bis(2-methylpropyl) perylene-dicarboxylate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 June 2016 - 26 July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

adopted 22 July 2010

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

06 July 2012

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

3,10-bis(2-methylpropyl) perylene-3,10-dicarboxylate; 3,9-bis(2-methylpropyl) perylene-3,9-dicarboxylate

EC Number:

616-749-7

Cas Number:

79869-59-3

Molecular formula:

C30H28O4

IUPAC Name:

3,10-bis(2-methylpropyl) perylene-3,10-dicarboxylate; 3,9-bis(2-methylpropyl) perylene-3,9-dicarboxylate

Details on test material:

- State of aggregation: orange solid- State of aggregation:

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

In vivo test system

Test animals

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
- Age at study initiation: Pre-test: 10 - 11 weeks; Main study: 8 - 9 weeks
- Weight at study initiation: 19-21.8 g
- Housing: group in Makrolon Type II (pre-test) / III (main study), with wire mesh top
- Diet: 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
- Water, tap water, ad libitum
- Acclimation period: At least 5 days
- Indication of any skin lesions:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 45-65
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From: To:

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

5, 10, and 25% (w/w)

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: 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 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).
- Irritation: 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 (for detailed results see Appendix 1).
At the tested concentrations the animals did not show any signs of local skin irritation or systemic toxicity. Redness of the ear skin could partly not be examined, 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 be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed in the pre-experiment.

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response:
A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
• First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index.
• Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was placed into a mortar on a tared balance and PG was added whilst continuously grinding.
The different test item concentrations were prepared individually. Homogeneity of the test item in vehicle was maintained during treatment by manual stirring of the suspensions.
The preparations were made freshly and used within two hours before each dosing occasion. Concentrations were in terms of material as supplied.
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 5, 10, and 25% in PG. The application volume, 25 µL/ear/day, was spread over the entire dorsal surface (diameter 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).
Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline containing 20.1 µCi of 3H-methyl thymidine (equivalent to 80.5 µCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.

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. 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. No outlier value was detected in both outlier tests. However, both biological and statistical significance were considered together.

Results and discussion

Positive control results:

The sensitivity and reliability of the experimental technique employed was assessed by use of α-hexyl cinnamaldehyde dissolved in acetone/olive oil (4+1, v/v) (compound listed in OECD 429 Guideline) which is known to have skin sensitisation properties in mice. The periodic positive control experiment was performed using CBA/CaOlaHsd mice in April 2016.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

0.8

Test group / Remarks:

5% test article

Results 2

Parameter:

SI

Value:

0.8

Test group / Remarks:

10% test article

Results 3

Parameter:

SI

Value:

1.1

Test group / Remarks:

25% test article

Cellular proliferation data / Observations:

LYMPH NODE WEIGHS AND CELL COUNTS
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 or –cell counts was not observed in any of the test item treated groups 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.
 
EAR WEIGHTS
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.

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

CLINICAL OBSERVATIONS:
No signs of systemic toxicity or local skin irritation were observed during the study period.

BODY WEIGHTS
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.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Executive summary:

In order to assess the test item's potential for skin sensitising a local lymph node assay inmice 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. The animals neither showed any signs of systemic toxicity or local skin irritation nor mortality during the course of the study. A statistically significant or biologically relevant increase in ear weights was not observed in any treated group in comparison to the vehicle control group. 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 0.8, 0.8 and 1.1 were determined with the test item at concentrations of 5, 10, and 25% (w/w) in PG, respectively. A statistically significant and biologically relevant increase in DPM value and also in lymph node weight and -cell count was not observed in any dose group in comparison to the vehicle control group. Furthermore, thecut-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 was thus not a skin sensitiser under the test conditions of this study.