Poly[oxy(methyl-1,2-ethanediyl)], α-hydro-ω-hydroxy-, ether with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol (3:1), 2-propenoate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From June 07, 2017 to July 04, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

see ''Principles of the mehod if other than guideline''

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

see ''Principles of the mehod if other than guideline''

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Chemicals Act of the Federal Republic of Germany, ‘’Anhang 1’’ (Annex 1) in its currently valid version

Deviations:

not specified

Principles of method if other than guideline:

The following deviations from study plan occurred:
The relative humidity in the animal room was between approximately 29-65% instead of 45-65% and the temperature was 22±4°C instead of 22±2°C for several hours. The age of the animals was up to 13 weeks (beginning of treatment). The incubation of the lymph nodes was less than 18 hours (17h and 45min).
These deviations to the study plan, however, did not affect the validity of the study.

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:

Propylidynetrimethanol, propoxylated

EC Number:

500-041-9

EC Name:

Propylidynetrimethanol, propoxylated

Cas Number:

25723-16-4

Molecular formula:

C9 H20 O4

IUPAC Name:

Poly[oxy(methyl-1,2-ethanediyl)], a-hydro-w-hydroxy-, ether with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol

Constituent 2

Reference substance name:

Poly[oxy(methyl-1,2-ethanediyl)], a-hydro-w-hydroxy-, ether with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol (3:1), mono-2-propenoate

Cas Number:

1378240-17-5

Molecular formula:

(C3H6O)n(C3H6O)n(C3H6O)nC9H16O4

IUPAC Name:

Poly[oxy(methyl-1,2-ethanediyl)], a-hydro-w-hydroxy-, ether with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol (3:1), mono-2-propenoate

Constituent 3

Reference substance name:

Poly[oxy(methyl-1,2-ethanediyl)], α-hydro-ω-hydroxy-, ether with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol (3:1), di-2-propenoate

Cas Number:

104956-81-2

Molecular formula:

(C3H6O)n(C3H6O)n(C3H6O)nC12H18O5

IUPAC Name:

Poly[oxy(methyl-1,2-ethanediyl)], α-hydro-ω-hydroxy-, ether with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol (3:1), di-2-propenoate

Constituent 4

Reference substance name:

Poly[oxy(methyl-1,2-ethanediyl)], a-hydro-w-hydroxy-, ether with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol (3:1), mono[3-[(1-oxo-2-propen-1-yl)oxy]propanoate] mono-2-propenoate

Cas Number:

1378240-19-7

Molecular formula:

(C3H6O)n(C3H6O)n(C3H6O)nC12H18O5 + C3H4O2

IUPAC Name:

Poly[oxy(methyl-1,2-ethanediyl)], a-hydro-w-hydroxy-, ether with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol (3:1), mono[3-[(1-oxo-2-propen-1-yl)oxy]propanoate] mono-2-propenoate

Constituent 5

Reference substance name:

Propylidynetrimethanol, propoxylated, esters with acrylic acid

EC Number:

500-123-4

EC Name:

Propylidynetrimethanol, propoxylated, esters with acrylic acid

Cas Number:

53879-54-2

Molecular formula:

(C3H6O)n(C3H6O)n(C3H6O)nC15H20O6

IUPAC Name:

Poly[oxy(methyl-1,2-ethanediyl)], .alpha.-hydro-.omega.-[(1-oxo-2-propenyl)oxy]-, ether with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol

Constituent 6

Reference substance name:

Poly[oxy(methyl-1,2-ethanediyl)], a-hydro-w-hydroxy-, ether with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol (3:1), mono[3-[(1-oxo-2-propen-1-yl)oxy]propanoate] di-2-propenoate

Cas Number:

1378240-20-0

Molecular formula:

(C3H6O)n(C3H6O)n(C3H6O)nC15H20O6 + C3H4O2

IUPAC Name:

Poly[oxy(methyl-1,2-ethanediyl)], a-hydro-w-hydroxy-, ether with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol (3:1), mono[3-[(1-oxo-2-propen-1-yl)oxy]propanoate] di-2-propenoate

Constituent 7

Reference substance name:

Poly[oxy(methyl-1,2-ethanediyl)], a-hydro-w-hydroxy-, ether with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol, ester ether with 3-hydroxypropanoic acid (6:2:1), [3-[(1-oxo-2-propen-1-yl)oxy]propanoate] tri-2-propenoate

Cas Number:

1378240-22-2

Molecular formula:

(C3H6O)n(C3H6O)n(C3H6O)nC12H18O5 + (C3H6O)n(C3H6O)n(C3H6O)nC15H20O6

IUPAC Name:

Poly[oxy(methyl-1,2-ethanediyl)], a-hydro-w-hydroxy-, ether with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol, ester ether with 3-hydroxypropanoic acid (6:2:1), [3-[(1-oxo-2-propen-1-yl)oxy]propanoate] tri-2-propenoate

Constituent 8

Reference substance name:

Michaël adduct of propoxylated trimethylolpropane monoacrylate (TMP(PO)MA) with propoxylated trimethylolpropane triacrylate (TMP(PO)TA) and propoxylated trimethylolpropane monoacrylate (TMP(PO)MA)

Molecular formula:

Not available (UVCB substance)

IUPAC Name:

Michaël adduct of propoxylated trimethylolpropane monoacrylate (TMP(PO)MA) with propoxylated trimethylolpropane triacrylate (TMP(PO)TA) and propoxylated trimethylolpropane monoacrylate (TMP(PO)MA)

Constituent 9

Reference substance name:

Sum of other constituents

Molecular formula:

Not applicable (UVCB substance)

IUPAC Name:

Sum of other constituents

Test material form:

liquid

Details on test material:

appearance: yellowish liquid

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

Test System
Test system: Mice, CBA/CaOlaHsd
Source: Envigo RMS B.V., Inc Postbus 6174 5960 AD Horst / The Netherlands
Number of animals for the pre-tests: 6 females (2 for each pre-test)
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): 1st pre-test: 11 - 12 weeks; 2nd pre-test: 12 - 13 weeks; 3rd pre-test: 9 - 10 week; Main study: 10 - 11 weeks
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.

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 %, artificial light 6.00 a.m. - 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

The concentrations of 0.5, 1, and 2.5% were used. The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by three pre-experiments.

No. of animals per dose:

4

Details on study design:

Vehicle and dose selection

A solubility experiment was performed according to the recommendations given by OECD 429. The highest test substance concentration, which could be technically used was 100% of the undiluted test substance. Test substance solution at different concentrations was prepared using acetone/olive oil (4:1, v/v) as vehicle. Vortexing was used to formulate the test substance.

To determine the highest non-irritant test concentration that at the same time did not induce signs of systemic toxicity, three pre-tests were performed.

1st pre-experiment:
Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test substance concentrations of 50 and 100% once daily each on three consecutive days. The first pre-test was terminated on day 2 after both animals showed an excessive erythema of ear skin and sore skin on the ears.

2nd pre-experiment:
Therefore, a second pre-test was performed using test substance concentrations of 5 and 10%. The second pre-test was terminated on day 3 after both animals showed an excessive erythema of ear skin (score 3) and starting eschar formation (score 4).

3rd pre-experiment:
A third pre-test was performed using test substance concentrations of 1 and 2.5%. At the tested concentrations the animals showed an erythema of ear skin. A very minor spot of eschar was observed on one ear of the animal treated with 2.5% of the test substance. As all other parameters (ear weights, ear thickness, erythema score), did not indicate excessive skin irritation, and as the skin beneath the eschar was found to be intact, this observation was considered to be not biologically relevant.

Main study:
Test substance administration:
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test substance concentrations of 0.5, 1, and 2.5% in acetone/olive oil (4:1, v/v). The application volume, 25 μL/ear/day, was spread over the entire dorsal surface (∅ ∼ 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 vehicle alone.

Administration of 3H-methyl-thymidine:
Five days after the first topical application (day 6) 250 μL of phosphate-buffered saline containing 19.6 μCi of 3H-methyl thymidine (equivalent to 78.3 μCi/mL 3HTdR) were injected into each test and control mouse via the tail vein. Approximately five hours after treatment with 3HTdR all mice were euthanized by using CO2, which was, after harvesting of the lymph nodes, followed by cervical dislocation to ensure death.

Preparation of Single Cell Suspensions:
The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group). 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 β-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL-aliquots of 5 % trichloroacetic acid. The β-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

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.
Determination of Ear Thickness
In the pre-test, the ear thickness was determined prior to the first application of the test substance (day 1), on day 3, and on day 6 prior to sacrifice using a micrometer.
Determination of ear weights
In the pre-test, after the lymph nodes have been excised, both ears 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 per animal using an analytical balance. The values obtained were taken down manually. The results are described in the report.
Determination of 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).

Positive control substance(s):

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

Results and discussion

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

0.7

Remarks on result:

other: 0.5% acetone/olive oil (4:1, v/v)

Results 2

Key result

Parameter:

SI

Value:

1.58

Remarks on result:

other: 1% acetone/olive oil (4:1, v/v)

Results 3

Key result

Parameter:

SI

Value:

1.5

Remarks on result:

other: 2.5% acetone/olive oil (4:1, v/v)

Cellular proliferation data / Observations:

Calculation of the EC3 value:
The EC3 value could not be calculated, since all S.I.´s are below the threshold value of 3.
Viability / mortality:
No deaths occurred during the study period.
Clinical Signs:
No signs of systemic toxicity were observed during the study period. From day 1 to 5, the animals showed an erythema of the ear skin.
Body weights:
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.

Any other information on results incl. tables

Calculation and results of individual data:

 

Test sub concentration %	Group	Measurement DPM	DPM-BG(a)	number of lymph nodes	DPM per lymph node(b)	SI
---	BG I	17	---	---	---	---
---	BG II	21	---	---	---	---
0	1	6338	6319	8	789.9	1.00
0.5	2	4470	4451	8	556.4	0.70
1	3	9979	9960	8	1245	1.58
2.5	4	9523	9504	8	1188	1.50

 

1 = Control Group

2-4 = Test Group

BG = Background

(a) - The mean value was taken from the figures BG I and BG II

(b) -Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the study conditions, the test substance was not a skin sensitizer.

Executive summary:

A study was conducted to determine the skin sensitisation potential of the test substance according to OECD Guideline 429 and EU Method B.42 (Local Lymph Node Assay), in compliance with GLP. Three groups of 4 female CBA/CaOlaHsd mice were treated at concentrations of 0.5, 1, and 2.5% w/w on three consecutive days by open application on the ears. Four control animals were similarly treated, but with vehicle alone (acetone/olive oil (4:1 v/v)). Three days after the last exposure, all animals were injected with 3H-methyl thymidine and, after 5 h, the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitation of the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as Disintegrations Per Minute (DPM) and a Stimulation Index (SI) was subsequently calculated for each group. The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by three pre-experiments. The animals did not show any signs of systemic toxicity during the course of the study and no cases of mortality were observed. From day 1 to 5, animals showed an erythema of the ear skin (Score 1 - 2). The Stimulation Indices (S.I.) determined with the test substance were of 0.7, 1.58, 1.5 at concentrations of 0.5, 1, and 2.5% in acetone/olive oil (4:1, v/v), respectively. Under the study conditions, the test substance was not a skin sensitizer (Dony, 2017).