Reaction products of diazotised 2-amino-5-{[2-(sulfooxy)ethyl]sulfonyl}benzenesulfonic acid coupled with 4-amino-5-hydroxynaphthalene-2,7-disulfonic acid under acidic conditions, further coupled with diazotised reaction products of 2,4,6-trifluoro-1,3,5-triazine with 2-[(2-anilinoethyl)sulfonyl]ethyl hydrogen sulfate and 2,4-diaminobenzenesulfonic acid (1:1:1) under alkaline conditions, potassium sodium salts

Endpoint summary

Description of key information

The test item was found to be a weak skin sensitiser under the test conditions of the mouse local lymphnode assay.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

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:

guideline study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

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%, artificial light 6.00 a.m. - 6.00 p.m.

Vehicle:

other: ethanol/water (3+7 v/v)

Concentration:

5, 10, and 25%

No. of animals per dose:

5

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 5, 10, and 25% in ethanol/water (3+7 v/v). 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
Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline containing 20.3 µCi of 3H-methyl thymidine (equivalent to 81 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 per animal (2 nodes per animal). 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 approximately 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.

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 and main experiment, 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 was recorded on day 1 (prior to dosing) and prior to sacrifice (pre-test) or prior to treatment with 3HTdR (main experiment)

Positive control substance(s):

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

Positive control results:

October 2018 (study 1921100): S.I. of 1.41, 2.70, and 10.33 were derived at tested concentrations of 5, 10, 25%, respectively and an EC3 value of 10.6% was calculated.

Key result

Parameter:

EC3

Value:

17.5

Parameter:

SI

Value:

1.6

Test group / Remarks:

5%

Parameter:

SI

Value:

2.8

Test group / Remarks:

10%

Parameter:

SI

Value:

3.2

Test group / Remarks:

25%

Cellular proliferation data / Observations:

In this study Stimulation Indices of 1.6, 2.8, and 3.2 were determined with the test item at concentrations of 5, 10, and 25% in ethanol/water (3+7 v/v). A clear dose response was observed. The EC3 value calculated was 17.5%.

Calculation and Results of Individual Data

Vehicle: ethanol/water (3+7 v/v)

Test item concentration			DPM values measured	DPM-BG per animal (2 lymph nodes)^a)	S.I.^b)
%	Group no.	Animal no.
---	---	BG I	26	---	---
---	---	BG II	15	---	---
0	1	1	1044	1023.5	---
0	1	2	913	892.5	---
0	1	3	1192	1171.5	---
0	1	4	1142	1121.5	---
0	1	5	2037	2016.5*	---
5	2	6	1658	1637.5	1.3
5	2	7	1646	1625.5	1.3
5	2	8	2049	2028.5	1.6
5	2	9	2028	2007.5	1.6
5	2	10	2388	2367.5	1.9
10	3	11	3268	3247.5	2.6
10	3	12	2872	2851.5	2.3
10	3	13	2785	2764.5	2.2
10	3	14	2471	2450.5	2.2
10	3	15	6091	6070.5*	4.9
25	4	16	2857	2836.5	2.3
25	4	17	2963	2942.5	2.4
25	4	18	3195	3174.5	2.5
25	4	19	6198	6177.5	5.5
25	4	20	4807	4786.5	3.8

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

1      =   Control Group for the test item and for the positive control item

3-4 =   Test Groups

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)

*      =   statistical outlier value

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 (ethanol/water (3+7 v/v))	1245.1	444.2	1.0
5% Reactive Blue F07-0195	1933.3	310.4	1.6
10% Reactive Blue F07-0195	3476.9	1477.5	2.8*
25% Reactive Blue F07-0195	3983.5	1458.8	3.2*

^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)

*      statistically significant if compared to vehicle control (p<0.05).

 

Calculation of the EC3 value

	Test item concentration %	S.I.
Test Group 3	10 (a)	2.8 (b)
Test Group 4	25 (c)	3.2 (d)
EC3 = (a-c) [(3-d)/(b-d)] + c = 17.5%

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.

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.

 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.

The individual body weight values are included in Appendix 2.

Ear Weights

The measured ear weight of all animals treated was recorded on test day 6 (after necropsy). A biologically relevant or statistically significant increase in ear weights was not observed.

The individual data are included in Appendix 3.

Interpretation of results:

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

Conclusions:

The test item was found to be a weak skin sensitiser under the test conditions of this study.

Executive summary:

In order to study a possible skin sensitising potential of the test item, three groups each of five female mice were treated once daily with the test item at concentrations of 5, 10, and 25% in ethanol/water (3+7 v/v) 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 five mice was treated with the vehicle (ethanol/water (3+7 v/v)) 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 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. 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 1.6, 2.8, and 3.2 were determined with the test item at concentrations of 5, 10, and 25% in ethanol/water (3+7 v/v). A clear dose response was observed. The EC3 value calculated was 17.5%.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Due to the weak skin sensitising effects in the mouse local lymphnode assay, the substance is classified as Skin Sens. 1B (H317) in accordance with Regulation (EC) 1272/2008.