Salts of 9-[2-(ethoxycarbonyl)phenyl]-3,6-bis(ethylamino)-2,7-dimethylxanthenium chloride and 4-{[1-hydroxy-6-({[5-hydroxy-6-(phenyldiazo)-7-sulfonaphthalen-2-yl]carbamoyl}amino)-3-sulfonaphthalen-2-yl]diazo}benzoic acid (2:1)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2008-04-16 to 2008-04-22

Reliability:

1 (reliable without restriction)

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

not applicable

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

Species and strain: CBA/Ca Ola Hsd mice
Source: Harlan Hungarian Supplier: WOBE Kereskedelmi Kft. H-1164, Budapest, Garmada u. 10.
Hygienic level at arrival: SPF
Hygienic level during the study: Good conventional
Justification of strain: On the basis of comparative investigations in other laboratories, mice of the CBA/Ca strain were found to exhibit a more marked response than other strains. 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: Young adult, 10-11 weeks old, age-matched within one week
Body weight range at starting: The weight variation in animals involved in the study did not exceed 20 % of the mean weight.
Acclimatisation time: 28 days

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

2.5; 5; 10 % (w/v) (25μl)

No. of animals per dose:

4

Details on study design:

16 female CBA/Ca mice were allocated to 4 groups of 4 animals each: 3 groups received the appropriate formulation of F 213 Red at concentrations of 10 %, 5 % or 2.5 %, - 1 negative control group received the solvent (DMF). Each substance was applied on the external surface of each ear (25 µl/ear) for three consecutive days (Day 0, 1 and 2) at the appropriate concentrations. On Day 5, 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).

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

not applicable

Results and discussion

Positive control results:

The positive control substance -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 7.1. Although in this study the proliferation value increased by the positive control in DMF was slightly lower than the historical minimum value, the difference was not significant and the effect was considered to be adequate, demonstrating the test reliability.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: Stimulation index (SI = DPN of a treated group divided by DPN of the negative control group) for each treatment group was also calculated.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: DPM was measured for each group. The results were expressed as DPN (DPM divided by the number of lymph nodes) following the industry standard for data presentation.

Any other information on results incl. tables

no remarks

Applicant's summary and conclusion

Interpretation of results:

other: moderate sensitiser

Conclusions:

Under the conditions of the present assay F 213 Red, dissolved in an appropriate solvent up to the maximum feasible concentration (10 %), was shown to have moderate sensitisation potential (moderate-sensitiser) in the Local Lymph Node Assay.

Executive summary:

Local Lymph Node Assay according to OECD 429 and Commission Directive 2004/73/EC, B.42 was performed with CBA/Ca Ola Hsd mice. The aim of this study was to determine the skin sensitisation potential of F 213 Red following dermal exposure in the Local Lymph Node Assay. The maximum concentration of test item in an acceptable solvent was established according to the principles of the relevant guidelines. The maximum available concentration was 10 % (% w/v) in N,N-Dimethylformamide (DMF). A preliminary irritation/toxicity test was performed showing that the maximum concentration of test item of 10 % in DMF was acceptable. In the main assay sixteen female CBA/Ca mice were allocated to four groups of four animals each: - three groups received the appropriate formulation of F 213 Red at concentrations of 10 %, 5 % or 2.5 %, - the negative control group received the solvent (DMF). Each substance was applied on the external surface of each ear (25 µl/ear) for three consecutive days (Day 0, 1 and 2) at the appropriate concentrations. On Day 5, 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 effect on the body weights was observed. No sign of irritation was observed in any of the treated groups, although the test item discoloured the skin of animals, which render the observation of any cutaneous effect more difficult. A lymphoproliferative response (SI 3) was noted for F 213 Red with stimulation index values (SI) of 5.1 and 6.0 at concentrations of 10 % and 5 %, respectively. No lymphoproliferative response was observed at concentration of 2.5 %, the stimulation index was 2.6 at this concentration. The stimulation index values obtained were compatible with the conventional biological dose-response. The result of the latest reliability check (performed within an interval of no longer than six months) was used to demonstrate the appropriate performance of the assay in accordance with the OECD guideline 429 (Ref. 1). The positive control substance -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 7.1, the result confirms the validity of the LLNA in this laboratory. Under the conditions of the present assay F 213 Red, dissolved in an appropriate solvent up to the maximum feasible concentration (10 %), was shown to have moderate sensitisation potential (moderate-sensitiser) in the Local Lymph Node Assay.