m,m'-[(9,10-dihydro-9,10-dioxo-1,4-anthrylene)diimino]bis[2,4,6-trimethylbenzenesulphonic] acid, compound with hexane-1,6-diamine (1:1)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 November 2016 - 13 December 2016

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)

Version / remarks:

2010

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2012

GLP compliance:

yes (incl. QA statement)

Remarks:

Envigo CRS GmbH

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

m,m'-[(9,10-dihydro-9,10-dioxo-1,4-anthrylene)diimino]bis[2,4,6-trimethylbenzenesulphonic] acid, compound with hexane-1,6-diamine (1:1)

EC Number:

279-213-8

EC Name:

m,m'-[(9,10-dihydro-9,10-dioxo-1,4-anthrylene)diimino]bis[2,4,6-trimethylbenzenesulphonic] acid, compound with hexane-1,6-diamine (1:1)

Cas Number:

79665-26-2

Molecular formula:

C32 H30 N2 O8 S2 . C6 H16 N2

IUPAC Name:

m,m'-[(9,10-dihydro-9,10-dioxo-1,4-anthrylene)diimino]bis[2,4,6-trimethylbenzenesulphonic] acid, compound with hexane-1,6-diamine (1:1)

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS B.V., Inc.
- Females (if applicable) nulliparous and non-pregnant: [yes]
- Age at study initiation: 8 - 9 weeks
- Weight at study initiation: 19.0 g (average)
- Housing: group
- Diet: 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
- Water: tap water, ad libitum
- Acclimation period: At least 5 days prior to the start of dosing

ENVIRONMENTAL CONDITIONS
- Temperature: 22 + 2°C
- Humidity: 45-65%:
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

2, 5, and 10% (w/w)

No. of animals per dose:

5 females

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: 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% suspension in DMSO. Vortexing was used to formulate the test item. At higher concentrations, an applicable formulation of the test item was not achieved.
- Irritation: 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. 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 cm³) 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.
- Systemic toxicity: At the tested concentrations the animals did not show any signs of systemic toxicity.
- Ear thickness measurements: On day 6, visible swelling of the ears was observed in both animals, however this ear swelling was only reflected by an excessive increase in ear thickness above the threshold (increase of 26.1% on day 3, 32.6% on day 6) in the animal treated with 25%
- Erythema scores: Redness of the ear skin could not be determined, due to the colour of the test item.

MAIN STUDY
The test item in the main study was assayed at 2, 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.

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: The animals were distributed into the test groups at random.
- Criteria used to consider a positive response: according to OECD 429

TREATMENT PREPARATION AND ADMINISTRATION:
The different test item concentrations were prepared individually. Homogeneity of the test item in the vehicle was maintained during treatment using a magnetic stirrer. The preparations were made freshly and used within two hours before each dosing occasion. Concentrations were in terms of material as supplied.
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, and 10% in DMSO. 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).

Positive control substance(s):

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

Statistics:

The mean values and standard deviations were calculated in the body weight tables, for the ear weights, the lymph node weights and lymph node cell count, and for the DPM values (group mean DPM ± standard deviation).
All calculations conducted on the DPM values, the ear weights, the lymph node weights and the lymph node cell count were performed with a validated test script of “R”, a language and environment for statistical computing and graphics.
Within the program a statistical analysis conducted on the DPM values, the ear weights, the lymph node weights and the lymph node cell count to assess whether the difference was statistically significant between the test item groups and negative control group. Statistical significance was set at the five per cent level (p < 0.05). Additionally, the Dean-Dixon-Test and Grubb’s Test were used for identification of possible outliers. No outlier was detected.
However, both biological and statistical significance were considered together.

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

0.7

Test group / Remarks:

2%

Results 2

Parameter:

SI

Value:

1.1

Test group / Remarks:

5%

Results 3

Parameter:

SI

Value:

1.2

Test group / Remarks:

10%

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
The measured lymph node weights and –cell counts of all animals treated were recorded after sacrifice. A statistically significant or biologically relevant increase in lymph node weight and -cell counts was not observed in any of the test item treated groups in comparison to the vehicle control group. For BALB/c mice, a cut-off value for the lymph node cell count index of 1.55 was reported for a positive response. The indices determined for the lymph node cell count did not exceed this threshold.

EAR WEIGHTS
The measured ear weight of all animals treated was recorded on test day 6 (after necropsy). A statistically significant increase in ear weights was observed in the high dose group in comparison to the vehicle control group (p<0.05). For BALB/c mice, a cut-off value of 1.1 for the ear weight index was reported for a positive response regarding ear skin irritation. None of the indices determined for the test item treated groups reached or exceeded this threshold.

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

CLINICAL OBSERVATIONS:
No signs of systemic toxicity were observed during the study period. Redness of the ear skin could not be determined due to the colour of the test item. On day 6, during preparation, slightly scaly ears were observed on all dose groups. However assessment of skin alterations was difficult due to the blue colour of the test item.

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 of Stimulation Indices per Dose Group

	Mean DPM per animal (2 lymph nodes)	SD	S.I.
Vehicle Control Group (DMSO)	4265.6	1499.6	1.0
2% test article	2797.8	304.4	0.7
5% test article	4849.2	1561.6	1.1
10% test article	5295.4	1769.1	1.2

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)

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

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

Executive summary:

In this study the test item was assessed for its skin sensitising potential using the Local Lymph Node Assay (LLNA) in mice. Test item suspension at different concentrations were prepared in the vehicle dimethylformamide (DMSO). The local lymph node assay was performed using test item concentrations of 2, 5, and 10% (w/w). The highest concentration tested was the highest concentration that could technically be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by a pre-experiment. The animals neither showed any signs of systemic toxicity nor mortality during the course of the study. Redness of the ear skin could not be determined due to the colour of the test item. On day 6, during preparation, slightly scaly ears were observed in all dose groups. However assessment of skin alterations was difficult due to the blue colour of the test item. A statistically significant increase in ear weights was observed in the high dose group in comparison to the vehicle control group (p<0.05). For BALB/c mice, a cut-off value of 1.1 for the ear weight index was reported for a positive response regarding ear skin irritation. None of the indices determined for the test item treated groups reached or exceeded this threshold. A test item is regarded as a sensitiser in the LLNA if exposure to one or more test item concentration results 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 test item concentration required to produce a S.I. of 3 is referred to as the EC3 value. In this study Stimulation Indices (S.I.) of 0.7, 1.1, and 1.2 were determined with the test item at concentrations of 2, 5, and 10% (w/w) in DMSO, respectively. A dose response was observed. An outlier was not identified. A statistically significant or biologically relevant increase in DPM value and also in lymph node weight and -cell count was not observed in any treated group in comparison to the vehicle control group. Furthermore, the cut-off value of 1.55 for a positive response regarding the lymph node cell count index reported for BALB/c mice was not reached or exceeded in any dose group. The test item was thus not a skin sensitiser under the test conditions of this study.