p-(1,1-dimethylpropyl)phenol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11-09-2012 to 18-01-2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline study done to GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (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

Reference substance name:

p-(1,1-dimethylpropyl)phenol

EC Number:

201-280-9

EC Name:

p-(1,1-dimethylpropyl)phenol

Cas Number:

80-46-6

Molecular formula:

C11H16O

IUPAC Name:

4-(2-methylbutan-2-yl)phenol

Test material form:

solid: crystalline

Details on test material:

- Name of test material (as cited in study report): phenol, 4-(1,1-dimethylpropyl)-
- Lot/batch No.: 20409018

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories, Indianapolis, IN
- Age at study initiation: 7-12 weeks of age (adult)
- Weight at study initiation: 18.5-22.9 grams
- Housing: polycarbonate cages, group housed (5 per cage of same sex)
- Diet: Teklad 7012 Rodent Diet, Harlan Laboratories, Madison, WI, ad libitum
- Water: ad libitum
- Acclimation period: minimum 5 days, under same conditions as for the actual test

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 68 ± 5 oF
- Humidity (%): 30-70%
- Air changes (per hr): a minimum of 10 changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour light/dark cycle, full spectrum fluorescent lights

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

100%, 50% and 25% w/v/ dosing solutions.

No. of animals per dose:

n =5

Details on study design:

- Compound solubility: Max soluble concentration 3.41 g/ml

A fresh dose solution was prepared on each day of dosing

TREATMENT PREPARATION AND ADMINISTRATION:
On Day 1, 25 microlitres of the test article or control article was applied to the dorsum of both ears.
The same procedure performed on Day 1 was repeated on Days 2 and 3.
There was no treatment on Days 4 and 5.

The animals were observed for clinical signs daily after dosing. Observations
conducted included all clinical and toxicological signs, including local irritation at the
application site or systemic toxicity. None of the animals died or exhibited adverse clinical
signs needing euthanasia prior to termination. No animals were replaced.
All animals were weighed at the end of the observation period.
On Day 6, 5 hours prior to sacrifice, all animals were injected intravenously with
250 microlitres of an 80 microCurie/mL solution of 3H-methyl thymidine in PBS (v/v). The concentration of
radio labeled tracer was verified before use.
The animals were sacrificed by carbon dioxide (C02) inhalation and the draining
auricular lymph nodes were excised from each animal.
A single cell suspension of the lymph node cells from each animal was prepared by
gentle mechanical desegregation.

The cell suspension was centrifuged, re-suspended in cold 5% TCA, allowed to
precipitate at 2 to 6 degrees C for 18 ± 1 hours. After precipitation, the cells were centrifuged and
re-suspended in fresh 5% TCA.
The level of radioactivity in the cells was measured using a scintillation counter. Each vial was counted three times.


- Criteria used to consider a positive response: The proliferative response of lymph node cells is expressed as the number of radioactive
disintegrations per minute per mouse (DPM/MOUSE) and as the Stimulation Index (SI). Sl is
obtained by comparing the proliferation of lymph node cells from test animals with the lymph
node cells from the control animals. That is, the ratio of radiolabeled thymidine incorporation in
the lymph node cells, expressed as a group mean DPM, relative to that for control lymph node
cells (TEST/CONTROL RATIO).
The test article is regarded as positive if the test article at the test concentration produces a
test/control ratio equal to or greater than 3.0 and is statistically significant relative to the
negative control.

Positive control substance(s):

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

Statistics:

Appropriate statistical methods are performed to aid in the interpretation of radioactivity
results. Groups differing from the negative control at the level of p < 0.05 are considered
statistically significant.

Results and discussion

Positive control results:

The Stimulation Index of the positive control animals when compared to the negative control animals was 10.05 (p < 0.0001).
Hexylcinnamaldehyde was clearly positive in the assay.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Remarks on result:

other: The Stimulation Index of the test animals treated with the 100%, 50%, and 25% concentrations of the test article when compared to the negative control animals were: 8.19 (p = 0.0113), 9.66 (p = 0.0010), and 6.91 (p < 0.0001).

Results 2

Key result

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Data provided in tabular form below

Any other information on results incl. tables

Disintegrations per minute (DPM) for each of the test substances (n=5 animals per group), with mean and SD

	DMSO	Hexylcinnamaldehyde	25% PTAP	50% PTAP	100% PTAP
	675.99	4584.96	3344.62	2280.50	1265.70
	551.36	4081.94	2770.56	3232.55	2525.20
	613.92	5164.41	3581.27	6357.37	5007.96
	410.02	5245.38	4408.53	5715.82	7601.05
	227.85	5843.58	3027.46	6360.64	3905.08
Mean	495.83	4984.05	3426.49	4789.38	4061.00
SD	179.35	673.18	629.39	1904.20	2430.30

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

At all test concentrations (25%, 50% and 100% w/v PTAP in DMSO), the stimulation index was greater than 3-fold higher than the negative control.
P-tert amylphenol was sensitising at all concentrations tested, and can be regarded as a 'moderate' sensitiser.

Executive summary:

In an OECD guideline 429 Murine Local Lymph Node Assay (LLNA), the test substance p-tert amyl phenol was dissolved in dimethyl sulphoxide (DMSO) at concentrations of 25%, 50% and 100% w/v, and was found to be sensitising (SI > 3-fold above vehicle control) at all concentrations tested. P-tert amyl phenol is a sensitiser under the conditions of this study.