N-(4-ethoxyphenyl)-3-hydroxy-4-[(2,4,5-trichlorophenyl)azo]naphthalene-2-carboxamide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

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:

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

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Pigment Additiv FGR

IUPAC Name:

Pigment Additiv FGR

Constituent 2

Reference substance name:

N-(4-ethoxyphenyl)-3-hydroxy-4-[(2,4,5-trichlorophenyl)azo]naphthalene-2-carboxamide

EC Number:

225-691-8

EC Name:

N-(4-ethoxyphenyl)-3-hydroxy-4-[(2,4,5-trichlorophenyl)azo]naphthalene-2-carboxamide

Cas Number:

5012-29-3

Molecular formula:

C25H18Cl3N3O3

IUPAC Name:

N-(4-ethoxyphenyl)-3-hydroxy-4-[(2,4,5-trichlorophenyl)diazenyl]-2-naphthamide

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Identification: Pigment Additiv FGR
Batch: FB3212.013
CAS-No.: 5012-29-3
EINECS / EC-No.: 225-691-8
Purity: 94.51% (w/w), dose calculation was not be adjusted to purity
Appearance: Brown powder
Expiry Date: 10.05.2020 (Statement of producer)
Storage Conditions: At room temperature
Stability in Solvent: > 72 h in DMSO at room temperature
> 72 h in 1,2-propylene glycol at room temperature
Certificate of Analysis: AZ 577/Toxd2, dated 11. December 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: Envigo RMS B.V., Inc, Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: 11 - 12 weeks
- Weight at study initiation: 19 - 21.7 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:

2.5, 5, and 10%

No. of animals per dose:

4

Details on study design:

Vehicle and Dose Selection
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 10% suspension in propylene glycol. Vortexing was used to formulate the test item. 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 at the same time did not induce signs of systemic toxicity, a pre-test was performed in two animals and stated in raw data and report. Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 5 and 10% 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 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. Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch (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 (for detailed results see Appendix 1).
At the tested concentrations the animals did not show any signs of local skin irritation or systemic toxicity. Possible redness of the ear skin could not be examined, due to the colour of the test item.
Thus, the test item in the main study was assayed at 2.5, 5, and 10%. The highest concentration tested was the highest level that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed in the pre-experiment.
.

Test Item Preparation
The test item was placed into an appropriate container on a tared balance and PG was added.
The different test item concentrations were prepared individually. Homogeneity of the test item in vehicle was maintained using a magnetic stirrer.
The preparations were made freshly before each dosing occasion.


TOPICAL APPLICATION:
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 2.5, 5, and 10% in PG. The application volume, 25 µL/ear/day, was spread over the entire dorsal surface (  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 THYMIDINE AND DETERMINATION OF INCORPORATED 3H-METHYL THYMIDINE
Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline containing 20.4 µCi of 3H-methyl thymidine (equivalent to 81.5 µCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.

Determination of incorporated 3HTdR
Approximately five hours after treatment with 3HTdR all mice were euthanized by using CO2, which was, after harvesting of the lymph nodes, followed by cervical dislocation to ensure death.
The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing two times 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 scintillation vials with 10 mL of scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured in a -scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL aliquots of 5 % trichloroacetic acid. The -scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute.

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 experiment after sacrifice; biopsy punches were taken from each ear.
Clinical signs (local / systemic): Clinical signs (local irritation at the application site or systemic toxicity) 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 October 2015 (Harlan study number 1728500) using concentrations of 5, 10, and 25 % alpha-hexyl cinnamic aldehyde in acetone:olive oil (4:1). These concentrations yielded S.I.´s of 1.62, 4.23, and 17.56, respectively.
The EC3 value calculated was 7.6 % (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

Key result

Parameter:

SI

Value:

> 1.32 - < 1.82

Remarks on result:

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

Any other information on results incl. tables

Calculation and results of individual data

Vehicle: PG

Test item concentration %	Group	Measurement DPM	Calculation			Result
			DPM-BG^a)	number of lymph nodes	DPM per lymph node^b)	S.I.
---	BG I	15	---	---	---	---
---	BG II	15	---	---	---	---
0	1	5169	8	5154	644.2	1.00
2.5	2	7389	8	7374	921.8	1.43
5	3	6802	8	6787	848.4	1.32
10	4	9393	8	9378	1172.2	1.82

1    =  Control Group

2-4=  Test Group

^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 the threshold value of 3.

Viability / Mortality

No deaths occurred during the study period.

Clinical Signs

No symptoms of local skin irritation at the ears of the animals and no signs of systemic toxicity were observed during the study period. Possible redness of the ear skin could not be examined, due to the colour of the test item.

Body Weights

The body weight of the animals, recordedprior to the first application and prior to treatment with³HTdR, was within the range commonly recorded for animals of this strain and age.

Ear Weights

The measured ear weight of all animals treated was recorded on test day 6 (after necropsy), except for the ear weight of animal 5 which was accidentally not taken down. A relevant increase in ear weights was not observed.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

The test item Pigment Additiv FGR was not a skin sensitiser under the test conditions of this study and if the concentration of Naphtol AS is below 3%.

Executive summary:

In order to study a possible skin sensitising potential of Pigment Additiv FGR, three groups each of four female mice were treated once daily with the test item at concentrations of 2.5, 5, and 10% in PG by topical application to the dorsum of each ear for three consecutive days. The highest concentration tested was the highest concentration that could technically be achieved. A control group of four mice was treated with the vehicle (PG) 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 group. 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 of³H-methyl thymidine measured in ab-scintillation counter.

All treated animals survived the scheduled study period and no signs of systemic toxicity or local skin irritation were observed Possible redness of the ear skin could not be examined, due to the colour of the test item. A relevant increase in ear weights was not observed.

A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in a 3-fold or greater increase in incorporation of³HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value.

In this study Stimulation Indices of 1.43, 1.32, and 1.82 were determined with the test item at concentrations of 2.5, 5, and 10% in PG. The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3.