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Administrative data

Description of key information

An OECD guideline 429 Murine Local Lymph Node Assay (LLNA)
An OECD guideline 406 Buehler test

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11-09-2012 to 18-01-2013
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline study done to GLP
according to guideline
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Details on test animals and environmental conditions:
- 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

- 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

dimethyl sulphoxide
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

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
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)
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.
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.
Key result
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).
Key result
other: disintegrations per minute (DPM)
Remarks on result:
other: Data provided in tabular form below

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
Interpretation of results:
Criteria used for interpretation of results: EU
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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Two studies, an LLNA and a Buehler test both indicate that p-tert amyl phenol is a sensitiser.

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.

In a dermal sensitization study according to OECD guideline 406, p-tert-amylphenol (> 99.5 %) in vaseline was tested in female guinea pigs (strain Dunkin-Hartley)(20 test animals and 10 control animals for the main experiment; 6 animals for the pre-test) using the method of Buehler. Based on the result of a pre-test the induction concentration used was 50 %. The induction phase led to severe skin reactions from the second phase on. However, individual application of 10% during the pre-tests and 50% during the induction phase did not show significant skin reaction after 30h - while 10% application on intact skin during the challenge phase resulted in clear skin reaction (erythema, pustules).
Justification for selection of skin sensitisation endpoint:
OECD guideline 429 study performed to GLP

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based upon the results of both the LLNA study and the Buehler test on p-tert amyl phenol, this material should be classified as a skin sensitiser