Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 April 2010 - 27 April 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP 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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/J Rj
Sex:
female
Details on test animals and environmental conditions:
Source: ELEVAGE JANVIER Route des Chènes Secs B.P. 4105 53940 LE GENEST-ST-ISLE, France
Hygienic level at arrival: SPF
Hygienic level during the study: Standard housing conditions
Justification of strain: On the basis of OECD Guideline, mice of CBA/Ca or CBA/J strain can be used. Females are used because the existing database is predominantly based on females.
Number of animals: 4 animals / treatment group
Sex: female, nulliparous, non pregnant
Age of animals at starting: 10 – 11 weeks old
Body weight range at starting: 21.4 – 22.8 grams (The weight variation in animals involved in the study did not exceed ± 20 % of the mean weight)
Acclimatization time: 34 days
Animal health: Only healthy animals were used for the study. Health status was certified by the veterinarian.
Housing / Enrichment: Individual caging / mice were provided with glass tunnel-tubes
Cage type: Type II. polypropylene/ polycarbonate
Bedding: Bedding was available to animals during the study
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 22 ± 3 °C
Relative humidity: 30 - 70 %
Ventilation: 15-20 air exchange/hour

The temperature and relative humidity were recorded twice every day during the acclimatisation and experimental phases.
Room/Cabinet (non-radioactive phase): 244/5
Room/Cabinet (radioactive phase): 139 - 140

Food and feeding: Animals received ssniff SM R/M-Z+H "Autoclavable complete diet for rats and mice – breeding and maintenance" (Batch number: 476 3413 Expiry Date: June 2010 and Batch number: 928 3711 Expiry Date: September 2010) produced by ssniff Spezialdiäten GmbH (Ferdinand-Gabriel-Weg 16, D-59494 Soest, Germany), ad libitum.

Water supply: Animals received tap water from the municipal supply from 500 ml bottle, ad libitum. Water quality control analysis was performed once every three months and microbiological assessment was performed monthly, by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A.u.36., Hungary). Copies of the relevant Certificates of Analysis are retained in the Archive at LAB Research Ltd.
Bedding: Lignocel(R) Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (D-73494 Rosenberger (Germany) Holzmühle 1) was available to animals during the study.

Identification: A unique number written on the tail with a permanent marker identified each animal. The animal number was assigned on the basis of LAB Research Ltd.’s master file. The cages were marked with identity cards with information including study code, cage number, and dose group, sex and individual animal number. The animals were randomised and allocated to the experimental groups. The randomisation was checked by computer software according to the actual body weights, verifying the homogeneity and variability between the groups.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
The Preliminary Irritation/Toxicity Test was performed in CBA/J Rj mice using two doses (test item concentrations of 50 and 25 (w/v) %). This preliminary experiment was conducted in a similar experimental manner to the main study, but it was terminated on Day 6 with a body weight measurement (radioactive proliferation assay was not performed). During the Preliminary Irritation/Toxicity Test no mortality was observed in any treatment groups. No significant body weight loss was observed in the treated groups.
As such, the animals were dosed for the main study at test concentrations of 50%, 25% and 10% (w/v). A group of animals dosed with just the vehicle solvent containing no test substance was used as a vehicle control group.
No. of animals per dose:
4 animals per dose
Details on study design:
During the Preliminary Compatibility Test the solubility of the test item was examined in acetone/olive oil 4:1 (v/v) mixture (AOO). Since the maximum solubility was greater than 50 (w/w) % in AOO, it was chosen as vehicle for the study. The test item was weighed and formulations prepared daily on a weight: volume basis in the Central Dispensary Unit of LAB Research Ltd.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Interpretation of Results
The test item is regarded as a sensitizer if both of the following criteria are fulfilled:
- That exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index.
- The data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

Results and discussion

Positive control results:
In the reliability check experiment, no mortality, cutaneous reactions or signs of toxicity were observed for the positive control substance. A significant lymphoproliferative response (SI ≥ 3) was noted for HCA dissolved in AOO with stimulation index value of 9.8. This value was considered to demonstrate the appropriate performance of the assay.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: See table below
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: See table below

Any other information on results incl. tables

DPM, DPN and Stimulation Index Values for all Groups

Test Group Name

Measured DPM/group

DPM

No. of Node

DPN

Stimulation Index Values

Background (% (w/v)% TCA)

32.5

 

-

 

 

Negative control AOO

1102

1069.5

8

133.7

1.0

Test Material 50% in AOO

3776

3743.5

8

467.9

3.5

Test Material 25% in AOO

867

834.5

8

104.3

0.8

Test Material 10% in AOO

1316

1283.5

8

160.4

1.2

No mortality or signs of local or systemic toxicity were observed during the study.

No treatment related effects were observed on animal body weights.

The test item was a clear/pale yellow liquid, which was dissolved in AOO.

Since there were no confounding effects of irritation or systemic toxicity at the applied concentrations, the proliferation values obtained are considered to reflect the real potential of the test item to cause lymphoproliferation in the Local Lymph Node Assay.

A significant lymphoproliferative response (SI ≥ 3) was noted for the test material at a concentration of 50 (w/v) %. The stimulation index values of the test item were 3.5, 0.8 and 1.2 at treatment concentrations of 50 %, 25 % and 10 (w/v) %, respectively. The stimulation index values were compatible with a biological dose-related response with a weak positive response.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information
Conclusions:
In conclusion, under the conditions of the present assay the test material, tested in a suitable vehicle, was shown to have a weak sensitization potential (sensitizer) in the Local Lymph Node Assay.
Executive summary:

The aim of this study was to determine the skin sensitization potential of the test material following dermal exposure using OECD Guideline 429.

Based on the results of the Preliminary Compatibility Test and on the recommendations of the OECD Guideline, the test item was suspended in acetone/olive oil 4:1 (v/v) mixture (AOO). The maximum attainable concentration was greater than 50 (w/v) %.

The Preliminary Irritation/Toxicity Test was performed in CBA/J Rj mice using two doses (test item concentrations of 50 and 25 (w/v) %) in the selected vehicle. The applicability and biocompatibility of the test item on the ears of animals at the maximum concentration of test item of 50 (w/v)% was acceptable.

In the main assay, sixteen female CBA/J Rj mice were allocated to four groups of four animals each:

- three groups received the appropriate formulation of the test material at concentrations of 50 %, 25 % and 10 (w/v) %,

- the negative control group received AOO.

The suspensions of the test item were applied on the dorsal surface of ears of experimental animals (25 μl/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6, the cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI).

No mortality or systemic clinical signs were observed during the study. No treatment related effects were observed on animal body weights in any treated groups.

Stimulation index values of the test item were 3.5, 0.8 and 1.2 at treatment concentrations of 50 %, 25 % and 10 (w/v) %, respectively. The result of the latest reliability check (performed within an interval of no longer than six months, Study code: 10/073-037E) was used to demonstrate the appropriate performance of the assay in accordance with the OECD Guideline 429. The positive control substance α-Hexylcinnamaldehyde (HCA) was examined at a concentration of 25 % in the relevant vehicle. A significant lymphoproliferative response (SI ≥ 3) was noted for HCA with stimulation index value of 9.8, the result confirms the validity of the LLNA in this laboratory.

In conclusion, under the conditions of the present assay the test material, tested in a suitable vehicle, was shown to have a weak sensitization potential (sensitizer) in the Local Lymph Node Assay.