di-tert-butyl-dimethoxy-({3,3',5,5'-tetramethyl-2'-[(tetramethyldisubstitutedheteropolycyclyl)oxy]biphenyl-2-yl}oxy) disubstituted heteropolycycle

Skin sensitisation

Administrative data

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:

other: GLP guidline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

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)

Test material

Test material information

Constituent 1

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/J Rj mice

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Age at study initiation: 8 weeks old (age-matched, within one week)
- Weight at study initiation: 19.1-20.8 g
- Housing: 4 animals / group
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 20 days (preliminary experiment); 8 days (main experiment)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.2 - 25.1°C
- Humidity (%): 30-79%
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:

methyl ethyl ketone

Concentration:

2.5, 5, 10, 25 and 50 % w/v

No. of animals per dose:

4

Details on study design:

The Preliminary Irritation/Toxicity Test was started according to the Study Plan on CBA/J Rj mice using two doses (2 animals/dose) at test item
concentrations of 50 and 25% (w/v) in MEK. The preliminary experiment was conducted in a similar experimental manner to the main study, but it was terminated on Day 6 and the radioactive proliferation assay was not performed.

During the study, animals were topically dosed with 25 μL of the appropriate formulation using a pipette on the dorsal surface of each ear. Each
animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.

During the study (Day 1 to Day 6) each animal was observed daily for any clinical signs, including local irritation and systemic toxicity. Clinical
observations were performed twice a day (before and after treatments) on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Individual records were maintained. Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to 3HTdR injection).

On Day 6, animals were taken to the radioactive suite and each mouse was intravenously injected via the tail vein with 250 μL of sterile PBS (phosphate buffered saline) containing approximately 20 μCi of 3HTdR. Once injected, the mice were left for 5 hours (± 30 minutes). Five hours (± 30 minutes) after intravenous injection the mice were euthanized by asphyxiation with ascending doses of carbon dioxide.
The draining auricular lymph nodes were excised and removed using forceps. Once removed, the nodes of mice from each test group was pooled and collected in separate Petri dishes containing a small amount of PBS to keep the nodes wet before processing. A single cell suspension of pooled lymph node cells (LNCs) was prepared and pelleted by centrifugation. Pellets were washed with PBS.
After the final washing step, supernatants were removed. Pellets were gently agitated resuspended and 5% (w/v) TCA solution was added to the tubes for precipitation of macromolecules. After overnight (approximately 18 hours) incubation at 2-8 °C, precipitates were centrifuged and supernatants were removed. Pellets were resuspended in 5% (w/v) TCA solution and dispersed by using an ultrasonic bath. Samples were transferred into a suitable sized scintillation vial filled with 10 mL of scintillation liquid and thoroughly mixed. The vials were loaded into a β-scintillation counter and 3HTdR incorporation was measured (10-minute measurement).
Stimulation index (SI = DPN value of a treated group divided by the DPN value of the negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result.

Positive control substance(s):

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

Results and discussion

Any other information on results incl. tables

DPM, DPN and Stimulation Index Values for all Groups

Test Group Name	Measured DPM / group	DPM	Number of lymph nodes	DPN	Stimulation Index
Background (5% (w/v) TCA)	34 33	-	-	-	-
Negative (vehicle) control (MEK)	4611	4577.5	8	572.2	1.0
Oxophos 64i 50% (w/v) in MEK	2468	2434.5	8	304.3	0.5
Oxophos 64i 25% (w/v) in MEK	2339	2305.5	8	288.2	0.5
Oxophos 64i 10% (w/v) in MEK	2735	2701.5	8	337.7	0.6
Oxophos 64i 5% (w/v) in MEK	2424	2390.5	8	298.8	0.5
Oxophos 64i 2.5% (w/v) in MEK	2371	2337.5	8	292.2	0.5
Positive control (25% (w/v) HCA in MEK)	27992	27958.5	8	3494.8	6.1

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Migrated information

Conclusions:

In conclusion, under the conditions of the present assay Oxophos 64i, tested in a suitable vehicle, was shown to have no sensitisation potential (non-sensitizer) in the Local Lymph Node Assay.