Methyl 4-[[(2,5-dichlorophenyl)amino]carbonyl]-2-[[2-hydroxy-3-[[(2-methoxyphenyl)amino]carbonyl]-1-naphthyl]azo]benzoate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 1 AUG 2012 to 4 SEP 2012

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:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Methyl 4-[[(2,5-dichlorophenyl)amino]carbonyl]-2-[[2-hydroxy-3-[[(2-methoxyphenyl)amino]carbonyl]-1-naphthyl]azo]benzoate

EC Number:

263-272-1

EC Name:

Methyl 4-[[(2,5-dichlorophenyl)amino]carbonyl]-2-[[2-hydroxy-3-[[(2-methoxyphenyl)amino]carbonyl]-1-naphthyl]azo]benzoate

Cas Number:

61847-48-1

Molecular formula:

C33H24Cl2N4O6

IUPAC Name:

methyl 4-[(2,5-dichlorophenyl)carbamoyl]-2-({2-hydroxy-3-[(2-methoxyphenyl)carbamoyl]-1-naphthyl}diazenyl)benzoate

Test material form:

not specified

Details on test material:

Name of test material (as cited in study report): Local Lymph Node Assay (LLNA) in Mice: Pigment Red 188
Analytical purity: Not specified

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: 9 - 11 weeks
- Weight at study initiation: 19.7 - 22.5 g
- Housing: group caging
- 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

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2°C
- Humidity (%): 45-65 %
- 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:

0%, 20%

No. of animals per dose:

4 (5 animals were treated, one outlier was excluded from calculation)

Details on study design:

RANGE FINDING TESTS:
A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration which could be technically used was a 20% (w/w) suspension in propylene glycol. At higher concentrations, an applicable formulation of the test item was not achieved, neither by the use of other vehicles nor by using additional methods to formulate the test item (e.g. vortexing, sonicating, warming to 37°C).
To determine the highest non-irritant test concentration that did at the same time not induce signs of systemic toxicity, a pre-test was performed in four animals and stated in raw data and report. Four mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 0, 5, 10, and 20% once daily each on three consecutive days. Prior to the first application of the test item and before sacrifice the body weight was determined. Clinical signs were recorded at least once daily. Eventual signs of local skin irritation were documented and a score was used to grade a possible erythema of the ear skin. Furthermore, prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6) the ear thickness was determined using a micrometer (S0247 Kroeplin, 36381 Schlüchtern, Germany). Additionally, for all animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch (Stiefel, Ø 8 mm corresponding to 0.5 cm2) and were immediately pooled per animal and weighed using an analytical balance. Eventual ear irritation was considered to be excessive if an erythema of the ear skin of a score value =3 was observed at any observation time and/or if an increase in ear thickness of =25% was recorded on day 3 or day 6. At the tested concentrations the animals did not show any signs of local skin irritation or systemic toxicity.
The test item in the main study was thus assayed at 20% test item concentration. Since a negative prediction is made based on test results of substances with similar chemical structure (such as C.I. Pigment Red 147, 170, or 187) the rLLNA protocol is considered to be appropriate. The highest concentration to be tested was the highest level that can be achieved whilst avoiding systemic toxicity and excessive local irritation as confirmed in the pre-experiment.


MAIN STUDY:
TOPICAL APPLICATION:
The test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear (left and right) with a test item concentration of 20 % (w/w) in propylene glycol. The application volume, 25 µL was spread over the entire dorsal surface (Ø ~ 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 beta-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). 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 At least 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 and main test after sacrifice; biopsy punches were taken from each ear.
Clinical signs (local / systemic) Clinical signs (systemic toxicity or local skin irritation) 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 April 2012 (Harlan study number 1486301) using concentrations of 5, 10, and 25 % alpha-hexyl cinnamic aldehyde in acetone:olive oil (4:1). These concentrations yielded S.I.´s of 0.88, 1.51, and 3.73, respectively.
The EC3 value calculated was 20.1 % (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

Value:

ca. 1.29

Test group / Remarks:

Group 1
Concentration 20%

Remarks on result:

other: Group Calculation S.I. Control Group: 1.00 20% Test Item: 1.29

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Group Calculation Mean DPM per animal (2 lymph nodes) Control Group: 547.9 20% Test Item: 709.0

Any other information on results incl. tables

Calculation and results of individual data; Vehicle: propylene glycol

Test item concentration % (w/w)	Group no.	Animal no.	DPM values measured	DPM-BG per animal (2 lymph nodes)^a)	S.I.^b)
---	---	BG I	21	---	---
---	---	BG II	22	---	---
0	1	1	661	639.5	---
0	1	2	447	425.5	---
0	1	3	375	353.5	---
0	1	4	526	504.5	---
0	1	5	838	816.5	---
20	2	6	775	753.5	1.4
20	2	7	1181*	1159.5*	---
20	2	8	771	749.5	1.4
20	2	9	692	670.5	1.2
20	2	10	684	662.5	1.2

* outlier animal, was therefore excluded from calculation of mean, SD and S.I.

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

1    =  Control Group

2   =  Test Group

S.I. =  Stimulation Index

^a)   =  values corrected for mean background value (BGI and BGII).

^b)   =  Stimulation Indices relative to the mean of the control group (Group 1)

 

Table 2: Calculation of Stimulation Indices per Dose Group

Test item concentration	Group Calculation
	Mean DPM per animal (2 lymph nodes)^a)	SD^b)	S.I.
Vehicle Control Group (propylene glycol)	547.9	183.8	1.00
20% Test Item	709.0	49.2	1.29

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 for vehicle group, 4 animals for treatment group)

b)      SD = Standard deviation

An EC3 value could not be calculated, because only one test item concentration was tested. Furthermore, the determined Stimulation Index was below the threshold value of 3 for a positive response.

VIABILITY / MORTALITY

No deaths occurred during the study period.

CLINICAL SIGNS

No signs of systemic toxicity or excessive irritation were observed during the study period. Erythema of the ears could not be evaluated due to the colour of the test item.

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.

 Ear Weight

The measured ear weight of all animals treated was recorded on day 6 (after necropsy). A statistically significant increase in ear weight was observed in the test group in comparison to the vehicle control group (p<0.05).

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 under the test conditions of this study.

Executive summary:

In order to study a possible skin sensitising potential of the test item, one group of five female mice was treated once daily with the test item at a concentration of 20% (w/w) suspended in propylene glycol by topical application to the dorsum of each ear for three consecutive days (according to OECD 429 reduced LLNA). This concentration tested was the highest concentration which could be technically used and did not cause systemic toxicity and local skin irritation as confirmed by a pre-experiment. A control group of five mice was treated with the vehicle (propylene glycol) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per animal. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a beta-scintillation counter.

All treated animals survived the scheduled study period and no signs of systemic toxicity or local skin irritation were observed. An outlier animal (animal 7) was detected and excluded from all calculations and statistical evaluations.

Eventual erythema of the ears could not be evaluated due to the colour of the test item. A statistically significant increase in ear weight was observed in the test group in comparison to the vehicle control group (p<0.05), indicating a possible irritation potential of the test item.

A test item is regarded as a sensitiser in the rLLNA if the exposure to the test item concentration resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index.

In this study a Stimulation Index (S.I.) of 1.29 was determined with the test item at a concentration of 20% suspended in propylene glycol. A statistically significant increase in comparison to the vehicle control group was not observed.

An EC3 value could not be calculated, because only one test item concentration was tested. Furthermore, the determined Stimulation Index was below the threshold value of 3 for a positive response.