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Administrative data

Description of key information

Under the given test conditions, the animals exposed to the test substance, Direct Brown 103, do not elicit sensitising response in LLNA assay. Negative results in cell proliferation revealed that the test substance Direct Brown 103 is not a contact allergen in mice.

The test substance Direct Brown 103, provides negative sensitising response in LLNA assay.

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:
1.3. – 14. 3. 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
Council Regulation (EC) No.640/2012, published in O.J. L 193, 2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)
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., Lysolajské údolí 15/53, 165 00 Prague 6, Czech Republic, RČH CZ 11760500- Females (if applicable) nulliparous and non-pregnant: yes- Age at study initiation: 8 to 10 weeks- Weight at study initiation: 16.13 - 18.25 g- Housing: macrolon cages with sterilized softwood shavings, monitored conditions, microbiologically defined background, according to internal SOP No.40Cleaning and disinfection of animal room was regularly performed, as it is described in internal SOP No.10.- Diet (e.g. ad libitum): ad libitum- Water (e.g. ad libitum): ad libitum- Acclimation period: 7 days- Indication of any skin lesions: no clinical changes, all animals were examined during the acclimatisation periodENVIRONMENTAL 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- Air changes (per hr): not specifiedIN-LIFE DATES: from: 22. 2. 2017 (acclimatization)to: 13. 3. 2017 (necropsy)
Vehicle:
other: DAE 433 (mixture of 40% dimethylacetamide, 30% acetone and 30% ethanol)
Concentration:
The test substance was administered in the form of suspension in DAE 433. Concentrations of test substance in application form:75% (w/v)750 mg/mL 7.5% (w/v)75 mg /mL0.75% (w/v)7.5 mg /mL
No. of animals per dose:
Exposed groups – 15 females (5 animals in three groups)Positive control group – 5 femalesNegative control group – 5 females
Details on study design:
PRE-SCREEN TESTS:- Compound solubility: The appropriate suspensions of the test substance (75%, 7.5%, 0.75% w/v) was applied to three animals in volume 25 l to the dorsum of each ear once a day morning for 3 consecutive days. The suspensions were prepared before the start of application by mixing on magnetic stirrer and then were still mixed during application. The application was performed very slowly by micropipette. The route of administration was the same as in the main study. - Irritation: no erythema and skin reaction- Systemic toxicity: no clinical symptoms of systemic toxicity- Ear thickness measurements: without any changes, during the pathological examination the auricular lymph nodes enlargement was not detected in all animals- Erythema scores: no erythema and skin reactionMAIN STUDYNo symptoms of toxicity and no erythema on application site were observed in all animals from the negative control group and all animals administered by the test substance.ANIMAL 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 (acc. to internal SOP No.42) and assigned animal numbers.- Criteria used to consider a positive response: Cell proliferationPositive response: the stimulation index (SI) is ≥ 3, and the response increases in dose-related mannerNegative response: the stimulation index (SI) is < 3 without the dose – response relationshipAmbiguous response: the stimulation index is < 3, but the response increases in dose-related manner (dose–response relationship), and eventually statistical significance is observed.Ear weight – irritation effectIf 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.Positive result in cell proliferation reveals that the test substance could be a contact allergen. When positive irritation effect in animals is demonstrated simultaneously, the possibility can not be ruled out that the evaluation based on cell proliferation could be a false positive.TREATMENT PREPARATION AND ADMINISTRATION:the same as in the pre-screen test (see above)
Positive control substance(s):
other: Dinitrochlorobenzene (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:
All animals in the positive control group showed symptoms caused by the application of DNCB: hyperaemia of skin with well defined erythema on application site, clonospasm and increased response to stimuli.
Key result
Parameter:
SI
Value:
< 3
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATAThe value of DPM and SI for positive control group was increased. The SI was ≥ 3 (16.41) – the LLNA was efficient. 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. Statistically significant increase of DPM for all dose levels was not observed.DETAILS ON STIMULATION INDEX CALCULATIONStimulation index (for incorporation of 3H-methyl thymidine) was calculated by dividing mean values from exposed groups and the positive control group by the corresponding mean value of the vehicle control group. The index for the vehicle control group was set at 1 by definition.SI = DPM mean values from exposed groups and the positive control group/DPM mean value of the vehicle control groupCLINICAL OBSERVATIONS:No symptoms of toxicity and no erythema on application site were observed in all animals from the negative control group and all animals administered by the test substance.All animals in the positive control group showed symptoms caused by the application of DNCB: hyperaemia of skin with well defined erythema on application site, clonospasm and increased response to stimuli.BODY WEIGHTSIndividual body weight of females before administration and before necropsy was relatively well balanced (result of random selection of animals into groups). Body weight increment was calculated from values of day 6 just before necropsy and day 1 before first application. Very slight reduction of body weight (in tenths of grams) was recorded in one animal at the negative control group.
Interpretation of results:
GHS criteria not met
Conclusions:
Under the given test conditions, the animals exposed to the test substance, Direct Brown 103, do not elicit sensitising response in LLNA assay. Negative results in cell proliferation revealed that the test substance Direct Brown 103 is not a contact allergen in mice.The test substance Direct Brown 103, provides negative sensitising response in LLNA assay.
Executive summary:

The test substance, Direct Brown 103, 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 Method 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 sensitization: Local Lymph Node Assay, Adopted 22th July 2010.

 

In this study the contact allergenic potential of Direct Brown 103 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 concentrations of test substance in application forms were used: 75 %, 7.5 %, 0.75 % (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.

Statistically significant increase of ear weight was not recorded at the all dose levels. 

Stimulation Index at the all dose levels was < 3 so the result of LLNA assay is negative (see criteria in chapter 3.8. of this report).

 

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, Direct Brown 103, do not elicit sensitising response in LLNA assay. Negative results in cell proliferation revealed that the test substance Direct Brown 103 is not a contact allergen in mice.

The test substance Direct Brown 103, provides negative sensitising response in LLNA assay.

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

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification