Disodium 3,3'-[carbonylbis[imino(3-methoxy-4,1-phenylene)azo]]bis[benzenesulphonate]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 08 March 2017. Experimental completion date: 04 April 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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:

Disodium 3,3'-[carbonylbis[imino(3-methoxy-4,1-phenylene)azo]]bis[benzenesulphonate]

EC Number:

233-316-4

EC Name:

Disodium 3,3'-[carbonylbis[imino(3-methoxy-4,1-phenylene)azo]]bis[benzenesulphonate]

Cas Number:

10114-86-0

Molecular formula:

C27H22N6Na2O9S2

IUPAC Name:

disodium 3,3'-[carbonylbis[imino(3-methoxy-4,1-phenylene)azo]]bis[benzenesulphonate]

Test material form:

solid: particulate/powder

Details on test material:

Identification: CIM-42(Direct Yellow 132)
Appearance/physical state: dark orange colored powder
Batch: 161011K
Purity: 94.0%
Expiry / Retest date: 01 December 2017
Storage conditions: Room temperature, in the dark over silica

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

Test System and Supporting Information
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: 2 females
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): Pre-test: 12 - 13 weeks Main study: 9 - 10 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 identified by tail tags. 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.

Husbandry:
The animals were kept conventionally. The experiment was conducted under standard laboratory 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.

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

2.5, 5, and 10% in DMF

No. of animals per dose:

4 animals per dose

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 25% solution in DMF. Warming to 37°C and 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 10 and 25% 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.

At the tested concentrations the animals did not show any signs of systemic toxicity. Ears, fur and feet were coloured by the test item on several days, but a possible redness of the ear skin could have been determined. On day 6, upon preparation, the animal treated with 25% test item concentration, showed slight eschar formation on the ears.

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 DMF was added (w/w). Warming to 37°C and vortexing was used to formulate the test item.
The different test item concentrations were prepared individually.
The preparations were made freshly before each dosing occasion.

Experimental Design and Study Conduct:
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 2.5, 5, and 10% in DMF. The application volume, 25 μL/ear/day, was spread over the entire dorsal surface (~ Ø 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 20.1 μCi of 3H-methyl thymidine (equivalent to 80.4 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. The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group).

Preparation of Single Cell Suspensions:
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 β-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL-aliquots of 5 % trichloroacetic acid. The β-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.

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)

Positive control substance(s):

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

Results and discussion

Positive control results:

Positive control substance: α-Hexylcinnamaldehyde
Vehicle: acetone:olive oil (4+1, v/v)

Test item concentration %: 0
SI: 1.00

Test item concentration %: 5
SI: 1.50

Test item concentration %: 10
SI: 3.84

Test item concentration %: 25
SI: 11.76

EC3: 8.2% (w/v)

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.15

Test group / Remarks:

Test item concentration 2.5 %

Results 2

Parameter:

SI

Value:

0.99

Test group / Remarks:

Test item concentration 5 %

Results 3

Parameter:

SI

Value:

0.89

Test group / Remarks:

Test item concentration 10 %

Cellular proliferation data / Observations:

:Calculation of the EC3 value
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.

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.

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	10	-	-	-	-
-	BG II	13	-	-	-	-
0	1	8611	8599.5	8	1074.9	1
2.5	2	9886	9874.5	8	1234.3	1.15
5	3	8553	8541.5	8	1067.7	0.99
10	4	7666	7654.5	8	956.8	0.89

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.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item CIM-42 was not a skin sensitiser under the test conditions of this study.

Executive summary:

In the study the test item CIM-42 formulated in DMF (dimethylformamide) was assessed for its possible skin sensitising potential.

For this purpose a local lymph node assay was performed using test item concentrations of 2.5, 5, and 10% (w/w). The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by a pre-experiment.

The animals did not show any signs of systemic toxicity or local skin irritation during the course of the study and no cases of mortality were observed.

In this study Stimulation Indices (S.I.) of 1.15, 0.99, and 0.89 were determined with the test item at concentrations of 2.5, 5, and 10% in DMF, respectively.

The test item CIM-42 was not a skin sensitiser under the test conditions of this study.