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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:
08 June 1995 to 07 July 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guidelines and the study was conducted under GLP conditions.
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 334 ± 17 g for males and 330 ± 14 g for females.
- Housing: Individually in polycarbonate cages (48 x 27 x 20 cm).
- Diet: ad libitum.
- Water: filtered drinking water, ad libitum.
- Acclimation period: At least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2 °C
- Humidity (%): 30 – 70 %
- Air changes (per hr): 12 cycles/hour of filtered, non-recycled air.
- Photoperiod: 12 hour light/dark cycle.
Route:
intradermal and epicutaneous
Vehicle:
other: sterile isotonic saline solution (0.9 % NaCl)
Concentration / amount:
Intradermal induction exposure: 10 % (w/w) in vehicle
Epicutaneous induction exposure: 20 % (w/w) in vehicle
Challenge exposure: 5 % (w/w) in vehicle
Route:
epicutaneous, occlusive
Vehicle:
other: sterile isotonic saline solution (0.9 % NaCl)
Concentration / amount:
Intradermal induction exposure: 10 % (w/w) in vehicle
Epicutaneous induction exposure: 20 % (w/w) in vehicle
Challenge exposure: 5 % (w/w) in vehicle
No. of animals per dose:
10 males and 10 females in the treated group; 5 males and 5 females in the control group.
Details on study design:
RANGE FINDING TESTS: Preliminary tests were conducted to define the concentrations to be used in the definitive test.
- The minimum irritation concentration was defined via the intradermal route as follows:
24 hours before treatment, the dorsal region of the animal was clipped and the test material was prepared in an appropriate vehicle. Intradermal administration of the test material (0.1 mL) at increasing concentrations was performed in order to determine the concentration which caused an irritation. Evaluation of the potential cutaneous reaction was performed at 24, 48 hours and at day 6 after injection.
The following concentrations were tested: 0, 5 and 10 % (w/w) in vehicle.
- The minimum irritation concentration and maximum non-irritant concentration was defined via the epicutaneous route as follows:
24 hours before treatment, the flanks of the animals were clipped, if necessary the test material was prepared in an appropriate vehicle. 0.5 mL of each concentration was applied to a dry gauze pad of approximately 4 cm² and then held in place by an occlusive dressing for 24 hours. Potential cutaneous reactions were evaluated 24 and 48 hours after removal of the gauze pads.
The following concentrations were tested: 2.5, 5 and 20 % (w/w) in vehicle.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: One intradermal and one epicutaneous induction was performed.
- Exposure period: Day 1 and day 7
- Site: An area of 4 x 2 cm in the scapular region.

Intradermal induction: On day 1 the intradermal injections were made on the clipped test site.
- No. of exposures: A total of 6 0.1 mL injections were made, 3 in each side of the animal as detailed below.
- Test group injections consisted of the following:
Freund’s complete adjuvant diluted to 50 % (v/v) with sterile isotonic saline solution (0.9 % NaCl)
Test material 10 % w/w in vehicle.
A mixture of 50/50 (w/v) Freund’s complete adjuvant diluted to 50 % (v/v) with sterile isotonic saline solution and the test material at 10 % w/w in the vehicle.
- Control group injections consisted of the following:
Freund’s complete adjuvant diluted to 50 % (v/v) with sterile isotonic saline solution (0.9 % NaCl)
Vehicle (sterile isotonic saline solution, 0.9 % NaCl)
A mixture of 50/50 (w/v) Freund’s complete adjuvant diluted to 50 % (v/v) with sterile isotonic saline solution and the vehicle

Epicutaneous induction: On day 7 the clipped treatment site was treated with 0.5 mL of sodium lauryl-sulphate (10 %) in Vaseline to provoke a local irritation. Then on day 8 the epicutaneous application was made to the test site as follows:
- Test group application: 0.5 mL of the test material 20 % (w/w) prepared in the vehicle (non-irritant concentration)
- Control group application: 0.5 mL of the vehicle.
- Duration of exposure: 48 hours.
- Type of wrap used: The test material and the vehicle were prepared of a dry gauze pad, applied to the scapular region and then held in place with an adhesive hypoallergenic dressing and an adhesive anallergenic waterproof plaster.
- Washing: On removal of the dressing, any residual test material was removed by means of a gauze pad moistened with water.
- Evaluation: Dermal reactions were recorded one hour after the dressing was removed.

B. CHALLENGE EXPOSURE
- No. of exposures: A single challenge exposure was performed.
- Day(s) of challenge: Challenge exposure was performed after a rest period on Day 22.
- Exposure period: 24 hours.
- Test and control groups: 0.5 mL of the test material was applied at a concentration of 5 % (w/w) in the vehicle, and 0.5 mL of the vehicle.
- Site: The test material was applied to the posterior right flank and the vehicle to the posterior left flank.
- Concentration: 5 % (w/w) in vehicle.
- Type of wrap used: The test material and the vehicle were prepared of a dry gauze pad, applied to the scapular region and then held in place with an occlusive hypoallergenic dressing and an adhesive anallergenic waterproof plaster.
- Washing: On removal of the dressing, any residual test material was removed by means of a gauze pad moistened with water.
- Evaluation (hr after challenge): Cutaneous examinations were performed 24 and 48 hours after the removal of the dressing from the challenge application sites.

SCORING SYSTEM:
Both treated and control flanks were observed and scored according to the scale shown in Table 1.
Challenge controls:
The vehicle was applied to the posterior left flank as a control for the challenge exposure, as detailed above.
Positive control substance(s):
yes
Remarks:
Dinitro 2.4 Chlorobenzene (1 % w/w)
Positive control results:
The positive control at a concentration of 1 % (w/w) induced a positive skin sensitization reaction in 95 % of the test animals.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
5 % w/w
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 5 % w/w. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: none.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
5 % w/w
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 5 % w/w. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: none.

PRELIMINARY TEST

- Intradermal route: Several tests were performed to determine the minimal irritant concentration which did not provoke necrosis or ulceration. All readings taken at 24 and 48 hours were masked by orange colouration due to the test material. The test material was tested up to a concentration of 10 % (w/w). This was selected as the concentration to be used for the main study.

- Epicutaneous route: The maximum test material concentration that could be achieved in the vehicle was 20 % (w/w). Several tests were performed to determine the maximum non-irritant concentration after application of the test material covered by an occlusive dressing for 4 hours.

The orange colouration observed at 5 and 20 % (w/w) could mask an eventual erythema reading of grade 1 to 2 at the 24 hour reading. At the 48 hour reading the orange colouration at 20 % (w/w) could also mask erythema readings.

A concentration of 5 % (w/w) was selected for the main test, to try to avoid masking of effects by the test material colouration.

MAIN TEST

- Clinical signs: No clinical signs or mortality was observed during the study.

- Body weight: Body weight gain was normal when compared to that of control animals.

- Induction exposure: On day 10, after removal of the dressing, signs of irritation were observed at the intradermal injection sites in the control group. An orange colouration which could mask an eventual irritation was observed at the intradermal injection site in the treated group.

- Challenge exposure: No cutaneous reactions other than dryness of the skin (3/10 control and 3/20 treated animals 48 hours after removal of the dressing only) were observed 24 and 48 hours after removal of the dressing of the challenge epicutaneous application of the test material. The dryness of the skin was due to shaving performed before the 24 hour reading.

Orange colouration of the test site was observed in all animals. It could mask a well-defined or slight erythema in 1/20 and 12/20 treated animals, respectively. This masking effect was only noted at the 24 hour reading.

Table 2: Mean Body Weight and Body Weight Gain (g)

Group

Sex

Days

-1

1

Gain

8

Gain

15

Gain

25

Control

Male

322 (± 16)

325 (± 15)

72 (± 9)

397 (± 19)

60 (± 20)

457 (± 37)

71(± 20)

527 (± 25)

Female

318 (± 8)

321 (± 7)

57 (± 16)

378 (± 13)

48 (± 7)

426 (± 19)

75 (± 14)

502 (± 16)

Treated

Male

355 (± 17)

338 (± 17)

61 (± 15)

399 (± 26)

55 (± 16)

453 (± 31)

82 (± 44)

535 (± 57)

Female

329 (± 11)

335 (± 14)

53 (± 16)

388 (± 22)

49 (± 18)

437 (± 25)

74 (± 16)

511 (± 34)

(± Standard Deviation)

 

Table 3: Cutaneous Reaction

Group

Sex

Animal No.

Day 24 Scoring Period (after 24 hours)

Day 25 Scoring Period (after 48 hours)

Erythema

Edema

Erythema

Edema

LF

RF

LF

RF

LF

RF

LF

RF

Control

Male

16

0

C1

0

0

0

0/C

0

0

17

0

0/C

0

0

0

0/C

0

0

18

0

C1

0

0

0

0/C

0

0

19

0

0/C

0

0

0

0/C

0

0

20

0

C1

0

0

0

0/C/S

0

0

Female

31

0

C1

0

0

0

0/C/S

0

0

32

0

C1

0

0

0

0/C/S

0

0

33

0

C1

0

0

0

0/C

0

0

34

0

C1

0

0

0

0/C

0

0

35

0

C1

0

0

0

0/C

0

0

Treated

Male

21

0

C1

0

0

0

0/C/S

0

0

22

0

C1

0

0

0

0/C

0

0

23

0

0/C

0

0

0

0/C

0

0

24

0

C1

0

0

0

0/C

0

0

25

0

C1

0

0

0

0/C

0

0

26

0

C1

0

0

0

0/C

0

0

27

0

C1

0

0

0

0/C

0

0

28

0

C1

0

0

0

0/C

0

0

29

0

C1

0

0

0

0/C/S

0

0

30

0

C2

0

0

0

0/C/S

0

0

Female

36

0

0/C

0

0

0

0/C

0

0

37

0

C1

0

0

0

0/C

0

0

38

0

0/C

0

0

0

0/C

0

0

39

0

0/C

0

0

0

0/C

0

0

40

0

0/C

0

0

0

0/C

0

0

41

0

C1

0

0

0

0/C

0

0

42

0

C1

0

0

0

0/C

0

0

43

0

C1

0

0

0

0/C

0

0

44

0

0/C

0

0

0

0/C

0

0

45

0

0/C

0

0

0

0/C

0

0

LF = left flank; RF = right flank

C = Yellowish colouration of the skin

C1 = Orange colouration of the skin that could mask an eventual erythema at grade 1

C2 = Orange colouration of the skin that could mask an eventual erythema at grade 1 to 2

S = Dryness of the skin (due to shaving before the 24 hour reading)

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of the test, the test material was determined to be non-sensitizing since none of the animals displayed a reaction at concentrations of 5 % (w/w) of the test material in vehicle.
Executive summary:

The skin sensitization potential of the test material was determined in a guinea pig maximisation test according to the method of method of Magnusson, B. and Kligman, A.M. The study was conducted under GLP conditions and in accordance with the standardised guidelines OECD 406 and EU Method B.6.

Twenty (10 male and 10 female) Dunkin-Hartley guinea pigs were exposed to the test material in an intradermal induction exposure of 10 % (w/w) in vehicle, followed by an epicutaneous induction exposure at 20 % (w/w) in vehicle. Following a rest period animal were subjected to an occlusive epicutaneous challenge exposure at 5 % (w/w) in vehicle. Sterile isotonic saline solution (0.9 % NaCl) was used as the vehicle. Concentrations were selected based on the findings of preliminary testing.Observations were also made after removal of the dressing for the epicutaneous induction exposure. The sensitization potential in terms of dermal irritation was observed 24 and 48 hours after the removal of the dressing for the challenge exposure, and scored according to the Draize scale.

On day 10, after removal of the induction exposure dressing, signs of irritation were observed at the intradermal injection sites in the control group. An orange colouration which could mask an eventual irritation was observed at the intradermal injection site in the treated group. No cutaneous reactions other than dryness of the skin (3/10 control and 3/20 treated animals 48 hours after removal of the dressing only) were observed 24 and 48 hours after removal of the dressing of the challenge epicutaneous application of the test material. The dryness of the skin was due to shaving performed before the 24 hour reading. Orange colouration of the test site was observed in all animals. It could mask a well-defined or slight erythema in 1/20 and 12/20 treated animals, respectively. This masking effect was only noted at the 24 hour reading.

Under the conditions of the test, the test material was determined to be non-sensitizing since none of the animals displayed a reaction at concentrations of 5 % (w/w) of the test material in vehicle.

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

The skin sensitization potential of the test material was determined a key study by de Jouffery (1995), in a guinea pig maximisation test according to the method of method of Magnusson, B. and Kligman, A.M. The study was conducted under GLP conditions and in accordance with the standardised guidelines OECD 406 and EU Method B.6.

Twenty (10 male and 10 female) Dunkin-Hartley guinea pigs were exposed to the test material in an intradermal induction exposure of 10 % (w/w) in vehicle, followed by an epicutaneous induction exposure at 20 % (w/w) in vehicle. Following a rest period animal were subjected to an occlusive epicutaneous challenge exposure at 5 % (w/w) in vehicle. Sterile isotonic saline solution (0.9 % NaCl) was used as the vehicle. Concentrations were selected based on the findings of preliminary testing.Observations were also made after removal of the dressing for the epicutaneous induction exposure. The sensitization potential in terms of dermal irritation was observed 24 and 48 hours after the removal of the dressing for the challenge exposure, and scored according to the Draize scale.

On day 10, after removal of the induction exposure dressing, signs of irritation were observed at the intradermal injection sites in the control group. An orange colouration which could mask an eventual irritation was observed at the intradermal injection site in the treated group. No cutaneous reactions other than dryness of the skin (3/10 control and 3/20 treated animals 48 hours after removal of the dressing only) were observed 24 and 48 hours after removal of the dressing of the challenge epicutaneous application of the test material. The dryness of the skin was due to shaving performed before the 24 hour reading. Orange colouration of the test site was observed in all animals. It could mask a well-defined or slight erythema in 1/20 and 12/20 treated animals, respectively. This masking effect was only noted at the 24 hour reading.

Under the conditions of the test, the test material was determined to be non-sensitizing since none of the animals displayed a reaction at concentrations of 5 % (w/w) of the test material in vehicle.


Migrated from Short description of key information:
Skin sensitisation: Non-sensitizing, OECD 406, EU Method B.6, de Jouffery 1995.

Justification for selection of skin sensitisation endpoint:
This is the only avaliable study that addresses acute skin irritation of the test material. The study was performed under GLP conditions to standardised guidelines. The report is conclusive and reported to a sufficient standard for classification. In accordance with the principles for assessing data quality defined by Klimisch et al (1997), this study was assigned a reliability score of 1.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

In accordance with criteria for classification as defined in Annex I, Regulation 1272/2008, the test material does not require classification for skin sensitisation.