Reaction mass of aluminium tris(5,7,12,14-tetrahydro-7,14-dioxoquino[2,3-b]acridine-2-sulphonate) and 5,12-dihydro-quino[2,3-b]acridine-7,14-dione

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

Remarks:

according to OECD and GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

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:

Aluminium tris(5,7,12,14-tetrahydro-7,14-dioxoquino[2,3-b]acridine-2-sulphonate)

EC Number:

278-207-2

EC Name:

Aluminium tris(5,7,12,14-tetrahydro-7,14-dioxoquino[2,3-b]acridine-2-sulphonate)

Cas Number:

75431-69-5

Molecular formula:

C20H12N2O5S.1/3Al

IUPAC Name:

aluminum tris(7,14-dioxo-5,7,12,14-tetrahydroquino[2,3-b]acridine-2-sulfonate)

Constituent 2

Reference substance name:

5,12-dihydroquino[2,3-b]acridine-7,14-dione

EC Number:

213-879-2

EC Name:

5,12-dihydroquino[2,3-b]acridine-7,14-dione

Cas Number:

1047-16-1

Molecular formula:

C20H12N2O2

IUPAC Name:

5,12-dihydroquino[2,3-b]acridine-7,14-dione

Constituent 3

Reference substance name:

Dialuminium tris(5,7,12,14-tetrahydro-7,14-dioxoquino[2,3-b]acridine-2,9-disulphonate)

EC Number:

243-319-2

EC Name:

Dialuminium tris(5,7,12,14-tetrahydro-7,14-dioxoquino[2,3-b]acridine-2,9-disulphonate)

Cas Number:

19795-24-5

Molecular formula:

C20H12N2O8S2.2/3Al

IUPAC Name:

dialuminium tris(5,7,12,14-tetrahydro-7,14-dioxoquino[2,3-b]acridine-2,9-disulphonate)

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., Postbus 6174, 5960 AD Horst, The Netherlands
Age at study initiation: 8 weeks (pre- and main test)
Weight at study initiation: 17.1 g - 20.4 g (pretest); 17.3 - 21.6 g (main test)

Housing: 1 animal per cage
Cage Type: polycarbonate cages type M II, with wire mesh tops
Enrichment: PLEXX mouse tunnel and nest building material Nestlets NES 3600
Bedding: dust free wooden bedding
Feed: Kliba mouse/rate maintenance diet "GLP", Provimi Kliba SA, Kaiseraugst, Basel, Switzerland, ad libitum
Water: tap water, ad libitum
Environment: temperature 22 ± 2°C, relative humidity 30-70%, artificial light 6.00 a.m. - 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

5, 10, and 30%

No. of animals per dose:

5

Details on study design:

Vehicle and Dose Selection

A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which can be technically used, was a 30% suspension in PG.
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 5 and 30% once daily each on three consecutive days. Prior to the first application of the test item (day 0) and before sacrifice (day 5) the ear thickness was determined by using a micrometer. Furthermore, the ears were punched after sacrifice at the apical area by using a biopsy punch (0.8 cm) and were immediately pooled per animal and weighed by using an analytical balance. Additionally, the weight of the pooled lymph nodes from both sides were determined for both animals.
No signs of systemic toxicity were observed in the pretest. At the tested concentrations, the animals did not show relevant signs of local irrations as confirmed by the determination of the ear weights and ear thickness measurements (compared to current vehicle status). During the observation period, slight swelling of the ear skin was noted at both tested concentrations. In addition, very slight erythema was observed in the animals treated with the 30% test substance concentration on study day 2.

Therefore, the following dose levels were selected for the main study: 5%, 10% and 30%.

Experimental Design and Study Conduct

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 5, 10, and 30% in PG. The application volume, 25 µL/ear/day, was spread over the entire dorsal surface 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 5) 250 µL of sterile saline containing 20 µCi of 3H-methyl thymidine were injected into each test and control mouse via the tail vein.
Terminal Procedure
Approximately five hours after treatment with 3HTdR all mice were sacrifice by cervical dislocation under Isoflurane anesthesia.
Immediately after the death of each animal, a circular piece of tissue (diameter 0.8 cm) was punched out ot the apical part of each ear of all animals. The weight of the pooled punches was determined for each animal. These measurements serve for detecting a potential inflammatory ear swelling.
Immediately after removal of the ear punches, the left and right auricular lymph nodes were dissected.The weight ofthe pooled lymph nodes from both sides was determined for each animal.

Preparation of Single Cell Suspensions
Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by carefully passing the lymph nodes through an iron mesh (200 µm mesh size) into 6 mL phosphate-buffered physiological saline. For determination of cell counts, an aliquot of each suspension was further diluted with Casy(R) ton in a ratio 1:500. The cell count was determined by using a Casi (R) counter.
The remaining cell suspensions were washed two times with phosphate buffered saline and precipitated with 5 % trichloroacetic acid. Each precipitate was transferred to scintillation fluid and incorporation of 3HTdR into the cells was measured in a ß-scintillation counter.

Observations
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.
Ear thickness: In the pre-test, the ear thickness was determined prior to the first application (day 1), on day 3, and prior to sacrifice on day 6.
Ear weights: In the pre-test and main experiment, the ear weight was determined after sacrifice (biopsy punches will be taken from each ear).
Body Weights
The body weights were recorded on day 0 (prior to dosing) and prior to sacrifice (pre-test) or prior to treatment with 3HTdR (main experiment).

Positive control substance(s):

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

Results and discussion

Positive control results:

alpha-hexyl-cinnamylaldehyde: Jan 2017 (study 58V0288/98A009): S.I. of 1.56, 3.20 and 9.16 were derived at tested concentrations of 1, 5, 15%, respectively.

In vivo (LLNA)

Results

Results 1

Parameter:

EC3

Remarks on result:

not determinable

Remarks:

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

Results 2

Key result

Parameter:

SI

Value:

1.003

Variability:

0.73 - 1.15

Test group / Remarks:

mean of all treatment groups

Results 3

Parameter:

SI

Value:

0.73

Test group / Remarks:

5 % test material in propyleneglycol

Results 4

Parameter:

SI

Value:

1.15

Test group / Remarks:

10 % test material in propyleneglycol

Results 5

Parameter:

SI

Value:

1.13

Test group / Remarks:

30 % test material in propyleneglycol

Any other information on results incl. tables

Calculation and Results of Individual Data

Vehicle: propyleneglycol (PG)

		^3H-thymidine incorporation [DPM / lymph node pair]
Test Group	Treatment	Mean	SD	Stimulation Index
1	vehicle control	324.5	122.7	1.00
2	5 % in PG	235.6	100.8	0.73
3	10 % in PG	373.0	110.0	1.15
4	30 % in PG	367.8	207.9	1.13

Calculation of the EC3 value

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

Viability / Mortality

No deaths occurred during the study period.

Clinical Signs

No signs of systemic toxicity were noticed in all animals during general observation.

Body Weights

The body weight of the animals, recorded prior to the first application and prior to treatment with³HTdR, 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 5 (after necropsy)

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Regulation (EC) no 1272/2008

Conclusions:

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

Executive summary:

In order to study a possible skin sensitising potential of the test item, three groups each of five female mice were treated once daily with the test item at concentrations of 5, 10, and 30% in Propylene Glycol (PG) 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 four mice was treated with the vehicle (PG) 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 and the auricular lymph nodes were removed. Lymph node response was evaluated by measuring ³H-thymidine incorporation. Cell counts and weights of each animal's pooled lymph nodes were also determined. In addition, a 0.8 cm diameter sample was punched out of the apical part of each ear and for each animal the weight of the pooled punches was determined to obtain an indication of possible skin irritation.

All treated animals survived the scheduled study period and no signs of systemic toxicity were observed. A statistically significant or biologically relevant increase in ear weight was not observed in any dose group in comparison to the vehicle control group.

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 of³HTdR 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 1.01, 1.00, and 1.03 were determined with the test item at concentrations of 5, 10, and 30% in PG. The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3.

Statistically significant increases in lymph node weights were noted at the 10% and 30% concentration. The observed increases were not biologically relevant as the corresponding S.I.s and cell count did not reach or exceed the cut-off values.

Thus, it is concluded that the test substance does not exhibit a skin sensitizing potential in the LLNA.