Propanoic acid, 2-hydroxy-, compd. with 7-[[4,6-bis[[3-(diethylamino)propyl]amino]-1,3,5-triazin-2-yl]amino]-3-[2-(2,3-dihydro-2-oxo-1H-benzimidazol-6-yl)diazenyl]-4-hydroxy-2-naphthalenesulfonic acid (1:1)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline Study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

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:

registered substance

IUPAC Name:

registered substance

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands, B.V. Postbus 6174, NL - 5960 AD Horst / The Netherlands
- Age at study initiation: 8-12 weeks
- Housing:Individual in Makrolon type-2 cages with standard softwood bedding
- Diet (e.g. ad libitum): Pelleted standard Kliba 3433, batch no. 42/04 mouse maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst) available ad libitum.
- Water (e.g. ad libitum): Community tap water from Füllinsdorf ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3°C
- Humidity (%): 30-70%
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

5%, 10%, 25%

No. of animals per dose:

4 animals per dose group

Details on study design:

RANGE FINDING TESTS:
To determine the highest non-irritant and technically applicable test item concentration, a non-GLP pretest was performed in two mice with concentrations of 2.5 %, 5 %, 10 % and 25 % (w/v), on one ear each. 24 hours after a single topical application, the pre-test results determined that 25 % (w/v) was the highest technically applicable concentration in the chosen vehicle. No severe irritant effects were tolerated choosing the test
concentrations.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: A test item is regarded as a sensitizer in the LLNA if the following criteria are fulfilled:
First, 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 (S.I.).
Second, 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 APPLICATION
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 5 %, 10 % and 25% (w/v) in acetone/olive oil (4/1, v/v). The application volume, 25 µl, was spread over the entire dorsal surface (0 - 8 mm) of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals). A hair dryer was passed briefly over the ear's surface to prevent the loss of any of the test item applied.
ADMINISTRATION OF 3H-METHYL THYMIDINE
Five days after the first topical application, all mice were administered with 250 µl of 75.91 µCi/mI 3HTdR (equal to 19.0 µCi 3HTdR) by intravenous injection via a tail vein.

DETERMINATION OF INCORPORATED 3HTDR
Approximately five hours after treatment with 3HTdR all mice were euthanized by inhalation of carbon dioxide (dry ice).
The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group with the exception of Group 3, in which only 7 nodes were excised). Single cell suspensions (phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing twice with phosphate buffered saline (approx. 10 ml) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 ml) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 ml) and transferred to glass scintillation vials with 10 ml of `Irga-Safe Plus' scintillation liquid and thoroughly mixed.
The level of 3HTdR incorporation was then measured on a beta-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 ml-aliquots of 5 % trichloroacetic acid. The beta-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

INTERPRETATION OF RAW DATA
The proliferative 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 group relative to that recorded for control group (STIMULATION INDEX) (S.I.). Before DPM/node values were determined, mean scintillation-background DPM was subtracted from test and control raw data.
(STIMULATION INDEX) (S.I.) is calculated according to the following formula:

S.I = dpm/LN of treated group / dpm/LN of control group

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:

In the study STIMULATION INDICES of 2.7, 3.4 and 12.4 were determined with the test item at concentrations of 5 %, 10 % and 25 % (w/v), respectively, in acetone/olive oil (4/1, v/v). alpha-hexylcinnamaldehyde was therefore found to be a skin sensitiser.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: Group 2 (5%): 1.5 Group 3 (10%): 1.2 Group 4 (25%): 1.0

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Background 1: 0 DPM Background 2: 0 DPM Group 1 (control): 2677 DPM Group 2 (5%): 4031 DPM Group 3 (10%): 2796 DPM Group 4 (25%): 2654 DPM

Any other information on results incl. tables

Calculation and results of individual data

Test itemconcentration% (w/v)		Measurement [dpm]	Calculation			Result
			dpm -BG^a)	number of lymph nodes	dpm per lymph node^b)	S.I.
-	BG1	0	-	-	-	-
-	BG2	0	-	-	-	-
-	CG1	2677	2677	8	335	-
5	TG2	4031	4031	8	504	1.5
10	TG3	2796	2796	7*	399	1.2
25	TG4	2654	2654	8	332	1.0

* In this group only 7 nodes were excised

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

CG = Control Group

TG = 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

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

not relevant for classification and labelling

Executive summary:

In order to assess the cutaneous allergenic potential of the substance a valid LLNA performed with the test substance registered is available. In this study the SI (Stimulation Incidences) of 1.5, 1.2 and 1.0 were determined with the test substance registered at concentrations of 5%, 10% and 25% (w/v), respectively, in acetone/olive oil (4/1, v/v). The substance is considered to be a non-skin sensitizer under the conditions of this test.