Biphenyl-4-yl-(9,9-dimethyl-9H-fluoren-2-yl)-[2-(9,9-diphenyl-9H-fluoren-4-yl)-phenyl]-amine

Endpoint summary

Description of key information

The test item did not show any skin sensitizing potential in the local lymph node assay according to OECD 429.

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:

16 Nov 2016 - 08 Dec 2016

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:

22 July 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

06 July 2012

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 nulliparous and non-pregnant: no
- Weight at study initiation: 16.6 - 21.0 g
- Housing: group
- Diet: 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 45 - 65
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

propylene glycol

Concentration:

5, 10, and 25%

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: The highest test item concentration, which can be technically used, was 25% suspension in propylene glycol.
- Irritation and systemic toxicity: On day 3 and 4, the animal treated with 25% test item concentration showed an erythema of the ear skin (Score 1). Both animals showed ruffled fur (on day 2 and within 1 hour after the third application) and eyelid closure (within 1 hour after the second application). However, no body weight loss or other specific signs of toxicity were observed in both animals.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.
- Ear thickness measurements: Prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6).

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response:
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 an appropriate container on a tared balance and PG was added (weight per weight). Grinding of the test item in a mortar was used to formulate the test item. The different test item concentrations were prepared individually. Homogeneity of the test item in vehicle was maintained during treatment using a magnetic stirrer. The preparations were made freshly before each dosing occasion.

- Topical application: 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).

- Administration of 3H-methyl-thymidine: Five days after the first topical application (day 6) 250 μL of phosphate-buffered saline containing 20.5 μCi of 3H-methyl thymidine (equivalent to 81.9 μCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

A statistical analysis was carried out on the DPM values 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.

Positive control results:

SI = 11.76 (25% positive control)

Key result

Parameter:

SI

Value:

1.3

Test group / Remarks:

5% test item

Key result

Parameter:

SI

Value:

1.4

Test group / Remarks:

10% test item

Key result

Parameter:

SI

Value:

1.3

Test group / Remarks:

25% test item

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

vehicle control

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION
Mean DPM/animal (2 lymph nodes) = 808.2 (Vehicle control)
Mean DPM/animal (2 lymph nodes) = 1080.0 (5% test item)
Mean DPM/animal (2 lymph nodes) = 1160.2 (10% test item)
Mean DPM/animal (2 lymph nodes) = 1043.0 (25% test item)

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

CLINICAL OBSERVATIONS:
No deaths occurred during the study period. No symptoms of local skin irritation at the ears of the animals and no signs of systemic toxicity 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.

EAR THICKNESS
The measured ear thickness of all animals treated was recorded prior to the st application, on study day 3 and prior to necropsy (day 6). A relevant increase in ear thickness was not observed.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was not a skin sensitiser under the test conditions of this study.

Executive summary:

In the study the test item formulated in propylene glycol (PG) was assessed for its possible skin sensitising potential. 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.

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. A relevant increase of ear thickness was not observed. In this study Stimulation Indices (S.I.) of 1.3, 1.4, and 1.3 were determined with the test item at concentrations of 5, 10, and 25% (w/w) in PG, respectively.

The test item was not a skin sensitiser under the test conditions of this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

In the study the test item formulated in propylene glycol (PG) was assessed for its possible skin sensitising potential. 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.

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. A relevant increase of ear thickness was not observed. In this study Stimulation Indices (S.I.) of 1.3, 1.4, and 1.3 were determined with the test item at concentrations of 5, 10, and 25% (w/w) in PG, respectively.

The test item was not a skin sensitiser under the test conditions of this study.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Justification for classification or non-classification

Based on the available data which are sufficient and reliable, the test item is not considered to be classified and labelled as skin sensitizer according to Regulation (EC) No 1272/2008.