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Administrative data

Description of key information

The test item is not a skin senstitiser based on the LLNA.

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:
04 March 2020 to 03 April 2020
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)
Version / remarks:
2010
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS B.V., Inc Postbus 6174, 5960 AD Horst / The Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Pre-test: 14 - 15 weeks, Main study: 8 - 9 weeks
- Weight at study initiation: 18.1 (mean), 16-19.5 (range)
- Housing: 2 - 4 animals per cage, in Makrolon Type II (pre-screen test) / III (main study), with wire mesh top and granulated soft wood bedding
- Diet: 2018C Teklad Global 18% protein rodent diet, ad libitum
- Water: tap water, ad libitum- Acclimation period: 5 days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): approx. 45 – 65
- Photoperiod (hrs dark / hrs light): 12 / 12
Vehicle:
propylene glycol
Concentration:
2.5, 5, and 10%.
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TESTS:
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 10% suspension in PG. Grinding of the test item in a mortar 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 and stated in raw data and report. Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 5 and 10% 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. Thus, the test item in the main study was assayed at 2.5, 5, and 10%. The highest concentration tested was the highest level that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed in the pre-experiment.

MAIN STUDY

TREATMENT PREPARATION AND ADMINISTRATION:
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 2.5, 5, and 10% in PG. The application volume, 25 µL/ear/day, was spread over the entire dorsal surface (∅ ∼ 8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone (control animals).
Positive control substance(s):
other:
Statistics:
All calculations conducted on the DPM values, were performed with a validated test script of “R”, a language and environment for statistical computing and graphics. Within the program the Dean-Dixon-Test and Grubb’s Test were used for identification of possible outliers. An outlier (DPM value determined for animal 7) was detected in the All calculations conducted on the DPM values, were performed with a validated test script of “R”, a language and environment for statistical computing and graphics. Within the program the Dean-Dixon-Test and Grubb’s Test were used for identification of possible outliers. An outlier (DPM value determined for animal 7) was detected in the Grubb’s, but not in the Dean-Dixon-Test, but was not excluded from calculations, since exclusion of the outlier would not change the overall test result.
Positive control results:
The historical control data range of the last 10 positive control experiments was 5.7- 17.6 (= S.I. values).
Key result
Parameter:
SI
Value:
0.7
Test group / Remarks:
2.5 % test substance
Key result
Parameter:
SI
Value:
0.9
Test group / Remarks:
5 % test substance
Key result
Parameter:
SI
Value:
0.7
Test group / Remarks:
10 % test substance
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
vehicle control group

Table 1: Results of the GLP Positive Control




















































































Test item concentration %



Group



Measurement DPM



Calculation



Result



DPM-BGa)



number of lymph nodes



DPM per lymph nodeb)



S.I.



---



BG I



10



---



---



---



---



---



BG II



10



---



---



---



---



0



1



6901



6891



8



861.4



1.00



5



2



11583



11573



8



1446.6



1.68



10



3



12260



12250



8



1531.2



1.78



25



4



56477



56467



8



7058.4



8.19


         

 


Table 2: Stimulation Indices per Dose Group







































Test item concentration



Group Calculation



Mean DPM per animal (2 lymph nodes)



SD



S.I.



Vehicle Control Group (PG)



1529.7



1069.0



1.0



2.5% Art. 697548 (Diazo PW 1186)



1130.7



377.9



0.7



5% Art. 697548 (Diazo PW 1186)



1351.5



200.8



0.9



10% Art. 697548 (Diazo PW 1186)



1019.7



356.7



0.7


Interpretation of results:
GHS criteria not met
Conclusions:
The test item was not a skin sensitizer under the test conditions of the study. 
Executive summary:

In the study the test item formulated in PG (propylene glycol) was assessed for its possible skin sensitising potential. For this purpose a local lymph node assay was performed using test item concentrations of 2.5, 5, and 10%. The highest concentration tested was the highest concentration that could technically be achieved. The animals did not show any signs of systemic toxicity or local skin irritation during the course of the study and no cases of mortality were observed. In this study Stimulation Indices (S.I.) of 0.7, 0.9, and 0.7 were determined with the test item at concentrations of 2.5, 5, and 10% in PG, respectively. The test item is therefore 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

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on skin sensitization, the test item is not considered to be classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the seventeenth time in Regulation (EU) 2021/849.