6-{4-[(1E)-3-methoxy-3-oxoprop-1-en-1-yl]phenoxy}hexyl methacrylate

Endpoint summary

Description of key information

The test item ROC-601 was not a skin sensitiser under the test conditions of this study.

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:

Sept. 2016 - Jan. 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

Identification: ROC-601
Batch: S23PSG0816
Purity: 99.5% by LC-UV-MS
Appearance: White, crystalline powder
Expiry Date: 01 July 2017
Storage Conditions: (provided by the Sponsor) At room temperature, light protected

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

Test system: Mice, CBA/CaOlaHsd
Rationale: Recognised as the recommended test system.
Source: Envigo RMS B.V., Inc, Postbus 6174, 5960 AD Horst / The Netherlands
Number of animals for the pre-test: 2 females
Number of animals for the main study: 16 females
Number of animals per group: 4 females (nulliparous and non-pregnant)
Number of test groups: 3
Number of control (vehicle) groups: 1
Age (beginning of treatment): Pre-test and main study: 8 - 9 weeks
Body weight: see Appendix 1 and 2
Identification: The animals were distributed into the test groups at random. All animals belonging to the same experimental group were kept in one cage. In the main experiment, the animals were identified by tail tags. In the pre-experiment, animals were identified by cage number.
Acclimation: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.
Husbandry: The animals were kept conventionally. The experiment was conducted under standard laboratory conditions.
Housing: group
Cage Type: Makrolon Type II (pre-test) / III (main study), with wire mesh top
Bedding: granulated soft wood bedding
Feed: 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
Water: tap water, ad libitum
Environment: temperature 22 ± 2 °C, relative humidity approx. 45 - 65% (except for deviation) artificial light 6.00 a.m. - 6.00 p.m.

Vehicle:

methyl ethyl ketone

Concentration:

Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 10, 25, and 50% in MEK. The application volume, 25 µL/ear/day, was spread over the entire dorsal surface of each ear once daily for three consecutive days.

No. of animals per dose:

The animals were distributed to the different test groups as follows:
(Group Concentration %, Number of Animals per Group, Animal Numbers/Group Housing)
Group 1 (Control Group) 0% test substance (MEK as vehicle), 4 animals, No. 1 - 4
Group 2 (Low Dose) 10% test substance, 4 animals, No. 5 - 8
Group 3 (Mid Dose) 25% test substance, 4 animals, No. 9 - 12
Group 4 (High Dose) 50% test substance, 4 animals, No. 13 - 16

Details on study design:

Determination of Ear Thickness: Prior to the first and third application of the test item (study days 1 and 3) and prior to treatment with 3HTdR (study day 6), the ear thickness was determined using a micrometer.
Test Item Administration: Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 10, 25, and 50% in MEK. The application volume, 25 µL/ear/day, was spread over the entire dorsal surface (diameter: 8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone (control animals).
Administration of 3H-methyl-thymidine: Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline containing 20.1 µCi of 3H-methyl thymidine (equivalent to 80.3 µCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.
Terminal Procedure: Approximately five hours after treatment with 3HTdR all mice were euthanized by using CO2, which, after harvesting of the lymph nodes, was followed by cervical dislocation to ensure death. The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group).
Preparation of Single Cell Suspensions: Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing two times with phosphate buffered saline (approx. 10 mL) the lymph node cells were resuspended in 5% trichloroacetic acid (approx. 3 mL) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules.
Determination of cellular proliferation (incorporation of 3HTdR): The precipitates were then resuspended in 5% trichloroacetic acid (1 mL) and transferred to scintillation vials with 10 mL of scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured in a -scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL aliquots of 5% trichloroacetic acid. The -scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).
Determination of Ear Weights: After the lymph nodes had been excised, both ears (left and right) of mice were punched at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2). For each animal both punches were immediately weighed (pooled per animal) using an analytical balance.
Observations
Clinical Observations: All animals were observed on a daily basis, including pre- and post-dose observations on days 1, 2 and 3. Any clinical signs of systemic toxicity, local skin irritation or signs of ill health during the study were recorded.
Ear thickness: In the pre-test, the ear thickness was determined prior to the first application (day 1), on day 3, and prior to sacrifice on day 6.
Ear weights: In the pre-test, the ear weight was determined after sacrifice (biopsy punches will be taken from each ear).
Body Weights: The body weights were recorded on day 1 (prior to dosing) and prior to sacrifice (pre-test) or prior to treatment with 3HTdR (main experiment).
Data Evaluation
Interpretation of raw data: The proliferative response of the lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/lymph node) and as the ratio of 3HTdR incorporated into lymph node cells of test animals relative to that recorded for lymph nodes of control animals (Stimulation Index; S.I.). Before DPM/lymph node values were determined, mean scintillation-background DPM was subtracted from test and control raw data.

Positive control substance(s):

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

Positive control results:

The sensitivity and reliability of the experimental technique employed was assessed by use of α-hexyl cinnamaldehyde dissolved in acetone/olive oil (4+1 v/v) (compound listed in OECD 429 Guideline) which is known to have skin sensitisation properties in mice. The periodic positive control experiment was performed using CBA/CaOlaHsd mice in October 2016 and results, showing the test system being sensitive, were:
S.I. 1.00 at 0% concentration α-hexyl cinnamaldehyde,
S.I. 1.50 at 5% concentration α-hexyl cinnamaldehyde,
S.I. 3.84 at 10% concentration α-hexyl cinnamaldehyde, and
S.I. 11.76 at 25% concentration α-hexyl cinnamaldehyde.

Parameter:

SI

Value:

1.45

Test group / Remarks:

10% test item in methyl ethyl ketone (MEK)

Parameter:

SI

Value:

1.5

Test group / Remarks:

25% test item in methyl ethyl ketone (MEK)

Key result

Parameter:

SI

Value:

1.43

Test group / Remarks:

50% test item in methyl ethyl ketone (MEK)

Cellular proliferation data / Observations:

The EC3 value could not be calculated, since all S.I.´s are below the threshold value of 3.

No deaths occurred during the study period.

No symptoms of local skin irritation at the ears of the animals and no signs of systemic toxicity were observed during the study period.

The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item ROC-601 was not a skin sensitiser under the test conditions of this study.

Executive summary:

In order to study a possible skin sensitising potential of ROC-601, three groups each of four female mice were treated once daily with the test item at concentrations of 10, 25, and 50% in MEK by topical application to the dorsum of each ear for three consecutive days. A control group of four mice was treated with the vehicle (MEK) only. Five days after the first topical application radio-labelled thymidine (³H-methyl thymidine) was injected into all mice via the tail vein. Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes 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 of³H-methyl thymidine measured in ab-scintillation counter.

All treated animals survived the scheduled study period and no signs of systemic toxicity or local skin irritation were observed.

In this study Stimulation Indices of 1.45, 1.50, and 1.43 were determined with the test item at concentrations of 10, 25, and 50% in methyl ethyl ketone. The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3. Thus, the test substance is considered not being a skin sensitiser in this test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

A possible skin sensitising potential of ROC-601 was investigated, using three groups each of four female mice being treated once daily with concentrations of 10, 25, and 50% test item in MEK by topical application according to OECD 429 study design (LLNA). A control group of four mice was treated with the vehicle (MEK) only. Five days after the first topical application radio-labelled thymidine (³H-methyl thymidine) was injected into all mice via the tail vein. Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes 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 of³H-methyl thymidine measured in ab-scintillation counter.

All treated animals survived the scheduled study period and no signs of systemic toxicity or local skin irritation were observed.

In this study Stimulation Indices of 1.45, 1.50, and 1.43 were determined with the test item at concentrations of 10, 25, and 50% in MEK. The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3. Thus, the test substance is considered not being a skin sensitiser in this test.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The substance was tested for skin sensitisation according to OECD 429 (LLNA study) under GLP. No sensitisation response was observed and thus the substance is not subject to classificaton according to GHS and/or CLP (Commission Regulation EC No. 1272/2008) for skin sensitisation.

Data for respiratory sensitisation are not available and no observation for respiratory sensitisation were made so far.