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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11.01. – 22. 02. 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
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)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Test material form:
solid: particulate/powder
Details on test material:
Batch No.: 7031/2007Stability/Expiration: Feb 2018Storage: The test substance should be stored in dry room in dark in supplied 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., 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: 16.89 - 19.14 g (at start of dosing), in pilot experiment 16.79 - 18.61 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: 4.1.2017 (start of acclimatization)to: 23.1.2017 (necropsy)

Study design: in vivo (LLNA)

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. Losses caused by draining from the ear must be minimized.The route of administration was the same 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- Erythema scores: no erythema and skin reactionMAIN STUDYNote: 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.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 ≥ 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.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 cannot 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.

Results and discussion

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.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.

In vivo (LLNA)

Results
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 (4.72) – 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. 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 animal died during the main study.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). Very slight reduction of body weight (in tenths of grams) was recorded only in two animals at the negative control group.Body weight increment was calculated from values of day 6 just before necropsy and day 1 before first application.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the given test conditions, the animals exposed to the test substance, Direct Blue 85, do not elicit sensitising response in LLNA assay. Negative results in cell proliferation revealed that the test substance Direct Blue 85 could not be a contact allergen in mice.The test substance Direct Blue 85, provides negative sensitising response in LLNA assay.
Executive summary:

The test substance, Direct Blue 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 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 sensitisation: Local Lymph Node Assay, Adopted 22th July 2010

In this study the contact allergenic potential of Direct Blue 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 comparison of the Stimulation Indexes and values of DPM between the treated groups and the control group revealed that the test substance did not cause a significant increase in radioisotope incorporation into the DNA of dividing lymphocytes so the result of LLNA assay is negative.

Statistically significant increase of ear weight was recorded at the middle and lower dose levels. Residues of the test substance on the ears were visible during whole study so it could cause this weight increase. 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, Direct Blue 85, do not elicit sensitising response in LLNA assay. Negative results in cell proliferation revealed that the test substance Direct Blue 85 could not be a contact allergen in mice.

The test substance Direct Blue 85, provides negative sensitising response in LLNA assay.