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Administrative data

Description of key information

In an in vivo OECD 429 study, under GLP conditions, the Stimulation Index of 2-Propenoic acid, 2-[[(octadecylamino)carbonyl]oxy]ethyl ester is <1.90 resulting in a negative result and is, therefore, not sensitising to skin (Envigo, 2016k).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
Identification: 2-Propenoic acid, 2-[[(octadecylamino)carbonyl]oxy]ethyl ester
Purity: >98%
Physical state/Appearance: white solid
Storage Conditions: room temperature in the dark
Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
On receipt the animals were randomly allocated to cages. The animals were nulliparous and non pregnant. After an acclimatization period of at least 5 days the animals were selected at random and given a number unique within the study by indelible ink marking on the tail and a number written on a cage card. At the start of the study the animals were in the weight range of 15 to 23 g, and were 8 to 12 weeks old.

The animals were housed in suspended solid floor polypropylene cages furnished with softwood woodflakes. Free access to mains tap water and food was allowed throughout the study.

The temperature and relative humidity were set to achieve limits of 19 to 25C and 30 to 70%, respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness.

The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
Vehicle:
propylene glycol
Concentration:
10%, 5% or 2.5% w/w in propylene glycol.
No. of animals per dose:
Preliminary study - 1
Main test - 4 per dose
Vehicle only - 4
Details on study design:
Test Item Administration
Groups of four mice were treated with the test item at concentrations of 10%, 5% or 2.5% w/w in propylene glycol. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local skin irritation at the highest suitable concentration. The mice were treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.

A further group of four mice received the vehicle alone in the same manner.

H-Methyl Thymidine Administration
Five days following the first topical application of the test item or vehicle (Day 6) all mice were injected via the tail vein with 250 µL of phosphate buffered saline (PBS) containing 3H methyl thymidine (3HTdR: 80 µCi/mL, specific activity 2.0 Ci/mmoL, ARC UK Ltd) giving a total of 20 µCi to each mouse.

Terminal Procedures
Termination: Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. For each group 1 mL of PBS was added to the pooled lymph nodes.

Preparation of Single Cell Suspension: A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through a 200 mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 mL of PBS into a petri dish labeled with the study number and dose concentration. The lymph node cell suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 mL of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The pooled lymph node cells were pelleted at 1400 rpm (approximately 190 g) for 10 minutes. The pellet was re suspended in 10 mL of PBS and re pelleted. To precipitate out the radioactive material, the pellet was re suspended in 3 mL of 5% Trichloroacetic acid (TCA).

Determination of 3HTdR Incorporation: After approximately 18 hours incubation at approximately 4C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for 10 minutes, re suspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. 3HTdR incorporation was measured by scintillation counting. The "Poly Q™" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left to stand in darkness for approximately 20 minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately 20 minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system.
Positive control substance(s):
other: Phenylacetaldehyde (CAS 112-78-1)
Positive control results:
Results
The Stimulation Index expressed as the mean radioactive incorporation for the treatment group divided by the mean radioactive incorporation of the vehicle control group is as follows:
Concentration (% v/v) in propylene glycol: 5
Stimulation Index: 9.89
Result: Positive
Conclusion
Phenylacetaldehyde (>90%) was considered to be a sensitizer under the conditions of the test.
Key result
Parameter:
SI
Value:
1.7
Test group / Remarks:
2.5% in propylene glycol
Key result
Parameter:
SI
Value:
1.84
Test group / Remarks:
5% in propylene glycol
Key result
Parameter:
SI
Value:
1.9
Test group / Remarks:
10% in propylene glycol
Cellular proliferation data / Observations:
There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

Preliminary Screening Test

No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted.

Based on this information the dose levels selected for the main test were10%,5% and2.5% w/winpropylene glycol.

Interpretation of results:
GHS criteria not met
Conclusions:
The test item was considered to be a non-sensitizer under the conditions of the test.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

An in vivo skin sensitisation study has been submitted in place of an in vitro study as the substance (monomer) is manufactured outside of the European Union thus the manufacturer, and data owner, has obligations to submit in vivo data in other jurisdictions other than EU REACH.

In addition, contracts with the chosen testing laboratory were signed on 15th June 2016 following a data gap analysis completed in March 2016. The requirement to submit in vitro data under REACH came into force on 11th October 2016, therefore testing had already commenced.

The in vitro skin sensitisation endpoint is, therefore, waived.

Justification for classification or non-classification

Skin Sensitisation:

In an in vivo OECD 429 study, under GLP conditions, the Stimulation Index of 2-Propenoic acid, 2-[[(octadecylamino)carbonyl]oxy]ethyl ester is <1.90 resulting in a negative result and is, therefore, not sensitising to skin (Envigo, 2016k).

According to the ECHA Guidance on the Application of the CLP Criteria (version 4.1, June 2015), a substance is considered a skin sensitiser is the Stimulation Index is ≥ 3.

The Stimulation Index of 2-Propenoic acid, 2-[[(octadecylamino)carbonyl]oxy]ethyl ester is <1.90, therefore, is not classified for skin sensitisation.