2-[2-[4-[(2-cyanoethyl)methylamino]phenyl]vinyl]-1,3,3-trimethyl-3H-indolium hydrogen sulphate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 May 2016 to 21 June 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:

22 July 2010

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

6 July 2012

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:

2-[2-[4-[(2-cyanoethyl)methylamino]phenyl]vinyl]-1,3,3-trimethyl-3H-indolium hydrogen sulphate

EC Number:

276-586-9

EC Name:

2-[2-[4-[(2-cyanoethyl)methylamino]phenyl]vinyl]-1,3,3-trimethyl-3H-indolium hydrogen sulphate

Cas Number:

72319-18-7

Molecular formula:

C23H26N3.HO4S

IUPAC Name:

2-[2-[4-[(2-cyanoethyl)methylamino]phenyl]vinyl]-1,3,3-trimethyl-3H-indolium hydrogen sulphate

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Name of test substance as used in the report: Basic Red 14 sulfate
- Test Item No.: 16/0122-1
- Batch: Lot 4030158820
- Expiry Date: March 2018
- Content: 100 g/100 g

RADIOLABELLING INFORMATION
- Chemical: ³H-Methyl thymidine (aqueous solution)
- Source: Perkin Elmer
- Specific activity: 74 GBq/mmol (2 Ci/mmol)
- Concentration: 37 MBq/mL (1 mCi/mL)

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

OTHER SPECIFICS:
- Physical state, appearance: Violet to sparkling dark blue solid

TEST ITEM PREPARATION
- The test item was placed into a mortar on a tared balance and ethanol/water (3+7, v/v) was added whilst grinding.
- The different test item concentrations were prepared individually. Homogeneity of the test item in 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.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

OlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS B.V., Inc., Postbus 6174, 5960 AD Horst / The Netherlands
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Pre-test: 11 - 12 weeks; Main study: 10 - 11 weeks
- Weight at study initiation: Pre-test: 18.7-19.2 g; Main study: 17.4-21.2 g, mean 19.2 ± 1.1 g
- Housing: group
- Cage Type: Makrolon Type II (pre-test) / III (main study), with wire mesh top
- Bedding: granulated soft wood bedding
- 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 under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 45-65 (deviation: The relative humidity in the animal room was between approximately 41-65% instead of 45–65% for several hours.This deviation to the study plan, however, does not affect the validity of the study)
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

PRE-TEST
10 and 25%

MAIN STUDY
5, 10, and 25% (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)

No. of animals per dose:

5

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 ethanol/water (3+7, v/v). Grinding of the test item in a mortar 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).
- Irritation: At the tested concentrations the animals did not show any signs of excessive local skin irritation.
- Systemic toxicity: At the tested concentrations the animals did not show any signs of systemic toxicity.
- Ear thickness measurements: increase was not ≥25%
- Erythema scores: possible erythema of the ear skin could not be examined due to the colour of the test item.

MAIN STUDY
TREATMENT PREPARATION AND ADMINISTRATION
- Topical application: Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item. The application volume, 25 μL/ear/day, was spread over the entire dorsal surface (diameter approximately 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 19.8 μCi of 3H-methyl thymidine (equivalent to 79.1 μCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.

DETERMINATION OF INCORPORATED 3HTDR
- 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 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 at least 18 hours for precipitation of macromolecules. 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.

DETERMINATION OF LYMPH NODE WEIGHT AND CELL COUNT
- After excision, the lymph nodes were pooled per animal and weighed immediately using an analytical balance.
- The lymph node cell count was determined for each animal. For this, the volume of the cell suspensions was adjusted to an equal final volume and vortexed. Subsequently, individual cell counts were determined using a cell counter (CASY® DT, Schärfe System). The values obtained were taken down manually.

DETERMINATION OF EAR WEIGHTS
After the lymph nodes had been excised, both ears (left and right) 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 (pooled per animal) using an analytical balance

INTERPRETATION OF RAW DATA
The proliferative response of the lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph nodes of each animal (DPM/animal) 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/animal values were determined, mean scintillation-background DPM was subtracted from test and control raw data.

CRITERIA USED TO CONSIDER A POSITIVE RESPONSE
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.
Furthermore, an index was calculated for the lymph node weight and –cell count as well as for the ear weight by dividing the mean values of the test item treated groups by the mean of the vehicle treated 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 (Ehling et al., 2005) and the cut-off value for the ear weight index regarding a positive response (ear irritation) was reported to be 1.1 (Ulrich et al., 2001). However, these cut-off values mentioned in the respective papers have been determined using a different strain of mice and can thus not be implicitly adopted.

Positive control substance(s):

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

Statistics:

- The mean values and standard deviations were calculated for the body weight, 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 validated program R Script.
- Within the program a statistical analysis was 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 value was detected in both outlier tests. However, both biological and statistical significance were considered together.

Results and discussion

Positive control results:

The results of the most recent study is included in the study report

In vivo (LLNA)

Results

Key result

Parameter:

SI

Value:

1.5

Test group / Remarks:

5%: SI of 1.5; 10 and 25%: SI of 1.4

Cellular proliferation data / Observations:

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 examined, due to the color of the test item. On day 2, a red discoloration of the urine was observed in the high dose group.

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.

LYMPH NODE WEIGHTS AND CELL COUNTS
- The measured lymph node weights and –cell counts of all animals treated were recorded after sacrifice.
- A statistically significant but biologically not relevant increase in lymph node weight was observed in the low dose group in comparison to the vehicle control group.
- A statistically significant or biologically relevant increase in lymph node cell counts was not observed in any of the test item treated groups in comparison to the vehicle control group.

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 in any dose group in comparison to the vehicle control group.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met