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Description of key information

Skin sensitisation: Split Adjuvant Test, 20 female guinea pigs at 25%, 10% and 5% w/w in 1% aqueous methyl cellulose, results: one animal showed positive skin reactions (sensitisation rate of 5%). It can be expected that the weak sensitisation resulted from the Thiram impurity of the test item.

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:
1987-11-16 - 1987-12-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EPA OPP 81-6 (Skin Sensitisation)
Version / remarks:
revised Nov. 1984
Deviations:
no
GLP compliance:
yes
Remarks:
US FDA (21 CFR 58) and US EPA (40 CFR 160 and 40 CFR 792)
Type of study:
split adjuvant test
Justification for non-LLNA method:
The guinea pig is the classical animal for determining the potential of substance to induce delayed contact hypersensitivity.
The dermal route of administration was selected because the test substance may accidentally come into contact with the skin during manufacture, handling or use.
Freunds Complete Adjuvant (FCA) is intracutaneoulsy injected to attract circulating immunocompetent cells to the epicutaneously exposed area.
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
Thirty-five young adult female guinea pigs of the Dunkin-Hartley strain, approximately 2 months old at the start of the experiment, SPF-quality, were obtained from Charles River Wiga, FRG. Date of arrival at the animal house: November 3, 1987. The animals were nulliparous and non-pregnant; they were identified by means of tattoo behind the ears. A few days before the start of the experiment, the body weights ranged from 357 to 443 g (see Tables 1 and 2). The animals were housed in metal cages with wire-mesh floors (ITL, Bergen, The Netherlands: 2 animals per cage). They were fed standard guinea pig diet, including ascorbic acid (1600 mg/kg) obtained from Hope Farms, Woerden (LC 23-B, pellet diameter 4 mm) and had free access to tap-water. In addition, once a week hay was provided.

ENVIRONMENTAL CONDITIONS
The animal room temperature was between 19 and 23 °C and the relative humidity between 55 and 95 per cent. The artificial light sequence was 12 hours light, 12 hours dark. A combined quarantine/acclimation period of 13 days was allowed (7 days for the animals of the primary irritation experiment).
Route:
epicutaneous, occlusive
Vehicle:
other: 1% aqueous methyl cellulose
Concentration / amount:
25% in 0.2 mL
Day(s)/duration:
20 days
Adequacy of induction:
other: highest concentration used causing no substantial skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
other: 1% aqueous methyl cellulose
Concentration / amount:
25%, 10%, 5%, 0% in 0.5 mL
Day(s)/duration:
1 - 3 days
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Primary irritation experiment: 4 (+1evaluation of toxicity)
Experimental group: 20 females
Control group: 10 females
Details on study design:
RANGE FINDING TESTS:

Primary irritation experiment
A primary irritation test was carried out using five guinea pigs to determine the concentrations to be applied in the challenge phase (these should not produce any substantial irritation). Four guinea pigs were shaved and their flank was exposed for 24 hours to 50%, 25%, 10% and 5% (W/W) of the test substance in 1% aqueous methyl cellulose, occlusively administered by means of four Square chambers (v.d. Bend, Brielle, The Netherlands). None of the animals reacted to these test substance concentrations.
One additional animal received 0.5 ml of the test substance, 50% in 1% methyl cellulose, on a patch of Metalline (Lohmann. W.-Germany) for evaluation of the toxicity of the compound. In this case, also no irritation was observed. No systemic toxic effects resulted from these treatments in any of the animals.

MAIN STUDY
A. INDUCTION EXPOSURE
The experimental animals were clipped and shaved in an area of skin on the back just behind the right shoulder girdle. Each animal was wrapped in a "window dressing" which consisted of Coban elastic bandage (Smith and Nephew, England). A 2 x 2 cm opening was cut in the dressing over the exposure site. Dry ice was applied to the test site for approximately 5 sec with firm pressure. Then 0.2 ml of test substance, 25% (W/W) in 1% methyl cellulose was applied, and this was covered with a Metalline-patch (Lohmann). Then the patch was covered with water-impervious tape (Sleek, Smith and Nephew, England).
On day 2, the dressing that covered the window was removed, 0.2 ml of the test material in 1% methyl cellulose was applied, and the window was reclosed with the same dressing.
On day 4, the window was again opened and 0.1 ml of Freund's complete adjuvant (Difco, Detroit, USA) was injected intradermally on both sides of the exposure site. This was followed by a further application of 0.2 ml of the test substance in 1% aqueous methyl cellulose, and the window was closed. On day 7, the test material was again applied. On day 9, all wrappings were removed. On day 4, the negative control animals received i.d. injections with Freund's complete adjuvant to exclude interpretation difficulties in the challenge phase caused by aspecific lowering of the skin irritation threshold by this substance.

B. CHALLENGE EXPOSURE
On day 21, the flank was carefully shaved and the protruding long hairs from the edges were cut away with a pair of scissors. The test substance, suspended in 1% methyl cellulose, was applied in a volume of 0.05 ml on Square chambers (v.d. Bend, Brielle, The Netherlands), mounted on Micropore-tape. Each animal received 4 different doses of test substance; all animals were treated similarly. The concentrations tested were: 25%, 10%, 5% and 0% (W/W) of the test substance in 1% aqueous methyl cellulose. The elastic bandage (Coban) was kept in place for 24 hours. Twenty-four hours after removal of the dressings the first reading was made after closely shaving the skin in the morning.
Further readings were made 48 and 72 hours after removal of the bandage. If necessary for evaluation of the skin reactions, shaving was repeated.
Positive control substance(s):
yes
Remarks:
formaldehyde solution 37%
Positive control results:
A sensitisation rate of 50 per cent was obtained to the 0.5% concentration.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
25 %, 10 %, 5 %
No. with + reactions:
1
Total no. in group:
20
Clinical observations:
First challenge: Skin reactions of grade 1 were observed in one experimental animal. No skin reactions were evident in the control animals.
Remarks on result:
other: weak sensitising properties
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
25 %, 10 %, 5 %
No. with + reactions:
1
Total no. in group:
20
Clinical observations:
Second challenge: Skin reactions of grade 1 were observed in one experimental animal. No skin reactions were evident in the control animals.
Remarks on result:
other: weak sensitising properties
Key result
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
test group
Dose level:
25 %, 10 %, 5 %
No. with + reactions:
1
Total no. in group:
20
Clinical observations:
Third challenge: Skin reactions of grade 1 were observed in one experimental animal. No skin reactions were evident in the control animals.
Remarks on result:
other: weak sensitising properties
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0 %
No. with + reactions:
1
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:
0 %
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
negative control
Dose level:
0 %
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Group:
positive control
Remarks on result:
not measured/tested

The test substance, suspended in 1% aqueous methyl cellulose at several concentrations (25%, 10% and 5%) and administered epicutaneously to guinea pigs in the challenge phase, resulted in positive skin reactions (grade 2) in one experimental animal (no. 49). No positive reactions were observed in animals of the control group.

Table 1: Primary Irritation Experiment: Erythema* from exposure of the guinea pig skin
to the test material for 24 hours
Animal # Day -6 BW** (gram) 24 Hours readings*** 48 Hours readings Day 2 BW (gram)
50**** 25 10 5 50**** 25 10 5
66 381 0 0 0 0 0 0 0 0 423
67 368 0 0 0 0 0 0 0 0 407
68 359 0 0 0 0 0 0 0 0 384
69 384 0 0 0 0 0 0 0 0 400
* Grading scheme: see below
** body weight
*** Time after removal of dressings
**** Concentration in 1 % aqueous methyl cellulose (% w/w)
Erythema Grade
no erythema 0
very slight erythema 1
well-defined erythema 2
moderate to severe erythema 3
severe erythema (beet redness) 4

Table 2: Individual body weights and skin readings of guinea pigs challenged with the test material
Group Animal # Day - 3 BW* (gram) Day 23 Readings Day 24 Readings Day 25 Readings Day 25 BW* (gram) Comments
25** 10 5 0 25** 10 5 0 25** 10 5 0
Expr. 36 383 0 0 0 0 0 0 0 0 0 0 0 0 532 not sensitised
37 381 0 0 0 0 0 0 0 0 0 0 0 0 516 not sensitised
38 429 0 0 0 0 0 0 0 0 0 0 0 0 587 not sensitised
39 378 0 0 0 0 0 0 0 0 0 0 0 0 503 not sensitised
40 413 0 0 0 0 0 0 0 0 0 0 0 0 563 not sensitised
41 430 0 0 0 0 0 0 0 0 0 0 0 0 594 not sensitised
42 406 0 0 0 0 0 0 0 0 0 0 0 0 598 not sensitised
43 423 0 0 0 0 0 0 0 0 0 0 0 0 578 not sensitised
44 357 0 0 0 0 0 0 0 0 0 0 0 0 747 not sensitised
45 358 0 0 0 0 0 0 0 0 0 0 0 0 500 not sensitised
46 373 0 0 0 0 0 0 0 0 0*** 0 0 0 494 not sensitised
47 379 0 0 0 0 0 0 0 0 0 0 0 0 525 not sensitised
48 370 0 0 0 0 0 0 0 0 0 0 0 0 477 not sensitised
49 366 0 1 1 0 2 2 2 0 2*** 2 2 0 516 sensitised
50 399 0 0 0 0 0 0 0 0 0 0 0 0 554 not sensitised
51 372 0 0 0 0 0 0 0 0 0 0 0 0 510 not sensitised
52 415 0 0 0 0 0 0 0 0 0 0 0 0 571 not sensitised
53 430 0 0 0 0 0 0 0 0 0 0 0 0 568 not sensitised
54 367 0 0 1 0 0 0 0 0 0 0 0 0 504 not sensitised
55 408 0 0 1 0 0 0 0 0 0 0 0 0 556 not sensitised
Contr. 56 404 0 0 0 0 0 0 0 0 0 0 0 0 580
57 383 1 0 0 0 0 0 0 0 0 0 0 0 566
58 357 0 0 0 0 0 0 0 0 0 0 0 0 448
59 367 0 0 1 0 0 0 0 0 0 0 0 0 494
60 406 0 0 0 0 0 0 0 0 0 0 0 0 585
61 398 0 0 1 0 0 0 0 0 0 0 0 0 482
62 387 0 0 0 0 0 0 0 0 0 0 0 0 529
63 387 0 0 0 0 0 0 0 0 0 0 0 0 552
64 443 0 0 0 0 0 0 0 0 0 0 0 0 478
65 363 0 0 0 0 0 0 0 0 0 0 0 0 575
* body weight
** Concentration in 1 % aqueous methyl cellulose (% w/w)
*** this site had ascaly appearance
Interpretation of results:
other: Category 1 (skin sensitising) based on EU GHS criteria
Conclusions:
A sensitisation rate of 5 % was obtained. Based on this sensitisation rate the test substance should be considered to have weak sensitising properties.
Executive summary:

The test material was tested in the Split Adjuvant Test according to EPA OPP 81-6 (Skin Sensitisation) and in compliance with GLP to determine its sensitizing potential to induce delayed contact hypersensitivity in the guinea pig.

In a primary irritation experiment with five adult female guinea pigs the concentrations to be applied in the challenge phase was determined (these should not produce any substantial irritation) at concentrations of 5%, 10%, 25% and 50% w/w.

In the main experiment 20 adult female guinea pigs (Dunkin-Hartley strain) were induced by epicutaneous applications of the test substance, 25% (w/w) in 1% aqueous methyl cellulose. After being challenged with a series of test substance concentrations (25 %, 10% and 5%, w/w) in 1% aqueous methyl cellulose, one of the experimental animals showed a weak positive skin reaction to all three concentrations tested.

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

Skin sensitisation

The test material was tested in the Split Adjuvant Test (Weterings, 1987c) according to EPA OPP 81-6 (Skin Sensitisation) and in compliance with GLP to determine its sensitising potential to induce delayed contact hypersensitivity in the guinea pig.

In a primary irritation experiment with five adult female guinea pigs the concentrations to be applied in the challenge phase was determined (these should not produce any substantial irritation) at concentrations of 5%, 10%, 25% and 50% w/w. in the main experiment 20 adult female guinea pigs (Dunkin-Hartley strain) were induced by epicutaneous applications of the test substance, 25% (w/w) in 1% aqueous methyl cellulose. After being challenged with a series of test substance concentrations (25 %, 10% and 5%, w/w) in 1% aqueous methyl cellulose, one of the experimental animals showed a positive skin reaction to all three concentrations tested. Thus, a sensitisation rate of 5 % was obtained which is most likely caused by the Thiram impurity which is a known skin sensitiser (Cat 1 harmonised classification - Annex VI of Regulation (EC) No 1272/2008 (CLP Regulation)).

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The test material showed a sensitisation rate of 5% in an GLP compliant Split Adjuvant Test.

Therefore, the test item is regarded as a weak sensitser and meets the criteria for classification and labelling for skin sensitisation (Cat.1) in accordance with Regulation (EC) No 1272/2008 (CLP Regulation).