Sodium 3,3'-(9,10-dioxoanthracene-1,4-diyldiimino)bis(2,4,6-trimethylbenzenesulphonate)

Endpoint summary

Description of key information

Non skin sensitising

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Remarks:

an in vitro or in chemico skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From October 09 to 23, 2002

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source:Harlan Netherlands
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 18.5 g -22.4 g
- Housing: ln groups of four in Makrolon type-3 cages with standard softwood bedding.
- Diet: Pelleted standard Kliba 3433, batch no. 57102 mouse maintenance diet ad libitum.
- Water: Community tap water from ltingen, available ad libitum.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3'C,
- Humidity (%): 30 -70 %
- Air changes (per hr): 10 - 15 air change per hour
- Photoperiod (hrs dark / hrs light): There was a 12 hour fluorescent light / 12 hour dark cycle with at least 8 hours music during the light period.

Vehicle:

other: ethanol:water, 1:1 (v/v)

Concentration:

1 %, 2.5 %, 5 % and 10 % (w/v)

No. of animals per dose:

groups concentration %(w/v) N° per group
1 control / 4 females
2 2.5 4 females
3 5 4 females
4 10 4 females

Details on study design:

RANGE FINDING TESTS
ln a non-GLP conform pre{est in two mice, test item concentrations ol 1%, 2.5 %, 5% and 10 % (%) were tested on one ear each. No irritation effects were observed at the concentrations applied.
10 % (w/v) was the highest technically applicable concentration that could sufficiently be dosed to achieve optimal skin contact.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method:
- Criteria used to consider a positive response:

TREATMENT PREPARATION AND ADMINISTRATION:
TOPICALAPPLICATION
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 2.5 %, 5 % and 10% (wlv) in ethanol:water, 1:1 (v/v). The application volume, 25 µl, was spread over the entire dorsal surface (Ø - 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). A hair dryer was used to dry the ear's surface as quickly as possible to avoid loss of test item applied.

ADMINISTRATION OF 3H.METHYL THYMIDINE
H-methyl thymidine (HTdR) was purchased from Amersham lnternational (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 82.49 µCi/mltHTdR (equalto 20.6 µCi HTdR) by intravenous injection via a tail vein.
DETERMINATION OF INCORPORATED HTDR
Approximately five hours after treatment with HTdR all mice were euthanized by intraperitoneal injection of VETANARCOL (Veterinaria AG, Zürich).
The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group). Single cell suspensions (phosphate buffered saline) of pooled lymphnode cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 pm mesh size). After washing three 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 about 42 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 HTdR incorporation was then measured on a p-scintillation counter. Similarly, background HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid.
The p-scintillation counter expresses HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

OTHER:
ln addition to the sensitizing reactions the following observations and data were recorded during the test and observation period:
Mortality / Viability: Twice daily from acclimatization start to the termination of in-life phase.
Body weights: At acclimatization start and prior to necropsy.
Clinical signs (local/ systemic): Daily from acclimatization start to the termination of in-life phase. Especially the treatment sites were recorded carefully.

Positive control substance(s):

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

Statistics:

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

Parameter:

SI

Value:

ca. 1.8

Test group / Remarks:

Group 2

Remarks on result:

other: test item concentration: 2.5 % w/v

Parameter:

SI

Value:

ca. 1.5

Test group / Remarks:

Group 3

Remarks on result:

other: test item concentration: 5 % w/v

Parameter:

SI

Value:

ca. 1.7

Test group / Remarks:

Group 4

Remarks on result:

other: test item concentration: 10 % w/v

The proliferative capacity of pooled lymph node cells was determined by the incorporation of H-methyl thymidine measured on a p-scintillation counter. A test item is regarded as a sensitizer if the exposure to at least one concentration resulted in an incorporation of HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the STIMULATION INDEX.

An exact EC3 value could not be calculated because this calculation requires data lying immediately above and below S.l. value of 3.

VIABILITY / MORTALITY

No deaths occurred during the study period

CLINICAL SIGNS

No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

BODY WEIGHTS

The body weight of the animals, recorded at the start of acclimatization period and prior to necropsy, was within the range commonly recorded for animals of this strain and age.

Interpretation of results:

other: CLP criteria not met

Conclusions:

Non sensitising

Executive summary:

Method

ln order to study a possible allergenic potential of the test item three groups of four female mice were each treated with the test item at concentrations of 2.5 %, 5 % and 10 % (w/v) in ethanol:water, 1:1 (v/v) by topical application to the dorsum of each ear lobe (left and right) on three consecutive days. A control group of four mice was treated with the vehicle (ethanol:water, 1:1 (v/v)) 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 group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were washed subsequently 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 pscintillation counter.

Results

No test item-related clinical signs were observed. All treated animals survived the scheduled study period. A test item is regarded as a sensitizer in the LLNA if the exposure to at least one concentration of the test item resulted in an incorporation of "HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the STIMULATION INDEX (S.I.).

Test item concentration           %(w/v)         S.I

Group 2                                   2.5              1.8

Group 3                                   5                1.5

Group 4                                 10                 1.7

An exact EC3 value could not be calculated because this calculation requires data lying immediately above and below S.l. value of 3.

Conclusion

The test item was found to be a nonsensitizer when tested up to 10 % (wlv) in ethanol:water, 1:1 (v/v).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

An experiment was conducted according to the guideline OECD 406 and OECD 429 (in vitro test).

The basic principle underlying the LLNA is that sensitizers induce proliferation of lymphocytes in the lymph nodes draining the site of test substance application. This proliferation is proportional to the dose and to the potency of the applied allergen and provides a simple means of obtaining a quantitative measurement of sensitization. Proliferation is measured by comparing the mean proliferation in each test group to the mean proliferation in the vehicle treated control (VC) group. The ratio of the mean proliferation in each treated group to that in the concurrent VC group, termed the Stimulation Index (SI), is determined, and should be ≥ 3 before classification of the test substance as a potential skin sensitizer is warranted. The methods described here are based on the use of in vivo radioactive labelling to measure an increased number of proliferating cells in the draining auricular lymph nodes. However, other endpoints for assessment of the number of proliferating cells may be employed provided the PS requirements are fully met.

Under the test conditions stimulation indices of 1.8, 1.5 and 1.7 were determined with the test item at concentrations of 2.5 %, 5 % and 10 % (w/v) in ethanol:water, 1:1 (v/v). A test item is regarded as a sensitizer in the LLNA if the exposure to at least one concentration of the test item resulted in an incorporation of HTdR at least 3 -fold or greater than that recorded in control mice, as indicated by the SI. Based on these criteria, the test item was found to be a non-sensitizer when tested up to 10 % (wlv) in ethanol: water, 1:1 (v/v).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

SKIN SENSITIZATION

Category 1

Substances shall be classified as skin sensitizers in category 1 where data are not sufficient for sub-categorisation in accordance with the following criteria:

(a) if there is evidence in humans that the substance can lead to sensitisation by skin contact in a substantial number of persons; or

(b) if there are positive results from an appropriate animal test.

Specific criteria of animal test:

when an adjuvant type test method for skin sensitisation is used, a response of at least 30 % of the animals is considered as positive.

For a non-adjuvant Guinea pig test method a response of at least 15 % of the animals is considered positive.

Furthermore, stimulation index of three or more is considered a positive response in the local lymph node assay.

Sub-category 1A

Substances showing a high frequency of occurrence in humans and/or a high potency in animals can be presumed to have the potential to produce significant sensitisation in humans. Severity of reaction may also be considered.

Specific criteria:

Local lymph node assay-EC3 value ≤ 2 %

Guinea pig maximisation test-≥ 30 % responding at ≤ 0,1 % intradermal induction dose or ≥ 60 % responding at > 0,1 % to ≤ 1 % intradermal induction dose

Buehler assay - ≥ 15 % responding at ≤ 0,2 % topical induction dose or ≥ 60 % responding at > 0,2 % to ≤ 20 % topical induction dose

Sub-category 1B

Substances showing a low to moderate frequency of occurrence in humans and/or a low to moderate potency in animals can be presumed to have the potential to produce sensitisation in humans. Severity of reaction may also be considered.

Local lymph node assay - EC3 value > 2 %

Guinea pig maximisation test- ≥ 30 % to < 60 % responding at > 0,1 % to ≤ 1 % intradermal induction dose or ≥ 30 % responding at > 1 % intradermal induction dose.

Buehler assay - ≥ 15 % to < 60 % responding at > 0,2 % to ≤ 20 % topical induction dose or ≥ 15 % responding at > 20 % topical induction dose.

Based on the results obtained in the LLNA study, the test item is Not classified as the SI, calculated in all groups of animals analyzed, is always below the threshold of classification (SI < 3).