2-Naphthalenol, heptyl-1-[2-[3-methyl-4-[2-(3-methylphenyl)diazenyl]phenyl]diazenyl]-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from October 5 to October 26, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

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:

Reaction products of 3-Methylaniline with heptyl naphthalen-2-ol

Cas Number:

1619917-05-3

Molecular formula:

Not applicable (UVCB Substance)

IUPAC Name:

Reaction products of 3-Methylaniline with heptyl naphthalen-2-ol

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- batch No.of test material: 78-231-15
- Expiration date of the batch: July 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, protected from light

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo (formed partially from Harlan in September 2015), 5800 AN Venray, The Netherlands
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 8-9 weeks

Housing and Feeding Conditions
- Full barrier in an air-conditioned room
- Temperature: 22 ± 3 °C
- Relative humidity: 55 ± 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: at least 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice
- Free access to tap water, sulphur acidified to a pH value of approx. 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- The animals were kept in groups of 5 animals in IVC cages, type II L, polysulphone cages on Altromin saw fibre bedding
- Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich
- Adequate acclimatisation period (at least five days) under laboratory conditions

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

Based on the results observed in the prescreen test the following test item concentrations were selected for the main study:
3%, 6% and 12% (w/v)
The preparations were made immediately prior to each dosing.

No. of animals per dose:

15 / main test
5 / prescreen test

Details on study design:

PRE-SCREEN TESTS:
The maximum technically applicable concentration of the test item in the vehicle was found to be 50% in AOO (Acetone, Sigma-Aldrich, lot no. SZBF3480V, expiry date: 08/2021; olive oil highly refined, Sigma-Aldrich, lot no. BCBQ4885V, expiry date: 17/10/2016).
In order to determine the highest tolerated and not excessively irritant test concentration a prescreen test was performed which was conducted under the same conditions as the main study, except there was no assessment of lymph node proliferation. The mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Body weights were recorded pre-test and prior to termination. Both ears were observed for erythema and scored.
Ear thickness measurements were performed on day 1 (pre-dose), day 3 (approximately 48 hours after the first dose) and day 6. Excessive local irritation was indicated by an erythema score ≥ 3 and/or ear swelling of ≥ 25%.
Two animals were treated by topical application with the test item on three consecutive days at a concentration of 50% (diluted with AOO) to the entire dorsal surface of each ear.
Two animals were treated by topical application with the test item on three consecutive days at a concentration of 12.5% (diluted with AOO) to the entire dorsal surface of each ear.
One further animal was treated with 100% AOO and served as negative control.
Immediately before the first application, approximately 48 hours after the first application and shortly before sacrificing the thickness of both ears of all animals was measured

During this period also all clinical signs were recorded.
Cageside observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge).
The animals treated with the test item at a concentration of 50% in AOO showed signs of systemic toxicity from day 2 to day 5 (moving the bedding) and signs of excessive irritation (increased ear thickness > 25%) at both application sites on day 3 and day 6.
The animals treated with the test item at a concentration of 12.5% in AOO showed slight signs of irritation at both application sites on day 6.
As in all animals both application sites were coloured red due to residual test item from day 2 onwards, skin irritation in terms of erythema could not be evaluated.
No signs of irritation were detected in the animal treated with the negative control.

All animals showed the expected weight development, which includes a weight loss of up to 2 g throughout the duration of the prescreen test

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
The animals were randomly selected using the validated departmental computerised system E WorkBook (version 9.4.0, ID Business Solutions Ltd.).
Identification was ensured by cage number and individual marking (tail).

- Criteria used to consider a positive response:
The proliferative response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (DPM/NODE) and as the ratio of 3H-methyl thymidine - incorporation into lymph node cells of test group animals relative to that recorded for control group animals (STIMULATION INDEX). Before DPM/NODE values were determined, background values were subtracted.
EC3 values, calculated concentrations which induce stimulation indices of three, are determined by linear interpolation, EC3=c+[(3-d)/(b-d)]x(a c), between two points of the stimulation indices axis, one above (a,b) and one below (c,d) the stimulation index of three [10], [11]. If all measured points are above or below the stimulation index of three, no EC3 value can be stated.
In certain situations where the dose response does not incorporate a data point lying below the SI value of three, provided the data are of good quality (relatively close to an SI of three and evidence of a dose response), an EC3 value may be estimated by using the two doses closest to the SI value of three. The EC3 value is estimated by log-linear interpolation between these two points on a plane where the x-axis represents the dose level and the y-axis represents the SI. The point with the higher SI is denoted (a,b) and the point with the lower SI is denoted (c,d). The formula for the EC3 estimate is as follows: EC3=2^{(log2(c)+(3-d)/(b-d)*[(log2(a)-log2(c)]}, by log-transforming the doses, EC3 estimates will never fall below zero [11], [12].
A substance is regarded as a 'sensitiser' in the LLNA if at least one concentration of the test item results in a 3-fold or greater increase in 3H-methyl thymidine - incorporation into lymph node cells of the test group animals, relative to that recorded for the lymph nodes of control group animals (Stimulation Index equal to or greater than 3.0).
On the basis of the test results, the test substance may be classified into one of the following categories in conformity with the criteria given in Commission Regulation (EU) No 286/2011 [8] as well as in GHS - Globally Harmonised System of Classification and Labelling of Chemicals, sixth revised edition, 2015 [10]:

Skin sensitiser
Category 1:
A substance is classified as a skin sensitiser
a) if there is evidence in humans that the substance can lead to sensitisation by skin contact in a substantial number of persons, or
b) if there are positive results from an appropriate animal test.
WARNING, exclamation mark. May cause an allergic skin reaction.

Sub-category 1A:
Substances showing a high frequency of occurrence in humans and/or a high potency in animals can be presumed to have the potential to produce significant sensitisation in humans. Severity of reaction may also be considered.
EC3 value ≤ 2%
WARNING, exclamation mark. May cause an allergic skin reaction.

Sub-category 1B:
Substances showing a low to moderate frequency of occurrence in humans and/or a low to moderate potency in animals can be presumed to have the potential to produce sensitisation in humans. Severity of reaction may also be considered.
EC3 value > 2%
WARNING, exclamation mark. May cause an allergic skin reaction.


TREATMENT PREPARATION AND ADMINISTRATION:
Topical Application
Each mouse was treated by topical application of 25 µL of the selected solution to the entire dorsal surface of each ear.
Topical applications were performed once daily over three consecutive days. The first treatment day is defined as study day 1.

Administration of 3H-Methyl Thymidine
Five days after the first topical application all mice were dosed with 20 µCi 3H-methyl thymidine by intravenous injection (tail vein) of 250µL of 3H-methyl thymidine, diluted with PBS to a working concentration of 80µCi/mL.

Preparation of Cell Suspension
Approximately 5 hours after the injection of 3H-methyl thymidine all mice were sacrificed by cervical dislocation. The draining auricular lymph nodes were excised, individually pooled for each animal (2 lymph nodes per animal) and collected in phosphate buffered saline (PBS). A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through polyamide gauze (200 mesh size). After washing the gauze with PBS the cell suspension was pelleted in a centrifuge. The supernatant was discarded and the pellets were resuspended with PBS. This washing procedure was repeated.
After the final wash each pellet was resuspended in approx. 1 mL 5% TCA at approx. 4° C for approximately 18 hours for precipitation of macromolecules. Each precipitate was once washed again, resuspended in 1 mL 5% TCA and 7 mL scintillation fluid was added. Then this solution was transferred into scintillation vials and stored at room temperature overnight.

Determination of Incorporated 3H -Methyl Thymidine
The 3H-methyl thymidine – incorporation was measured in a ß-counter and expressed as the number of disintegrations per minute (DPM). Similarly, background 3H-methyl thymidine levels were also measured (5% TCA). Determination of radioactivity was performed individually for each animal.

Positive control substance(s):

other: phenylene-diamine in AOO

Results and discussion

Positive control results:

The positive-control substance exceeded the stimulation index of 3 confirming the reliability of the test system .

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

5

Test group / Remarks:

at a concentration of 3%

Results 2

Parameter:

SI

Value:

10.3

Test group / Remarks:

at concentration of 6%

Results 3

Parameter:

SI

Value:

16.4

Test group / Remarks:

at concentration of 12%

Cellular proliferation data / Observations:

Each of the three tested concentrations of the test item exceeded the stimulation index of 3.


DETAILS ON STIMULATION INDEX CALCULATION
Ratio of 3H-methyl thymidine - incorporation into lymph node cells of test group animals relative to that recorded for control group animals

EC3 CALCULATION
EC3 values, calculated concentrations which induce stimulation indices of three, are determined by linear interpolation, EC3=c+[(3-d)/(b-d)]x(a c), between two points of the stimulation indices axis, one above (a,b) and one below (c,d) the stimulation index of three [10], [11]. If all measured points are above or below the stimulation index of three, no EC3 value can be stated.
In certain situations where the dose response does not incorporate a data point lying below the SI value of three, provided the data are of good quality (relatively close to an SI of three and evidence of a dose response), an EC3 value may be estimated by using the two doses closest to the SI value of three. The EC3 value is estimated by log-linear interpolation between these two points on a plane where the x-axis represents the dose level and the y-axis represents the SI. The point with the higher SI is denoted (a,b) and the point with the lower SI is denoted (c,d). The formula for the EC3 estimate is as follows: EC3=2^{(log2(c)+(3-d)/(b-d)*[(log2(a)-log2(c)]}, by log-transforming the doses, EC3 estimates will never fall below zero

CLINICAL OBSERVATIONS:
All animals survived throughout the test period without showing any clinical signs.

BODY WEIGHTS
All animals showed the expected weight development, which includes a weight loss of up to 2 g throughout the study.

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

There was no mortality and there were no significant clinical observations or effects on body weights.
Each of the three tested concentrations exceeded the stimulation index of 3.
The stimulation index at a concentration of 3% was 5.0
The stimulation index at a concentration of 6% was 10.3
The stimulation index at a concentration of 12% was 16.4

The EC3 value (estimated by linear interpolation) was calculated to be at a test item concentration of 2.3%.
Consequently, according to OECD 429 solutions or preparations containing more than 2.3% RED 2596 are expected to have a stimulation index of >3 and are therefore considered to be dermal sensitisers.
According to Commission Regulation (EU) No 286/2011 [7] as well as GHS (Globally Harmonized Classification System) [10] the test item RED 2596 has obligatory labelling requirement for skin sensitisation and is classified into Category 1B.

Executive summary:

Based on the results of the prescreen test the test item was assessed for sensitising properties at concentrations of 3%, 6% and 12% (w/v), each diluted with AOO 4:1 (v/v), 4 parts acetone and 1 part olive oil.

Species/strain:                     Mice, CBA/CaOlaHsd

Number of animals:              15 / main test; 5 / prescreen test

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

There was no mortality and there were no significant clinical observations or effects on body weights.

Each of the three tested concentrations exceeded the stimulation index of 3.

The stimulation index at a concentration       of       3%       was       5.0

The stimulation index at a concentration       of       6%       was       10.3

The stimulation index at a concentration       of       12%       was       16.4

The EC3 value (estimated by linear interpolation) was calculated to be at a test item concentration of 2.3%.

Consequently, according to OECD 429 solutions or preparations containing more than 2.3% RED 2596 are expected to have a stimulation index of >3 and are therefore considered to be dermal sensitisers.

According to Commission Regulation (EU) No 286/2011 [7] as well as GHS (Globally Harmonized Classification System) [10] the test item RED 2596 has obligatory labelling requirement for skin sensitisation and is classified into Category 1B.