Propanenitrile, 3-[[4-[[5,6(or 6,7)-dichloro-2-benzothiazolyl]azo]phenyl]ethylamino]-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 February 2022 to 12 April 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

1

Reference substance name:

Reaction mass of 3-({4-[2-(5,6-dichloro-1,3-benzothiazol-2-yl)diazen-1-yl]phenyl}(ethyl)amino)propanenitrile and 3-({4-[2-(6,7-dichloro-1,3-benzothiazol-2-yl)diazen-1-yl]phenyl}(ethyl)amino)propanenitrile

EC Number:

616-632-0

Cas Number:

78564-87-1

Molecular formula:

C18H15Cl2N5S

IUPAC Name:

Reaction mass of 3-({4-[2-(5,6-dichloro-1,3-benzothiazol-2-yl)diazen-1-yl]phenyl}(ethyl)amino)propanenitrile and 3-({4-[2-(6,7-dichloro-1,3-benzothiazol-2-yl)diazen-1-yl]phenyl}(ethyl)amino)propanenitrile

Test material form:

solid: particulate/powder

Details on test material:

Identification: Disperse Red 153

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

Test system: Mice, CBA/CaOlaHsd
Rationale: Recognised as the recommended test system.
Source: Envigo RMS B.V., Inc
Postbus 6174
5960 AD Horst / The Netherlands
Number of animals for
the pre-test: 4 females (2 females for each pre-test)
Number of animals for
the main study: 16 females
Number of animals per group: 4 females (nulliparous and non-pregnant)
Number of test groups: 3
Number of control (vehicle) groups: 1
Age (beginning of treatment): 8 - 12 weeks
Identification: The animals were distributed into the test groups at random. All animals belonging to the same experimental group were kept in one cage. In the main experiment, the animals were individually marked. In the pre-experiment, animals were identified by cage number.
Acclimation: 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.
Housing: group
Cage Type: Makrolon Type II (pre-test) / III (main study), with wire mesh top
Bedding: granulated soft wood bedding
Feed: 2018C Teklad Global 18% protein rodent diet
(certified), ad libitum
Water: tap water, ad libitum
Environment: temperature 22 ± 2°C
relative humidity approx. 45-65% (except for deviation)
artificial light 6.00 a.m. - 6.00 p.m.
ventilation: at least eight air changes per hour

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

0.5, 1, and 2.5%

No. of animals per dose:

4

Details on study design:

Test Item Administration
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 0.5, 1, and 2.5% in DMF. 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).
3.6.2 Administration of 3H-methyl-thymidine
Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline containing 19.8 µCi of 3H-methyl thymidine (equivalent to 79.3 µCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.

Terminal Procedure
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.

Preparation of Single Cell Suspensions
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.

Determination of cellular proliferation (incorporation of 3HTdR)
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 (DPM).

Observations
Clinical Observations
All animals were observed on a daily basis, including pre- and post-dose observations on days 1, 2 and 3. Any clinical signs of systemic toxicity, local skin irritation or signs of ill health during the study were recorded.
Determination of Ear Thickness
In the pre-test, the ear thickness was determined prior to the first application of the test item (day 1), on day 3, and on day 6 prior to sacrifice using a micrometer.
Determination of Ear Weights
In the pre-test, after the lymph nodes have been excised, both ears of mice were punched at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2). For each animal both punches were immediately weighed per animal using an analytical balance. The values obtained were taken down manually. The results are described in the report.
Determination of Body Weights
The body weights were recorded on day 1 (prior to dosing) and prior to sacrifice (pre-test) or prior to treatment with 3HTdR (main experiment)

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

General Calculations
The mean values and standard deviations were calculated in the body weight tables.
Where appropriate, the EC3 value were calculated according to the equation
EC3 = (a-c) [(3-d)/(b-d)] + c
where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot.
All calculations conducted on the DPM values were performed with a validated test script of “R”, a language and environment for statistical computing and graphics.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:

Periodic Positive Control November 2021
Calculation of Stimulation Indices per Dose Group

Test item concentration Group Calculation
Mean DPM per
animal (2 lymph nodes) a) SD S.I.
Vehicle Control Group
(acetone/olive oil (4+1, v/v)) 1360.8 684.2 1.0
5% alpha-Hexylcinnamaldehyde 3249.0 392.8 2.4
10% alpha-Hexylcinnamaldehyde 4598.2 761.0 3.4
25% alpha-Hexylcinnamaldehyde 13378.2 3966.6 9.8
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 (4 animals)

Calculation of the EC3 value
Test item concentration % S.I.
Test Group 2 5 (a) 2.4 (b)
Test Group 3 10 (c) 3.4 (d)
EC3 = (a-c) [(3-d)/(b-d)] + c = 8% (w/v)
a,b,c,d = Co-ordinates of the two pairs of data lying immediately above and below the S.I. value of 3 on the LLNA dose response plot.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

2.61

Test group / Remarks:

2.5%

Results 2

Parameter:

SI

Value:

1.33

Test group / Remarks:

1%

Results 3

Parameter:

SI

Value:

1.27

Test group / Remarks:

0.5%

Any other information on results incl. tables

Calculation and Results of Individual Data

Vehicle: DMF

Test item concentration %	Group	Measurement DPM	Calculation			Result
			DPM-BG^a)	number of lymph nodes	DPM per lymph node^b)	S.I.
---	BG I	19	---	---	---	---
---	BG II	19	---	---	---	---
0	1	6892	6873	8	859.1	1.00
0.5	2	8717	8698	8	1087.2	1.27
1	3	9159	9140	8	1142.5	1.33
2.5	4	17987	17968	8	2246	2.61

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

1 = Control Group

2-4 = Test Group

1. a) = The mean value was taken from the figures BG I and BG II and subtracted form measured DPM values for the pooled lymph nodes


2. 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

Calculation of the EC3 value

The EC3 value could not be calculated, since all S.I. values are below the threshold value of 3.

Viability / Mortality

No deaths occurred during the study period.

 Clinical Signs

Possible redness of the ear skin could not be determined due to the colour of the test item and test item residuals on the ears. The animals treated with a test item concentration of 2.5% showed mild and transient, unspecific signs of discomfort on day 2 and 3 following application, namely, partially closed eyes, piloerection, and decreased activity. Animals treated with 0.5 and 1% test item concentration did not show any clinical signs.

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:

GHS criteria not met

Conclusions:

The test item Disperse Red 153 was not a skin sensitiser under the test conditions of this study.

Executive summary:

In order to study a possible skin sensitising potential of Disperse Red 153, three groups each of four female mice were treated once daily with the test item at concentrations of 0.5, 1, and 2.5% (w/w) in DMF by topical application to the dorsum of each ear for three consecutive days. The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by two
pre-experiments. A control group of four mice was treated with the vehicle (DMF) 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 of ³H-methyl thymidine measured in a b-scintillation counter.

No cases of mortality were observed. Possible redness of the ear skin could not be determined due to the colour of the test item and test item residuals on the ears. The animals treated with a test item concentration of 2.5% showed mild and unspecific signs of discomfort, such as partially closed eyes, piloerection, and decreased activity only at the post-dose observation time points on days 2 and 3. Animals treated with 0.5 and 1% test item concentration did not show any clinical signs.

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 of ³HTdR 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 1.27, 1.33, and 2.61 were determined with the test item at concentrations of 0.5, 1, and 2.5% (w/w) in DMF, respectively. The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3.