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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:
03 - 27 Aug 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
/ (temperature and relative humidity above ranges from OECD 406)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
17 July 1992
Deviations:
yes
Remarks:
/ (temperature and relative humidity above ranges from OECD 406)
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
30 May 2008
Deviations:
yes
Remarks:
/ (temperature and relative humidity above ranges from OECD 406)
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
March 2003
Deviations:
yes
Remarks:
/ (temperature and relative humidity above ranges from OECD 406)
GLP compliance:
yes (incl. certificate)
Remarks:
OGYÉI, Országos Gyógyszerészeti és Élelmezés-egészségügyi Intézet, Budapest, Hungary
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Based on the physico-chemical properties, the test substance is suspected to be a surface active substance. Thus, according to OECD TG 429 (§5), a GPMT was performed instead of a LLNA due to the known limitations of the LLNA that may necessitate the use of OECD TG 406 (e.g. false positive findings with certain skin irritants [such as some surfactant type chemicals].

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, protected from light and humidity

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: CRL:HA Guinea pig
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: about 10 weeks old
- Weight at study initiation: 462 – 517 g
- Housing: 5 animals of the same sex per cage in macrolon cages, laboratory bedding, Lignocel 3/4-S (J. Rettenmaier & Söhne GmbH+CO.KG, Rosenberg, Germany)
- Diet: Cunigra Diet for Rabbits (# NN16201718, Bonafarm-Bábolna Takarmány Ltd., Hungary), ad libitum
- Water: tap water (containing 50 mg/100 mL ascorbic acid), ad libitum
- Acclimation period: 41 days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.4 - 25.0
- Humidity (%): 31 - 84%
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12/ 12
- IN-LIFE DATES: From: 28 June To: 27 Aug 2016

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
other: 5% (w/v) ethanol and 0.1% (w/v) Polysorbate 80 in physiological saline
Concentration / amount:
0.5%
0.1 mL/ injection site
Day(s)/duration:
single injection
Adequacy of induction:
other: 0.5% (w/v) test item in the vehicle caused no more than mild-to-moderate erythema (graded with score 1), therefore this concentration was chosen in the main study.
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100%
closed patch saturated with 0.5 g of test item
Day(s)/duration:
48 h
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100% and 50% (safeguard dose, dissolved in ethanol:sesame oil 1:3 mixture with 1% (w/v) Polysorbate 80)
0.5 mL
Day(s)/duration:
24 h
Adequacy of challenge:
other: non irritant concentration
No. of animals per dose:
Testgroup: 10 females
Control group: 5 females
Range finding test: 8 females
Details on study design:
RANGE FINDING TESTS:
- intradermal injections:
site 1: 0.5% test substance (in intradermal vehicle = 5% (w/v) ethanol and 0.1% (w/v) Polysorbate 80 in physiological saline)
site 2: 1% test substance (in intradermal vehicle)
site 3: 2.5% test substance (in intradermal vehicle)
site 4: 5% test substance (in intradermal vehicle)
site 5: 5% test item was also tested in a 1:1 mixture (v/v) of Freund's Complete Adjuvant and physiological saline solution
0.1 mL of each concentration were injected into shaved skin of two guinea pigs.

- epidermal applications: 25, 50, 75 % (in dermal vehicle = ethanol:sesame oil 1:3 (v/v) mixture with 1% (w/v) Polysorbate 80) and 100% (undiluted). 0.5 mL of the test article (diluted) or 0.5 g (undiluted) were applied to hair-free scapular area of each guinea pig via a sterile gauze patch (2.5x2.5 cm, 4 layers of porous gauze pads). A surrounding adhesive hypoallergenic plaster held the patches in place. The treated areas were covered for 48 h with a fully occlusive foil (Closed Patch Test).
After the patch removal any remaining test substance was removed with 70% (w/v) ethanol using a gauze swab.

1, 2.5 and 5% (w/v) test item (in vehicle) caused haemorrhagic skin lesions at the treatment site that developed to wounds, therefore 0.5% (w/v) test item (in vehicle), which caused no more than mild-to-moderate erythema (graded with score 1) was chosen for the main study.
It was found that all the dermal treatments at the tested concentrations produced no reaction on the skin of guinea pigs.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (intradermal and epicutaneous, respectively)
- Exposure period: single injection (intradermal) and 48 h (epicutaneous)

- Test groups:
Intradermal (three pairs of injections; 0.1 mL/site):
Injection 1: 1:1 mixture (v/v) of FCA and physiological saline solution
Injection 2: 0.5% test substance (in intradermal vehicle)
Injection 3: 0.5 % test substance, emulsified in a 1:1 mixture (v/v) of FCA and physiological saline solution

Epicutaneous:
Since the undiluted test item was not skin irritating in the dermal dose range-finding study, the test area was painted with 0.5 mL of 10% sodium dodecyl sulphate in Vaseline 24 h prior to the topical induction application. A 2.5x2.5 cm sterile gauze patch (4 layers of porous gauze pads) was saturated with approximately 0.5 g of the undiluted test item and placed over the injection sites. A surrounding adhesive hypoallergenic plaster held the patches in place. The treated areas were covered for 48 h with a fully occlusive foil (Closed Patch Test). After the patch removal any remaining test substance was removed with 70% (w/v) ethanol using a gauze swab.

- Control group:
Intradermal (three pairs of injections, 0.1 mL/site):
Injection 1: 1:1 mixture (v/v) of Freund's Complete Adjuvant abd physiological saline solution
Injection 2: intradermal vehicle
Injection 3: intradermal vehicle, emulsified in a 1:1 mixture (v/v) of Freund's Complete Adjuvant abd physiological saline solution

Epicutaneous:
The test area was painted with 0.5 mL of 10% sodium dodecyl sulphate in Vaseline 24 h prior to the topical induction application. A 2.5x2.5 cm sterile gauze patch (4 layers of porous gauze pads) was saturated with 0.5 mL of the dermal vehicle only and placed over the injection sites. A surrounding adhesive hypoallergenic plaster held the patches in place. The treated areas were covered for 48 h with a fully occlusive foil (Closed Patch Test).

Local effects were examined and scored 1, 24, 48 and 72 hours after the treatment or after patch removal.

- Site: scapular region
- Frequency of applications: every 7 days
- Duration: days 0-14
- Concentrations: 0.5% (intradermal) and 100% (epicutaneous)

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 22
- Exposure period: 24 h
- Test groups: 100 and 50% test substance
- Control group: 100 and 50% test substance
- Site: shaved left and right side of respective animal
- Concentrations: 100% (left side) 50% (right side; safeguard dose)
- Evaluation (hr after challenge): 24 and 48 h
Challenge controls:
the control group is actually a challenge control
Positive control substance(s):
yes
Remarks:
2-Mercaptobenzothiazole (reliability study)

Results and discussion

Positive control results:
The selection of dose levels for 2-Mercaptobenzothiazole was set on the basis of the previous reliability study.
Intradermal induction exposure: 1% (w/v) (in vehicle (1% methyl cellulose in distilled water)
Dermal induction exposure: 75% (w/v) (in vehicle)
Challenge treatment: 50% (w/v) (in vehicle)

Challenge with 2-Mercaptobenzothiazole resulted in a positive response in test animals previously sensitised. The net response values at the 24 and 48 h observations represented an incidence rate of 80% and 70% and net score values of 0.80 and 0.70 respectively. In the control animals no visible changes were found after 24 or 48 h of examinations following challenge with the reference item. The dermal scores represented discrete erythema (score 1) developed on the skin of sensitised guinea pigs.

On the basis of the results of the reliability check study, the reference item 2-Mercaptobenzothiazole was classified as a skin sensitizer and thus, the results proved the validity of the experimental procedure and the test system used.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
intradermal induction: 0%; challenge: 100%
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Animals were treated with a 10 % Sodium Dodecyl Sulphate solution 24 h prior to the epicutaneous induction.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
intradermal induction: 0.5% challenge: 100%
No. with + reactions:
9
Total no. in group:
10
Remarks on result:
other: Animals were treated with a 10 % Sodium Dodecyl Sulphate solution 24 h prior to the epicutaneous induction.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
intradermal induction: 0% challenge: 100%
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Animals were treated with a 10 % Sodium Dodecyl Sulphate solution 24 h prior to the epicutaneous induction.
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
intradermal induction: 0.5% challenge: 100%
No. with + reactions:
9
Total no. in group:
10
Remarks on result:
other: Animals were treated with a 10 % Sodium Dodecyl Sulphate solution 24 h prior to the epicutaneous induction.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
intradermal induction: 0.5% challenge: 50%
No. with + reactions:
7
Total no. in group:
10
Remarks on result:
other: Animals were treated with a 10 % Sodium Dodecyl Sulphate solution 24 h prior to the epicutaneous induction.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
intradermal induction: 0% challenge: 50%
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Animals were treated with a 10 % Sodium Dodecyl Sulphate solution 24 h prior to the epicutaneous induction.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
intradermal induction: 0% challenge: 50%
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Animals were treated with a 10 % Sodium Dodecyl Sulphate solution 24 h prior to the epicutaneous induction.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
intradermal induction: 0.5% challenge: 50%
No. with + reactions:
3
Total no. in group:
10
Remarks on result:
other: Animals were treated with a 10 % Sodium Dodecyl Sulphate solution 24 h prior to the epicutaneous induction.

Any other information on results incl. tables

Range finding test

Table 1: Results after intradermal injection (Range finding test)

Animal No.

Concentration (w/v) [%]

Reaction scores after [h]

1

24

48

72

E

O

E

O

E

O

E

O

1

5 (in FCA:saline)

2

2

2

1

2

0

2

0

2

2

2

2

1

2

0

2

0

1

5 (in intradermal vehicle)

0

0

1

0

1

0

1

0

2

0

0

1

0

1

0

1

0

1

2.5 (in intradermal vehicle)

0

0

1

0

1

0

1

0

2

0

0

1

0

1

0

1

0

1

1 (in intradermal vehicle)

0

0

1

0

1

0

1

0

2

0

0

1

0

1

0

1

0

1

0.5 (in intradermal vehicle)

0

0

1

0

1

0

1

0

2

0

0

1

0

1

0

1

0

E = erythema

O = oedema

Intradermal vehicle = 5% (w/v) ethanol and 0.1% (w/v) Polysorbate 80 in physiological saline

Additional information on results (Range finding test)

A) Intradermal Induction

In addition to the erythema and/or oedema observed (see Table 1), whitish to brownish skin lesions were observed in 2/2 animals treated with 5% (in FCA:saline) at any reading time point.

Haemorrhagic skin lesions and/or wounds were observed in the 2/2 animals treated with 2.5 and 5% (in intradermal vehicle) and 1/2 animals treated with 1% of the test substance at 24 h persisting at least until 72 h after treatment.

No local reactions (oedema or erythema) were visible after treatment with theb lowest concentration (0.5% test substance). Thus, a concentration of 0.5% was selected for intradermal induction.

B) Epicutaneous Induction

No local reactions (oedema or erythema) were visible after topical application of 25, 50, 75 (in dermal vehicle*) or 100% test substance (undiluted), neither after 1 nor after 24 or 48 or 72 h after the removal of the dressing.

*Dermal vehicle = ethanol:sesame oil 1:3 (v/v) mixture with 1% (w/v) Polysorbate 80 in physiological saline

Main study

Table 2: Skin response after intradermal induction with intradermal vehicle (control group) or test substance (100 % in intradermal vehicle, test group)

Animal No.

Group

Reaction scores after 24 h

E

O

1

control

0

0

2

0

0

3

0

0

4

0

0

5

0

0

6

test

1

0

7

1

0

8

1

0

9

1

0

10

1

0

11

1

0

12

1

0

13

1

0

14

1

0

15

1

0

Additional information on results (Main study)

A) Skin response after intradermal induction with intradermal vehicle (control group) or test substance (100 % in intradermal vehicle, test group)

All animals of the test group showed local reactions visible as erythema (graded with score 1),. No local reactions (oedema or erythema) were observed in the control group.

B) Skin response after epidermal induction of the dermal vehicle or test substance (100 %)

Animals were treated with a 10 % sodium-lauryl-sulphate 24 h prior to the epidermal induction application.

Except after the reading time point of 1 h, where 6/10 animals of the test group showed skin erythema (graded with score 1), no local reactions (oedema or erythema) were observed after topical application of the dermal vehicle (control group) or 100% test substance (test group), neither after 24, 48 or 72 h after removal of the dressing.

Challenge

Table 3: Skin response (Erythema) after challenge (treated with 100% or 50% test substance)

 

Group

Reaction scores after removal of the dressing

Reading time point

24 h

48 h

Concentration [%]

100

50

100

50

Animal No.

 

1

control

0

0

0

0

2

0

0

0

0

3

0

0

0

0

4

0

0

0

0

5

0

0

0

0

6

test

1

1

1

0

7

1

0

1

0

8

1

2

1

1

9

1

0

1

0

10

1

1

1

0

11

1

1

1

0

12

2

2

2*

2*

13

2

2

1

0

14

2

2

1

1

15

0

0

0

0

* dry skin

Mortality /viability/systemic effects

No mortalities or signs indicative for systemic or local toxicity were observed.

No histopathological evaluation was performed after the sacrifice of the animals.

Clinical observations showed no abnormalities.

Body weights

Table 4: Body weights [g]

 

Animal No.

Day(s) relative to start date

-1

7

14

21

25

Control group

1

478

562

507

516

513

2

496

523

547

571

575

3

467

490

456

499

494

4

487

481

546

563

545

5

517

479

588

612

620

Mean

 

489

507

528.8

552.2

549.4

SD

19

35.5

49.8

45.2

50.1

Test group

6

490

511

498

593

564

7

462

493

471

527

521

8

501

529

499

570

553

9

501

522

517

579

558

10

477

512

493

565

546

11

470

472

504

534

540

12

482

491

510

532

525

13

475

500

489

526

501

14

485

488

490

534

516

15

514

482

486

536

523

Mean

 

485.7

500

495.7

549.6

534.7

SD

16

18.2

13

24.6

20.5

The body weight gain of the animals was not affected adversely during the study.

Applicant's summary and conclusion

Interpretation of results:
Category 1A (indication of significant skin sensitising potential) based on GHS criteria
Conclusions:
CLP: Skin sens 1A