1-amino-4-hydroxy-2-(2-phenoxyethoxy)anthraquinone

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 May 2021 to 18 May 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

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

Version / remarks:

2008

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

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:

1-amino-4-hydroxy-2-(2-phenoxyethoxy)anthraquinone

EC Number:

241-443-1

EC Name:

1-amino-4-hydroxy-2-(2-phenoxyethoxy)anthraquinone

Cas Number:

17418-59-6

Molecular formula:

C22H17NO5

IUPAC Name:

1-amino-4-hydroxy-2-(2-phenoxyethoxy)-9,10-dihydroanthracene-9,10-dione

Test material form:

solid: particulate/powder

Details on test material:

Physical state: Red powder
Storage condition: at room temperature

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: checked but status not specified
- Age at study initiation: 12 weeks old (at start of the DRF); 11 weeks old (at start of main test)
- Weight at study initiation: 19.6 – 23.3 g (main test). The weight variation in animals involved in the study did not exceed ± 20 % of the mean weight.
- Housing: Grouped caging in small groups in Type II. Polypropylene / polycarbonate cages with laboratory bedding. Mice were group-housed to allow social interaction, and with deep wood sawdust bedding, to allow digging and other normal rodent activities.
- Diet: ad libitum.
- Water: Animals received tap water from watering bottles, ad libitum.
- Acclimation period: 21 days (DRF test); 7 days (main test)
- Indication of any skin lesions: Only healthy animals (and not showing any sign of skin lesion) were used.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 – 70 %
- Air changes (per hr): not specified
- Photoperiod: In light 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

25 %, 10 %, 5 % and 2.5 % (w/v) formulations of test material in vehicle (DMF)

No. of animals per dose:

4 animals per concentration

Details on study design:

RANDOMISATION
The animals were set in order of their body weight. The animals were randomly assigned to control and test groups using a randomization scheme. The randomization was checked by computer software [SPSS/PC+ (4.0.1)] according to the actual body weights verifying the homogeneity and deviations between the groups.

DOSE RANGE FINDING (DRF) TEST
The maximum dose selection was performed according to the relevant guidelines. The preliminary irritation/toxicity screen was conducted in a similar experimental manner to the exposure phase of the main test except there was no assessment of lymph node proliferation and fewer animals were used.
The test material was formulated in DMF and evaluated at concentrations of 25 %, 10 % and 5 % (w/v) in the DRF. All formulations (apparently suspension/solution) were adequately applicable on the ears of animals. Groups of 2 CBA/Ca mice were treated with the appropriate formulations once daily for 3 consecutive days. All animals were observed for any clinical signs of systemic toxicity or local irritation at the application site during the test. Body weights were recorded prior to the first treatment (on Day 1) and prior to termination (on Day 6). Both ears of each mouse were observed for erythema and scored. Measurement of ear thickness was taken using digital micrometer on Day 1 (pre-dose), Day 3 (prior to the treatment) and Day 6.
No mortality, significant, treatment related effect on body weights or any other sign of systemic toxicity were observed. No sign of significant irritation (indicated by an erythema score ≥ 3 and/or an increase of ≥ 25 % of ear thickness observed on any day of measurement). It should be noted that intensive colour of the test material formulations (red) made observation of erythema difficult in some cases (especially on the treatment days), but it was considered that a severe effect (an erythema scored as ≥ 3) would have been visible. No other local effect was observed in any dose group during the test.
Based on the DRF data, the 25 % (w/v) concentration was used as the maximum in the main test with the aim of testing the highest possible concentration. The test material was tested also at three additional, lower concentrations (10 %, 5 % and 2.5 %, w/v) to evaluate dose-response relationship and ensure validity of the test in accordance with the relevant guidelines.

MAIN TEST DESIGN
The test material was administered at four different concentrations.

IN VIVO TREATMENT
Each mouse was topically treated with 25 μL of the appropriate formulations of the test material, the positive control substance or the vehicles using a pipette, on the dorsal surface of each ear. After the treatment animals were returned to their cages. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.

PROLIFERATION ASSAY
No animals showed symptoms of systemic toxicity or excessive skin irritation toxicity (significant loss of body weights or other significant clinical symptoms) or excessive skin irritation (erythema scored as ≥ 3) and no technical treatment failures were observed during the test. All animals treated were processed and therefore no treatment group was excluded from the evaluation.
- Injection of 3HTdR - On Day 6 each mouse was intravenously injected via the tail vein with 250 μL of sterile PBS (1 x PBS, diluted from 10x concentrate) containing approximately 20 μCi# of 3H-methyl-thymidine using a hypodermic needle with 1 mL sterile syringe. Once injected, mice were left for 5 hours (± 30 minutes).
- Removal and Preparation of Draining Auricular Lymph Nodes - Five hours (± 30 minutes) after intravenous injection the mice were sacrificed by cervical dislocation. The draining auricular lymph nodes were excised by making a small incision in the skin between the jaw and the sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. Then the nodes were removed using forceps. Once removed, the nodes of the mice from each test group were pooled and collected separately in a Petri dish containing a small amount (1-2 mL) of PBS to keep the nodes wet before processing.
- Preparation of Single Cell Suspension of Lymph Node Cells - A single cell suspension (SCS) of lymph node cells (LNCs), pooled according to groups, was prepared and collected in disposable tubes by gentle mechanical disaggregating of the lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was washed with PBS (up to 10 mL). LNCs were pelleted with a relative centrifugal force (RCF) of approximately 190 x g for 10 minutes at 4 °C. After centrifugation, the supernatant was removed, leaving 1-2 mL supernatant above each pellet. The pellets were gently agitated before making up to 10 mL with PBS and re-suspending the LNCs. The washing procedure was repeated twice. This procedure was repeated for each group of pooled lymph nodes.
- Determination of Incorporated 3HTdR - After the final wash, each supernatant was removed leaving a small volume (< 0.5 mL) of supernatant above each pellet. The pellets were gently agitated before suspending the LNCs in 3 mL of 5 % (w/v) trichloroacetic acid (TCA, dissolved in purified water) for precipitation of the macromolecules. After incubation with 5 % TCA at 2-8°C overnight (approx. 18 hrs), each precipitate was removed by centrifugation of the samples at approximately 190 x g for 10 minutes at 4°C and decanting the supernatants. Then the pellets were re-suspended in 1 mL of 5 % TCA, dispersed using an ultrasonic water bath and stored at 2-8°C until measurement. On the day of the measurement sample were dispersed using an ultrasonic water bath, transferred to suitable sized scintillation vials containing 10 mL of scintillation liquid, gently mixed and loaded into the β-scintillation counter. 3HTdR incorporation was measured for up to 10 minutes per sample.
The β-counter expressed the 3HTdR incorporation as the amount of radioactive disintegration per minute (DPM). Similarly, background 3HTdR levels were measured in two 1 mL aliquots of 5 % TCA. Instrument used for the measurement:
Name: Tri-Carb®4910TR Liquid Scintillation Analyzer

OBSERVATIONS IN THE MAIN TEST
- Clinical Observations - During the main test (from Day 1 to Day 6) all animals were observed at least once a day for any clinical signs, including systemic toxicity and local irritation. Irritation was monitored by erythema scoring and by measurement of ear thickness (in the test material treated groups). Measurement of ear thickness was taken using digital micrometer on Day 1 (pre-dose), Day 3 (prior to the treatment) and Day 6. An increase of ≥ 25 % (compared to the initial value) is an indication of a significant adverse effect. Individual records were maintained for the observations.
- Measurement of Body Weight - Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to 3HTdR injection) with a precision of ± 0.1 g.
- Evaluation of the Results - DPM (disintegration per minute) was measured for each treatment group. The measured DPM values were corrected with the background DPM value: the average of the two measured DPM values of 5 % (w/v) TCA solutions was used as the background DPM value. The results were expressed as DPM/group and as DPM/mouse (mean DPM). The stimulation index (SI = the DPM/mouse of a treated (positive control or test material) group divided by the DPM/mouse of the respective negative control group) for each treatment group was also calculated. A SI value of 3 or greater is an indication of a positive result. Dose-response relationship was evaluated by linear regression using SI values. Calculations were made by Microsoft Excel Software. Based on the results EC3 value (dose calculated to induce a stimulation index of 3) of the test material was not calculated.
- Interpretation of the Results - The test material is considered as a skin sensitizer, if: Exposure to at least one concentration of the test material resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index (SI ≥ 3). However, the strength of the dose-response, the statistical significance and the consistency of the solvent/vehicle and positive control responses may also be used when determining whether a borderline result is declared positive.

Positive control substance(s):

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

Results and discussion

Positive control results:

A significant lymphoproliferative response (SI ≥ 3) was noted for HCA (SI = 13.4). The results of the positive control item demonstrated an appropriate performance of the test in accordance with the relevant guidelines and confirmed the validity of the assay.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1

Test group / Remarks:

Vehicle control for the positive control: AOO

Results 2

Parameter:

SI

Value:

13.4

Test group / Remarks:

Positive control: 25 % HCA in AOO

Results 3

Parameter:

SI

Value:

1

Test group / Remarks:

Vehicle control for the test material: DMF

Results 4

Key result

Parameter:

SI

Value:

3.5

Test group / Remarks:

Test item 2.5 % (w/v) in DMF

Results 5

Key result

Parameter:

SI

Value:

4.7

Test group / Remarks:

Test item 5 % (w/v) in DMF

Results 6

Key result

Parameter:

SI

Value:

4

Test group / Remarks:

Test item 10 % (w/v) in DMF

Results 7

Key result

Parameter:

SI

Value:

2.2

Test group / Remarks:

Test item 25 % (w/v) in DMF

Cellular proliferation data / Observations:

BODY WEIGHT MEASUREMENT
Body weight decrease by > 5 % was observed in the following groups: AOO vehicle control group (1 of the 4 animals, 9 % decrease); 5 % (w/v) dose group (1 of the 4 animals, 7 % decrease); 2.5 % (w/v) dose group (1 of the 4 animals, 10 % decrease). Group mean body weights did not change significantly in any test group. Based on this no significant treatment related effect on the body weights was considered during the test.

CLINICAL OBSERVATIONS, SIGNS OF IRRITATION
No mortality or symptoms of systemic toxicity were observed in any treatment group. No sign of irritation (indicated by an erythema score ≥ 3) or any other local effect were observed in any treatment group. It should be noted that intensive colour of the test material formulations (red) made observation of erythema difficult, but it is considered that a significant effect (an erythema scored as ≥ 3) would have been visible. Ear thicknesses were measured in the test material treated groups. No significant effect (i. e. ≥ 25 % increase compared to the initial values) was observed in any of the dose groups.

PROLIFERATION ASSAY
Visually larger lymph nodes compared to the relevant vehicle control (AOO) were observed in the positive control group only. The appearance of the lymph nodes was normal in both vehicle control groups (AOO or DMF). No visually larger lymph nodes compared to the relevant control (DMF) were observed in the test material treated groups. Significantly increased lymphoproliferation (indicated by a stimulation index (SI) ≥ 3) compared to the relevant control (DMF) was noted for the test material at test concentrations of 10 %, 5 % and 2.5 % (w/v). No significant effect on the lymphoproliferation was observed in the 25 % (w/v) dose group. The observed SI values were 2.2, 4.0, 4.7 and 3.5 at test material concentrations of 25 %, 10 %, 5 % and 2.5 % (w/v), respectively. Significance of the dose-response was evaluated by linear regression using the SI values obtained. No statistical significance for monotonic dose-response correlation was observed (p = 0.19, r² = 0.65).

RELIABILITY OF THE TEST
The positive control group animals were treated with 25 % (w/v) HCA solution (formulated in AOO) concurrent to the test material groups. No mortality, cutaneous reactions or signs of toxicity were observed in the positive control group. A significant lymphoproliferative response (SI ≥ 3) was noted for HCA (SI = 13.4). The results of the positive control item demonstrated an appropriate performance of the test in accordance with the relevant guidelines and confirmed the validity of the assay.

INTERPRETATION OF OBSERVATIONS
The maximum dose selection was based on results of the formulation evaluation and the dose range finding test (DRF). Poor solubility of the test material was observed in standard LLNA vehicles. The maximum concentration producing adequate homogeneous formulation suitable for application on the dorsum of ears of animals was 25 % (w/v) in Dimethylformamide (DMF). The physical appearance of the formulation was suspension although partial dissolution of the test material was also visible. With the aim of testing the highest possible concentration the test material was examined in the LLNA as suspension formulation (in accordance with the guideline).
As no significant adverse effects (systemic toxicity or irritation) were observed in the DRF up to this maximum achievable concentration the test material was examined in the main test as 25 %, 10 %, 5 % and 2.5 % (w/v) formulations in DMF.
Based on the positive control result the test was valid. No confounding effects of irritation or systemic toxicity were observed during the main test. According to this the proliferation values obtained are considered to reflect the real potential of the test material to cause/not cause lymphoproliferation in the Local Lymph Node Assay.
Significantly increased lymphoproliferation (indicated by an SI ≥ 3) compared to the relevant control (DMF) was noted for the test material at test concentrations of 10 %, 5 % and 2.5 % (w/v). No significant effect on the lymphoproliferation was observed in the 25 % (w/v) dose group, however increased value (compared to the relevant control) was observed. The observed SI values were 2.2, 4.0, 4.7 and 3.5 at test material concentrations of 25 %, 10 %, 5 % and 2.5 % (w/v), respectively. No monotonic linear dose-response curve was observed.
It is believed that the nature of the formulations (suspension/solution) may have contributed to the lack of a clear dose dependence (although it is known that real sensitisation has strong linear dose-response correlation). On the other hand, since no systemic toxicity or irritation interfered with the test result the proliferation values obtained are considered reliable.
Based on this and in accordance with criteria of the relevant guideline regarding the evaluation of the test material as a sensitizer, the significantly increased lymphoproliferations (indicated by an SI ≥ 3) observed at three of the four tested concentrations are considered evidence that the test material is a potential skin sensitizer.
Chemicals can be classified according to their relative skin-sensitization potency using an EC3 value (dose calculated to induce a stimulation index of 3) calculated by linear interpolation using data points lying immediately above and below the SI value of 3 on the LLNA dose-response curve according to published method. In this LLNA no EC3 could be calculated hence the test material cannot be classified according to the published data for classification of contact allergens.
Based on the positive LLNA result, the test material is in GHS Category 1. According to the current regulation, the sub-category cannot be given because EC3 could not be calculated in this LLNA.

Any other information on results incl. tables

DPM, DPN and Stimulation Index Values for all Groups

Dose Group	Measured DPM/ Group	Group DPM	DPN/Mouse	Stimulation Index
Vehicle control for the positive control: AOO	4719	4707	1176.8	1.0
Positive control: 25 % HCA in AOO	62857	62845	15711.3	13.4
Vehicle control for the test item: DMF	2232	2220	555.0	1.0
Test material 25 % w/v in DMF	4875	4863	1215.8	2.2
Test material 10 % w/v in DMF	8818	8806	2201.5	4.0
Test material 5 % w/v in DMF	10503	10491	2622.8	4.7
Test material 2.5 % w/v in DMF	7884	7872	1968.0	3.5

Applicant's summary and conclusion

Interpretation of results:

other: Classified (Category 1) according to EU criteria. The OECD TG 442 (DPRA) study could not conducted due to the poor solubility, and it would not be possible to confirm without LLNA that the substance is not skin sensitiser from the "Bottom up 3 out of 3".

Conclusions:

Under the conditions of the present assay, the test material was tested as 25 %, 10 %, 5 % or 2.5 % (w/v) formulations [apparently suspension/solution, prepared with Dimethylformamide (DMF) as vehicle] was shown to have skin sensitization potential in the Local Lymph Node Assay. Based on the results the test item is in GHS Category 1 according to the effective regulation.

Executive summary:

The skin sensitisation potential of the test material was investigated in accordance with the standardised guidelines OECD 429 and EU Method B.42 in accordance with GLP.

The study utilised the minimum number of animals (mice), corresponding to the regulatory guidelines being followed. The best vehicle taking into account the test material characteristics, and the requirements of the relevant OECD guideline was considered to be DMF. 25 % (w/v) was the highest concentration which was suitable for the test. The formulations appeared to be a solution/suspensions by visual examination. A preliminary dose range finding test was performed at concentrations of 25 %, 10 % and 5 % (w/v) in DMF and based on the results, 25 % (w/v) was selected as the top dose for the main test.

In the main assay, 28 female CBA/CaOlaHsd mice were allocated to 7 groups, each group comprised four animals: groups (four) of animals received the test material (formulated in DMF) at either 25, 10, 5 or 2.5 % (w/v), a vehicle control group received the vehicle (DMF) only, a positive control group received 25 % (w/v) HCA (dissolved in AOO) and a positive control vehicle group received AOO. The test material suspensions were applied to the dorsal surface of the ears of the experimental animals (25 μL/ear) for three consecutive days (Days 1, 2 and 3) and then maintained on study for an additional 3 days. Cell proliferation in the (local) lymph nodes was assessed by measuring disintegrations per minute after the incorporation of tritiated methyl thymidine (3HTdR) into the lymph nodes and the values obtained were used to calculate stimulation indices (SI) in comparison with the control group.

There was no mortality or signs of systemic toxicity observed during the study. No test material related marked body weight losses (≥ 5 %) was observed on the mean body weight changes. The SI values were 2.2, 4.0, 4.7 and 3.5 at test material concentrations of 25 %, 10 %, 5 % and 2.5 % (w/v), respectively. The SI value for the positive control substance α-Hexylcinnamaldehyde (HCA) dissolved in DMF was 13.4 therefore demonstrating the appropriate performance of the assay.

Under the conditions of this study, the test material did show skin sensitisation potential. Therefore, based on the results the test item is in Category 1 according to Regulation (EC) No 1272/2008 (CLP).