1-hydroxy-4-(p-toluidino)anthraquinone

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

Three groups each of five female mice were treated with different concentrations of the test item by topical application at the dorsum of each ear once daily each on three consecutive days. A control group of five mice was treated with the vehicle only. Five days after the first topical application, the mice were intravenously injected into a tail vein with radio-labelled thymidine (³H-methyl thymidine; ³HTdR). Approximately five hours after intravenous injection, the mice were sacrificed and 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 were then determined by the incorporation of ³H-methyl thymidine measured in a β-scintillation counter.

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

1-hydroxy-4-(p-toluidino)anthraquinone

EC Number:

201-353-5

EC Name:

1-hydroxy-4-(p-toluidino)anthraquinone

Cas Number:

81-48-1

Molecular formula:

C21H15NO3

IUPAC Name:

1-hydroxy-4-[(4-methylphenyl)amino]-9,10-anthraquinone

Specific details on test material used for the study:

Identification: Macrolex Violett B
CAS-No.: 81-48-1
Purity: 97.0%
Appearance: Violet powder

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

Test System
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 animalsfor the main study: 20 females
Number of animals per group: 5 females (nulliparous and non-pregnant)
Number of test groups: 3
Number of control (vehicle) groups: 1
Age (beginning of treatment): Pre-test: 10 - 11 weeks Main study: 11 - 12 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:

0 (control group), 5, 10 or 25%

No. of animals per dose:

Number of animals for the pre-test: 2 females
Number of animals for the main study: 20 females

Details on study design:

Experimental Design and Study Conduct
Topical application
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 5, 10, and 25% in DMF. The application volume, 25 μL/ear/day, was spread over the entire dorsal surface (diameter ~ 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).
Determination of Ear Thickness
Prior to the first and third application of the test item (study days 1 and 3) and prior to treatment with ³HTdR (study day 6), the ear thickness was determined using a micrometer.
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 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.
Observations
In addition to the sensitising reactions the following observations and data were recorded during the test and observation period:
Mortality / Viability: At least once daily from experimental start to necropsy.
Body weights: In the pre-test: prior to the first application and prior to sacrifice. In the main experiment: prior to the first application and prior to treatment with ³HTdR.
Ear thickness: In the pre-test prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6). In the main experiment prior to the first application (day 1), on study day 3 and prior to treatment with ³HTdR (day 6)
Ear weights: In the pre-test and main experiment after sacrifice; biopsy punches were taken from each ear.
Clinical signs (local /
systemic): Clinical signs (local irritation at the application site or systemic toxicity) were recorded at least once daily. Especially the treatment sites were observed carefully.

Positive control substance(s):

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

Statistics:

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.
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.
Within the program a statistical analysis conducted the ear weights 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 statistical outlier values were detected.

Results and discussion

Positive control results:

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 2016.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

2.6

Test group / Remarks:

Test Group 3

Remarks on result:

other: Test item concentration: 10 %

Results 2

Key result

Parameter:

SI

Value:

3.5

Test group / Remarks:

Test Group 4

Remarks on result:

other: Test item concentration: 25 %

Results 3

Key result

Parameter:

EC3

Remarks on result:

other: EC3 = 16.7% (w/w)

Cellular proliferation data / Observations:

General Calculations
The mean values and standard deviations were calculated.
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.
Within the program a statistical analysis conducted the ear weights 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 statistical outlier values were detected.

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. Redness of the ear skin could not 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.
Ear Weights
The measured ear weight of all animals treated was recorded on test day 6 (after necropsy). A relevant increase in ear weights was not observed.
Ear Thickness
The measured ear thickness of all animals treated was recorded prior to the 1st application, on study day 3 and prior to necropsy (day 6). A relevant increase in ear thickness was not observed.

Any other information on results incl. tables

In this study Stimulation Indices of 2.1, 2.6, and 3.5 were determined with the test item at concentrations of 5, 10, and 25% in DMF. A dose response was observed. The EC3 value calculated was 16.7 %.

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

In this study Stimulation Indices of 2.1, 2.6, and 3.5 were determined with the test item at concentrations of 5, 10, and 25% in DMF. A dose response was observed. The EC3 value calculated was 16.7 %. Therefore Category 1B (indication of skin sensitising potential) based on GHS criteria ist justified.

Executive summary:

Three groups each of five female mice were treated with different concentrations of the test item by topical application at the dorsum of each ear once daily each on three consecutive days. A control group of five mice was treated with the vehicle only. Five days after the first topical application, the mice were intravenously injected into a tail vein with radio-labelled thymidine (³H-methyl thymidine; ³HTdR). Approximately five hours after intravenous injection, the mice were sacrificed and 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 were then determined by the incorporation of ³H-methyl thymidine measured in a β-scintillation counter.

Stimulation Indices of 2.1, 2.6, and 3.5 were determined with the test item at concentrations of 5, 10, and 25% in DMF. A dose response was observed. The EC3 value calculated was 16.7 %. Therefore Category 1B (indication of skin sensitising potential) based on GHS criteria ist justified.