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Description of key information

The test substance, Acid Yellow 25 , was tested for the assessment of skin sensitisation potential with the murine local lymph node assay.

The testing was conducted according toMethod B.42 – Skin Sensitisation: Local Lymph Node Assay, Council Regulation (EC) No.640/2012, published in O.J. L 193, 2012 with respect to: OECD Test Guideline No. 429, Skin sensitisation: Local Lymph Node Assay, Adopted 22thJuly 2010

Only one study is available.

GLP study.

Klimish score 1.

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:
02.03.-15.03.2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
Adopted 22th July 2010
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
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 - Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle: 6am-6pm/6pm-6am
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 femalesExposed groups – 15 females (5 animals in three groups)Positive control group – 5 femalesNegative control group – 5 femalesTotal: 28 animals
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.
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.
Key result
Parameter:
SI
Value:
< 3
Cellular proliferation data / Observations:
The value of DPM and SI for positive control group was increased. The SI was ≥ 3 (5.57) – the LLNA was efficient (see Table 9). The SI for the test groups treated by the test substance at the all dose levels was below the threshold, stimulation index (SI) is < 3.

Table 9. Individual Activities and Calculated Values

Group

(Anim.No.)

NC

(1-5)

PC

(6-10)

50%

(11-15)

5%

(16-20)

0.5%

(21-25)

IndividualActivity (DPM)

493.21

3191.50

409.00

437.65

460.48

521.53

2791.09

411.21

295.04

488.90

535.33

2478.21

446.00

531.61

535.60

613.31

3651.30

377.89

815.39

507.27

502.65

2750.26

369.62

344.36

676.98

mean

533.21

2972.47

402.74

484.81

533.85

median

521.53

2791.09*

409.00*↓

437.65

507.27

SD

47.67

456.99

30.40

205.81

84.56

SI

1.00

5.57

0.76

0.91

1.00

Note:   

* = statistically significant on probability level < 0.05 (Mann-Whitney test)

SI = DPMmean values from exposed groups and the positive control group/DPM mean value of the vehicle control group

Interpretation of results:
GHS criteria not met
Conclusions:
Under the given test conditions, the animals exposed to the test substance, Acid Yellow 25, do not elicit sensitising response in LLNA assay. Negative results in cell proliferation revealed that the test substance Acid Yellow 25 could not be a contact allergen in mice.The test substance Acid Yellow 25, provides negative sensitising response in LLNA assay.
Executive summary:

The test substance, Acid Yellow 25, 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 toMethod B.42 – Skin Sensitisation: Local Lymph Node Assay, Council Regulation (EC) No.640/2012, published in O.J. L 193, 2012 with respect to: OECD Test Guideline No. 429, Skin sensitisation: Local Lymph Node Assay, Adopted 22thJuly 2010

 

In this study the contact allergenic potential of Acid Yellow 25 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 %, 0.5 % (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 comparison of the Stimulation Indexes and values of DPM between the treated groups and the control group revealed that the test substance did notcause a significant increase in radioisotope incorporation into the DNA of dividing lymphocytes so the result of LLNA assay isnegative.

Statistically significant increase of ear weight was not recorded in the treated animals. Residues of the test substance on the ears were visible during whole study so it could cause slightly weight increase at the highest dose level. The test substance did not cause of irritation to skin at all dose levels.

 

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.

 

Under the given test conditions, the animals exposed to the test substance, Acid Yellow 25,do not elicit sensitising response in LLNA assay. Negative results in cell proliferation revealed that thetest substance Acid Yellow 25 could not be a contact allergen in mice.

The test substanceAcid Yellow 25, provides negative sensitising response in LLNA assay.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Justification for classification or non-classification

Based on the available results of the test substance Acid Yellow 25 is not classified.