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Toxicological information

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:
10 May - 09 Jun 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report date:
1988

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
adopted in 1981
Deviations:
yes
Remarks:
GPMT is the preferred technique.
Qualifier:
according to guideline
Guideline:
EPA OPP 81-6 (Skin Sensitisation)
Version / remarks:
adopted in 1984
Deviations:
no
Remarks:
No deviations to the Split Adjuvant Test.
GLP compliance:
yes
Type of study:
split adjuvant test
Justification for non-LLNA method:
The study was initiated prior to the implementation of the LLNA method.

Test material

Constituent 1
Chemical structure
Reference substance name:
Ziram
EC Number:
205-288-3
EC Name:
Ziram
Cas Number:
137-30-4
Molecular formula:
C6H12N2S4Zn
IUPAC Name:
ziram

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Wiga, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approximately 2 month
- Weight at study initiation: 287 - 413 g
- Housing: Groups of two animals were housed in metal cages with wire-mesh floors.
- Diet: standard diet (Hope Farms, Woerden, Netherlands) including ascorbic acid (1600 mg/kg bw), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 13 days (7 days for the animals of the primary experiment)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 60 - 85
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Induction
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
0.2 mL at a concentration of 25% (w/w) in corn oil
Day(s)/duration:
patches were renewed on Day 1, 2, 4 and 7 and removed on Day 9
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
1, 5 and 10% in corn oil
Day(s)/duration:
24 h (starting on Day 21)
No. of animals per dose:
20 females (treatment group) and 10 fmelaes (control group)
Details on study design:
RANGE FINDING TESTS:
A primary irritation experiment was fconducted first. The purpose of this test was to determine the concentrations to be applied in the challenge phase. Four guinea pigs were shaved and their flank was exposed for 24 h to an amount of 0.05 mL at 5, 10, 25 and 50% (w/w) of the test substance in corn oil occlusively administered by means of four Square chambers. 24 h after removal of the dressings, no erythema was seen. 48 h after removal of the dressings, most animals showed very slight erythema and one animal at the concentration of 10% and 20% severe erythema. One additional animal received 0.5 mL of the test substance, 50% in corn oil for 24 h, on a patch of Metalline for the evaluation of the toxicity of the compound. In this case, very slight erythema and scaliness was observed. No systemic toxic effects resulted from these treatments with ziram in any of the animals.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 4 (epicutaneous)
- Exposure period: continiously up to 9 days
- Test groups:
Epicutaneous 1: 0.2 mL of test substance, 25% (w/w) in corn oil was applied on Day 1
Epicutaneous 2: 0.2 mL of test substance, 25% (w/w) in corn oil was applied on Day 2
Epicutaneous 3: 0.2 mL of test substance, 25% (w/w) in corn oil was applied on Day 4
Injection 1: 0.1 mL of Freund’s complete adjuvant was injected intradermally in both sides of the exposure site.
Epicutaneous 4: 0.2 mL of test substance, 25% (w/w) in corn oil was applied on Day 7
- Control group: Animals of the control group received injections of Freund’s complete adjuvant on Day 4 only.
- Site: back
- Frequency of applications: 4 times
- Duration: continiously up to Day 9
- Concentrations: 25% (w/w) in corn oil

B. CHALLENGE EXPOSURE
- No. of exposures: 1 (challenge)
- Day(s) of challenge: Challenge was performed three weeks after intradermal induction on Day 21.
- Exposure period: 24 h
- Test groups: test substance and vehicle only
- Control group: test substance and vehicle only
- Site: flank
- Concentrations: 0, 1, 5 and 10% (w/w) in corn oil
- Evaluation (hr after challenge): 24 and 48 h after removal of the dressing

OTHER:
The test animals were observed for mortality/moribundity. Clinical signs were recorded.
Challenge controls:
Yes, the treatment group, which were induced with the test substance but treated only with corn oil in the challenge phase.
Positive control substance(s):
yes
Remarks:
The assay was checked for reliability in a separate study on guinea pigs using formaldehyde solution as a positive control substance.

Results and discussion

Positive control results:
A positive control experiment was carried out in February 1987 in order to validate the animals and the test procedure. Test substance was formaldehyde solution 37%. In the induction phase, 5% (w/w) test solution in Milli-Ro water was applied. In the challenge phase, 0.5, 2 and 5% (w/w) test substance in Milli-Ro water was applied. A sensitization rate of 50% was obtained to the 0.5% concentration.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
induction: 25% (epicutaneous); challenge: 0% (epicutaneous)
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
induction: 25% (epicutaneous); challenge: 1% (epicutaneous)
No. with + reactions:
2
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Remarks:
any skin reaction of grade 2 or more where counted
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
induction: 25% (epicutaneous); challenge: 5% (epicutaneous)
No. with + reactions:
5
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Remarks:
any skin reaction of grade 2 or more where counted
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
induction: 25% (epicutaneous); challenge: 10% (epicutaneous)
No. with + reactions:
6
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Remarks:
any skin reaction of grade 2 or more where counted
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
induction: 0% (epicutaneous); challenge: 0% (epicutaneous)
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
induction: 0% (epicutaneous); challenge: 1% (epicutaneous)
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
induction: 0% (epicutaneous); challenge: 5% (epicutaneous)
No. with + reactions:
1
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Remarks:
skin reaction with grade 1; no skin reaction with a grade > 1 observed
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
induction: 0% (epicutaneous); challenge: 10% (epicutaneous)
No. with + reactions:
1
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Remarks:
skin reaction with grade 1; no skin reaction with a grade > 1 observed
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
induction: 25% (epicutaneous); challenge: 0% (epicutaneous)
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
induction: 25% (epicutaneous); challenge: 1% (epicutaneous)
No. with + reactions:
2
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Remarks:
any skin reaction of grade 2 or more where counted
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
induction: 25% (epicutaneous); challenge: 5% (epicutaneous)
No. with + reactions:
3
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Remarks:
any skin reaction of grade 2 or more where counted
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
induction: 25% (epicutaneous); challenge: 10% (epicutaneous)
No. with + reactions:
3
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Remarks:
any skin reaction of grade 2 or more where counted
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
induction: 0% (epicutaneous); challenge: 0% (epicutaneous)
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
induction: 0% (epicutaneous); challenge: 1% (epicutaneous)
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
induction: 0% (epicutaneous); challenge: 5% (epicutaneous)
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
induction: 0% (epicutaneous); challenge: 10% (epicutaneous)
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
other: not specified
Group:
positive control
Dose level:
induction: 5% (epicutaneous); challenge: 0.5, 2 and 5% (epicutaneous)
Remarks on result:
other: A sensitization rate of 50 % was obtained to the 0.5 % concentration.
Remarks:
The positive control was previosuly investigated, no challenge duration, number of animals and number reactions were not provided.

Any other information on results incl. tables

Clinical signs and fatalities
Other than dermal irritation during induction, clinical signs related to exposure of the test substance were not observed. All animals survived to the scheduled termination of the in life phase.

Skin reactions
Control group
There were no skin reactions observed in the control animals, except for one animal which showed red spots in reaction to the 5% and 10% test substance concentration.
Test substance group
The test substance, suspended in corn oil at several concentrations (0, 1, 5 and 10%) and administered epicutaneously to guinea pigs in the challenge phase, resulted in a skin response (considering any skin reaction at grade 2 or more) to the 0, 1, 5 and 10% concentration in 0, 2, 5 and 6 animals, respectively 24 h after the challenge phase. This corresponded to 0, 10, 25 and 30% sensitization rate in the animals challenged with 0, 1, 5 and 10% of the test material, respectively at the 24 h reading time point. At the 48 h reading time point, skin responses to the 0, 1, 5 and 10% concentration were observed in 0, 2, 3 and 3 animals, respectively. This corresponded to 0, 10, 15 and 10% sensitization rate in the animals challenged with 0, 1, 5 and 10% of the test material, respectively at the 48 h reading time point.

Applicant's summary and conclusion

Interpretation of results:
other: According to the classification criteria of the CLP Regulation (EU) No. 1272/2008, classification for skin sensitisation Category 1 (H317) is warranted.
Conclusions:
The study (Split Adjuvant Test) was conducted according to EPA OPP 81-6 and OECD guideline 406, under GLP and is considered valid and reliable. Adult female guinea pigs were induced by epicutaneous applications of the test substance, 25% (w/w) in corn oil and afterwards challenged with a series of test substance concentrations (10%, 5%, and 1%, w/w). Under the test conditions, the test substance produced a sensitizing reaction to the skin of guinea pigs with a skin sensitisation rat of 30 and 15% at the higest test substance concentration 24 and 48 h after patch removal. According to the classification criteria of the CLP Regulation (EU) No. 1272/2008, classification for skin sensitisation Category 1 (H317) is warranted.