Registration Dossier

Administrative data

Description of key information

The test substance, Reactive Yellow 85, provides ambiguous 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:
10.5. – 23.5. 2017
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:
Council Regulation (EC) No.640/2012, published in O.J. L 193, 2012
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., 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: 16.94 - 18.67 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: 3.5.2017 (acclimatization)to: 22.5.2017 (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 ul 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.Both ears of each mouse were observed for erythema and scored and subsequently ear thickness was measured using digital thickness gauge - Irritation: no erythema and skin reaction- Systemic toxicity: no clinical symptoms of systemic toxicity - Ear thickness measurements: without change, during the pathological examination the auricular lymph nodes enlargement was not detected- Erythema scores: no erythema and skin reactionMAIN 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 (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 ≥ 3Negative 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.Test validity criteria The test is considered valid, if the positive control substance DNCB produce a clear positive LLNA response and if the stimulation index SI is ≥ 3 over the negative control group. 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 by applying the parametric 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.Statistical evaluation of the body weightAs the first step the test for normality (Shapiro-Wilk test) was used. Since the smallest P-value amongst the tests performed is greater than 0,05, we can not reject the idea that data comes from a normal distribution with 95% confidence. For normally distributed data the variance check was performed (Levene´s test) to verify if standard deviations within each group are equal. One-Way ANOVA (probability level 0.05) was used to detect whether there are any significant differences amongst the means. Statistical evaluation of ears weightsAs the first step the test for normality (Shapiro-Wilk test) was used. Since the smallest P-value amongst the tests performed is lower than 0,05, we can reject the idea that data comes from a normal distribution with 95% confidence. The transformation of data was performed (Box-Cox transformation). Because the normal distributed distribution was not achieved after transformation of data then the non-parametric tests (Kruskal-Wallis Test, Mann-Whitney test) were used. Statistical evaluation of DPMNon-parametric two-group Mann-Whitney rank test (probability level 0.05) for two-group comparisons was used for statistical evaluation of the value of DPM.
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 (12.45) – the LLNA was efficient. 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. Statistically significant increase of DPM was observed at the middle dose level.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.CLINICAL 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. In mice of the highest dose level the wiping of the test substance from the ears to the shaving was observed.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. Negative body weight increment was recorded at the middle dose level.

SI at the all dose levels was < 3, but the value of DPM was significantly increased against negative control group at the middle dose level and exposure time was short at the highest dose level (the mice wiped the test substance from ears into shaving after application), so the result of LLNA assay is ambiguous (see criteria in Details on study design).

Interpretation of results:
study cannot be used for classification
Conclusions:
Under the given test conditions, the animals exposed to the tested concentrations of the test substance Reactive Yellow 85 in LLNA assay, elicited ambiguous results in cell proliferation revealed that the test substance Reactive Yellow 85 could be a contact allergen in mice.The test substance, Reactive Yellow 85, provides ambiguous sensitising response in LLNA assay.
Executive summary:

The test substance, Reactive Yellow 85, 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 the 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 Reactive Yellow 85 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 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.

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 recorded at the highest dose level.

 

SI at the all dose levels was < 3, but the value of DPM was significantly increased against negative control group at the middle dose level and exposure time was short at the highest dose level (the mice wiped the test substance from ears into shaving after application), so the result of LLNA assay is ambiguous (see criteria in Details on study design).

 

Under the given test conditions, the animals exposed to the tested concentrations of the test substance Reactive Yellow 85 in LLNA assay, elicited ambiguous results in cell proliferation revealed that the test substance Reactive Yellow 85 could be a contact allergen in mice, but it should be proved by further testing.

The test substance Reactive Yellow 85, provides ambiguous sensitising response in LLNA assay.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

The results from positive and negative controls demonstrate that the method performed had sufficient reliability .

The animals exposed to the tested concentrations of the test substance Reactive Yellow 85 (75%, 7.5% and 0.75% w/v) showed no skin reactions and clinical symptoms of intoxication throughout the experiment.

The values of SI for the test groups treated by the test substance Reactive Yellow 85 at the all dose levels were below the threshold, stimulation index (SI) was < 3. The value of DPM at the highest dose level was probably affected by the shorter time of exposure, because the mice wiped the test substance from ears into shaving after application.

The value of DPM of the middle dose level was significantly increased against negative control group at, so the result of LLNA assay is ambiguous.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Under the given test conditions, the animals exposed to the tested concentrations of the test substance Reactive Yellow 85 in LLNA assay, elicited ambiguous results in cell proliferation revealed that the test substance Reactive Yellow 85 could be a contact allergen in mice.

The test substance, Reactive Yellow 85, provides ambiguous sensitising response in LLNA assay and it is concluded to classify the substance as skin sensitiser category 1B.