2,9-dimethylanthra[2,1,9-def:6,5,10-d'e'f']diisoquinoline-1,3,8,10(2H,9H)-tetrone

Endpoint summary

Description of key information

Skin sensitization: not sensitizing in LLNA, according OECD TG 429, GLP-compliant, 3.5 %, 7 %, 14 % test substance in DMSO, mouse, 2005, K1

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Remarks:

RCC Cytotest Cell Research GmbH

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

- Physical state: solid
- Storage condition of test material: at room temperature protected from moisture

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands
- Age at study initiation: 7 weeks (beginning of acclimatization)
- Housing: Makrolon Type I, with wire mesh top (EHRET GmbH, D-79302 Emmendingen) with granulated soft wood bedding (Harlan Winkelmann GmbH, D-33178 Borchen), housed individually
- Diet (e.g. ad libitum): pelleted standard diet (Harlan Winkelmann GmbH, D-33178 Borchen), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: Under test conditions after health examination. Only animals without any visible signs of illness will be used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

dimethyl sulphoxide

Concentration:

0% (vehicle control), 3.5%, 7% and 14% (based a non-GLP local toxicity pre-test, 14% was the highest technically applicable concentration in the chosen vehicle)

No. of animals per dose:

4 females

Details on study design:

RANGE FINDING TESTS:
- Irritation: In a non-GLP conform pre-test in two mice, test item concentrations of 1.75, 3.5, 7 and 14 % (w/v) were tested on one ear each. No irritation effects were observed at these concentrations after a single application.

MAIN STUDY
TREATMENT PREPARATION AND ADMINISTRATION:
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 3.5, 7 and 14% (w/v) in DMSO. The application volume, 25 μl, was spread over the entire dorsal surface (∅ ∼ 8 mm) of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).
3H-methyl thymidine (3HTdR) was purchased from Amersham International (Amersham product code no. TRA 310; specific activity, 2 Ci/mmol; concentration, 1 mCi/ml). Five days after the first topical application, all mice were administered with 250 μl of 82 μCi/ml 3HTdR (corresponds to 20.5 μCi 3HTdR per mouse) by intravenous injection via a tail vein.
Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Na-thiopental (Trapanal, Altana, D-78467 Konstanz).
The draining lymph nodes were rapidly excised and pooled per group (8 nodes per group). 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 glass scintillation vials with 10 ml of ‘Ultima Gold’ scintillation liquid and thoroughly mixed.
The level of 3HTdR incorporation was then measured on a β-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid. The β-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

- 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.

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was placed into a volumetric flask glass beaker on a tared balance and the vehicle (DMSO) was quantitatively added. The test item concentrations were prepared individually. Homogeneity of the test item in the vehicle was maintained during treatment with the magnetic stirrer. The preparations were made freshly before each dosing occasion.

Positive control substance(s):

other: α-Hexylcinnamaldehyde in acetone:olive oil, 4:1 (v/v)

Statistics:

The mean values and standard deviations were calculated in the body weight tables.

Parameter:

SI

Value:

1.4

Test group / Remarks:

3.5%

Parameter:

SI

Value:

1.3

Test group / Remarks:

7%

Parameter:

SI

Value:

2

Test group / Remarks:

14%

No deaths occurred during the study period. No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period. Due to the intense red colour of the test item local irritation reactions such as ear redness could not be detected. 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			Result
Test item concentration	Group	Measurement DPM	DPM-BG a)	number of lymph nodes	DPM/lymph node b)	S.I.
--	BG I	23.6	--	--	--	--
--	BG II	34.2	--	--	--	--
--	CG 1	3467.18	3438.3	8	429.8
3.5 % (w/v)	TG 2	4733.81	4704.9	8	588.1	1.4
7 % (w/v)	TG 3	4615.95	4587.1	8	573.4	1.3
14 % (w/v)	TG 4	6932.97	6904.1	8	863	2

 

BG = Background (1 ml 5% trichloroacetic acid) in duplicate

CG = Control Group

TG = Test Group

S.I. = Stimulation Index

a) = The mean value was taken from the figures BG I and BG II

b) = Since the lymph nodes of the animals of a dose group were pooled,

DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

 

A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentrations resulted in 3-fold or greater increase in incorporation of 3HTdR compared to the concurrent control, as indicated by the stimulation index. The estimated concentration of test item required to produce an S.I. of 3 is referred to as the EC3 value. In this study the EC3 value could not be calculated, since none of the tested concentrations induced an S.I. greater than 3. The test item was found to be not a skin sensitiser.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was found not to be a skin sensitiser.

Executive summary:

In GLP compliant study following OECD guideline 429 the test item dissolved in DMSO was assessed for its potential to induce a contact allergy. For this purpose a local lymph node assay was performed using test item concentrations of 3.5, 7 and 14%. No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period. Due to the intense red colour of the test item local irritation reactions such as ear redness could not be detected. In this study Stimulation Indices (S.I.) of 1.4, 1.3 and 2.0 were determined with the test item at concentrations of 3.5, 7 and 14% (w/v) in DMSO, respectively. The test item was not a skin sensitiser in this assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

In a GLP-compliant LLNA study conducted according to OECD guideline 429, groups of female CBA/CaOlaHsd mice (4/dose) were treated with a 3.5%, 7% and 14% (w/v) preparation of the test substance in DMSO (CCR, 2005). The dorsal surface of both ear lobes in each test animal was treated with 25 µL of the test substance preparation for three consecutive days. A control group was treated with the vehicle alone. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labeled thymidine (³H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed and the draining lymph nodes were rapidly excised and pooled per group. The level of ³H-methyl thymidine incorporation was then measured on a β-scintillation counter. No deaths occurred during the study period. No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period. Due to the intense red colour of the test item local irritation reactions such as ear redness could not be detected. The body weight of the animals, recorded prior to the first application and prior to treatment with ³H-methyl thymidine, was within the range commonly recorded for animals of this strain and age. Stimulation indices of 1.4, 1.3 and 2.0 were determined with the test item at concentrations of 3.5%, 7% and 14% in DMSO (w/v), respectively. The test article does not have a skin sensitizing effect in the Local Lymph Node Assay under the test conditions chosen.

 

Further toxicological data of category members:

This finding was supported by the data obtained for additional members of the same substance category. In three LLNA assays conducted according to OECD guideline 429 and in compliance with GLP, three additional members of the same category were analyzed for their sensitization potential. None of the tests gave a positive response, therefore all tested substances were considered as not sensitizing.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. No increase in the stimulation index was observed in the LLNA (OECD 429). Therefore, the substance does not require classification as a skin sensitizer under Regulation (EC) No. 1272/2008, as amended for the fourteenth time in Regulation (EC) No. 2020/217.