pentasodium 4-amino-3-[{4-[(2,4-disulfonatophenyl)diazenyl]-3-methylphenyl}diazenyl]-5-hydroxy-6-[(4-nitro-2-sulfonatophenyl)diazenyl]naphthalene-1,7-disulfonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 15 APRIL 2021 (study initiation date) to 24 JUNE 2021 (experimental completion date)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Justification for in vivo testing

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 22 July 2010

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

impurity 1

Reference substance name:

Unknown impurities

IUPAC Name:

Unknown impurities

impurity 2

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Dihydrogen oxide

Constituent 1

Reference substance name:

pentasodium 4-amino-3-(2-{4-[2-(2,4-disulfonatophenyl)diazen-1-yl]-3-methylphenyl}diazen-1-yl)-5-hydroxy-6-[2-(4-nitro-2-sulfonatophenyl)diazen-1-yl]naphthalene-1,7-disulfonate

Cas Number:

2259360-19-3

Molecular formula:

C29H17N8Na5O18S5

IUPAC Name:

pentasodium 4-amino-3-(2-{4-[2-(2,4-disulfonatophenyl)diazen-1-yl]-3-methylphenyl}diazen-1-yl)-5-hydroxy-6-[2-(4-nitro-2-sulfonatophenyl)diazen-1-yl]naphthalene-1,7-disulfonate

Test material form:

solid: particulate/powder

Details on test material:

Batch XMEF 1476

Specific details on test material used for the study:

solid black/brown powder

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

Animal Specifications and Acclimatisation
Nulliparous, non-pregnant female CBA/CaOlaHsd strain mice were obtained from Envigo RMS BV, Inc, AD Horst (NL).

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 fur clippings. In the pre-experiment, animals were individually marked.

Age at beginning of treatment: 8-12 weeks

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.

Animals in the main study were in a body weight range of 19.9 to 20.7 g at the start of experiment.

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 (except for deviations)
relative humidity approx. 45-65% (except for deviations)
artificial light 6.00 a.m. - 6.00 p.m.
ventilation: at least eight air changes per hour

The relative humidity in the animal room was between approximately 15 to 65% instead of 45 to 65% (due to a defective air humidifier) and the temperature was 20 to 26°C instead of 20 to 24°C (due to insufficient capacity of the air conditioning system during a summer heat wave) for several hours on several days.

These deviations to the study plan, however, did not affect the validity of the study

Study design: in vivo (LLNA)

Vehicle:

other: 1% aqueous Pluronic

Concentration:

Preliminary study: 10 and 25%

Main study: 2, 10, 25%

Dose calculation was adjusted to the purity of the test item and a correction factor of 1.24 was used

No. of animals per dose:

Five

Details on study design:

Test Article Formulation - Vehicle and Dose Selection
A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which could bewas found to be technically suitableused was a 25% suspension in 1% aqueous Pluronic®. Vortexing was used to formulate the test item. At higher concentrations, an applicable formulation of the test item was not achieved.

Pre-test and dose selection for the main study
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 (for detailed results see Appendix 1).
At the tested concentrations the animals did not show any signs of local skin irritation or systemic toxicity. Possible redness of the ear skin could not always be examined due to the colour of the test item.
Thus, the test item in the main study was assayed at 2, 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.

Test item preparation
The test item was placed into an appropriate container on a tared balance and 1% aqueous Pluronic® was added (weight per weight).
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 before each dosing occasion.

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, 10, and 25% in 1% aqueous Pluronic®. 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 H³-methyl-thymidine
Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline containing approximately 20 µCi of 3H-methyl thymidine (equivalent to approximately 80 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
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.

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. The results are described in the report.

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)

Data evaluation
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.
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.

General Calculations
The mean values and standard deviations were calculated in the body weight tables.
Where appropriate, the EC3 value were calculated according to the equation
EC3 = (a-c) [(3-d)/(b-d)] + c
where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot.
All calculations conducted on the DPM values were performed with a validated test script of “R”, a language and environment for statistical computing and graphics.
Within the program the Dean-Dixon-Test and Grubb’s Test were used for identification of possible outliers. No statistical outliers were detected.

Positive control data
The sensitivity and reliability of the experimental technique employed was assessed by use of α-hexyl cinnamaldehyde dissolved in acetone/olive oil (4+1 v/v) (compound listed in OECD 429 Guideline) which is known to have skin sensitisation properties in mice. The periodic positive control experiment was performed using CBA/CaOlaHsd mice in April 2021 as part of the current experiment.

Positive control substance(s):

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

Statistics:

All calculations conducted on the DPM values were performed with a validated test script of “R”, a language and environment for statistical computing and graphics.
Within the program the Dean-Dixon-Test and Grubb’s Test were used for identification of possible outliers. No statistical outliers were detected.

Results and discussion

Positive control results:

The positive control article (25% alpha-hexylcinnamaldehyde) produced a Stimulation Index of 7.9, demonstrating adequate performance of the assay.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1

Test group / Remarks:

vehicle control (1% aqueous Pluronic)

Remarks on result:

other: mean DPM per animal: 880.4 (SD 202.3), SI 1.0

Results 2

Key result

Parameter:

SI

Value:

1.7

Test group / Remarks:

2% w/v in applied formulation

Remarks on result:

other: mean DPM per animal: 1496.0 (SD 317.1), SI 1.7

Results 3

Key result

Parameter:

SI

Value:

1.3

Test group / Remarks:

10% w/v in applied formulation

Remarks on result:

other: mean DPM per animal: 1113.6 (SD 288.3), SI 1.3

Results 4

Key result

Parameter:

SI

Value:

2.2

Test group / Remarks:

25% w/v in applied formulation

Remarks on result:

other: mean DPM per animal: 1914.4 (SD 906.6), SI 2.2

Cellular proliferation data / Observations:

Preliminary Screening Test
Death or signs of systemic toxicity/excessive irritation were not noted.

Main Test
Mortality
All animals survived treatment with the test item

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. Possible redness of the ear skin could not always be examined due to the 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.

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

Any other information on results incl. tables

The test item is manufactured and handled as aqueous solution, therefore, stability and homogeneity can be presumed.

 

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).

 

For Calculation and Results of Individual Data (Background DPM of 11 is substracted form the DPM per animal) see Attachment 1.

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The positive control article produced a Stimulation Index of 7.9, demonstrating adequate performance of the assay.

The Local Lymph Node Assay demonstrated that Bayscript Black TP LXS 51134 was not a skin sensitizer under the test conditions of this study.

Executive summary:

In order to study a possible skin sensitising potential of Bayscript Black TP LXS 51134, three groups each of five female mice were treated once daily with the test item at concentrations of 2, 10, and 25% in 1% aqueous Pluronic® 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 (1% aqueous Pluronic®) 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 of 3H-methyl thymidine measured in a -scintillation counter.
All treated animals survived the scheduled study period and no signs of systemic toxicity or local skin irritation were observed. Possible redness of the ear skin could not always be examined due to the colour of the test item.
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 of 3HTdR 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 1.7, 1.3, and 2.2 were determined with the test item at concentrations of 2, 10, and 25% in 1% aqueous Pluronic®. The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3.

In conclusion, the test item Bayscript Black TP LXS 51134 was not a skin sensitiser under the test conditions of this study.