3,3'-[ethylenebis[(3-sulpho-p-phenylene)azo]]bis[5-amino-4-hydroxynaphthalene-2,7-disulphonic acid] hexasodium salt AND 3,3'-[ethylenebis[(3-sulpho-p-phenylene)azo]]bis[5-amino-4-hydroxynaphthalene-2,7-disulphonic] acid, compound with 2,2',2''-nitrilotriethanol (1:6)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 March 2017 to 04 April 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

contract for this study was signed in August 2016

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

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)

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:

3,3'-[ethylenebis[(3-sulpho-p-phenylene)azo]]bis[5-amino-4-hydroxynaphthalene-2,7-disulphonic] acid, compound with 2,2',2''-nitrilotriethanol (1:6)

Cas Number:

75701-36-9

IUPAC Name:

3,3'-[ethylenebis[(3-sulpho-p-phenylene)azo]]bis[5-amino-4-hydroxynaphthalene-2,7-disulphonic] acid, compound with 2,2',2''-nitrilotriethanol (1:6)

Test material form:

solid: granular

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS B.V., Inc, Postbus 6174, 5960 AD Horst / The Netherlands
- Strain: CBA/CaOlaHsd
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 9-10 weeks
- Weight at study initiation: 18.5-21 g
- Housing: per dosing group in Makrolon Type III with wire mesh top
- Diet: 2018C Teklad Global 18% protein rodent diet ad libitum
- Drinkng water: tap water ad libitum
- Acclimation period: at least 5 days
- Indication of any skin lesions: not mentioned

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 45-65%
- Air changes (per hr): not indicated
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

10, 25 and 50%

No. of animals per dose:

4 females/concentration

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: max solubility 50%
- Irritation: no signs of irritation (1 female/concentration at 25 and 50%)
- Systemic toxicity: non observed
- Ear thickness measurements: no treatment related effects
- Erythema scores: max score : 1 (Draize)

MAIN STUDY: at 10, 25 and 50%

TREATMENT PREPARATION AND ADMINISTRATION:
Test substance or vehicle (25 µL/ear/day) was spread over the entire dorsal surface (ca 8 mm) of each ear once daily for three consecutive days. On day 6 250 µL of phosphate-buffered saline containing 20.1 µCi of 3H-methyl thymidine was injected via the tail vein. Five hours thereafter mice were euthanized and lymphnodes were harvested and pooled (per dose group). Single cell suspensions of pooled lymphnodes were prepared and, after incubation in 5 % trichloroacetic acid for 18 hours at 4 °C, the 3HTdR incorporation was then measured in a β-scintillation counter.

Positive control substance(s):

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

Statistics:

NA

Results and discussion

Positive control results:

--> periodically investigated SI (25% 11.8; EC3 8.2%)

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.77

Test group / Remarks:

10%

Results 2

Key result

Parameter:

SI

Value:

2.23

Test group / Remarks:

25%

Results 3

Key result

Parameter:

SI

Value:

13.78

Test group / Remarks:

50%

Remarks on result:

other: focal crust formation in 2/4 animals (due to removal of residual substance)

Remarks:

this cannot completely be ruled out as the cause for some non-specific stimulation of the lymphocytes

Results 4

Key result

Parameter:

EC3

Value:

26.7

Cellular proliferation data / Observations:

CLINICAL OBSERVATIONS: none observed (very slight erythema and slight hairloss)

BODY WEIGHTS: within normal ranges

Any other information on results incl. tables

Test item concentration %	Group	Measurement DPM	Calculation			Result
			DPM-BG^a)	number of lymph nodes	DPM per lymph node^b)	S.I.
---	BG I	10	---	---	---	---
---	BG II	13	---	---	---	---
0	1	4421	4409.5	8	551.2	1.00
10	2	7810	7798.5	8	974.8	1.77
25	3	9850	9838.5	8	1229.8	2.23
50	4	60782	60770.5	8	7596.3	13.78

1 = Control Group

2-4 = Test Group

^a)= The mean value was taken from the figures BG I and BG II (BG = back ground)

^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

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

The substance is considered to be sensitizing to the skin

Executive summary:

In an LLNA test 4 female mouse/concentration were dermally (back of the ears) exposed to the substance at 0, 10, 25 and 50% during 3 consecutive days. After injection with ³H-thymidine, 3HTdR incorporation was measured in a β-scintillation counter in cell suspensions of pooled lymphnodes (per concentration). The EC3 calculated from the stimulation indices was 26.7%. The substance is considered sensitizing to the skin.