Registration Dossier

Administrative data

Description of key information

The test substance is not a skin sensitiser.

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:Envigo RMS B.V., Inc. Horst, The Netherlands
- Females (if applicable) nulliparous and non-pregnant:yes
- Age at study initiation:Pre-test: 8 - 10 weeks; Main study: 8 -9 weeks
- Housing:group (Makrolon Type II (pretest)/ III (main study) with wire mesh top
- Diet (e.g. ad libitum): tap water, ad libitum
- Water (e.g. ad libitum):2018C Teklad Global 18% protein rodent diet (certified), ad libitum
- Acclimation period: al least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2°C
- Humidity (%): 45-65%
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
other: 1% aqueous Pluroic
Concentration:
5, 10 and 25%
No. of animals per dose:
5
Details on study design:
Vehicle and Dose Selection: A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which could be technically used was a 25% suspension in 1% aqueous Pluronic®. Vortexing was used to formulate the test item. At higher concentrations, an applicable formulation of the test item was not achieved, neither by the use of other vehicles nor by using additional methods to formulate the test item (e.g. vortexing, sonicating, warming to 37°C).
To determine the highest non-irritant test concentration that at the same time did not induce signs of systemic toxicity, a pre-test was performed in two animals.
Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 10 and 25% once daily each on three consecutive days. Prior to the first application of the test item and before sacrifice the body weight was determined. Clinical signs were recorded at least once daily. Eventual signs of local irritation were documented and a score was used to grade a possible erythema of the ear skin. Furthermore, prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6) the ear thickness was determined using a micrometer. Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm²) and were immediately pooled per animal and weighed using an analytical balance. Eventual ear irritation was considered to be excessive if an erythema of the ear skin of a score value ≥3 was observed at any observation time and/or if an increase in ear thickness of ≥25% was recorded on day 3 or day 6.
At the tested concentrations the animals did not show any signs of local skin irritation or systemic toxicity. Possible redness of the ear skin could partially not be determined due to the colour of the test item.
Thus, the test item in the main study was assayed at 5, 10, and 25%. The highest concentration tested was the highest level that could technically be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed in the pre-experiment.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated in the body weight tables, for the ear weights, the lymph node weights and lymph node cell count, and for the DPM values (group mean DPM ± standard deviation). All calculations conducted on the DPM values, the ear weights, the lymph node weights and the lymph node cell count were performed with validated program R Script STABW-mitStat.Rnw). Within the program a statistical analysis conducted on the DPM values, the ear weights, the lymph node weights and the lymph node cell count to assess whether the difference was statistically significant between the test item groups and negative control group. Statistical significance was set at the five per cent level (p < 0.05). Additionally, the Dean-Dixon-Test and Grubb’s Test were used for identification of possible outliers. An outlier (DPM value determined for animal no. 8) was detected in both outlier tests, but was not excluded from calculations, since exclusion of the value in question would not change the overall test result.
Parameter:
EC3
Remarks on result:
other: The EC3 value could not be calculated, since all S.I.`s are below the threshold value of 3.
Parameter:
SI
Value:
1.3
Test group / Remarks:
5% Direct Black 18L NA active dye
Parameter:
SI
Value:
1.1
Test group / Remarks:
10% Direct Black 18L NA active dye
Parameter:
SI
Value:
1.6
Test group / Remarks:
25% Direct Black 18L NA active dye

Calculation of Stimulation Indices per Dose Group

Test item concentration

Group Calculation

Mean DPM per
animal (2 lymph nodes)a)

SD

S.I.

Vehicle Control Group (1% aqueous Pluronic®)

1321.7

258.8

1.0

5% Direct Black 18L NA active dye

1722.9

472.1

1.3

10% Direct Black 18L NA active dye

1464.3

807.6

1.1

25% Direct Black 18L NA active dye

2123.5

822.5

1.6

a) Mean DPM/animal was determined by dividing the sum of the measured values from lymph nodes of all animals within a group by the number of animals in that group (5 animals)

The EC3 value could not be calculated, since all S.I.´s are below the threshold value of 3.

No deaths occurred during the study period.

No signs of systemic toxicity were observed during the study period. Possible redness of the ear skin could not be determined within 1h after each application due to the colour of the test item.

The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

The measured lymph node weights and –cell counts of all animals treated were recorded after sacrifice. A statistically significant or biologically relevant increase in lymph node weights was not observed in any of the test item treated groups in comparison to the vehicle control group. A statistically significant increase in lymph node cell count was observed in the high dose group in comparison to the vehicle control group. For BALB/c mice, a cut-off value for the lymph node cell count index of 1.55 was reported for a positive response. The indices determined for the lymph node cell count did not reach or exceed this threshold.

The measured ear weight of all animals treated was recorded on test day 6 (after necropsy). A biologically relevant or statistically significant increase in ear weights was not observed. Furthermore, the cut-off value (1.1) of the ear weight index for a positive response regarding ear skin irritation reported for BALB/c mice was not reached or exceeded in any of the treated groups.

Interpretation of results:
GHS criteria not met
Conclusions:
The test item Direct Black 18L NA active dye was not a skin sensitiser under the test conditions of this study.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

In this study the test item Direct Black 18L NA active dye was assessed for its skin sensitising potential using the Local Lymph Node Assay (LLNA) in mice. Test item suspensions at different concentrations were prepared in the vehicle 1% aqueous Pluronic®. The local lymph node assay is recommended by international test guidelines (e.g., OECD) as an animal test for predicting skin sensitisation in humans and provides a rational basis for risk assessment. The basic principle underlying the LLNA is that sensitisers induce a primary proliferation of lymphocytes in the lymph node draining the application site. The ratio of proliferation in test item treated groups compared to that in vehicle controls is termed the Stimulation Index (S.I.). Radioactive labeling is used to measure cell proliferations. For this purpose, a local lymph node assay was performed using test item concentrations of 5, 10, and 25% (w/w). The highest concentration tested was the highest concentration that could technically be achieved whilst avoiding systemic toxicity and excessive local skin irritation (as determined by a pre-experiment). The animals neither showed any signs of systemic toxicity nor mortality during the course of the study. Possible redness of the ear skin could not be determined within 1h after each application due to the colour of the test item. A statistically significant or biologically relevant increase in ear weights was not observed in any treated group in comparison to the vehicle control group. Furthermore, for BALB/c mice, a cut-off value of 1.1 for the ear weight index was reported for a positive response regarding ear skin irritation. None of the indices determined for the test item treated groups reached or exceeded this threshold. A test item is regarded as a sensitiser in the LLNA if exposure to one or more test item concentration results in a 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated test item concentration required to produce a S.I. of 3 is referred to as the EC3 value. In this study Stimulation Indices (S.I.) of 1.3, 1.1, and 1.6 were determined with the test item at concentrations of 5, 10, and 25% (w/w) in 1% aqueous Pluronic®, respectively. An outlier was identified in the low dose group (DPM value determined for animal number 8). However, as exclusion of the outlier did not change the overall test result, the value in question was not excluded from calculation. A statistically significant or biologically relevant increase in DPM value and lymph node weights was not observed in any treated group in comparison to the vehicle control group. A statistically significant, but biologically not relevant, increase in lymph node cell count was observed in the high dose group in comparison to the vehicle control group. However, the cut-off value of 1.55 for a positive response regarding the lymph node cell count index reported for BALB/c mice was not reached or exceeded in any dose group. The test item Direct Black 18L NA active dye was thus not a skin sensitiser under the test conditions of this study.

Justification for classification or non-classification

Based on the results, the test item is no subject to classification and labelling according to Regulation (EC) No 1272/2008 (CLP).