidity (%): 50 +/- 10
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12 hours

Study design: in vivo (non-LLNA)

Induction
Route:
intradermal and epicutaneous
Vehicle:
other: sesame oil
Concentration / amount:
1% for intradermal application in sesame oil and saline adjuvant mixture
30% for epidermal application (Induction) in vaseline ~ 0 . 4 g per patch
Day(s)/duration:
Induction, intradermal application: - Induction, epidermal application: after one week for 48 hours (occlusive)
Adequacy of induction:
not specified
Challengeopen allclose all
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: vaseline
Concentration / amount:
10% for epidermal application in vaseline ~ 0 , 2 g per patch
Day(s)/duration:
After 20-22 days; two weeks after epidermal induction for 24 hours
Adequacy of challenge:
not specified
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
other: vaseline
Concentration / amount:
10% for epidermal application in vaseline ~ 0 , 2 g per patch
Day(s)/duration:
48 hours after removing the dressing of the first challenge
Adequacy of challenge:
not specified
No. of animals per dose:
Control group:
vehicle application 20
test item application 10

Test group:
vehicle application 20
test item application 20
Details on study design:
Test procedure:
Induction, intradermal application:
Two intradermal injections (0,1 ml per injection) were made into the neck of the guinea pigs with a mixture of adjuvant and saline, with the test compound in sesame oil and with the test compound in the adjuvant saline mixture.
Induction, epidermal application: One week later the test compound was incorporated in vaseline and applied on a filterpaper patch to the neck of the animals (occlusive administration for 48 hours),

Challenge:
Two weeks after the epidermal induction application the animals were tested on the flank with 10% test compound in vaseline and the vehicle alone (24 h occlusive application), Twenty four hours after removing the dressings the challenge reactions were graded according the Draize scoring scale, The application sites were chemically depilated 3 hours before examination (Veet ^,'^^ 5 minutes), A second evaluation is made 4 8 hours after removing the dressings.

The concentrations of the test compound for induction and challenge periods were determined on separate animals.

A control group was treated with adjuvant and the vehicle during the induction period. During the challenge period the group was treated with the vehicle as well as with the test compound to control the maximal subirritant concentration of the test compound in adjuvant treated animals.
Challenge controls:
control group treated with test item in challenge phase
Test group treated just with vehicle in challenge phase
Positive control substance(s):
yes
Remarks:
The sensitivity of the strain is controlled every six month with p - phenylenediamine.

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
vehicle application
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
24
Group:
negative control
Dose level:
10 % test compound
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
vehicle application
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
10% test comound
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
10% test compound
No. with + reactions:
5
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
10% test compound
No. with + reactions:
3
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
vehicle application
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
vehicle application
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation
Reading:
other: 2nd challenge
Hours after challenge:
24
Group:
test chemical
Dose level:
vehicle application
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation
Reading:
other: 2nd challenge
Hours after challenge:
48
Group:
test chemical
Dose level:
vehicle application
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation
Reading:
other: 2nd challenge
Hours after challenge:
24
Group:
test chemical
Dose level:
10% test compound
No. with + reactions:
9
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Reading:
other: 2nd challenge
Hours after challenge:
48
Group:
test chemical
Dose level:
10% test compound
No. with + reactions:
6
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Reading:
other: no positive control tested in this study
Hours after challenge:
24
Group:
positive control
Dose level:
no positive control tested in this study
No. with + reactions:
0
Total no. in group:
0
Remarks on result:
not measured/tested

Applicant's summary and conclusion

Conclusions:
Up to 45% of the guinea pigs exhibited positive responses by the test compound 24 hours after the second challenge which previously have been treated with 1% of the test compound intradermaly and 30% epidermaly during the induction phase.
Therefore, the substance is regarded to have the potential to provoke skin sensitization.
Executive summary:

Male and female guinea pigs were exposed to the test compound (benzothiazol-2-ylthio)succinic acid.

The animals were treated with 1% test compound in vehicle by intradermal application and after one week with 30% test compound in vaseline by epicutaneous application in the induction phase. Thereafter, two challenge treatments were conducted by epicutaneous application, occlusive with 10% test compound for 24 hours.

The animals from the control groups that have been induced with the test compound but challenged with the vehicle as well as the animals that were induced just with the vehicle but challenged with the test compound showed no responses after the challenge.

In the groups induced and challenged with the test compound, up to 25% of the animals were sensitized after the first challenge and up to 45% of the animals were sensitized after the second challenge. After the first epicutaneous challenge with 10% test compound  5 out of 20 animals showed positive responses after 24 hours and 3 out of 20 animals after 48 hours. After the second challenge 9 out of 20 animals showed a positive response after 24 hours and 6 out of 20 animals after 48 hours.