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Toxicological information

Skin sensitisation

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Administrative data

skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
Type of study:
mouse local lymph node assay (LLNA)

Test material

Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
disodium 4-amino-6-{2-[4-({4-[2-(2,4-diaminophenyl)diazen-1-yl]phenyl}sulfamoyl)phenyl]diazen-1-yl}-5-hydroxy-3-[2-(4-nitrophenyl)diazen-1-yl]naphthalene-2,7-disulfonate
Test material form:
solid: particulate/powder

In vivo test system

Test animals

Details on test animals and environmental conditions:
Test system justification:
The mouse is the recommended species for this test method according to international test guidelines. The BALB/c strain is selected because of the availability of historical control data for this strain at this facility, and because it was regarded as a sensitive strain in the context of contact allergy.

Experimental animals:
Animals species and strain: Young adult female mice (nulliparous and non-pregnant), strain BALB/CBYJICO (referred to as BALB/c in this document)
Source: Breeding farm VELAZ s.r.o., Koleč u Kladna, Czech Republic, RČH CZ 21760118
Number of animals per group:
Pilot experiment – 3 females
Exposed groups - 15 females (5 animals in each three groups)
Positive control group – 5 females
Negative control group –5 females
Reserve group – 2 females
(Reserve animals were intended for possible replacement of animals in other groups during the acclimatisation period.)
Total: 30 animals
Age: 8 to 10 weeks (at start of dosing)
Body weight range: 18.14 to 21.82 g (at start of dosing)
Health examination: All animals were examined during the acclimatisation period
Acclimatisation: At least 5 days

Animal quarters/husbandry
Animal rooms: Monitored conditions, microbiologically defined background, according to SOP No.40
Microclimatic conditions: Room temperature 22 + 3C, permanently monitored
Relative humidity 30 – 70 %, permanently monitored
Light: 12 hours light/dark cycle: 6am-6pm/6pm-6am
Animal caging: Animals groups: group-wise five in macrolon cages with sterilized softwood shavings.
Water: Drinking tap water ad libitum. Water quality corresponded to Ministerial Decree No. 252/2004 Czech Coll. of Law
Diet: Pelleted standard diet for experimental animals ad libitum. Microbiological control and content of nutrients was perform ed according SOP No. 75
Prophylactic arrangement: Cleaning and disinfection of animal room was regularly performed, as it is described in corresponding SOP No.10.

Animal identification
Cage identification: By cage number, study number and group specific colour.
Animal identification: By felt tip marking (from 1 to 5 per each group and group specific colour)
Random selection: According to SOP No.42

Study design: in vivo (LLNA)

other: mixture of 40% dimethylacetamide, 30% acetone and 30% ethanolethylacetamide, 30% acetone and 30% ethanol
30% (w/v) or 300 mg/ml
3% (w/v) or 30 mg/ml
0.3% (w/v) or 3 mg/ml
No. of animals per dose:
Number of animals per group:
Pilot experiment – 3 females (30% w/v)
Exposed groups - 15 females, (5 animals in each three groups, i.e. 0.3, 3, 30% w/v)
Positive control group – 5 females
Negative control group –5 females
Reserve group – 2 females
(Reserve animals were intended for possible replacement of animals in other groups during the acclimatisation period.)

Total: 30 animals
Details on study design:
Positive control
For the demonstration of test efficiency a group of animals treated with a substance with prove positive effect is included in the test (positive effect = Stimulation Index SI ≥ 3).
The substance DNCB (dinitrochlorbenzene, 0.5% (w/v) solution) was used as the positive control. A dose level for DNCB was determined during the verification of the method. The vehicle and dosage volume were the same as in treated groups. An application form was prepared each day of administration by dissolving an appropriate amount of the positive control item in the vehicle to obtain a concentration of 5 mg/mL.
The solution was prepared before the start of application by mixing on magnetic stirrer and was still being mixed during application.
3. 7. Pilot experiment
The highest concentration 30% (maximum technically practicable concentration) was administered to three animals to assess and/or discard a possible systemic toxicity or high irritation of skin. The route of administration was the same as in the main study. During the pilot experiment, no clinical symptoms of systemic toxicity were observed. No macroscopic changes (after necropsy) were found in all three animals.

Animal check-in and allocation
Animals were subjected to a clinical examination (health check) shortly after arrival.
Study animals were randomly allocated to the dose groups manually and assigned animal numbers as per table 1.
3. 9. Application
The volume of the dose was constant for all groups of animals - 25 L of the appropriate dilution to the dorsum of each ear once a day morning for 3 consecutive days. The application was performed very slowly by micropipette to avoid losses caused by draining from the ear.

Experimental schedule
Day 1:
Open application of 25 μL (in the morning, by pipette) of appropriate solutions 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:
Injection of 250 μL of phosphate-buffered saline (PBS) containing 7.18 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 examinations

During working hours the animals were checked for general health whenever other activities were performed – at least twice daily during the application period.

Clinical observations
Clinical signs were assessed using a defined scoring system described in SOP M/8 – at least twice daily during the dosing period. Efforts were made to characterize onset and duration of signs observed.

Body weight
Individual body weights were measured using an electronic balance. Weighing was performed on the first day of treatment, before dosing, and on the day of necropsy before application of the radionuclide.

Third day after last administration (five hours after application of radionuclide), all test animal were sacrificed by injection of veterinary preparation T 61.

T 61* inj. a.u.v.
Manufacturer: Intervet International GmbH
Feldstrasse 1A
85716 Unterschleissheim
Composition: Embutramidum 200 mg, Mebenzonii iodidum 50 mg, Tetracaini hydrochloridum 5 mg in 1 mL of water for injections

Post mortem investigations

Ear weights
Immediately after death, both ears of one animal 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
Both lymph nodes of one animal were prepared by gentle mechanical disaggregation through 100 m-mesh nylon gauze with concomitant pooling in 1 mL PSB (Phosphate Buffered Saline). Cells were washed twice with an excess of PBS and precipitated with 5% trichloroacetic acid (TCA) at 4 oC for 18h. The pellets were re-suspended in 1 mL TCA and transferred to scintillation vials containing 10 mL of scintillation fluid. Incorporation of 3H-methyl thymidine were measured by β-scintillation counting (Beckmann LS 6500) as disintegrations per minute (DPM)/mouse.

Positive control substance(s):
other: dinitrochlorobenzene
Data analysis
Mean values and standard deviations of ear weights and incorporation of 3H-methyl thymidine into the adjacent lymph nodes were computed for the test substance treatment groups and for the positive as well as the vehicle control group. Results (incorporation of 3H-
methyl thymidine) for each treatment group were expressed as mean Stimulation Index (SI). The SI was the ratio of the mean dpm/mouse within each test substance treatment group versus the mean dpm/mouse for the vehicle treated control group. The index for the vehicle control group was set at 1 by definition.

Statistical evaluation of data
For statistical calculations the software Statgraphic ® Centurion (version XV, USA) was used. At first the global comparison of all three values of the concentration groups with vehicle control is performed by applying the non-parametric Kruskal-Wallis test, and then the non-parametric two-group Mann-Whitney rank test (probability level 0.05) was applied to all two-group comparisons.
Evaluation of results
Stimulation index
The SI is obtained by dividing the pooled radioactive incorporation for each treatment group by the incorporation of the pooled vehicle control group; this yields a mean SI.

Ear weight – irritation effect
If after treatment with the test substance a statistically significant increase of ear weight together with clear concentration dependence of the effect is recorded, the inflammatory effect is considered as irritation induced by the test substance.
A positive result in cell proliferation reveals that the test substance could be a contact allergen, but an irritation effect of test substance (increased ear weight) does not rule out the possibility that it can be a false positive result.

Results and discussion

Positive control results:
Body weight of animals
The individual body weights before administration were relatively well balanced. Body weight increments were calculated from values of day 6 before necropsy and day 1 before first application. There was no significant difference in body weight increment of treated groups in comparison to the vehicle control.

Clinical observation and result of macroscopic necropsy
No animal died during the main experiment.
No symptoms of toxicity throughout the experiment were observed at all dose levels. All animals in the positive control group showed these symptoms: hyperaemia of skin, clonospasm and decreased response on stimuli.

Examination of cell proliferation in lymph nodes
In the positive control group, the SI was ≥ 3 (11.55) – test LLNA was efficient.
The SI for the test groups treated with the test substance was decreased in dose-related manner. At the highest dose level, the SI was 1.87, at the middle dose level the SI was 1.13, and at the lowest dose level, the mean SI was 0.95. The stimulation indexes of all dose levels were below the threshold.
Evaluation of irritating effect of the test substance
In the positive control group, the weight of ear target was statistically significantly increased as compared to the negative control group – the test design used is efficient in the detection of irritation effect.
At the highest dose levels of the test substance, statistically significant increase in the weights of ears was recorded (residues of the test substance on the ears). At the middle and lowest dose levels increases in the weights of ears were not recorded.

In vivo (LLNA)

Resultsopen allclose all
Remarks on result:
other: negative control 1 positive control 11.55 30% 1.87 3% 1.13 0.3% 0.95
other: disintegrations per minute (DPM)
Remarks on result:
other: Negative control 601,28 dpm positive control 6945 dpm 30% 1125.22 dpm 3% 678.38 dpm 0.3% 570.01 dpm
ca. 1.87
Test group / Remarks:
ca. 1.13
Test group / Remarks:
ca. 0.95
Test group / Remarks:

Applicant's summary and conclusion

Interpretation of results:
other: Not classified according to the CLP Regulation (EC) No. 1272/2008
Under the given test conditions, the test substance, Acid Black 210, does not elicit sufficient sensitising response.
Executive summary:

The test substance, Acid Black 210, was tested for the assessment of skin sensitisation potential with the murine local lymphnode assay. This study is a part of the test substance health hazard evaluation.

The Local Lymph Node Assay (LLNA) with radionuclides was used. The testing was conducted according tothe EUMethod B.42, Skin sensitization: Local Lymph Node Assay, Council Regulation (EC) No. 440/2008, published in O.J. L142, 2008.

In this study the contact allergenic potential of the test substance was evaluated after topical application to female BALB/c mice. Five mice per group were exposed by test and control substances on the dorsum of both ears once a day during 3 consecutive days. Primary proliferation of lymphocytes in the lymph node draining the site of application was evaluated by using radioactive labelling. The ratio of the proliferation in treated groups to that in vehicle controls, termed the Stimulation Index (SI), was determined. Statistical evaluation of ear weight was performed for elimination of potentially false positive findings with certain skin irritants.

Concentrations: positive control DNCB (dinitrochlorobenzene): 0.5% (w/v) and Acid Black 210: 30%, 3%, 0.3% (w/v) in the solvent mixture, DAE 433.

The animals exposed to the test substance at all dose levels showed no pathological skin reactions. No symptoms of systemic toxicity were observed at all dose levels.The positive control substance DNCB elicited a reaction pattern with statistically significant increase in ear weight and cell proliferation, the Stimulation Index reaching 11.55, which was consistent with its expected mode of action as a contact allergen.

The test substance Black Acid 210 showed a tendency to increase of ear weight at the highest dose level but without cell proliferation. Residues of the test substance on the ears caused this increased weight.

The comparison of the Stimulation Indexes between the treated groups and the control group revealed that the test substance Acid Black 210 did not cause a significant increase in radioisotope incorporation into the DNA of proliferating lymphocytes.


Under the given test conditions,the test substance, Acid Black 210,does not elicit sufficient sensitising response.