Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Start of preliminary test: 04 May 2011; Start of experiment: 18 May 2011; End of experiment: 24 May 2011; Draft report: 15 June 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Performed according to GLP and OECD Guideline followed. Deviation not considered to adverseley affect the results or integrity of the study.
Justification for data waiving:
other:
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): KS-235
- Molecular formula (if other than submission substance): C14H14O2
- Molecular weight (if other than submission substance): 214.27
- Physical state: White crystalline powder
- Analytical purity: 99.9%
- Lot/batch No.: 91014
- Expiration date of the lot/batch: October 2014
- Storage condition of test material: Room temperature (15-25°C)
- Other:
- Manufacture date: October 2009
- Safety precautions: Routine safety precautions (lab coat, gloves, goggles, face mask) for unknown materials will be applied to assure personnel health and safety.

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/J Rj
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:
Elevage Janvier,
Route des Chènes Secs B.P. 4105,
53940 Le Genest-St-Isle,
France
- Age at study initiation: 9-10 weeks old (Female, nulliparous, non pregnant)
- Weight at study initiation: 22.0 - 22.9 g (The weight variation in animals involved in the study did not exceed ± 20 % of the mean weight)
- Housing:
Housing / Enrichment: Group caging / mice were provided with glass tunnel-tubes
Cage type: Type II. polypropylene/ polycarbonate
Bedding: Bedding was available to animals during the study
- Diet (e.g. ad libitum): Animals received ssniff SM R/M-Z+H "Autoclavable complete diet for rats and mice - breeding and maintenance" (Batch number: 555 5267 Expiry Date: August 2011) produced by ssniff Spezialdiaten GmbH (Ferdinand-Gabriel-Weg 16, D-59494 Soest, Germany), ad libitum.
- Water (e.g. ad libitum): 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.
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 30-70
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): Light 12 hours daily, from 6.00am to 6.00pm

Room/Cabinet (non-radioactive phase): 244/5
Room/Cabinet (radioactive phase): 139 – 140

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Remarks:
N,N-dimethylformamide
Concentration:
25, 10 and 5 (w/v)%
No. of animals per dose:
4 animals/treatment group
Details on study design:
RANGE FINDING TESTS: See Appendix 2 of attached report for results of preliminary irritation/toxicity test.

The Preliminary Irritation/Toxicity Test was performed in CBA/J Rj mice using two doses (test item concentrations of 25 and 10 (w/v)%) in the selected vehicle. The observations recorded in the preliminary test suggest that the 25 (w/v)% formulation is a suitable dose level for a valid LLNA.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: 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.

- 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.

TREATMENT PREPARATION AND ADMINISTRATION:
- ADMINISTRATION OF THE TEST ITEM
- Dose Selection and Justification of Dose Selection: A Preliminary Irritation/Toxicity Test was performed on CBA/J Rj mice using two doses, at test item concentrations of 25 and 10 (w/v)%, respectively. 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, systemic toxicity or local irritation were observed (score of erythema was 0 during the observation time). No treatment related effect on body weights was observed in the treated groups. Ear thickness was measured by using a thickness gauge on Days 1, 3 and 6 and the weight of an ear punch on Day 6. The observations recorded in this preliminary test suggest that the formulations, the application of the material and the local effects on the animal are acceptable for a valid LLNA. The observations are summarized in Appendix 2 of attached report. The experimental groups and dose levels are summarized in Table 2 of the attached report.

- Topical application: During the assay each mouse was topically dosed on the dorsal surface of each ear with 25 µl of the appropriate formulation applied using a pipette. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.
Positive control substance(s):
other: α-Hexylcinnamaldehyde, ≥95%
Statistics:
EVALUATION OF THE RESULTS
- DPM was measured for each pooled group of nodes. The measured DPM values were corrected with the background DPM value (‘’DPM’’). The average of the two measured DPM values of 5 (w/v)% TCA solutions was used as the background DPM value. The results were expressed as ‘’DPN’’ (DPM divided by the number of lymph nodes) following the industry standard for data presentation. Stimulation index (SI = DPN value of a treated group divided by the DPN value of the negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result.

Results and discussion

Positive control results:
Positive control 25% HCA in DMF:
Measured DPM/Group: 14806
Group DPM: 14764.5
No. of Nodes: 8
DPN: 1845.6
Stimulation Index Values: 7.8

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: Negative control DMF: 1.0 KS-235 25 (w/v)% in DMF: 1.6 KS-235 10 (w/v)% in DMF: 2.4 KS-235 5 (w/v)% in DMF: 2.0 Positive control 25% HCA in DMF: 7.8
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Measured DPM/group: - Background (5 (w/v)% TCA ): 41.5 - Negative control DMF: 1928 - KS-235 25 (w/v)% in DMF: 3099 - KS-235 10 (w/v)% in DMF: 4633 - KS-235 5 (w/v) in DMF: 3852 - Positive control 25% HCA in DMF: 14806 Group DPM: - Negative control DMF: 1886.5 - KS-235 25 (w/v)% in DMF: 3057.5 - KS-235 10 (w/v)% in DMF: 4591.5 - KS-235 5 (w/v)% in DMF: 3810.5 - Positive control 25% HCA in DMF: 14764.5

Any other information on results incl. tables

CLINICAL OBSERVATION: No mortality or signs of systemic toxicity were observed during the study. The results are given in Appendix 3 of attached report.

 

BODY WEIGHT MEASUREMENT: No treatment related effects were observed on animal body weights. Individual and mean body weights are given in Table 3 of attached report.

 

PROLIFERATION ASSAY: The results of the proliferation assay are summarized in Table 4 and Figure 1 of the attached report. Appearance of the lymph nodes was normal in the negative control group and in the test item treated groups. Larger than normal lymph nodes was observed in the positive control group.

 

INTERPRETATION OF OBSERVATIONS: The test item was a white crystalline powder, which was dissolved in N,N- dimethylformamide

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. The lack of any positive result under these exaggerated test conditions is considered to be good evidence that KC-235 is not a sensitizer (Figure 1 of attached report).

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
In conclusion, under the conditions of the present assay KS-235 (Batch No.: 91014), tested in a suitable vehicle, was shown to have no sensitisation potential (not-sensitizer) in the Local Lymph Node Assay.
Executive summary:

The aim of this study was to determine the skin sensitization potential of KS-235 following dermal exposure.

 

Based on the results of the Preliminary Compatibility Test and oil the recommendations of the OECD Guideline [1], the test item was dissolved in N, N-dimethylformamide (abbreviation: DMF). The test item formed an appropriate formulation in this vehicle at concentrations of 25 (w/v) %, therefore it was chosen as vehicle for the test.

 

The Preliminary Irritation/Toxicity Test was performed in CBA/J Rj mice using four doses (test item concentrations of 25 and 10 (w/v) %) in the selected vehicle. The observations recorded in the preliminary test suggest that the 25 (w/v) % formulation is a suitable dose level for a valid LLNA.

 

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

- three groups received the appropriate formulation of KS-235 at concentrations of 25, 10 and 5 (w/v) %,

- the negative control group received DMF,

- the positive control group received 25% α-Hexylcinnamaldehyde (HCA) in DMF.

                                                       

The solutions of the test item were applied on the dorsal surface of ears of experimental animals (25µl/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 Iymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI).

No mortality, systemic toxicity or local irritation was observed during the study. No treatment related effects were observed on animal body weights in any other treated groups. The observed clinical signs are summarized in Appendix 3 of attached report.

 

Stimulation index values of the test item were 1.6, 2.4 and 2.0 at treatment concentrations of 25, 10 and 5 (w/v) %, respectively.

 

α-Hexyleimsamaldehyde (25 (w/v) % dissolved in DMF) was used as a positive control to demonstrate the appropriate performance of the assay [1]. A significant lymphoproliferative response (stimulation index value of 7.8) was noted for the positive control chemical and this result confirmed the validity of the assay.

 

In conclusion, under the conditions of the present assay KS-235 (Batch No.: 91014), tested in a suitable vehicle, was shown to have no sensitisation potential (not-sensitizer) in the Local Lymph Node Assay.

 

[1] OECD Guidelines for Testing of Chemicals No. 429. Skin Sensitisation: Local Lymph Node Assay. Adopted: 22 July 2010.