Sodium 4-amino-5-hydroxy-3-(4-nitrophenylazo)-6-(phenylazo)naphthalene-2,7-disulphonate

Endpoint summary

Description of key information

Sensitizer

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

Study period:

From January 19th to February 21st, 2005

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

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 (beginning of acclimatization)
- Weight at study initiation: at the start of the experiment the mean initial weight of the animals was 18.9 g (17 to 20.3 g).
- Identification: single caging. The animals will be distributed into the test groups at random and identified by cage number.
-Acclimtisation: under test conditions after health examination.
- Housing: single caging. Cage type Makrolon Type I, with wire mesh top.
- Bedding: the bedding consisted of bedding of granulated soft wood (Harlan Winkelmann GmbH).
- Diet: the diet consisted of pelletted standard diet, ad libitum (Harlan Winkelmann)
- Water: tap water, ad libitum.
- Health status: Only animals without any visible signs of illness will be used for the study.

ENVIRONMENTAL CONDITIONS
- Temperatur: 22 ± 3 °C
- Humidity: 30 - 75 %
- Photoperiod: 12 hours light/dark cycle, artificial light from 6 a.m. to 6 p.m.

Vehicle:

other: DMSO test groups; Acetone: olive oil , 4:1 (v/v)

Concentration:

To determine the highest non-irritant test concentration or the highest technically applicable concentration, a non-GLP pretest was performed in two mice. The data showed that the highest test item concentration, which could be technically used was a 20 % suspension. The treatment of mice with 2.5, 5, 10 and 20 % test item in DMSO did not show any signs of systemic toxicitye. The data showed that the highest test item concentration, which could be technically used was a 20 % suspension. The treatment of mice with 2.5, 5, 10 and 20 % test item in DMSO did not show any signs of systemic toxicity.

No. of animals per dose:

Four groups of experimental animals were randomly established for the main study: one test substance group consisting of 4 experimental animals and one control groups consisting of 4 animals each.

Details on study design:

TEST ITEM FORMULATION
The test item was placed into a volumetric flask on a tared balance and vehicle (DMSO) was quantitatively added. The dilutions were prepared individually. Homogeneity of the test item in vehicle was maintained during treatment using a magnetic stirrer.
The preparations were made freshly before each dosing occasion.

DOSE SELECTION
To determine the highest non-irritant test concentration or the highest technically applicable concentration, a non-GLP pretest was performed in two mice. The data showed that the highest test item concentration, which could be technically used was a 20 % suspension. The treatment of mice with 2.5, 5, 10 and 20 % test item in DMSO did not show any signs of systemic toxicity. Due to the intense black colour of the test item local irritation reactions such as ear redness could not be detected . No swelling of the ears was observed.

RANGE FINDING TESTS
On request of the sponsor the test item in the main study was assayed at four consecutive concentrations (1, 5,10, and 20 %).

OUTLINE OF THE STUDY
In order to study a possible allergenic potential of Acid Black 1 (C.I.20470), four groups each of four female mice were treated with different concentrations of the test item by topical application at the dorsum of each ear lobe (Ieft and right) on three consecutive days. A control group of four 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-Iabelled thymidine (H-methyl thymidine) Approximately five hours after intravenous injection, the mice were sacrificed and 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 subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of H-methyl thymidine measured in a ß-scintillation counter.

TOPICAL APPLICATION
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (Ieft and right) with different test item concentrations of 1, 5, 10, and 20 % (w/v) in DMSO. 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
3H-methyl thymidine (3HTdR) was purchased from Amersham International (Amersham product code no.TRA 310; specific activity, 2 Cilmmol; concentration, 1 mCi/ml).
Five days after the first topical application, all mice were administered with 250 µl of 78 µCi/ml 3HTdR (corresponds to 19.5 µCi 3HTdR per mouse) by intravenous injection via a tail vein.

DETERMINATION OF INCORPORATED 3HTdR
Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Na-thiopental (Trapanal). 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 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 % tichloroacetic 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 ß-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 ml-aliquots of 5 % trichloroacetic acid. The ß-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

Positive control substance(s):

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

Statistics:

A statistical analysis was conducted for assessment of the dose-response relationship, and the EC3 value was 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.

Parameter:

SI

Value:

2.4

Test group / Remarks:

1%

Parameter:

SI

Value:

4.6

Test group / Remarks:

5%

Parameter:

SI

Value:

5.9

Test group / Remarks:

10%

Parameter:

SI

Value:

7.4

Test group / Remarks:

20%

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Test item concentration and Measured DPM 1.0 % (w/v) = 8742.1 5.0 % (w/v) = 16837.7 10.0 % (w/v) = 21565.5 20.0 % (w/v) = 27133.6

Cellular proliferation data / Observations:

Test item concentration and S.I. 1.0 % (w/v) = 2.4 5.0 % (w/v) = 4.6 10.0 % (w/v) = 5.9 20.0 % (w/v) = 7.4

Results of individual data

Test item concentraion % (w/v)	Group	Measurement DPM	Calculation			RESULT
			DPM-BGa	number of lymph nodes	DPM per lymph node b)	S.I.
--	BG I	2.9
--	BG II	3.7	--	--	--	--
--	CG 1	3657.4	3654.1	8	456.8
1.0	TG 2	8749.1	8745.8	8	1093.2	2.4
5.0	TG 3	16837.7	16834.4	8	2104.3	4.6
10.0	TG 4	21565.5	21562.2	8	2695.3	5.9
20.0	TG 5	27133.6	27130.3	8	3391.3	7.4

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 figuresBGl andBGII

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 nodespooled

	Test itemconcentration% (w/v)	S.I.
Group 2	1.0(а)	2.4 (b)
Group 3	5.0(c)	4.6 (d)
EC3  = (a-c)[(3-d)/(b-d)]+c=2.1% (w/v)

 

EC3 = Estimated concentration for aS.I.of 3.

a,b,c,d = Co-ordinates of the two pair of data lying immediately above and below theSI..value of 3 on the LLNA dose response plot.

Interpretation of results:

other: Category Skin Sens 1.B according to CLP Regulation (EC 1272/2008)

Conclusions:

Under the test conditions the test item was found to be skin sensitiser and EC3 value of 2.1 % (w/v) was derived.

Executive summary:

In order to study a possible contact allergenic potential of Acid Black 1 (C.1.,20470), four groups each of four female mice were treated daily with the test item at concentrations of 1, 5, 10, and 20 % (w/v) in DMSO by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. A control group of four mice was treated with the vehicle (DMSO) only. Five days after the first topical application the mice were injected intravenouslyinto atailvein withradio-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 subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph nodecells wasdeterminedby theincorporationof³H-methyl thymidinemeasured in a ß-scintillation counter. All treated animals survived the scheduled study period. In this study Stimulation Indices of 2.4, 4,6, 5.9, and 7.4 were determined with the test item at concentrations of 1, 5, 10, and 20 % (w/v) in DMSO, respectively.

Conclusion

Under the test conditions test item was found to be skin sensitiser and EC3 value of 2.1 % (w/v) was derived.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

The potential of the test item to cause skin sensitisation reactions following topical application to the skin of CBA mouse was assessed using the Skin Sensitization: Local Limph Node Assay according to the OECD Guideline for testing of chemicals n. 429.

An increase in cell proliferation of draining limph nodes was observed in any treatment group.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

According to the CLP Regulation (EC n. 1272/2008), respiratory or skin sensitisation section (3.4), skin sensitizer means a substance that will lead to an allergic response following skin contact.

The criteria to classify a substance as skin/respiratory sensitizer are reported into the second adaptation to technical progress.

For Category 1, a stimulation index of three or more is considered a positive response in the local lymph node assay, moreover if the data available are sufficient for the sub-categorization, the following thresholds will be used.

The EC3 value for the subcategorization 1A for the Local lymph node assay have to be <= 2% according to the Table 3.4.3 of the CLP Regulation.

The EC3 value for the subcategorization 1B for the Local lymph node assay have to be > 2% according to the Table 3.4.4 of the CLP Regulation.

 

The test substance under the test conditions shows a EC3 = 2.1% (w/v), therefore the substance is considered classified as Skin Sens. 1B.