Tetrasodium 2-[[4-[[4-[[1-hydroxy-6-(phenylamino)-3-sulphonato-2-naphthyl]azo]-1-naphthyl]azo]-6-sulphonato-1-naphthyl]azo]benzene-1,4-disulphonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02.03.-15.03.2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

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:

Tetrasodium 2-[[4-[[4-[[1-hydroxy-6-(phenylamino)-3-sulphonato-2-naphthyl]azo]-1-naphthyl]azo]-6-sulphonato-1-naphthyl]azo]benzene-1,4-disulphonate

EC Number:

219-708-8

EC Name:

Tetrasodium 2-[[4-[[4-[[1-hydroxy-6-(phenylamino)-3-sulphonato-2-naphthyl]azo]-1-naphthyl]azo]-6-sulphonato-1-naphthyl]azo]benzene-1,4-disulphonate

Cas Number:

2503-73-3

Molecular formula:

C42H29N7O13S4.4Na

IUPAC Name:

tetrasodium 2-[[4-[[4-[[1-hydroxy-6-(phenylamino)-3-sulphonato-2-naphthyl]azo]-1-naphthyl]azo]-6-sulphonato-1-naphthyl]azo]benzene-1,4-disulphonate

impurity 1

Reference substance name:

Sodium chloride

EC Number:

231-598-3

EC Name:

Sodium chloride

Cas Number:

7647-14-5

Molecular formula:

ClNa

IUPAC Name:

sodium chloride

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): Direct Blue 78- Physical state: solid, powder- Analytical purity: 95% (w/w)- Impurities (identity and concentrations): NaCl (CAS: 7647-14-5) 10% (w/w)- Lot/batch No.: 7013/207- Expiration date of the lot/batch: unlisted- Storage condition of test material: The test substance should be stored in dry room in dark in closed container at the room temperature.

In vivo test system

Test animals

Species:

mouse

Strain:

Balb/c

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS- Source: Breeding farm VELAZ s.r.o., Lysolaje, Czech Republic, RČH CZ 21760118- Sex: young adult females nulliparous and non-pregnant- Age at study initiation: 8 to 10 weeks- Weight at study initiation: 16.34 – 18.59 g (at start of dosing), in pilot experiment 16.11 – 16.83 g- Housing: Monitored conditions, microbiologically defined background, according to internal SOP No.40, Animals in groups in macrolon cages with sterilized softwood shavings- Diet: ad libitum, Pelleted standard diet for experimental animals (Altromin, manufacturer: Altromin Spezialfutter GmbH & Co. KG, Germany). Microbiological control and content of nutrients is performed according internal SOP No. 72.- Water: ad libitum, Drinking tap, water quality corresponded to Regulation No. 252/2004 Czech Coll. Of Law, Health Ministry- Acclimation period: 7daysENVIRONMENTAL CONDITIONS- Temperature (°C): 22 ± 3 °C, permanently monitored - Humidity (%): 30 – 70 %, permanently monitored - Air changes (per hr):- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle: 6am-6pm/6pm-6am

Study design: in vivo (LLNA)

Vehicle:

other: DAE 433 – mixture of 40% dimethylacetamide, 30% acetone and 30% ethanol

Concentration:

50% (w/v)500 mg/mL 5% (w/v)50 mg/mL0.5% (w/v)5 mg/mL

No. of animals per dose:

Pilot experiment – 3 femalesTotal: 28 animalsExposed groups – 15 females (5 animals in three groups)Positive control group – 5 femalesNegative control group – 5 females

Details on study design:

PILOT EXPERIMENTThe test item was administered to three animals to assess a possible systemic toxicity or high irritation to skin. The test item was administered in the form of suspension in DAE 433. The appropriate suspensions of the test item (50%, 5%, 0.5% w/v) was applied to three animals in volume 25 µl to the dorsum of each ear once a day morning for 3 consecutive days. During the pilot experiment no clinical symptoms of systemic toxicity were observed. In treated animals no erythema and skin reaction were observed. MAIN STUDYANIMAL ASSIGNMENT AND TREATMENTAnimals were subjected to a clinical examination (health check) shortly after arrival. No clinical changes were recorded.After acclimatization the animals have been randomly allocated to the dose groups and assigned animal numbersTREATMENT PREPARATION AND ADMINISTRATION:Dosage volume: 25 µl /ear/animalThe application forms of test item (suspension) were prepared immediately before administration.Experimental Schedule Day 1: Open application of 25μL (in the morning, by pipette) of appropriate suspension of the test substance, the vehicle alone or the positive control to the dorsum of each ear.Days 2 and 3: The application procedure repeated as carried out on day 1.Days 4 and 5: No treatment.Day 6: The weight of animals was recorded. Injection 250 μL of phosphate-buffered saline (PBS) containing 7.625 x105 Bq of 3H-methyl thymidine into all test and control mice via the tail vein. Five hours later, the animals were killed.IN VIVO EXAMINATION- mortality- clinical observation- body weightPOST MORTEM INVESTIGATIONS- ears weights: Immediately after scheduled humane kill of animals, the both ears were cut off and circular pieces from the apical area of each ear with a diameter of 8 mm (=0.5 cm2) were excised using a disposable punch and weighed together on analytical scales.- Incorporation of 3H-methyl Thymidine

Positive control substance(s):

other: DNCB

Statistics:

For statistical calculations the software Statgraphic ® Centurion (version XV, USA) was used. Statistical evaluation of measured parameters was performed at first by applying the non-parametric Kruskal-Wallis test for testing whether all group samples originate from the same distribution, and then the non-parametric two-group Mann-Whitney rank test (probability level 0.05) for two-group comparisons.

Results and discussion

Positive control results:

The positive control substance DNCB produced a positive LLNA response at the exposure level expected to give an increase in the Stimulation Index SI ≥ 3 over the negative control group, which was in congruence with the expected mode of action of a contact allergen. The positive control also elicited a reaction pattern with significant increase in ear weight. The negative control did not show any changes. These results demonstrate that the method performed in the conditions of our laboratory has sufficient reliability.

In vivo (LLNA)

Results

Key result

Parameter:

SI

Value:

< 3

Remarks on result:

other: ambiguous

Remarks:

Comparison of Stimulation Indexes between treated groups and control vehicle group revealed that the test substance Direct Blue 78 caused a significant and dose dependent increase in radioisotope incorporation into the DNA of dividing lymphocytes of animals treated by the test substance at the highest and the middle dose level. But the Stimulation Index in all treated groups was < 3.

Cellular proliferation data / Observations:

The value of DPM and SI for positive control group was increased. The SI was ≥ 3 (9.11) – the LLNA was efficient (see Table 8). The SI for the test groups treated with the test substance at all dose level is below the threshold, and stimulation index (SI) is < 3. The value of DPM at the highest and the middle dose levels was statistically significantly changed compared to negative control. Statistically significantly increased value of DPM was observed (see Table 8).

Any other information on results incl. tables

Table No. 8. Individual Activities and Calculated Values

Group (Anim.No.)	NC (1-5)	PC (6-10)	50% (11-15)	5.0% (16-20)	0.5% (21-25)
Activity (DPM)	241.32	2143.89	511.57	368.02	275.40
	250.80	1959.00	354.62	310.99	131.58
	173.55	1782.66	326.00	339.10	118.34
	207.03	1975.92	280.69	325.07	226.87
	207.37	1975.08	328.66	282.06	185.60
mean	216.01	1967.31	360.31	325.05	187.56
median	207.37	1975.08*	328.66*	325.07*	185.60
SD	30.86	127.94	88.64	31.98	65.56
SI	1.00	9.11	1.67	1.50	0.87

Table No. 10. Summary Table

Group	Radioisotope incorporation		Ear weight
	Median DPM	SI	Median (mg)
NC	207.37	1.00	23.60
PC	1975.08*	9.11+	34.70*
50%	328.66*	1.67	25.80*
5.0%	325.07*	1.50	24.50*
0.5%	185.60	0.87	24.60

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Under the given test conditions the animals exposed to the test substance, Direct Blue 78, provides ambiguous sensitising response in LLNA assay.

Executive summary:

The test substance, Direct Blue 78,was tested for the assessment of skin sensitisation potential with the murine local lymph node assay. This study is a part of the test substance health hazard evaluation.

 

The Local Lymph Node Assay (LLNA) with the incorporation of 3H-methyl thymidine radionuclide was used. The testing was conducted according to theMethod B.42 – Skin Sensitisation: Local Lymph Node Assay, Council Regulation (EC) No.640/2012, published in O.J. L 193, 2012.

 

In this study the contact allergenic potential of Direct Blue 78 was evaluated after topical application to female BALB/c mice. Mice were exposed to three concentrations of test substance suspended in vehicle DAE 433 (mixture of 40% dimethylacetamide, 30% acetone and 30% ethanol) for 3 consecutive days.

In pilot experiment the following concentrationsof test substance in application forms were used: 50 %, 5.0 %, 0.5 % (w/v).According to the results of pilot experiment the same doses were confirmed for main study.

Primary proliferation of lymphocytes in the lymph node draining the site of application was evaluated using radioactive labelling of proliferating cells. The ratio of the proliferation in treated groups to that in vehicular controls, termed the Stimulation Index, was determined. The evaluation of ear weight was performed for elimination of false positive findings with certain skin irritants.

The animals exposed to the test substance at all doses showed no pathological and no other negative clinical symptoms of intoxication throughout the experiment.

 

The positive control item Dinitrochlorobenzene (DNCB) as a contact allergen (concentration 0.5% (w/v) elicited the expected reaction pattern with significant increase in Stimulation Index of cell proliferation and of ear weight. Appropriate performance of the assay in the test laboratory was then demonstrated.

 

Comparison of Stimulation Indexes between treated groups and control vehicle group revealed that the test substance Direct Blue 78 caused a significant and dose dependent increase in radioisotope incorporation into the DNA of dividing lymphocytes of animals treated by the test substance at the highest and the middle dose level. But the Stimulation Index in all treated groups was< 3.