Disodium {sulfonylbis[4,1-phenylenediazene-2,1-diyl(1-ethyl-6-hydroxy-4-methyl-2-oxo-1,2-dihydropyridine-5,3-diyl)]}dimethanesulfonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-09-29 until 2010-10-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline-conform study under GLP without deviations

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Test guideline 2

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

Test material information

Constituent 1

Reference substance name:

Acid Yellow RN 2903

IUPAC Name:

Acid Yellow RN 2903

Details on test material:

Identity: Acid Yellow RN 2903
Batch No.: Vers. Kilo 6, 26.05.2009
Purity: Approx. 65% (dose calculation not adjusted to purity)
Stability in solvent: 4 days in water, PEG and CMC at room temperature
Storage: At room temperature
Expiration Date: August 01, 2015

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS: mice, CBA/CaOlaHsd
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 18.6 - 22.6 g
- Housing: group
- Diet: pelleted standard diet (Harlan Laboratories B.V., 5960 AD Horst, The Netherlands), ad libidum
- Water: tap water, (Gemeindewerke, 64380 Rossdorf, Germany), ad libitum
- Acclimation period: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible
signs of illness were used for the study
- Bedding: granulated soft wood bedding (Rettenmaier & Söhne GmbH + Co. KG, 73494 Rosenberg, Germany)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 2°C
- Humidity (%): 43-65 %
- Photoperiod (hrs dark / hrs light): artificial light 6:00 a.m. - 6:00 p.m.
- Bedding: granulated soft wood bedding (Rettenmaier & Söhne GmbH + Co. KG, 73494 Rosenberg, Germany)

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

The highest test item concentration, which can be technically used was a 25 % suspension in propylene glycol.
In the pre-test two mice were treated with test item concentrations of 10 % and 25 % (w/w). The test item in the main study was assayed at 5, 10, and 25 % (w/w).

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:

- Compound solubility: The highest test item concentration which can be technically used was a 25 % suspension in propylene glycol.
In the pre-test, two mice were treated with test item concentrations of 10 or 25 %. No signs of systemic toxicity or of excessive local skin irritation were observed using these concentrations. Thus, the test item in the main study was assayed at 5, 10, and 25 %.

MAIN STUDY:

TOPICAL APPLICATION:
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear (left and right) with different test item concentrations of 5, 10, and 25 % (w/w) in propylene glycol. The application volume, 25 µL was spread over the entire dorsal surface (~ 8 mm) of each ear once daily for three consecutive days. A futher group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).

ADMINISTRATION OF 3H-METHYL THYMIDINE AND DETERMINATION OF INCORPORATED 3H-METHYL THYMIDINE

Five days after the first topical application, all mice were intraveneously injected into a tail vein with radio-labelled thymidine (3HTdR). Approximately five hours after treatment with 3HTdR all mice were sacrificed and the draining lymph nodes were excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed with phosphate buffered saline and incubated with trichloroacetíc acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a ß-scintillation counter.

INTERPRETATION OF RAW DATA

The proliferation response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/node) and as the ratio of 3HTdR incorporated into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (stimulation index S.I.). Before DPM/node values were determined, mean scintillation-background DPM was subtracted from test and control raw data.
A test item is regarded as a sensitiliser 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.

OBSERVATIONS

In addition to the sensitising reactions the following observations and data were recorded during the test and observation period:
Mortality / Viability: Once daily from experimental start to necropsy.

Body weights: In the pre-test prior to the first application and prior to sacrifice. In the main experiment: prior to the first application
and prior to treatment with 3HTdR.

Ear thickness: In the pre-test prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6).

Ear weights: In the pre-test after sacrifice; biopsy punches were taken from each ear.

Clinical signs (local / systemic): In the pre-test and in the main experiment, clinical signs were recorded at least once daily. Especially the treatment
sites were observed carefully.

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.

Results and discussion

Positive control results:

Experiment performed in September 2010 using concentrations of 5, 10, and 25 % alpha-hexyl cinnamic aldehyde in acetone:olive oil (4:1).
These concentrations yielded S.I.´s of 1.06, 2.89, and 6.45, respectively.
The EC3 value calculated was 10.5 % (w/v).
The positive control substance alpha-hexyl cinnamic aldehyde was found to be a skin sensitizer under the described conditions, demonstrating the validity of the study.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

In this study Stimulation Indices (S.I.) of 1.34, 1.11 and 1.20 were determined with the test item at concentrations of 5, 10, and 25 % in propylene glycol, respectively. The EC3 value could not be calculated, since none of the tested concentrations induced an S.I. greater than 3.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see depicted table below

Any other information on results incl. tables

Calculation and Results of Individual Data  

Vehicle: propylene glycol

Test item concentration % (w/w)	Group	Measurement DPM	Calculation			Result
			DPM-BG^a)	number of lymph nodes	DPM per lymph node^b^)	S.I.
---	BG I	16	---	---	---	---
---	BG II	17	---	---	---	---
0.0	1	1216	1200	8	149.9	1.00
5	2	1622	1606	8	200.7	1.34
10	3	1352	1336	8	166.9	1.11
25	4	1457	1441	8	180.1	1.20

BG =  Background (1 ml 5% trichloroacetic acid) in duplicate

1    =  Control Group

2-4=  Test Group

S.I. =  Stimulation Index

^a)   =  The mean value was taken from the figures BG I and BG II

^b)    =  Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

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

VIABILITY / MORTALITY

No deaths occurred during the study period.

CLINICAL SIGNS

No symtoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

BODY WEIGHTS

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.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

In the study the test item Acid Yellow RN 2903 suspended in propylene glycol was assessed for its possible contact allergenic potential.
For this purpose a local lymph node assay was performed using test item concentrations of 5, 10, and 25 % (w/w).
The animals did not show any clinical signs during the course of the study and no cases of mortality were observed.
In this study Stimulation Indices (S.I.) of 1.34, 1.11 and 1.20 were determined with the test item at concentrations of 5, 10, and 25 % in propylene glycol, respectively.
The test item Acid Yellow RN 2903 was not a skin sensitiser in this assay.