Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

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

1
Chemical structure
Reference substance name:
[3-methoxycarbonyl-4-[6-(4-prop-2-enoyloxybutoxycarbonyloxy)naphthalene-2-carbonyl]oxy-phenyl] 6-(4-prop-2-enoyloxybutoxycarbonyloxy)naphthalene-2-carboxylate
Cas Number:
914918-26-6
Molecular formula:
C46H40O16
IUPAC Name:
[3-methoxycarbonyl-4-[6-(4-prop-2-enoyloxybutoxycarbonyloxy)naphthalene-2-carbonyl]oxy-phenyl] 6-(4-prop-2-enoyloxybutoxycarbonyloxy)naphthalene-2-carboxylate
Test material form:
solid
Details on test material:
Batch: 81184136WO/57698316KO = 1/1.47Expiry Date:2023Physical state, appearance: Off-white solid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL- Source and lot/batch No.of test material: 81184136WO/57698316KO = 1/1.47- Expiration date of the lot/batch: 2023STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL- Storage condition of test material: at room temperature

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS- Source: Envigo RMS B.V., Inc. Postbus 6174 5960 AD Horst / The Netherlands- Age at study initiation: Pre-test: 11 - 12 weeks, Main study: 8 - 9 weeks- Housing: in groups- Diet: ad libitum- Water: ad libitum- Acclimation period: 5 days- Indication of any skin lesions: noENVIRONMENTAL CONDITIONS- Temperature (°C): 22 + / - 2- Humidity (%): 45-65- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
2, 5, and 10%
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TESTS:- Compound solubility: 25% highest applicable concentration, tested was 10 and 25%- Systemic toxicity: no signs of systemic toxicity observed- Ear thickness measurements: not performed- Erythema scores: 10% erythema in one animal score 1 day 4, 25% eyrythema from days 3-6 score 1 and slight eschar formation on day 6MAIN STUDYANIMAL ASSIGNMENT AND TREATMENT- Name of test method:OECD 429- A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:• First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index. • Second, that 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. TREATMENT PREPARATION AND ADMINISTRATION: - Topical applicationEach test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 2, 5, and 10% in DMF. The application volume, 25 µL/ear/day, was spread over the entire dorsal surface (diameter ~ 8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone (control animals).- Administration of 3H-methyl-thymidineFive days after the first topical application (day 6) 250 µL of phosphate-buffered saline containing 19.7 µCi of 3H-methyl thymidine (equivalent to 78.9 µCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated in the body weight tables, for the ear weights, the lymph node weights and lymph node cell count, and for the DPM values (group mean DPM ± standard deviation). All calculations conducted on the DPM values, the ear weights, the lymph node weights and the lymph node cell count were performed with validated program R Script.Within the program a statistical analysis conducted on the DPM values, the ear weights, the lymph node weights and the lymph node cell count to assess whether the difference was statistically significant between the test item groups and negative control group. Statistical significance was set at the five per cent level (p < 0.05). Additionally, the Dean-Dixon-Test and Grubb’s Test were used for identification of possible outliers. No outlier values were observed in both statistical tests. However, both biological and statistical significance were considered together.

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
0.8
Test group / Remarks:
2%
Key result
Parameter:
SI
Value:
0.9
Test group / Remarks:
5%
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
10%
Cellular proliferation data / Observations:
- Viability / Mortality No deaths occurred during the study period. - Clinical SignsNo signs of systemic toxicity were observed during the study period. On day 4 and day 5, the animals treated with a test item concentration of 10% showed an erythema of the ear skin (score 1). The animals of all three dose groups showed slight scaly ears upon preparation on day 6.- Body WeightsThe body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.- Lymph Node Weights and Cell CountsThe measured lymph node weights and –cell counts of all animals treated were recorded after sacrifice. A statistically significant or biologically relevant increase in lymph node weights or –cell counts was not observed in any of the test item treated groups in comparison to the vehicle control group. For BALB/c mice, a cut-off value for the lymph node cell count index of 1.55 was reported for a positive response. The indices determined for the lymph node cell count did not reach or exceed this threshold.- Ear WeightsThe measured ear weight of all animals treated was recorded on test day 6 (after necropsy). A biologically relevant or statistically significant increase in ear weights was not observed. Furthermore, the cut-off value (1.1) of the ear weight index for a positive response regarding ear skin irritation reported for BALB/c mice was not reached or exceeded in any of the treated groups.

Any other information on results incl. tables

Calculation of Stimulation Indices per Dose Group

Test item concentration

Group Calculation

Mean DPM per
animal (2 lymph nodes)a)

SD

S.I.

Vehicle Control Group (DMF)

789.6

233.6

1.0

2% Paliocolor LC 1057

716.4

234.9

0.9

5% Paliocolor LC 1057

614.4

182.6

0.8

10% Paliocolor LC 1057

824.8

236.7

1.0

a)      Mean DPM/animal was determined by dividing the sum of the measured values from lymph nodes of all animals within a group by the number of animals in that group (5 animals)

The EC3 value could not be calculated, since all S.I.´s are below the threshold value of 3.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item was not a skin sensitiser under the test conditions of this study.