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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:
7 Sep - 1 Oct 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
GLP compliance:
yes
Type of study:
guinea pig maximisation test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Lanthancarbonate-octahydrate
- Molecular formula (if other than submission substance): La2(CO3)3*8H2O
- Molecular weight (if other than submission substance): 601.98 g/mol
- Physical state: white powder
- directly analysed: 42.9% (content of active ingredient La)
- Analytical purity: > 99.5%
- Lot/batch No.: MB04-016
- Expiration date of the lot/batch: 25 May 2005
- Storage condition of test material: at room temperature, light protection

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: Crl: HA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratory Animal Breeders, 88353 Kißligg, Germany
- Age at study initiation: 4-5 weeks
- Weight at study initiation: 286 - 383 g
- Housing: in groups of five during adaptation, in groups of two or three per cage throughout the study period
- Diet (e.g. ad libitum): PROVIMI KLIBA 3420 - Maintenance Diet for Guinea Pigs, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 5 days, state of health was monitored
- Healthy: Only healthy animals exhibiting no clinical signs were used for the study. The animals were not vaccinated or treated against infections either before receipt, or during the adaptation period or study period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 40 - 70
- Air changes (per hr): ≥ 10 times
- Photoperiod (hrs dark / hrs light): 12 / 12 (artificial lightning)

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
physiological saline
Concentration / amount:
as cited in study report
Intradermal induction: 5% (= 20 mg test item/animal)
Topical induction: 50% (= 250 mg test item/animal)
Challenge: 50% (=250 mg test item/animal)
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
as cited in study report
Intradermal induction: 5% (= 20 mg test item/animal)
Topical induction: 50% (= 250 mg test item/animal)
Challenge: 50% (=250 mg test item/animal)
No. of animals per dose:
treatment: 20 animals
control: 10 animals
Details on study design:
GENERAL:
- cages: type IV Makrolon cages, exchanged for ones with clean bedding at least two times a week.
- bedding: low-dust wood shavings; the beddings were spotchecked for contaminat levels.
- cleaning, disinfection of the animal room: each day: sept out; once weekly: thoroughly cleaned with water; once each months: disinfection with Terralin (working dilution 1%)
- test item formulation: as suspension in sterile pysiological saline solution, kept under light protection; suspensions were continuously homogeniezed on a magnetic stirrer during the treatments.
- general examinations: observation for clinical signs at least once daily throughout the entire study period
- body weight: recorded on day 1 before the first induction and at the end of the study (day 25 in the test item group and control group) and day 18 in the range finding group)

RANGE FINDING TESTS:
A. Dose-range finding study for intradermal induction
6 animals were given intradermal injections: each animal got two injections with vehicle and four injections with test item (per 0.1 mL). Following concentrations were tested: 0 and 1%, 0 and 2.5%, 0 and 5% (each with 2 animals). The injection sites were evaluated after 24 and 48 h.
B. Dose range finding study for topical induction
Three concentrations (12%, 25% and 50%) and the vehicle were tested on four guinea pigs. The patches moistened with 0.5 mL of the test item formulations or the vehicle were applied to each animal under occlusive conditions for 24 hours. At the end of the exposure period the remaining test item or vehicle was removed with phys. saline solution. 24 hours later the treated areas were shorn. Skin reactions were evaluated 48 and 74 h after the start of the application.
C. Dose range finding study for challenge
One week prior to the challenge, the challenge concentration was determind on 2 animals in the main study that were treated in the same manner as the control animals during the inductions. 4 patches each loaded with 0.5 mL test item formulation (concentration of test item formulations: 12%, 25% and 50%) or the vehicle were applied to each animal under occlusive conditions for 24 h. Further procedure: see range finding test B.

MAIN STUDY
A. INTRADERMAL INDUCTION
- Site: dorsal region and flanks of the animals were shorn one day prior to the application. Starting behind the nape of the neck, 3 injections each in a row were made on the left and right side of the spinal column.
- Volume applied: 0.1 mL
- Injections test item group:
complete Freund´s adjuvant diluted 1:1 with vehicle (cranial/bilateral injection)
5% Lanthancarbonat-octahydrat formulated in vehicle (medial/bilateral injection)
5% Lanthancarbonat-octahydrat formulated at equal parts in vehicle and complete Freund´s adjuvant (caudal/bilateral injection)
- Injections control group and range finding group:
in the same manner as the animals of the test item group; however, the injections did not contain any test item but undiluted vehicle instead of the test item.
- Observations: visual assessment 2 and 7 days after injection

B. TOPICAL INDUCTION
- Performed on week after the intradermal induction
- On the day prior to topical treatment, the test areas were shorn
- Hypoallergenic patches (2x4 cm) were placed between and on the injection sites, covered with aluminium foil and held securely in place on the skin using a ORABAND self adhesive tape.
- Treatment of patches: test item group: 0.5 mL 50% Lanthancarbonat-octahydrat; control: 0.5 mL vehicle
- exposure period: 48 h. After removal of the patch, the remaining test item was removed with vehicle.


B. CHALLENGE EXPOSURE
- Day(s) of challenge: three weeks after the intradermal induction
- Site: dorsal region and the right flank (shorn one day prior to challenge)
- Hypoallergenic patches (2x4 cm) loaded with the 50% test item formulation were placed on the right flank (caudal) of the animals of the test item group and the control group, covered with aluminium foil and held securely in place on the skin using a ORABAND self adhesive tape. A patch loaded with the vehicle was placed also on the right flank (cranial) as control.
- Applied volume: 0.5 mL
- Exposure period: 24 h. After removal of the patch, the remaining test item was removed with vehicle. 21 hours later the skin of the animals was shorn in the zone of the challenge area.
- Evaluation: visual assessment 48 and 72 h after the start of the challenge exposure

GRADING OF THE SKIN
0 = no reaction
1 = slight localized redness
2 = moderate confluent redness
3 = severe redness and swelling
Positive control substance(s):
yes
Remarks:
alpha hexyl cinnamic aldehyde formulated in polyethylene glycol 400. Intradermal induction: 5% test item formulation; topical induction: 25% test item formulation; challenge: 12% test item formulation.

Results and discussion

Positive control results:
100% of the test animals exhibited dermal reactions in the challenge treatment.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
50%
No. with + reactions:
1
Total no. in group:
18
Clinical observations:
18 animals were examined; 2 animals died on day 10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50% . No with. + reactions: 1.0. Total no. in groups: 18.0. Clinical observations: 18 animals were examined; 2 animals died on day 10.
Reading:
2nd reading
Hours after challenge:
72
Group:
test group
Dose level:
50%
No. with + reactions:
1
Total no. in group:
18
Clinical observations:
18 animals were examined; 2 animals died on day 10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 50%. No with. + reactions: 1.0. Total no. in groups: 18.0. Clinical observations: 18 animals were examined; 2 animals died on day 10.
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 0. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: negative control. Dose level: 0. No with. + reactions: 0.0. Total no. in groups: 10.0.

Any other information on results incl. tables

RANGE FINDING

A. Dose-range finding study for intradermal induction

- 0 and 1%: control: wheal after 24 h, no reaction after 48 h, treatment: red wheal after 24 h, wheal after 48 h

- 0 and 2.5%: control: wheal after 24 h, no reaction after 48 h, treatment: red wheal after 24 h, wheal after 48 h

- 0 and 5%: control: wheal after 24 h, no reaction after 48 h, treatment: red wheal after 24 h, wheal after 48 h

B. Dose range finding study for topical induction

No skin reactions were observed in control and treatment groups (12, 25 and 50 % test item) after 48 and 72 h.

C. Dose range finding study for challenge

No skin reactions were observed in control and treatment groups (12, 25 and 50 % test item) after 48 and 72 h.

Based on the results of the dose range-finding studies, the following concentrations were selected for the inductions and the challenge:

intradermal induction: 5%, topical induction: 50%, challenge: 50%.

MAIN STUDY

General:

- appearance and behaviour of the test item group were not different from the control group

- animal no 18 (out of the test item group) showed following clinical signs: at day 8: pale, piloerection, poor general condition; at day 9: pale, poor general condition, labored breathing, piloerection. At day 10 the animal died.

- animal no 17 (out of the test item group) showed following clinical signs: at day 9: pale, poor general condition. At day 10 the animal died.

- body weight: at the end of the study, the mean body weight of the treatment group animals was in the same range than that of the control group animals; control: 448 ± 45.7 g (on day 25); test item group: 471 ± 29.5 g (on day 25), challenge range finding group: 383 ± 8.5 g (on day 18).

- after the intradermal induction the animals of the control and the test item group showed red wheal after 48 hours. After 7 days at the injection sites of all animals (control and test item group) wheals and encrustations were observed.

Challenge

- there is a typing error in the report: In the table with the number of animals exhibiting skin effects the challenge concentration was given as 25%.

- one animal of the test item group showed skin effects (1: slight localized redness) after 48 and 72 h, no animal of the control group showed skin effects after 48 and 72 h.

- the control patches after challenge showed no skin reactions in test item group and control group.

Body weights of individual animals:

Animal No.

Body weight at the day of the first application (g)

Body weight at the end of the study* (g)

Control group

1

344

466

2

383

553

3

335

448

4

344

466

5

316

433

6

338

442

7

348

461

8

307

392

9

317

419

10

300

396

Mean and standard deviation

333 ± 24.3

448 ± 45.7

Test item group

11

348

533

12

347

470

13

326

517

14

316

469

15

326

468

16

332

478

17

327

died

18

321

died

19

332

468

20

292

451

21

374

482

22

311

482

23

349

447

24

334

445

25

350

458

26

311

417

27

328

487

28

358

480

29

365

500

30

338

420

Mean and standard deviation

334 ± 19.9

471 ± 29.5

Range finding group

31

301

377

32

286

389

Mean and standard deviation

294 ± 10.6

383 ± 8.5

*(control group and test item group: on day 25; range finding group: on day 18)

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information