2-Dibenzofuranyl-1-4,6-diphenyl-[1,3,5]-triazine

Endpoint summary

Description of key information

Skin sensitisation was investigated using in vitro studies and based on the inconclusive results obtained a further in vivo OECD 429 assay. The following results have been obtained:

• OECD 442C: technically impossible due to insolubility
• OECD 442D: technically impossible due to insolubility

The in vitro data are insufficient to conclude on the endpoint of skin sensitisation. Therefore, a further in vivo study was performed giving a clear negative response.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Feb 05 - Apr 07, 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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.42 (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

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: 8 - 12 weeks
- Weight : 18.0 +/- 1.1 g
- Housing: grouped per dose
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

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

IN-LIFE DATES: From: day 1 To: day 6

Vehicle:

methyl ethyl ketone

Concentration:

2.5, 5, and 10 (w/w)

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: 10% in Propylene glycol

Range Finding Exp. 1
- Concentration used: 5, 10 %
- Irritation:no irritation observed

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: OECD 429
- Concentration: 2.5, 5, and 10%
- Criteria used to consider a positive response: Stimulation index > 3

Positive control substance(s):

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

Statistics:

Standard statistical methods have been applied for data processing.

Positive control results:

Conc. SI
0%: 1.0
5% 1.68
10% 1.78
25% 8.19

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

Test Group: 2.5% in PG

Key result

Parameter:

SI

Value:

1.1

Test group / Remarks:

Test Group: 5% in PG

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

Test Group: 10% in PG

Cellular proliferation data / Observations:

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

CLINICAL OBSERVATIONS: 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.

Calculation of Stimulation Indices per Dose Group

Test item concentration	Group Calculation
	Mean DPM per animal (2 lymph nodes)	SD	S.I.
PG (Vehicle Control)	1037.7	177.9	1.0
2.5 % Test Item in PG	1044.7	324.1	1.0
5 % Test Item in PG	1147.9	273.0	1.1
10 % Test Item in PG	1045.9	417.2	1.0

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:

Objective

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

Study Design

For this purpose a local lymph node assay was performed using test item concentrations of 2.5, 5, and 10 (w/w). The highest concentration tested was the highest concentration that could technically be achieved. A control group of five mice was treated with the vehicle (methyl ethyl ketone) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per animal. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a β-scintillation counter.

Results

All treated animals survived the scheduled study period and no signs of systemic toxicity or local skin irritation were observed.

In this study Stimulation Indices of 1.0, 1.1, and 1.0 were determined with the test item at concentrations of 2.5, 5, and 10% in PG. The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3.

Conclusion

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

Reference 2

Endpoint:

skin sensitisation: in chemico

Study period:

Nov 15, 2019

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Justification for type of information:

This study was performed to assess the solubility of the test item in different solvents to conduct a DPRA.
An appropriate solvent should dissolve the test item completely.
For the solubility test, the solvents acetonitrile, methanol, ethanol and acetone were used. These solvents were shown to be suitable for the DPRA at ICCR-Roßdorf GmbH.
No solubility tests were conducted with the solvents water, 1:1 mixture water:acetonitrile and isopropanol, since these solvents are not compatible with the peptides (mainly not suitable for the cysteine peptide) as tested in the proficiency study at ICCR-Roßdorf GmbH.
To test the solubility, a test item concentration of 100 mM was prepared in the solvents acetonitrile, methanol, ethanol and acetone. All four approaches showed milky, white and inhomogeneous formulations. A further dilution to a test item concentration of 25 mM by adding the appropriate volume of the solvent to the formulations showed the same result. The solvents could not dissolve the test item completely, therefore the test item is considered not suitable for the DPRA at ICCR-Roßdorf GmbH.
In conclusion, the test item formulated in acetonitrile, methanol, ethanol or acetone is considered not suitable for the DPRA under the conditions of this study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

Solvents used: Acetonitrile, Fisher, Lot 1526907, Expiry date: 04.07.2022
Methanol, Fisher, Lot 176599, Expiry date: October 2022
Ethanol, Fisher, Lot 1710254, Expiry date: 06.09.2022
Acetone, Fisher, Lot 1331155, Expiry date: 06.09.2022

Positive control results:

Cinnamaldehyde
Cystein depletion: 71.8 %
Lysine depletion: 44.4 %

Run / experiment:

other: Run 1

Parameter:

other: Soubility

Value:

0

Vehicle controls validity:

not examined

Other effects / acceptance of results:

No solubility tests were conducted with the solvents water, 1:1 mixture water:acetonitrile and isopropanol, since these solvents are not compatible with the peptides (mainly not suitable for the cysteine peptide) as tested in the proficiency study at test facility.
To test the solubility, a test item concentration of 100 mM was prepared in the solvents acetonitrile, methanol, ethanol and acetone. All four approaches showed milky, white and inhomogeneous formulations. A further dilution to a test item concentration of 25 mM by adding the appropriate volume of the solvent to the formulations showed the same result. The solvents could not dissolve the test item completely, therefore the test item is considered not suitable for the DPRA at the test facility.

Interpretation of results:

study cannot be used for classification

Conclusions:

In conclusion, the test material formulated in acetonitrile, methanol, ethanol or acetone is considered not suitable for the DPRA under the conditions of this study.

Reference 3

Endpoint:

skin sensitisation: in vitro

Study period:

Feb 13, 2020

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro skin sensitisation study does not need to be conducted because the available in vitro test methods are not applicable for the substance and therefore an in vivo skin sensitisation study was conducted

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
The test item appeared to be insoluble in the required concentration in water. The test item was insoluble in DMSO at the required stock solution concentration of 200 mM and formed no stable suspension after addition of treatment medium.

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Qualifier:

according to guideline

Guideline:

other: KeratinoSens™, EURL ECVAM DB-ALM Protocol No. 155, July 1st, 2015

GLP compliance:

yes (incl. QA statement)

Details on the study design:

According to OECD 442D, the KeratinoSens test method is applicable to the testing of chemicals that are soluble or form a stable dispersion, either in water or DMSO.
The preliminary test (testing of the test material for solubility or suitability in the assay) was thus carried out with water and DMSO.

Key result

Run / experiment:

other: 1

Parameter:

other: solubility screening

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

other: insoluble in test solutions

Interpretation of results:

study cannot be used for classification

Conclusions:

The test item appeared to be insoluble in the required concentration in water. The test item was insoluble in DMSO at the required stock solution concentration of 200 mM and formed no stable suspension after addition of treatment medium. Since the KeratinoSens assay is not suitable for test items which precipitate into non stable suspensions or emulsions (defined by OECD 442D) the assay has to be considered as being technically impossible.

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

Based on the provided information there is no need for classification according to the EU Regulation (EC) No 1272/2008 on Classification, Labelling and Packaging of Substances and Mixtures.