Benzoic acid, 2-[[3-(4-hydroxyphenyl)-1-oxopropyl]amino]-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-09-30 to 2009-10-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

, adopted 2002-04-24

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

, 2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2008-11-12

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

470-130-4

EC Name:

-

Cas Number:

697235-49-7

Molecular formula:

Hill formula: C16H15NO4 CAS formula: C16H15NO4

IUPAC Name:

2-((3-(4-Hydroxyphenyl)propanoylamino)benzoic acid

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Physical state: solid
- Molecular formula: C16H15NO4
- Molecular weight: 285.3 g/mole

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS - CBA/CaOlaHsd
- Source: Harlan Nederland, Horst, The Netherlands
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 19.4 – 23.1 g
- Housing: group housing in Makrolon Type-II cages with standard softwood bedding.
- Diet (ad libitum): pelleted standard Kliba 3433 mouse maintenance diet (Provimi Kliba AG, Kaiseraugst/Switzerland)
- Water (ad libitum): community tap water from Itingen/Switzerland
- Acclimation period: 7 days prior to the start of the test

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3°C
- Relative humidity: 30 - 70%
- Air changes: 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

5, 10 and 25% (w/v) concentration of test item

No. of animals per dose:

4 female mice

Details on study design:

RANGE FINDING TESTS:
A solubility experiment was performed. The highest test item concentration which can be technically used was a 25% (w/v) solution in DMF.
A pre-test was performed in two animals. Two mice were treated with concentrations of 25% and 10% (w/v) each in DMF on three consecutive days. In the pre-test clinical signs were recorded within 4 ± 2 hours and 24 ± 4 hours after each application as well as on the day corresponding to the day of preparation of the main experiment.
Results:
At the tested concentrations the animals did not show any signs of irritation or systemic toxicity.
The test item in the main study was assayed at 5%, 10% and 25% (w/v) in DMF. The top dose is the highest technically achievable concentration while avoiding systemic toxicity and excessive local irritation.

MAIN STUDY
Each test group of mice was treated by topical application to the dorsal surface of each ear lobe with the test item at concentrations of 5%, 10% or 25% (w/v) in DMF. The application volume of 25 μL was spread over the entire dorsal surface of each ear lobe (Ø about 8 mm) once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle.
Five days after the first topical application, all mice were administered with 250 μL of phosphate-buffered saline (PBS) containing 81.73 μCi/mL 3HTdR (equal to 20.43 μCi 3HTdR) by intravenous injection via a tail vein.
Approximately five hours after treatment with 3HTdR all mice were euthanized by inhalation of CO2.
The draining lymph nodes were rapidly excised and pooled in PBS for each experimental group. Single cell suspensions of pooled lymph node cells were prepared by gentle mechanical disaggregation through a gauze. After washing twice with approximately 10 mL phosphate buffered saline the lymph node cells were resuspended in approximately 3 mL 5% trichloroacetic acid and incubated at approximately + 5 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 1 mL 5% trichloroacetic acid and transferred to glass scintillation vials with 10 mL of ‘Irga-Safe Plus’ scintillation liquid and thoroughly mixed.
The level of 3HTdR incorporation was measured on a β-scintillation counter. Background 3HTdR levels were also measured in two 1 mL aliquots of 5% trichloroacetic acid.

OBSERVATIONS
- Mortality / Viability: once daily from the start of acclimatisation to the termination of the in-life phase.
- Body weights: at the start of acclimatisation, on test day 2 (prior to the 2nd application) and on test day 6 (prior to 3HTdR injection).
- Clinical signs (local / systemic): on the day of animal delivery and once daily from test day 1 (after the 1st application) to the termination of in-life phase.

Positive control substance(s):

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

Statistics:

The mean values and standard deviations were calculated.

Results and discussion

Positive control results:

The S.I. of 1.0, 2.3 and 8.7 were determined with the test item dissolved in acetone/olive oil (4:1, v/v) at concentrations of 5, 10 and 25% (w/v), respectively.
The EC3 value calculated was 11.6%.
Based on the test results, the test item alpha-hexylcinnamaldehyde shows an allergenic potential when tested up to the concentration of 25% (w/v) in acetone/olive oil (4:1, v/v).

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: 5% (w/v) concentration: 1.9 10% (w/v) concentration: 2.8 25% (w/v) concentration: 2.3

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Control group: 1526 (dpm); 1485 (dpm-background) 5% (w/v) concentration: 2799 (dpm); 2758 (dpm-background) 10% (w/v) concentration: 4164 (dpm); 4123 (dpm-background) 25% (w/v) concentration: 3430 (dpm); 3389 (dpm-background)

Any other information on results incl. tables

A dose-response relationship was observed.

A calculation of the EC3 value was not performed because no test concentrations produced a S.I. of 3 or higher.

OBSERVATIONS:

- Mortality / Viability: no deaths occurred during the study period.

- Clinical signs: neither clinical signs on the ears of the animals nor systemic findings were observed during the study period.

- Body weights: the body weights of the animals, recorded at the start of acclimatisation, prior to the second application and prior to sacrifice, were within the range commonly recorded for animals of the strain and age.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item is not a skin sensitizer.
According to the EC-Commission directive 67/548/EEC and its subsequent amendments, the test substance is not classified as skin sensitizer.
According to the EC-Regulation 1272/2008 and subsequent regulations, the test item is not classified as skin sensitizer.