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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:
12 November 1987 to 20 December 1987
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
OECD Guidelines for Testing of Chemicals (1981) No. 406 "Skin Sensitisation" -Magnusson and Kligman Maximisation Test.
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Available study data over 12 years old.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
No further details sepcified in the study report.

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
Thirty-eight female, albino Dunkin-Hartley guinea pigs were supplied by Interfauna (UK) Limited, Wyton, Huntingdon, Cambridgeshire. At the start of the main study the animals weighed 308 - 405g, and were approximately six to ten weeks old. After a minimum acclimatisation period of five days, each animal was selected at random and given a number unique within the study which was written both on a small area of clipped rump using a black waterproof marker-pen, and on the cage card.
The animals were housed in groups of up to four in solid-floor polypropylene cages furnished with softwood shavings. Free access to mains tap water and food (Guinea Pig FD1 Diet, Special Diet Services Limited, Witham, Essex, U.K.) was allowed throughout the study.
The animal room was maintained at a temperature of 17 – 23 °C and relative humidity of 40 - 65%. The rate of air exchange was approximately 15 changes per hour and the lighting was controlled by a time switch to give 12 hours light and 12 hours darkness.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
arachis oil
Concentration / amount:
10% (w/v)
Day(s)/duration:
one administration on day 1
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
intradermal
Vehicle:
other: 1:1 preparation of Freund’s Complete Adjuvant plus arachis oil B.P.
Concentration / amount:
10% (w/v)
Day(s)/duration:
one administration on day 1
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
petrolatum
Concentration / amount:
50% w/w
Day(s)/duration:
day 8/48 hours
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
petrolatum
Concentration / amount:
25% w/w
Day(s)/duration:
day 22/24 hours
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
A group of thirty guinea pigs was used for the main study, twenty test and ten control.
Details on study design:
Procedure
The sensitising properties of the test material were assessed using the maximisation test of Magnusson B. & Kligman A.M., J. Invest. Dermatol. (1969) 52: 268 - 276.

The dose levels for each of the three stages of the main study were determined by a ‘sighting study’ in which groups of one or two guinea pigs were used and up to two dose levels were tested on each animal. The procedure was as follows:
i) Intradermal Injection (Induction):
Dilutions of the test material in arachis oil B.P. were tested to determine the highest practical level that could be intradermally injected and well tolerated both locally and systemically.
ii) Topical Application (Induction):
Dilutions of the test material in petroleum jelly B.P. were tested to determine the highest practical level which did not produce excessive inflammation on the flank of animals injected with Freund’s Complete Adjuvant (Difco Laboratories, Michigan, U.S.A.) at least seven days previously.
iii) Topical Application (Challenge):
Dilutions of the test material in petroleum jelly B.P. were tested to determine the highest practical level which did not produce inflammation or irritation on the flank of animals injected with Freund’s
Complete Adjuvant at least seven days previously.

A group of thirty guinea pigs was used for the main study, twenty test and ten control. The bodyweight of each animal was recorded at the start and end of the study.

Two main procedures were involved in the maximisation test; (a) an induction of a response and (b) a challenge of that response.

a) Induction
Induction of the Test Animals: The hair was removed from an area approximately 40 mm x 60 mm on the shoulder region of each animal with veterinary clippers and a row of three injections (0.1 ml each) was made on each side of the mid-line. The injections were:
i) Freund’s Complete Adjuvant plus arachis oil B.P. in the ratio 1:1.
ii) a 10% (w/v) dilution of test material in arachis oil B.P.
iii) a 10% (w/v) dilution of test material in a 1:1 preparation of Freund’s Complete Adjuvant plus arachis oil B.P.

One week later, the same area on the shoulder region used previously for intradermal injections was clipped again and treated with a topical application of the test material formulation (50% w/w in petroleum jelly B.P.).
The test material formulation (0.2 - 0.3 ml) was applied on filter paper (WHATMAN No. 4: approximate size 40 mm x 20 mm) which was held in place by a strip of surgical adhesive tape (BLENDERM: approximate size 60 mm x 25 mm) and covered with an overlapping length of aluminium foil. The patch and foil were further secured by a strip of elastic adhesive bandage (ELASTOPLAST: approximate size 250 mm x 35 mm) wound in a double layer around the torso of each animal. This occlusive dressing was kept in place for 48 hours.

Erythematous reactions were quantified immediately following removal of the patches using the 0 - 3 scale shown in Section b.

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 arachis oil B.P. in the ratio 1:1.
ii) arachis oil B.P.
iii) Freund's Complete Adjuvant plus arachis oil B.P. in the ratio 1:1.

The topical applications followed the same procedure as for the test animals except that the vehicle alone was applied to the filter paper. Skin reactions were quantified as for the test animals.

b) Challenge
Two weeks after the topical inductions, an area, approximately 50 - 70 mm x 50 mm on both flanks of each animal, was clipped free of hair' with veterinary clippers.
A quantity of 0.1 - 0.2 ml of the test material formulation (25% w/w in petroleum jelly B.P.) was applied to the shorn right flank of each animal on a 20 mm x 20 mm square of filter paper (WHATMAN No. 4) which was held in place by a strip of surgical adhesive tape (BLENDERM: approximate size 40 mm x 50 mm). The vehicle alone was similarly applied to the left shorn flank.
The patches were occluded with an overlapping length of aluminium foil and secured by .a strip of elastic adhesive bandage (ELASTOPLAST: approximate size 250 mm x 75 mm) wound in a double layer around the torso of each animal.

After 24 hours, the dressing was carefully cut using blunt-tipped scissors, removed and discarded. The position of the treatment sites was identified by using a black indelible marker-pen. After a further 24 and 48 hours, any erythematous reactions were quantified using the four-point scale shown and the number of positive responses recorded.
Scale
0 - no reaction
1 - scattered mild redness
2 - moderate and diffuse redness
3 - intense redness and swelling

To classify the sensitisation response, the percentage of test animals that showed a more severe reaction at the test material challenge site than the most severe reaction seen in the control animals, was compared with the following scale:
%of animals sensitised Classification of sensitisation potential
0 non-sensitiser
> 0 8 weak sensitiser
> 8 – 28 mild sensitiser
> 28 – 64 moderate sensitiser
> 64 – 80 strong sensitiser
> 80 – 100 extreme sensitiser
Challenge controls:
The vehicle alone was applied to the left shorn flank.
Positive control substance(s):
yes
Remarks:
FORMALDEHYDE (40% AQUEOUS SOLUTION)

Results and discussion

Positive control results:
The positive test material Formaldehyde (40% aqueous solution), produced a 95% (19/20) sensitisation rate and was classified as an extreme sensitiser to guinea pig skin.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
25%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
Heavy blue staining of the test material sites precluded evaluation of skin reactions.
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
25%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
Heavy blue staining of the test material sites precluded evaluation of skin reactions.
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
25%
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
25%
No. with + reactions:
0
Total no. in group:
10

Any other information on results incl. tables

INDIVIDUAL REACTIONS IN TEST ANIMALS AT CHALLENGE

TEST MATERIAL: DISPERSIONSBLAU F-60 768

CHALLENGE CONCENTRATION: 25% (w/w)

VEHICLE: PETROLEUM JELLY B.P.

ANIMAL NUMBER

SKIN REACTIONS

(HOURS AFTER REMOVAL OF DRESSING)

24

48

TEST

VEHICLE

TEST

VEHICLE

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

INDIVIDUAL REACTIONS IN CONTROL ANIMALS AT CHALLENGE

TEST MATERIAL: DISPERSIONSBLAU F-60 768

CHALLENGE CONCENTRATION: 25% (w/w)

VEHICLE: PETROLEUM JELLY B.P.

ANIMAL NUMBER

SKIN REACTIONS

(HOURS AFTER REMOVAL OF DRESSING)

24

48

TEST

VEHICLE

TEST

VEHICLE

21

22

23

24

25

26

27

28

29

30

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

INTRADERMAL SIGHTING TEST – SUMMARY OF RESULTS

TEST MATERIAL: DISPERSIONSBLAU F-60 768

ANIMAL IDENTIFICATION

TIME OF OBSERVATION

CONCENTRATION OF TEST MATERIAL *

(% w/v)

EVIDENCE OF NECROSIS

EVIDENCE OF SYSTEMIC TOXICITY

A

24 hours

48 hours

72 hours

7 days

1

None S

None S

None S

None S

None

None

None

None

B

24 hours

48 hours

72 hours

7 days

5

None S

None S

None S

None S

None

None

None

None

C

24 hours

48 hours

72 hours

7 days

10 <

None S

None S

None S

None S

None

None

None

None

*Vehicle = arachis oil B.P.

< = maximum practical concentration for intradermal dosing

S = heavy blue staining precluded assessment of erythema

 

The concentration of the test material to be used for the intradermal induction stage of the main study was selected as follows:

10% (w/v) in arachis oil B.P.

 

TOPICAL SIGHTING TEST – EVALUATION OF REACTIONS

TEST MATERIAL: DISPERSIONSBLAU F-60 768

ANIMAL IDENTIFICATION

CONCENTRATION OF TEST MATERIAL *

(% w/v)

EVALUATION OF APPLICATION SITES

(HOURS AFTER REMOVAL OF PATCHES)

1

24

48

E

50 <

25

2 S

1 S

0 S

0 S

0 S

0 S

F

50 <

25

2 S

1 S

1 S

0 S

0 S

0 S

G

25

10

1 S

0 S

0 S

0 S

0 S

0 S

H

25

10

1 S

0 S

0 S

0 S

0 S

0 S

*Vehicle = petroleum jelly B.P.

< = maximum practical concentration for topical dosing

S = blue-coloured staining of skin/hair not affected assessment of erythema

 

The concentrations of the test material to be used for the main study were selected as follows:

Topical Induction          :          50% (w/w) in petroleum jelly B.P.

Topical Challenge         :          25% (w/w) in petroleum jelly B.P.

 

TOPICAL INDUCTION – EVALUATION OF REACTIONS IN TEST ANIMALS

TEST MATERIAL: DISPERSIONSBLAU F-60 768

INDUCTION CONCENTRATION: 50% (w/w) IN PETROLEUM JELLY B.P.

ANIMAL NUMBER

SKIN REACTION

(IMMEDIATELY AFTER REMOVAL OF DRESSINGS)

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

? S

? S

? S

? S

? S

? S

? S

? S

? S

? S

? S

? S

? S

? S

? S

? S

? S

? S

? S

? S

?S = heavy blue-coloured staining precluded assessment of erythema

 

TOPICAL INDUCTION – EVALUATION OF REACTIONS IN CONTROL ANIMALS

VEHICLE: PETROLEUM JELLY B.P.

ANIMAL NUMBER

SKIN REACTION

(IMMEDIATELY AFTER REMOVAL OF DRESSINGS)

21

22

23

24

25

26

27

28

29

30

0

0

0

0

0

0

0

0

0

0

 

INDIVIDUAL BODYWEIGHTS OF TEST ANIMALS

TEST MATERIAL: DISPERSIONSBLAU F-60 768

ANIMAL NUMBER

BODYWEIGHT (g)

BODYWEIGHT (g) INCREASE

DAY 0

DAY 24

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

370

393

396

405

390

359

385

347

323

385

325

330

380

350

354

375

395

371

370

308

483

534

568

587

491

468

505

465

427

527

483

468

475

492

475

534

513

505

474

456

113

141

172

182

101

109

120

118

104

142

148

130

95

142

121

159

118

134

104

148

 

INDIVIDUAL BODYWEIGHTS OF CONTROL ANIMALS

TEST MATERIAL: DISPERSIONSBLAU F-60 768

ANIMAL NUMBER

BODYWEIGHT (g)

BODYWEIGHT (g) INCREASE

DAY 0

DAY 24

21

22

23

24

25

26

27

28

29

30

370

352

364

399

340

380

365

350

330

365

461

470

474

532

470

540

520

493

469

499

91

118

110

133

130

160

155

143

139

134

 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test material, DISPERSIONSBLAU F-60 768, was found to be a non-sensitiser to guinea pig skin.
The substance is not classifiable according to CLP criteria.
Executive summary:

A study was performed to assess the skin sensitisation potential of the test material in the albino guinea pig. The method used followed that described in the OECD Guidelines for Testing of Chemicals (1981) No. 406 "Skin Sensitisation" -Magnusson and Kligman Maximisation Test.

 

Twenty test and ten control animals were used for the main study.

Following sighting studies, the following concentrations were used in the induction and challenge phases:

Intradermal Induction :          10% (w/v)

Topical Induction        :          50% (w/w)

Topical Challenge      :          25% (w/w)

 

The test material produced a 0% (0/20) sensitisation rate and was classified as a non-sensitiser to guinea pig skin.

The substance is not classified in accordance with CLP criteria.