Registration Dossier

Diss Factsheets

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-05-30 to 2012-05-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (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

Constituent 1
Chemical structure
Reference substance name:
Sodium dimethyl 5-sulphonatoisophthalate
EC Number:
223-578-8
EC Name:
Sodium dimethyl 5-sulphonatoisophthalate
Cas Number:
3965-55-7
Molecular formula:
C10H10O7S.Na
IUPAC Name:
sodium 3,5-bis(methoxycarbonyl)benzenesulfonate
Test material form:
other: solide
Details on test material:
Name: Sodium dimethyl 5-sulphonatoisophthalate (SIM-Ester)

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V. Postbus 6174 5960 AD Horst / The Netherlands
- Age at study initiation: 9 – 10 weeks
- Weight at study initiation: 18.5 - 24 g
- Housing: group
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 2°C
- Humidity: 45-65%
- Photoperiod: artificial light 6.00 a.m. - 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Remarks:
(DMF)
Concentration:
2.5, 5, 10 %
No. of animals per dose:
5 females
Details on study design:
RANGE FINDING TESTS (Pre-test):
- Maximally achievable test item concentration: The highest test item concentration, which could be technically used, was a 10 % solution in dimethylformamide. Vortexing, sonicating, and warming to 37°C was used to formulate the test item. Applicable formulations of the test item at higher concentrations could not be achieved in other vehicles recommended by OECD 429, in Ethanol 70% (v/v) or in Ethanol 30% (v/v) and 1% Pluronic® L92.
- Treatment: Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item in concentrations of 5 and 10% once daily each on three consecutive days.
- Irritation: At the tested concentrations the animals did not show any signs of local skin irritation or systemic toxicity.


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
Groups of five mice was treated by topical (epidermal) application to the dorsal surface of each ear with different test item concentrations. The application volume, 25 µL/ear/day, was spread over the entire dorsal surface of each ear once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals). Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline (PBS) containing 20.5 µCi of 3HTdR (equivalent to 3HTdR 81.9 µCi/mL) were injected into each test and control mouse via the tail vein. Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection. The draining lymph nodes were rapidly excised and pooled per animal (2 nodes per animal). Single cell suspensions of pooled lymph node cells were prepared by gentle mechanical disaggregation. After washing two times with phosphate buffered saline the lymph node cells were resuspended in 5 % trichloroacetic acid and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid and with 10 mL of scintillation liquid. The level of 3HTdR incorporation was then measured on a beta-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid. The beta-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

After the lymph nodes have been excised, both ears (left and right) of mice were punched at the apical area using a biopsy punch. For each animal both punches were immediately weighed per animal using an analytical balance.

In addition to the sensitising reactions the following observations and data were recorded during the test and observation period: mortality / viability, body weights, ear thickness, ear weights, clinical signs (local / systemic).


- Criteria used to consider a positive response:

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.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
A One-Way-Analysis-of-Variance was conducted on the ear weights to assess whether the difference was statistically significant between test item groups and negative control (vehicle) group. In case of significant results of the One-Way-ANOVA, multiple comparisons were performed with the Dunnett test or the Student Newman Keuls test. Statistical significance was set at the five per cent level (p < 0.05). The Dean-Dixon-Test was used for identification of possible outliers.

Results and discussion

Positive control results:
The periodic positive control experiment performed with alpha-Hexyl cinnamaldehyde in acetone:olive oil (4+1 v/v) using CBA/CaOlaHsd mice demonstrated the sensitivity of the test system (EC3 = 20.1%).

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
EC3
Remarks on result:
not determinable
Key result
Parameter:
SI
Value:
1.12
Test group / Remarks:
10 % in DMF
Remarks on result:
other: Concentration of 10 % (w/v) in DMF represented the highest technically achievable concentration.
Key result
Parameter:
SI
Value:
1.18
Test group / Remarks:
5 % (w/v) in DMF
Key result
Parameter:
SI
Value:
1.15
Test group / Remarks:
2.5 % (w/v) in DMF
Remarks on result:
other:
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Concentration / mean DPM per animal (SD) vehicle control / 418 (104) 2.5 % / 480 (125) 5 % / 493 (104) 10 % / 468 (200)

Any other information on results incl. tables

VIABILITY/MORTALITY

No deaths occurred during the study period.

CLINICAL SIGNS

No systemic findings were observed during the study period. From day 4 to day 6, the animals treated with a test item concentration of 10% showed an erythema of the ear skin (Score 1). Animals treated with 2.5 and 5% test item concentration did not show any signs of local skin irritation.

BODY WEIGHT

The 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.

EAR WEIGHT

The measured ear weight of all animals treated was recorded on day 6 after necropsy. A relevant increase in ear weight was not observed.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item was not a skin sensitiser in the LLNA.
Executive summary:

In a Local Lymph Node Assay (Harlan Cytotest Cell Research GmbH, 2012, OECD 429, GLP compliance) mice (5/concentration) were treated with the test item in concentrations of 2.5, 5, and 10 % (w/v) and with an equivalent volume of the relevant vehicle (Dimethylformamide, DMF) alone. 10% represented the hihgest technically achievable concentration. A periodic positive control experiment was performed with alpha-Hexyl cinnamaldehyde in acetone:olive oil (4+1 v/v) using CBA/CaOlaHsd mice. Each substance was applied on the external surface of each ear (25 µL/ear) of the animals for three consecutive days (day 1,2 and 3). On day 6 animals were intravenously injected via the tail vein with tritiated methyl thymidine (3HTdR), than sacrificed approximately 5 hours after the injection. The draining lymph nodes were removed and processed (individual approach, 2 nodes per animal). The cell proliferation in the local lymph nodes was measured by incorporation of 3HTdR and the obtained values were used to calculate stimulation indices (SI). Ear punch weight determination method was also be used for measurement of ear thickness.


No mortality was observed during the test. No signs of systemic toxicity and no test item related effect on body weight gain occured. From day 4 to day 6, the animals treated with a test item concentration of 10% showed an erythema of the ear skin (score 1). Animals treated with 2.5 and 5% test item concentration did not show any signs of local skin irritation. A relevant increase in ear weight was not observed. No statistically significant lymphoproliferation was observed in any group treated with the test item Sodium dimethyl 5-sulphonatoisophthalate (SIM-Ester). The SI values were 1.15, 1.18 and 1.12 at concentrations of 2.5 %, 5% and 10 %, respectively. Under the conditions of the present Local Lymph Node Assay, SIM-Ester was shown to have no sensitization potential.