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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:
January 28, 1992 to February 28, 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report date:
1992

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
Organization for Economic Cooperation and Development (OECD) Guidelines for testing of chemicals
(Section 4, Subpart 406, Paris 1981).
Deviations:
not specified
GLP compliance:
yes
Type of study:
Buehler test
Justification for non-LLNA method:
Available study pre-dates LLNA method and is performed in accordance with GLP to international recognised guidelines.

Test material

Constituent 1
Test material form:
liquid
Specific details on test material used for the study:
No further details specified in the study report.

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
Species and strain: Dunkin Hartley albino guinea pigs
Justification for the selection of the test system: Dunkin Hartley albino guinea pig is the generally recommended species by Health Authorities in experimental model for skin sensitization studies
Supplier: Charles River Italia S.p.A., Via Indipendenza, 11, 22050 Calco (Como)
Shipping slip no. 00684, dated January 24, 1992 (20 animals)
Shipping slip no. 08363, dated October 31, 1991 (6 animals, preliminary test)

Number:
26 animals
10 animals/group
6 animals for the preliminary test (performed on December 11, 1991)

Body weight (and age): between 370-433 grams at the start of the experiment (corresponding to 2-3 months old)
Sex: male

Acclimatization: five days. Animals were observed daily to ascertain their fitness for the study.
Housing: (room T12C)
2 or 3 animals/cage in air-conditioned room.
-temperature: 22°C 2 2
-ail- changes: about 20/hour filtered on HEPA 99.97%
-relative humidity: 55% + 10
-light: 12 hours cycle (7 a.m. - 7 p.m.)
-cage: wire cages (40.5x38.5x18h) with stainelss steel feeder.
The waste that drops through the wire bottom on a removable paper was periodically disposed of.
An automatic standby power supply was not brought into operation as the main supply did not fail.

Animal identification: by coloring different area of the ears. A computerized randomization program was adopted allocate the animals to groups.
Cage identification: by cage card giving indelible experiment number, start day, the group and the subgroup (1a, 1b; 2a, 2b) identification.

Diet: the animals were fed a breeder standard GLP diet -certificate coded 8 GP 22, produced by Charles River Italia's feed licensee Mucedola S.r.l., Settimo bfilanese.
The declared contents, on the label, on dry matter basis (moisture 12%), were:
crude protein 19%
crude fat 4%
crude fiber 14.5%
ash 7.5%
The diet was supplemented by the Producer with vitamins and trace elements. According to the analytical certificates provided by the Supplier, the contents of the batch of diet used in this study were within ± 5% of the declared values and contaminants were within the limits proposed by EPA-TSCA (44FR:44053-44093, July 26, 1979).
The animal feed, in compliance with RBM SOPS is analyzed twice a year for bacterial contamination.
The diet was available to the animals "ad libitum".

Water: from the municipal water main system. Water is filtered and distributed "ad libitum" to the animals by an automatic valve system.
Periodically drinking water is analyzed for microbiologic count, heavy metals, other contaminants (e.g. solvents, pesticides) and other chemical and physical characteristics.
The acceptable limits of quality of the drinking water are those defined in EEC Directive 80/778.
Contaminants that might interfere with the objectives of the study are not expected to be present in the diet or in the drinking water.
The analytical certificates of animal feed and water are filed at RBM premises.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
50% (0.5 ml)
Day(s)/duration:
Day 0/6 hours
Adequacy of induction:
highest technically applicable concentration used
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
25% (0.5 ml)
Day(s)/duration:
Day 0/6 hours
Adequacy of induction:
highest technically applicable concentration used
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
0.5 ml
Day(s)/duration:
Day 27/24 hours
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
10 animals in test group
10 animal in negative control group
Details on study design:
Test article dosage levels
As reported in the protocol the preliminary test was performed in order to select a non irritating test article concentration.
The test article was assayed by occlusive patch on two animals for each concentration.
Three doses undiluted, 50% and 25% were tested.
0.5 ml of each test article concentration was applied onto the skin with a closed patch.
24 hours after the administration, the patches were removed, the animals were observed up to 48 hours for reaction at the skin area of the patch application.
All three doses were well tolerated and therefore the test article undiluted was used in the sensitization test.

Test article formulates
The test article formulate was used undiluted.

Test description
Administration route: topical exposure by occlusive patch
Dose administered: the test article was used undiluted
Observation of clinical sign: daily, be cage
Bodyweight recording: pre-trial and weekly thereafter
Experimental group: 1 of 10 animals
Negative control group: 1 of 10 animals.
The animals of this group were treated only at day 28 with the test article closed patch as the product was tested undiluted.

Experiment design
Induction
Day -1: the animals of group 1 were clipped on the shoulder region in an area of about 6x6 cm with an electric clipper, before treatment.

Day 0: the animals clipped the day before were treated with the test article. 0.5 ml of the product was held on contact with the skin of the shoulder region by mean of an occlusive patch for 6 hrs.

Day 7 and 14: the 6 hrs closed-patch was repeated (see day 0).

Challenge
Day 27: in the animals of the experimental or negative control group one flank was clipped in an area of about 2x2 cm.

Day 28: the challenge test was identical to the occlusive patch test of the inductive phase, unless the patch was held in place for 24 hrs.

Day 29: the patch was removed, the area of challenge marked, and the skin reaction was read.

Day 30: a second reading was done.

Assessment of skin reactivity
Evaluation of skin reaction:
0 normal skin
1 slight erythema
2 moderate-intense erythema, slight edema
3 marked erythema and edema
The results were expressed in terms of incidence and severity of response:
incidence: the number of animals showing responses of 1 or greater at either 24 or 48 hrs divided by the number of animals tested;
severity: the sum of the test grades divided by the number of animals tested.
Challenge controls:
the challenge test was identical to the occlusive patch test of the inductive phase
Positive control substance(s):
not required

Results and discussion

Positive control results:
Not required

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
50%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None specified
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
24
Group:
test chemical
Dose level:
25%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None specified
Remarks on result:
no indication of skin sensitisation

Any other information on results incl. tables

 Skin sensitisation test in guinea pigs (Buhler test)

Bodyweight (grams)

Group no.

Guinea pig no.

Days

-1

6

13

21

27

1

1

2

3

4

5

6

7

8

9

10

400

405

404

370

407

386

420

395

379

405

450

478

471

443

500

429

491

466

453

467

498

547

541

499

557

475

552

430

495

550

532

616

611

561

635

545

607

609

556

605

574

669

667

612

668

573

678

657

608

708

2

11

12

13

14

15

16

17

18

19

20

408

433

422

405

397

405

404

417

406

421

483

494

506

480

476

474

470

507

504

491

542

538

585

545

527

538

538

575

584

548

597

569

656

593

607

613

637

632

604

655

636

588

713

666

648

674

695

680

710

636

 

Skin sensitisation test in guinea pigs (Buhler test)

Experimental group No. 1

Substance: [ANOX BF]

 

Challenge

Days

Test article

29

Test article

30

Guinea pig no.

 

 

1

2

3

4

5

6

7

8

9

10

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

0: No reaction

1, 2, 3: skin reaction was assessed according to the scale described in the text. Animals showing reaction ≥ 1 were considered positive

No. of positive animals at the challenge: 0

Results: NEGATIVE

 

Skin sensitisation test in guinea pigs (Buhler test)

Experimental group No. 2

Substance: [ANOX BF]

 

Challenge

Days

Test article

29

Test article

30

Guinea pig no.

 

 

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: No reaction

No. of positive animals at the challenge: 0

Results: NOT IRRITANT

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The sensitizing potential of the test article [ANOX BF] was assessed in guinea pigs using the Buhler test as described by Klecak.
No animal treated with the test article showed positive reaction at the challenge.
On the basis of these results, under the experimental conditions applied, [ANOX BF] did not appear to possess sensitizing capacity.
Executive summary:

The sensitizing potential of the test article [ANOX BF] was assessed in guinea pigs using the Buhler test as described by Klecak.

In the group treated with the test article no animals showed positive reaction at the challenge.

Under the experimental conditions applied, [ANOX BF] did not appear to possess sensitizing properties.