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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:
22Feb1999 to 23Apr1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report date:
1999

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
OECD Guidelines for Testing of Chemicals, Number 406 "Skin Sensitization", adopted by the
Council on July 17, 1992 (reported Paris, April 29, 1993).
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
Directive 96/54/EEC, B.6. "Acute Toxicity - Skin Sensitization", July 30, 1996.
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The study using the maximization method was completed prior to the adaptation of OECD TG 429 in 2002.

Test material

Constituent 1
Chemical structure
Reference substance name:
Methyl 1-methyl-4-[(methylphenylhydrazono)methyl]pyridinium sulphate
EC Number:
269-503-2
EC Name:
Methyl 1-methyl-4-[(methylphenylhydrazono)methyl]pyridinium sulphate
Cas Number:
68259-00-7
Molecular formula:
C14H16N3.CH3O4S
IUPAC Name:
methyl 1-methyl-4-[(methylphenylhydrazono)methyl]pyridinium sulphate
Test material form:
solid: particulate/powder
Details on test material:
See study reports for batch and purity information
Specific details on test material used for the study:
Batch number:CGF-F016737/0016
Purity:>92%
Color/physical form: yellow / fine powder (according to RCC)

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: Ibm: GOHI; SPF-quality guinea pigs; (synonym: Himalayan spotted)
Sex:
female
Details on test animals and environmental conditions:
Test system: Ibm: GOHI; SPF-quality guinea pigs (synonym: Himalayan spotted)

Rationale: Recognized by the international guidelines as a recommended test system (e.g. OECD, EEC).

Source: RCC Ltd, Biotechnology & Animal Breeding Division, Wolferstrasse 4, CH-4414 Fiillinsdorf / Switzerland

Total number of animals: 15 female animals (nulliparous and non-pregnant)

Age at delivery: 4 - 6 weeks

Age at beginning of acclimatization period: 4 - 6 weeks

Body weight at beginning of acclimatization period: Control and test group; 298 - 340 g

Identification: By unique cage number and corresponding ear tags.

Randomization: Randomly selected at time of delivery.

Acclimatization: One week for the control and test group under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

Conditions
Standard Laboratory Conditions The animals were accommodated under standard laboratory conditions. The animal room was air-conditioned with 10-15 air changes per hour and the air was continuously monitored with a range for room temperature of 20-26 °C and for relative humidity between 40-64 %. The animals were provided with an automatically controlled light cycle of 12 hours light and 12 hours dark. Music was played during the light period.

Accommodation
Individually in Makrolon type-4 cages with standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz).

Diet
Pelleted standard Nafag Ecosan 845 25W4, batch no. 103/98, guinea pig breeding / maintenance diet ("Nafag", Nahr- und Futtermittel AG, CH-9202 Gossau), ad libitum. Results of analyses for contaminants are archived at RCC Itingen.

Water
Community tap water from Fullinsdorf, ad libitum. Once weekly additional supply of ascorbic acid (approx. 1 g/1) via the drinking water was provided. The water analysis Wcis performed at RCC Ltd, Environmental Chemistry & Pharmanalytics Division. Results of bacteriological, chemical and contaminant analyses are archived at RCC Itingen.

Acclimiatization: 22-FEB-1999 to 28-FEB-1999
Treatment: 01-MAR-1999 to 25-MAR-1999
Termination: 25-MAR-1999

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
physiological saline
Concentration / amount:
0.1 ml/site; test article concentration of 1 %
Day(s)/duration:
7 days prior to epidermal application
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
50 % in bidistilled water
Day(s)/duration:
48 hours exposure
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
highest non-irritating concentration of 50 %; approximately 0.2 ml
Day(s)/duration:
The dressings were left in place for 24 hours. Day 22 (21 days after Intradermal Injections; 14 days after Epidermal Application)
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
10 females
Details on study design:
The objective of this investigation was to identify a maximally tolerated concentration of the test article suitable for the induction phase of the main study. In addition, a suitable nonirritant concentration of the test article, by the topical route of administration (epidermal application), was identified for the challenge application. The concentrations tested for the epidermal application were chosen to assure an optimum technical application procedure. For intradermal injection concentrations were tested up to the recommended maximum of 5 % (The Guinea Pig Maximization-Test, page 270. Magnusson B.; Kligman A.M., 1969). The procedure employed for these investigations was as follows:

INTRADERMAL INJECTIONS:
Four intradermal injections (0.1 ml/site) of a 1:1 (v/v) mixture of Freund's Complete Adjuvant/physiological saline were made into the shaved neck of one guinea pig. One week later intradermal injections (0.1 ml/site) were made into the clipped flank of the same guinea pig at concentrations of 5, 3 and I % of the test article in bi-distilled water.

Dermal reactions were assessed 24 hours later.
Based on the results, a test article concentration of 5 % was selected for intradermal induction in the main study under the RCC project 720898. However, death occurred in four out of 10 test animals after the intradermal induction performed with FCA/physiological saline and the test article at 5 % in bi-distilled water. The remaining test animals were strongly sedated and killed for ethical reason. An internal non-GLP intradermal application was then performed with three animals in reserve, using two applications of FCA/physiological saline (1:1, v/v), of the test article at 1 % in bi-distilled water and of the test article at 1 % in FCA/physiological saline (1:1, v/v), 0.1 ml/site. No death occurred. Therefore, the test article concentration of 1 % was selected for the repeated study RCC 723633.

EPIDERMAL APPLICATIONS:
Four intradermal injections (0.1 ml/site) of a 1:1 (v/v) mixture of Freund's Complete Adjuvant/physiological saline were made into the shaved neck of two guinea pigs. One week later both flanks of each of the guinea pigs were clipped and shaved just prior to the application. Thereafter 4 patches of filter paper ( 3 x 3 cm) were saturated with the test article at A = 50 % (this concentration was found to be the maximum possible under the conditions of the procedure), B = 25%, C= 15% and D = 10 % in bi-distilled water and applied to the clipped and shaved flanks. The volume of test article preparation applied was approximately 0.2 ml. The patches were covered by a strip of aluminium foil and firmly secured by elastic plaster wrapped around the trunk and covered with impervious adhesive tape. This procedure ensured the intensive contact of the test article. The dressings were removed after an exposure period of 24 hours.

Approximately 21 hours after removal of the dressing the application site was depilated with an approved depilatory cream (VEET Cream, Reckitt & Colman AG, CH-4123 Allschwil) in order to visualize any resulting erythema.
The depilatory cream 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 returnd to their cages. The reaction sites were assessed 24 and 48 hours after removal of the bandage for erythema and oedema according to the method of Magnusson and Kligman (see 5.6).

The allocation of the different test dilutions to the sites (A, B, C, D) on the two animals was alternated in order to minimize site-to-site variation in responsiveness.

Based on the results obtained the concentration selected for induction and challenge in the main study was 50 %.
Positive control substance(s):
yes
Remarks:
2-MERCAPTOBENZOTHIAZOLE

Results and discussion

Positive control results:
In this study 90 % (at the 24-hour reading) and 100 % (at the 48-hour reading) of the animals of the test group were observed with very slight to moderate/severe erythematous reactions after treatment with a non-irritant test article concentration of 10 % in mineral oil. No skin reactions were observed in the control group.

A response of at least 30 % positive animals is considered positive "R43": may cause sensitization by skin contact according to the "Commission Directive 96/54/EEC, July 30, 1996 adapting to technical progress for the 22"'' time Council Directive 67/548/EEC on the approximation of the laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances".

Therefore, the test article 2-MERCAPTOBENZOTHL\ZOLE applied at a concentration of 10 % in mineral oil is considered to be an extreme sensitizer when used under the described test conditions.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
0.2 ml of 50% concentration in water
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No signs of systemic toxicity were observed in the animals.
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
0.2 ml of 50% concentration in water
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No signs of systemic toxicity were observed in the animals.
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
50 % IN BI-DISTILLED WATER
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
None
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
50 % IN BI-DISTILLED WATER
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
None
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
10% 2-Mercaptobenzothiazole in mineral oil
No. with + reactions:
9
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
10% 2-Mercaptobenzothiazole in mineral oil
No. with + reactions:
10
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In this study none of the animals of the control and test group were observed with skin reactions after challenge treatment with a non-irritant test article concentration of 50 % in bi-distilled water.

Under the test conditions the test item applied at a concentration of 50 % in bi-distilled water is considered not to be a sensitizer.
Executive summary:

In order to assess the cutaneous allergenic potential of the test item, the Maximization-Test was performed in 15 (10 test and 5 control) female albino guinea pigs, in accordance with OECD Guideline No. 406 and the Directive 96/54/EEC, B.6.

 

The intradermal induction of sensitization in the test group was performed with a 1 % dilution of the test article in bi-distilled water and in an emulsion of Freund's Complete Adjuvant (FCA) / physiological saline. The epidermal induction of sensitization was conducted under occlusion with the test article at 50 % in bi-distilled water one week after the intradermal induction and following pre-treatment of the test areas with 10 % Sodium-Lauryl-Sulphate (SLS) approximately 24 hours prior to application of the test article. The animals of the control group were intradermally induced with bi-distilled water and FCA/physiological saline and epidermally induced with bi-distilled water under occlusion following pre-treatment with 10 % SLS.

 

Two weeks after epidermal induction the control and test animals were challenged by epidermal application of the test article at 50 % in bi-distilled water and bi-distilled water alone under occlusive dressing. Cutaneous reactions were evaluated at 24 and 48 hours after removal of the dressing.

 

In this study none of the animals of the control and test group were observed with skin reactions after challenge treatment with a non-irritant test article concentration of 50 % in bi-distilled water.

 

Under the test conditions the test item applied at a concentration of 50 % in bi-distilled water is considered not to be a sensitizer.