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Description of key information

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:
1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
EPA OPP 81-6 (Skin Sensitisation)
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
This study was performed in 1990 before the requirement of LLNA study was mandated.
Specific details on test material used for the study:
NBW 8008/71 [7015/16 TIF]
The Test Substance employed was pre-dried technical grade active substance
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
Young adults weighing 313-368 g (main study)
Route:
intradermal and epicutaneous
Vehicle:
other: 3% w/v DMF in corn oil
Concentration / amount:
Intradermal: A row of three injections (0.05-0.1 mL each) was made on each side of the mid-line. The injections were:
i) Freund’s Complete Adjuvant plus 3% w/v DMF in corn oil in the ratio 1:1;
ii) 0.01% (w/v) preparation of the test sample in 3% w/v DMF in corn oil;
iii) 0.01% (w/v) preparation of the test sample in a 1:1 Freund’s Complete Adjuvant plus 3% w/v DMF in corn oil

Topical: One week later, the scapular area was clipped again and treated with a topical application of the test sample as a 30% (w/v) preparation in DMF.
Route:
intradermal and epicutaneous
Vehicle:
other: 3% w/v DMF in corn oil
Concentration / amount:
Induction of the control animals: intradermal injections were administered using an identical procedure to that used for the test animals, except that the injections were:
(i) Freund’s Complete Adjuvant plus 3% w/v DMF in corn oil in the ratio 1:1
(ii) 3% w/v DMF in corn oil only
(iii) Freund’s Complete Adjuvant plus 3% w/v DMF in corn oil in the ratio 1:1

The topical applications followed the same procedure as for the test animals except that DMF only was applied to the filter paper.
Vehicle:
other: 3% w/v DMF in corn oil
Concentration / amount:
10% (w/v) in DMF
3% (w/v) in DMF
No. of animals per dose:
20 test group, 10 negative control group, 20 positive control group
Details on study design:
1st application: Induction 0.01%; intracutaneous
2nd application: Induction 30%; epicutaneous
3rd application: Challenge 10%; epicutaneous
Challenge controls:
10% (w/v) in DMF
3% (w/v) in DMF
Positive control substance(s):
yes
Positive control results:
Positive control results: Following challenge with a 50% (w/v) dilution of the 40% (w/v) aqueous formaldehyde solution, scattered mild redness to moderate diffuse redness was seen in fourteen out of the seventeen test animals scored.No response was seen in any of the ten control animals. The net percentage response was calculated to be 82%.
Key result
Reading:
rechallenge
Hours after challenge:
24
Group:
test group
Dose level:
10% w/v
No. with + reactions:
13
Total no. in group:
20
Clinical observations:
Scattered mild redness to intense redness and swelling was seen in thirteen out of twenty test animals.
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
rechallenge
Hours after challenge:
24
Group:
test group
Dose level:
3% w/v
No. with + reactions:
2
Total no. in group:
20
Clinical observations:
Scattered mild redness was seen in two out of twenty test animals.
Remarks on result:
other: Weak sensitizer
Key result
Reading:
rechallenge
Hours after challenge:
48
Group:
test group
Dose level:
10% w/v
No. with + reactions:
13
Total no. in group:
20
Clinical observations:
Scattered mild redness to intense redness and swelling was seen in thirteen out of twenty test animals.
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
rechallenge
Hours after challenge:
48
Group:
test group
Dose level:
3% w/v
No. with + reactions:
1
Total no. in group:
20
Clinical observations:
Scattered mild redness was seen in one out of twenty test animals.
Remarks on result:
other: Weak sensitizer
Key result
Reading:
rechallenge
Hours after challenge:
24
Group:
positive control
Dose level:
20%
No. with + reactions:
13
Total no. in group:
20
Key result
Reading:
rechallenge
Hours after challenge:
48
Group:
positive control
Dose level:
20%
No. with + reactions:
13
Total no. in group:
20
Key result
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
10

24 h after challenge

High dose (10% w/v): 13/20

Low dose (3% w/v): 2/20

48 h after challenge

High dose (10% w/v): 13/20

Low dose (3% w/v): 1/20

Following challenge with a 10% (w/v) preparation of the test sample in DMF, scattered mild redness to intense redness and swelling was seen in thirteen out of twenty test animals.Scattered mild redness was seen in three out of ten control animals. The net percentage response was calculated to be 35%.

Following challenge with a 3% (w/v) preparation of the test sample in DMF, scattered mild redness was seen in two out of twenty test animals. No erythematous response was seen in any of the control animals. The net percentage response was calculated to be 10%.

% net response           description

0                                      not a sensitiser

1-8                                   weak sensitiser

9-28                                mild sensitiser

29-64                              moderate sensitiser

65-80                              strong sensitiser

81-100                           extreme sensitiser

Conclusions:

Materials and methods
The sensitising properties of the test sample were assessed using a method based on the maximisation test of Magnusson and Kligman (1970).
(a) Induction
The hair was removed from an area approximately 5cm x 5cm on the scapular region of each animal with a pair of veterinary clippers and a row of three injections (0.05-0.l mL each) was made on each side of the mid-line. The injections were:
i) Top: Freund’s Complete Adjuvant plus 3% w/v DMF in corn oil in the ratio 1:1;
ii) Middle: a 0.01% (w/v) preparation of the test sample in 3% w/v DMF in corn oil;
iii) Bottom: a 0.01% (w/v) preparation of the test sample in a 1:1 Freund’s Complete Adjuvant plus 3% w/v DMF in corn oil.
The injections were checked for any adverse effects for up to 48 hours.
One week later, the scapular area was clipped again and treated with a topical application of the test sample as a 30% (w/v) preparation in DMF. This preparation (0.2-0.3 mL) was applied on filter paper held in place by surgical tape. The tape was covered by an occlusive dressing which was kept in place for 48 hours.
The application sites were checked approximately 24 hours after removal of the dressings.
(b) Challenge
Two weeks after the topical inductions, an area, approximately 15cm x 5cm, on both flanks of all the test and control animals, was clipped free of hair with a pair of veterinary clippers. An occlusive dressing was prepared which consisted of two pieces of filter paper stitched to a piece of rubber sheeting.
A 10% (w/v) preparation of the test sample (0.05-0.l mL) in DMF was applied to one of the pieces of filter paper and a 3% (w/v) preparation in DMF (0.05-0.l mL) was applied to the second piece of filter paper. The dressing was placed on to the guinea pig so that the 10% (w/v) preparation was on the left shorn flank and the 3% (w/v) preparation was on the right shorn flank. It was then covered with a strip of adhesive bandage which was secured by a self-adhesive PVC tape.
After twenty four hours, the dressings were removed and discarded.
After a further 24 and 48 hours, any erythematous reactions were quantified and the number of positive responses was recorded.

Results and discussion
Challenge of previously induced guinea pigs with a 10% (w/v) preparation of benzisothiazolin-3-one in DMF elicited a moderate skin sensitisation response and challenge with a 3% (w/v) preparation elicited a mild skin sensitisation response.
Therefore, benzisothiazolin-3-one was a moderate skin sensitiser under the conditions of the test.
Executive summary:

A study was conducted to determine the skin sensitisation potential of the substance according to US EPA Guideline OPP 81-6. The method followed Magnusson and Kligman maximisation protocol closely. The test was performed in 50 (20 test, 10 negative control and 20 positive control) female albino Dunkin Hartley guinea pigs. The intradermal induction of sensitization in the test group was performed in the scapular region with a 0.01% dilution of the test substance in purified water in an emulsion of Freund’s Complete Adjuvant (FCA)/physiological saline. The epidermal induction of sensitization was conducted for 48 h under occlusion with the test substance at 30% in dimethyl formamide one week after the intradermal induction. The animals of the control group were intradermally induced with DMF under occlusion. Two weeks after epidermal induction the control and test animals were challenged by epidermal application of the test substance at 10% in DMF on the left flank and 3% in DMF on the right flank occlusive dressing. Cutaneous reactions were evaluated at 24 and 48 h after removal of the dressing. Challenge of previously induced guinea pigs with a 10% (w/v) preparation of the test substance in DMF elicited a moderate skin sensitisation response and challenge with a 3% (w/v) preparation elicited a mild skin sensitisation response. Under the study conditions, the substance was considered to be a moderate skin sensitiser (Botham, 1990).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

A study was conducted to determine the skin sensitisation potential of the substance according to US EPA Guideline OPP 81-6. The method followed Magnusson and Kligman maximisation protocol closely.The test was performed in 50 (20 test, 10 negative control and 20 positive control) female albino Dunkin Hartley guinea pigs. The intradermal induction of sensitization in the test group was performed in the scapular region with a 0.01% dilution of the test substance in purified water in an emulsion of Freund’s Complete Adjuvant (FCA)/physiological saline. The epidermal induction of sensitization was conducted for 48 h under occlusion with the test substance at 30% in dimethyl formamide one week after the intradermal induction. The animals of the control group were intradermally induced with DMF under occlusion. Two weeks after epidermal induction the control and test animals were challenged by epidermal application of the test substance at 10% in DMF on the left flank and 3% in DMF on the right flank occlusive dressing. Cutaneous reactions were evaluated at 24 and 48 h after removal of the dressing. Challenge of previously induced guinea pigs with a 10% (w/v) preparation of the test substance in DMF elicited a moderate skin sensitisation response and challenge with a 3% (w/v) preparation elicited a mild skin sensitisation response. Under the study conditions, the substance was considered to be a moderate skin sensitiser (Botham, 1990).

A study was conducted to determine the skin sensitisation potential of the substance in  guinea-pigs according to OECD Guideline 406 through the Magnusson-Kligman maximisation protocol. The closely-clipped dorsa of ten male and ten female Dunkin-Hartely  guinea-pigs were subject to intradermal injections of Freunds Complete  Adjuvant, 1% w/v of the test substance in purified water and 5% w/v of the substance in the adjuvant on  Day 1. Seven days later the same area of skin was treated by topical  application of 5% w/v of the test substance in purified water and the test site was covered  by an occlusive dressing for 48 hours. The same induction procedures were carried out on a contemporaneous control group of five male and five  female animals, except that the test substance was replaced by vehicle in  all doses. On Day 22, all animals were challenged by occluded application  of purified water to the left flank and 3% w/v and 0.5% w/v of the test substance in  purified water to two sites on the right flank. Intradermal injection at 1% w/v in purified water and 5% w/v in the adjuvant gave rise to moderate erythema, pallor and discolouration.  Occluded topical application at 5% w/v in purified water caused barely perceptible or slight erythema, exfoliation and low incidences of eschar formation or oedema. Challenge application at 3% w/v in purified water gave rise to a significant response (slight erythema or a more marked reaction) in ten test animals. A significant response was observed in four test animals following challenge application of 0.5% w/v of the substance in purified water. Under the study conditions, the substance was considered to be a skin sensitiser in guinea pigs (Rees, 1994).

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the in vivo skin sensitisation study results in guinea pigs, the substance warrants classification as Skin sens. 1B ( H317: May cause an allergic skin reaction) according to EU CLP (1272/2008) criteria.