Reaction mass of 2-hexadecyl-N-(2-hydroxyethyl)-3-oxoicosanamide, 2-hexadecyl-N-(2-hydroxyethyl)-3-oxo-, N-(2-hydroxyethyl)-3-oxo-2-tetradecylicosanamide and N-(2-hydroxyethyl)-3-oxo-2-tetradecyloctadecanamide.

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

between the 24th August and 13th December 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

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

Reference substance name:

PC-9S

IUPAC Name:

PC-9S

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Name: PC-9S
CAS Number: 1271738-25-0
Batch: PS101103
Physical Appearance: Off-white Powder
Purity: Over 99%
Melting Point: Approx. 95°C
Expiry Date: November 18, 2012
Storage Conditions: At room temperature (20+/- 5°C), avoid direct sun rays and extreme heat
Safety Precautions: Routine hygienic procedures

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

Test System: Mice, CBA/Ca
Source: Charles River Laboratories, Manston Road, Margate Kent CT9 4AL. United Kingdom [e-mail]
Age: 10-12 weeks at the beginning of treatment
Body Weight: 18.0 – 21.3 g at the beginning of treatment
Accommodation: Group housing in Makrolon Type-II cages with standard softwood bedding
Diet: Pelleted standard Harlan Teklad 2914C rodent maintenance diet (Provimi Kliba AG, 4303 Kaiseraugst/Switzerland), available ad libitum.
Water: Community tap water from Itingen/Switzerland, available ad libitum.

Environmental conditions:
Acclimatisation:
13 days prior to the start of the test under test conditions after health examination. Only animals with intact ears and without any visible signs of illness were used for the study.

Standard Laboratory Conditions. Air-conditioned with target ranges for room temperature 22 + /- 3 °C, relative humidity 30 - 70% and 10 - 15 air changes per hour. Room temperature and humidity was monitored continuously and values outside of these ranges may occasionally occur, usually following room cleaning. These transient variations were considered not to have any influence on the study and, therefore, these data were not reported but are retained at Harlan Laboratories Ltd. There was a 12 hour fluorescent light/12 hour dark cycle with at least 8 hours music during the light period.

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

Group concentration of test item (w/v) Number of animals per group Animal number
1 vehicle - 4 (1 - 4)
2 low dose 2.5 4 (5 - 8)
3 Mid dose 5 4 (9 - 12)
4 High dose 10 4 (13 - 16)

No. of animals per dose:

4

Details on study design:

A solubility experiment was performed according to the recommendations given by the guideline OECD 429. The highest test item concentration which can be technically used was a 10% (w/w) homogeneous suspension in DMF.

To determine the highest non-irritant test concentration, a pre-test was performed in two animals. Two mice were treated with concentrations of 5% and 10% (w/v) each in DMF on three consecutive days. In the pre-test clinical signs were recorded within approximately 1 hour and 24 ± 4 hours after each application as well as on the day corresponding to the day of lymph node preparation.

An erythema scoring was performed according to OECD guideline 429. Excessive local skin irritation is indicated by an erythema score ≥ 3.

Positive control substance(s):

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

Statistics:

For the body weight mean values and standard deviations were calculated.

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

In this study the stimulation index was calculated to be 0.9, 1.0 and 0.8 were determined with the test item, PC-9S, dissolved in DMF at concentrations of 2.5%, 5%, 10% (w/v), respectively. The EC3 value could not be calculated, since none of the tested concentrations induced a stimulation index greater than 3. Based on the test results, the test item PC-9S does not show an allergenic potential when tested up to the concentration of 10% (w/v) in DMF.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Not stated

Any other information on results incl. tables

Body weights:

The body weights of the animals, recorded at the start of acclimatization, prior to the first application and prior to sacrifice, were within the range commonly recorded for animals of the strain and age.

Clinical signs:

Neither clinical signs on the ears of the animals nor systemic findings were observed during the study period.

Mortality:

No deaths occurred during the study period.

	Test item concentration (%)	S.I.
Group 1	-	-
Group 2	2.5	0.9
Group 3	5.0	0.9
Group 4	10.0	0.8
EC3 = ---
A calculation of the EC3 value was not performed because no test concentrations produced a S.I. of 3 or higher.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

A test item is regarded as a sensitizer in the LLNA if the exposure to one or more test concentrations resulted in a 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the S.I.

In this study S.I. of 0.9, 1.0 and 0.8 were determined with the test item, PC-9S, dissolved in DMF at concentrations of 2.5%, 5%, 10% (w/v), respectively.

The EC3 value could not be calculated, since none of the tested concentrations induced an S.I. greater than 3.

Based on the test results, the test item PC-9S does not show an allergenic potential when tested up to the concentration of 10% (w/v) in DMF.

Executive summary:

In order to study the possible contact allergenic potential of PC-9S, three groups of four female mice each were treated daily with the test item at concentrations of 2.5%, 5%, and 10% (w/v) in DMF by topical application to the dorsum of each ear lobe for three consecutive days. A control group of four female mice was treated with the vehicle DMF only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine, 3HTdR). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes were excised and pooled per group. 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 3HTdR measured in a β-scintillation counter. All treated animals survived the scheduled study period. Neither clinical signs on the ears of the animals nor systemic findings were observed during the study period. The results obtained [Stimulation Index (S.I.)] are reported in the following table:

	Test item concentration (%)	S.I.
Group 1	-	-
Group 2	2.5	0.9
Group 3	5.0	0.9
Group 4	10.0	0.8
EC3 = ---
A calculation of the EC3 value was not performed because no test concentrations produced a S.I. of 3 or higher.

The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value.