Oxybis(2,1-ethanediyloxy-2,1-ethanediyl)diacrylate

Endpoint summary

Description of key information

No experimental data is available to evaluate the skin sensitisation potential of Tetraethylene Glycol Diacrylate.

A read-across is proposed with analogue substances : Tetraethylene Glycol Dimethacrylate and Triethylene Glycol Dimethacrylate, which are considered to be moderate skin sensitizers based on the in vitro and in vivo skin sensitisation studies.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 November 2013 - 19 November 2013

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:

individual approach (adopted 22 July 2010)

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

dated May 30, 2008

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: Mice, CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- CBA/CaOlaHsd mice, nulliparous, non-pregnant
- Age at study initiation (pre-test and main study): 8 - 9 weeks (beginning of treatment)
- Weight at study initiation (main test): 18.0 g - 22.2 g
- Housing: single; Makrolon Type II (pre-test)/ III (main study, with wire mesh top
- Diet (e.g. ad libitum):2018C Teklad Global 18% protein rodent diet (certified), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 5 days prior to start of dosing
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 2°C
- Humidity (%): 35-65 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 / 12

Vehicle:

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

Concentration:

1 (Control ) 0
2 (Low Dose) 25% (w/v)
3 (Mid Dose) 50% (w/v)
4 (High Dose) 100% (w/v)

No. of animals per dose:

Main study: 5 females (nulliparous and non-pregnant)
Pre-test: 2 females

Details on study design:

RANGE FINDING TESTS:
Concentrations of 50 and 100 % of the test substance were tested on two mice on one ear each once daily each on three consecutive days. The animals were sacrificed on day 6.
In the pre-test the tested animals did not show any signs of systemic toxicity.
From day 3 up to day 6, the animal treated with 50% test item concentration showed an erythema of the ear skin (Score 1). On day 2, 3 and day 6 the animals treated with 100% of the test item showed an erythema of the ear skin (Score 1), and on day 4 and 5 an erythema of the ear skin (Score 2). In addition the ears were scabby on day 5 and 6. No excessive increase in ear weights and/or ear thickness values were observed.
Thus, the test item in the main study was assayed at 25, 50 and 100%. The highest concentration tested was the highest level that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed in the pre-experiment.

Positive control substance(s):

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

Statistics:

The mean values and standard deviations were calculated in the body weight tables and for the DPM values (group mean DPM ± standard deviation).
The Dean-Dixon-Test and the Grubb's test were used for identification of possible outliers (performed with Microsoft Excel 2007).
However, both biological and statistical significance were considered together.

Positive control results:

A positive control performed with alpha-Hexylcinnamaldehyd in October 2016 resulted in an S.I. of 1.5 at 5% (w/v)
alpha-Hexylcinnamaldehyd in acetone/olive oil (4+1, v/v), 3.84 at 10 % and 11.76 at 25 %. An EC3 of 8.2 % (w/v) was calculated.

Parameter:

EC3

Value:

91.6

Parameter:

SI

Value:

1.4

Test group / Remarks:

25%

Parameter:

SI

Value:

1.51

Test group / Remarks:

50%

Parameter:

SI

Value:

3.3

Test group / Remarks:

100%

Cellular proliferation data / Observations:

disintegrations per minute (DPM):
Vehicle control group (negative control with vehicle, only): 999.4 DPM per lymph node (2 lymph nodes)
Test substance 25% DPM/lymph node: 1398.8
50% DPM/lymph node: 1510.2
100% DPM/lymph node: 3296.8

Calculation and Results of Individual Data

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

Test item concentration			DPM values measured	DPM-BG per animal (2 lymph nodes)^a)	S.I.^b)
%	Group no.	Animal no.
---	---	BG I	24	---	---
---	---	BG II	24	---	---
0	1	1	482	458.0	---
0	1	2	927	903.0	---
0	1	3	1007	983.0	---
0	1	4	1106	1082.0	---
0	1	5	1595	1571.0	---
25	2	6	1925	1901.0	1.9
25	2	7	1563	1539.0	1.5
25	2	8	1710	1686.0	1.7
25	2	9	1116	1092.0	1.1
25	2	10	800	776 .0	0.8
50	3	11	760	736.0	0.7
50	3	12	1918	1894.0	1.9
50	3	13	1625	1601.0	1.6
50	3	14	1823	1799.0	1.8
50	3	15	1545	1521.0	1.5
100	4	16	3100	3076.0	3.1
100	4	17	5249	5225.0	5.2
100	4	18	2036	2012.0	2.0
100	4	19	3746	3722.0	3.7
100	4	20	2473	2449.0	2.5

1    =  Control Group

2-4=  Test Group

^a)   =  values corrected for mean background value (BGI and BGII)

^b)    =  Stimulation Indices relative to the mean of the control group (Group 1)

Interpretation of results:

sensitising

Conclusions:

In this vaild Local Lymph Node Assay according to OECD 429 Triethyleneglycol dimethacrylate is a dermal sensitiser.
CLP EU GHS (Regulation (EC) No 1272/2008) classification: sensitizing category 1B (EC3 value > 2%)

Executive summary:

In this vaild dermal sensitisation study according to OECD Guideline 429 (adopted July 2010) Triethyleneglycol dimethacrylate (purity: 99.68%) in acetone:olive oil (4+1, v/v), groups of 5 female CBA/CaOlaHsd mice were tested using the LLNA method with the individual approach.

In this Local Lymph Node Assay Triethyleneglycol dimethacrylate is a dermal sensitiser.

 

No systemic findings were observed during the study period. Only the highest dose (100%) induced slight erythema on the ear skin on days 3 to 6 (Score 1). Animals treated with 25 and 50% of the test item did not show any signs of local skin irritation.

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. No cases of mortality were observed.

STIMULATION INDICES (S.I.) of 1.40, 1.51 and 3.30 were determined with the test substance at concentrations of 25%, 50% and 100% (w/v) in acetone:olive oil (4+1, v/v), respectively. The positive control substance was a-Hexylcinnamaldehyde, which gave an EC3 at 9.3 % (w/v). A result is regarded as positive when the S.I. is =3.

Based on these criteria, the test substance was found to be a sensitiser. The EC3 was calculated to be 91.6 %.

In this study, Triethyleneglycol dimethacrylate is a dermal sensitiser.

CLP EU GHS (Regulation (EC) No 1272/2008) classification: sensitizing category 1B (EC3 value > 2%)

 

 

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a

publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is

reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the

data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

August -October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

04 February 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

The objective of this study was to determine the reactivity of Tetraethylene glycol dimethacrylate towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL). After incubation of the test item with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and photodiode array (PDA) detection at 220 nm and 258 nm. SPCC and SPCL Percent Depletion Values were calculated and used in a prediction model which allows assigning the test item to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers.
The study procedures described in this report were based on the most recent OECD guideline.

At a concentration of 100 mM, Tetraethylene glycol dimethacrylate was soluble in ACN. Therefore this solvent was used to dissolve the test item in this DPRA study
Positive control: cinnamaldehyde (CAS No. 104-55-2)

Preparation of Test Item
No correction for the purity/composition of the test item was performed.
Test item stock solutions were prepared freshly for each reactivity assay.
For both the cysteine and lysine reactivity assay 56.26 mg of Tetraethylene glycol dimethacrylate was pre-weighed into a clean amber glass vial and dissolved, just before use,
in 1705 µL ACN to obtain a 100 mM solution. Visual inspection of the forming of a clear solution was considered sufficient to ascertain that the test item was dissolved. The test item,
positive control and peptide samples were prepared less than 4 hours before starting the incubation of the cysteine (cys) or lysine (lys) reactivity assay, respectively.
Any residual volumes were discarded.

Test systems :
Synthetic peptides containing cysteine (SPCC) (Ac-RFAACAA-COOH) or synthetic peptides containing lysine (SPCL) (Ac-RFAAKAA-COOH).
The molecular weight is 750.9 g/mol for SPCC and 775.9 g/mol for SPCL

ACCEPTABILITY CRITERIA
The following criteria had to be met for a run to be considered valid:
a) The standard calibration curve had to have an r2>0.99.
b) The mean Percent Peptide Depletion value of the three replicates for the positive control cinnamic aldehyde had to be between 60.8% and 100% for SPCC and between 40.2% and 69.0% for SPCL.
c) The maximum standard deviation (SD) for the positive control replicates had to be <14.9% for the Percent Cysteine Peptide Depletion and <11.6% for the Percent Lysine Peptide Depletion.
d) The mean peptide concentration of Reference Controls A had to be 0.50±0.05 mM.
e) The Coefficient of Variation (CV) of peptide areas for the nine Reference Controls B and C in ACN had to be <15.0%.

The following criteria had to be met for a test item’s results to be considered valid:
a) The maximum SD for the test item replicates had to be <14.9% for the Percent Cysteine Depletion and <11.6% for the Percent Lysine Depletion.
b) The mean peptide concentration of the three Reference Controls C in the appropriate solvent had to be 0.50±0.05 mM.

Data Evaluation
The concentration of SPCC or SPCL was photometrically determined at 220 nm in each sample by measuring the peak area of the appropriate peaks by peak integration and by calculating the concentration of peptide using the linear calibration curve derived from the standards.
In addition, the absorbance at 258 nm was determined in each sample by measuring the peak area of the appropriate peaks by peak integration. The ratio of the 220 nm peak area and the
258 nm peak was used as an indicator of co-elution. For each sample, a ratio in the range of 90% < mean area ratio of control samples < 110% gives a good indication that co-elution has
not occurred.

Data interpretation :
The mean Percent Cysteine Depletion and Percent Lysine Depletion were calculated for the test item. Negative depletion was considered as “0” when calculating the mean. By using the Cysteine 1:10 / Lysine 1:50 prediction model (see below), the threshold of 6.38% average peptide depletion was used to support the discrimination between a skin sensitizer and a non-sensitizer.

0% = Mean % depletion = 6.38% No or minimal reactivity Negative DPRA prediction
6.38% < Mean % depletion = 22.62% Low reactivity Positive DPRA prediction
22.62% < Mean % depletion = 42.47% Moderate reactivity Positive DPRA prediction
42.47% < Mean % depletion = 100% High reactivity Positive DPRA prediction

Positive control results:

Cysteine reactivity assay : The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 75.8% ± 0.9%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%).
Lysine reactivity assay : The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was 49.6% ± 4.4%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%).

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

mean cystein depletion

Value:

66.3 %

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

mean lysine depletion

Value:

12.2 %

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Other effects / acceptance of results:

Acceptability of the Cysteine Reactivity Assay
The correlation coefficient (r2) of the SPCC standard calibration curve was 0.9904. Since the r2 was >0.99, the SPCC standard calibration curve was accepted.
The mean peptide concentration of Reference Controls A was 0.500±0.005 mM while the mean peptide concentration of Reference Controls C was 0.503±0.017 mM. The means of Reference Control samples A and C were both within the acceptance criteria of 0.50±0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve the test item did not impact the Percent SPCC Depletion.
The Coefficient of Variation (CV) of the peptide areas for the nine Reference Controls B and C was 2.1%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time.
The mean area ratio (A220/A258) of the Reference Control samples was 17.37. The mean A220/A258 ratio ± 10% range was 15.63-19.10. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.
The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for the positive control cinnamic aldehyde (positive control) was 75.8% ± 0.9%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%).

Results Cysteine Reactivity Assay for Tetraethylene glycol dimethacrylate
Preparation of a 100 mM Tetraethylene glycol dimethacrylate stock solution in ACN showed that the test item was dissolved completely. Upon preparation and after incubation, both the co-elution control (CC) as well as the test item samples were visually inspected. No precipitate was observed in any of the samples.
In the CC sample no peak was observed at the retention time of SPCC. This demonstrated that there was no co-elution of the test item with SPCC. For the 208270/A-cys samples, the mean SPCC A220/A258 area ratio was 19.64. This was outside the 15.63-19.10 range. However, since the test item displayed high reactivity towards SPCC, accurate calculation of the peak purity was not possible due to the low SPCC signal at 258 nm. Overall, it can be concluded that the test item did not co-elute with SPCC.
The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for Tetraethylene glycol dimethacrylate was 66.3% ± 1.8%.

Acceptability of the Lysine Reactivity Assay
The correlation coefficient (r2) of the SPCL standard calibration curve was 0.998. Since the r2 was >0.99, the SPCL standard calibration curve was accepted.
The mean peptide concentration of Reference Controls A was 0.477±0.017 mM while the mean peptide concentration of Reference Controls C was 0.486±0.009 mM. The means of Reference Control samples A and C were both within the acceptance criteria of 0.50±0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve the test item did not impact the Percent SPCL Depletion.
The CV of the peptide areas for the nine Reference Controls B and C was 1.5%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time.
The mean area ratio (A220/A258) of the Reference Control samples was 13.91. The mean A220/A258 ratio ± 10% range was 12.52-15.30. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred. The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls C. The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was 49.6% ± 4.4%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%).

Results Lysine Reactivity Assay for Tetraethylene glycol dimethacrylate
Preparation of a 100 mM Tetraethylene glycol dimethacrylate stock solution in ACN showed that the test item was dissolved completely. Upon preparation and after incubation, both the
CC as well as the test item samples were visually inspected. No precipitate was observed in any of the samples.
In the CC sample no peak was observed at the retention time of SPCL. This demonstrated that there was no co-elution of the test item with SPCL. For the 208270/A-lys samples, the mean SPCL A220/A258 area ratio was 14.07. Since this was within the 12.52-15.30 range, this again indicated that there was no coelution of the test item with SPCL.
The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls C. The mean Percent SPCL Depletion for Tetraethylene glycol dimethacrylate was 12.2% ± 2.6%.

Interpretation of results:

other: considered positive in the DPRA assay

Remarks:

moderate reactivity class

Conclusions:

In the cysteine reactivity assay the test item showed 66.3% SPCC depletion while in the lysine reactivity assay the test item showed 12.2% SPCL depletion. The mean of the SPCC and SPCL
depletion was 39.3% and as a result the test item was positive in the DPRA and was classified in the “moderate reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction
model. Therefore, Tetraethylene glycol dimethacrylate was considered to be positive in the DPRA.

Executive summary:

The objective of this study was to determine the reactivity of Tetraethylene glycol dimethacrylate towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL). After incubation of the test item with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and photodiode array (PDA) detection at 220 nm and 258 nm. SPCC and SPCL Percent Depletion Values were calculated and used in a prediction model which allows assigning the test item to one of four reactivity classes used to support the discrimination

between sensitizers and non-sensitizers.

In the cysteine reactivity assay, the test item showed 66.3% SPCC depletion while in the lysine reactivity assay the test item showed 12.2% SPCL depletion. The mean of the SPCC and SPCL depletion was 39.3% and as a result Tetraethylene glycol dimethacrylate was considered to be positive in the DPRA and classified in the “moderate reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

November - December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

Februray 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Details on the study design:

The negative control is the vehicle of the test item : DMSO. The solvent control was 1% DMSO in exposure medium.
Positive control : Ethylene dimethacrylate glycol. The final concentration of the positive control ranges from 7.8 to 250 µM (final concentration DMSO of 1%).

Preparation of Test Item Stock, Spiking and Working Solutions
No correction was made for the composition/purity of the test item.
A solubility test was performed. The test item was dissolved in DMSO to a final concentration of 200 mM (clear colourless). The 100-fold dilution of the 200 mM DMSO stock in DMEM formed a clear solution (2000 µM). This concentration was selected as highest concentration for the main assay (highest dose required in the current guideline).
In the main experiments the test item was dissolved in dimethyl sulfoxide (DMSO) at 200 mM (colourless). From this stock 11 spike solutions in DMSO were prepared (2-fold dilution series). The stock and spike solution were diluted 25-fold with exposure medium. These solutions were diluted 4-fold in the assay resulting in final test concentrations of 2000, 1000, 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0 and 0.98 µM (final concentration DMSO of 1%). All concentrations of the test item were tested in triplicate. All formulations formed a clear solution. No precipitation was observed at the start and end of the incubation period in the 96-well plates.
Test item concentrations were used within 2.5 hours after preparation. Any residual volumes were discarded.

Test System
A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (e.g. the KeratinoSens™ cell line). The KeratinoSens™ cell line was generated by and obtained from Givaudan (Duebendorf, Switserland). Upon receipt, cells are propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock. Cells from this original stock can be propagated up to a maximum passage number from the frozen stock (i.e. 25) and are employed for routine testing using the appropriate maintenance medium.

All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 74 – 100 %), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.9 – 36.6°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature and humidity occurred due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.
Cells were subcultured upon reaching 80-90% confluency. To maintain the integrity of the response, the cells were grown for more than one passage from the frozen stock, and were not cultured for more than 25 passages from the frozen stock (P+25).

Experimental Design
For testing, cells were 80-90% confluent. One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. For each repetition, three replicates were used for the luciferase activity measurements, and one parallel replicate used for the MTT cell viability assay. The cells were incubated
The medium was removed and replaced with fresh culture medium (150 µL culture medium containing serum but without Geneticin) to which 50 µL of the 25-fold diluted test chemical and control items were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were then incubated for about 48 hours at 37±1.0oC in the presence of 5% CO2. In total 2 experiments were performed.
uciferase Activity Measurement : The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega were mixed together. The assay plates were removed from the incubator and the medium is removed. Then 200 µL of the Steady-Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 3 minutes at room temperature. Plates with the cell lysates were placed in the TECAN Infinite® M200 Pro Plate Reader to assess the quantity of luciferase (integration time two seconds).
ytotoxicity Assessment : For the KeratinoSensTM cell viability assay, medium was replaced after the 48 hour exposure time with fresh medium containing MTT and cells were incubated for 3 hours at 37°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution to each well. After shaking, the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.

ACCEPTABILITY CRITERIA
The KeratinoSensTM test is considered acceptable if it meets the following criteria:
a) The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, should be above the threshold of 1.5 in at least one of the tested concentrations (from 7.8 to 250 µM).
b) The EC1.5 should be between 5 and 125 µM. Moreover, the induction for Ethylene dimethacrylate glycol at 250 µM should be higher than 2-fold. If the latter criterion is not fulfilled, the dose-response of Ethylene dimethacrylate glycol should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.
c) Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO should be below 20% in each repetition which consists of 18 wells tested. If the variability is higher, results should be discarded.

INTERPRETATION OF DATA
The following parameters are calculated in the KeratinoSensTM test method:
•The maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the tested chemical and positive control
•The EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5 fold threshold (i.e. 50% enhanced luciferase activity) was obtained
•The IC50 and IC30 concentration values for 50% and 30% reduction of cellular viability.
In case the luciferase activity induction is larger than 1.5 fold, statistical significance is shown by using a two-tailed Student’s t-test, comparing the luminescence values for the three replicate samples with the luminescence values in the solvent (negative) control wells to determine whether the luciferase activity induction is statistically significant (p <0.05). ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis of the data. The lowest concentration with > 1.5 fold luciferase activity induction is the value determining the EC1.5 value. It is checked in each case whether this value is below the IC30 value, indicating that there is less than 30% reduction in cellular viability at the EC1.5 determining concentration.

A KeratinoSensTM prediction is considered positive if the following 4 conditions are all met in 2 of 2 or in the same 2 of 3 repetitions, otherwise the KeratinoSensTM prediction is considered negative:
1.The Imax is higher than (>) 1.5 fold and statistically significantly different as compared to the solvent (negative) control (as determined by a two-tailed, unpaired Student’s
t-test)
2. The cellular viability is higher than (>) 70% at the lowest concentration with induction of luciferase activity above 1.5 fold (i.e. at the EC1.5 determining concentration)
3.The EC1.5 value is less than (<) 1000 µM (or < 200 µg/mL for test chemicals with no defined MW)
4.There is an apparent overall dose-response for luciferase induction

Positive control results:

see below

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

Imax [442D]

Value:

23.17 %

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

Imax [442D]

Value:

19.77 %

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC 1.5 [442D]

Value:

49 µM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

EC 1.5 [442D]

Value:

18 µM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

IC30 [442D]

Value:

939 µM

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: cytotoxicity observed

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

IC30 [442D]

Value:

1 198 µM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: cytotoxicity observed

Other effects / acceptance of results:

Tetraethylene glycol dimethacrylate was evaluated for the ability to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway.
Two independent experiments were performed. The cells were in these experiments incubated with Tetraethylene glycol dimethacrylate in a concentration range of 0.98 –
2000 µM (2-fold dilution steps) for 48 hours. The activation of the ARE-dependent pathway was assessed by measuring the luminescence induction compared to the vehicle control. In addition, the viability was assessed with an MTT assay.

Experiment 1
•No precipitation was observed at the start and end of the incubation period in the 96-well plates.
•Tetraethylene glycol dimethacrylate showed toxicity. The calculated IC30 was 939 µM and the calculated IC50 was 1313 µM.
•A dose related luminescence activity induction was observed after treatment with Tetraethylene glycol dimethacrylate. The Imax was 23.17 and the EC1.5 49 µM.
•The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.19 and the EC1.5 110 µM.

Experiment 2
•No precipitation was observed at the start and end of the incubation period in the 96-well plates.
•Tetraethylene glycol dimethacrylate showed toxicity. The calculated IC30 was 1198 µM but 50% toxicity was not reached and thus no IC50 could be calculated.
•A dose related luminescence activity induction was observed after treatment with Tetraethylene glycol dimethacrylate. The Imax was 19.77 and the EC1.5 18 µM.
•The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.66 and the EC1.5 50 µM
.
Both tests passed the acceptance criteria:
•The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration.
•The EC1.5 of the positive control was between 5 and 125 µM (110 µM and 50 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (2.19-fold and 2.66-fold in experiment 1 and 2, respectively).
•Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (6.8% and 2.7% in experiment 1 and 2, respectively).
Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

Table1          
Overview Luminescence Induction and Cell Viability of Tetraethylene glycol dimethacrylate in Experiment 1 and 2

Concentration (µM)	0.98	2.0	3.9	7.8	16	31	63	125	250	500	1000	2000
Exp 1 luminescence	1.01	1.01	1.05	1.13	1.22	1.30	1.65***	2.31***	3.05***	3.85***	17.86***	23.17***
Exp 1 viability (%)	99.3	100.4	92.0	95.9	89.6	94.3	92.8	113.2	113.9	128.1	62.0	23.8
Exp 2 luminescence	1.32	1.59	1.25	1.26	1.30	2.39***	1.84***	3.14***	3.32***	6.32***	19.77***	19.77***
Exp 2 viability (%)	110.3	122.7	104.9	109.3	118.4	120.8	108.7	115.7	125.6	123.2	73.3	56.9

^***p<0.001 Student’s t test

 

Table2          
Overview Luminescence Induction and Cell Viability Positive Control EDMG in Experiment 1 and 2

Concentration (µM)	7.8	16	31	63	125	250
Exp 1 luminescence	0.86	0.97	1.09	1.32	1.56***	2.19***
Exp 1 viability (%)	101.3	104.1	105.1	102.5	109.0	114.7
Exp 2 luminescence	1.05	1.19	1.37	1.58***	1.88***	2.66***
Exp 2 viability (%)	118.6	111.3	119.3	116.2	120.3	119.0

^***p<0.001 Student’s t test

 

Table3          
Overview EC_1.5, I_max, IC₃₀and IC₅₀Values

	EC1.5(µM)	Imax	IC30(µM)	IC50(µM)
Test item Experiment 1	49	23.17	939	1313
Test item Experiment 2	18	19.77	1198	NA
Pos Control Experiment 1	110	2.19	NA	NA
Pos Control Experiment 2	50	2.66	NA	NA

NA = Not applicable

 

Interpretation of results:

other: positive in the KeratinoSens assay

Conclusions:

In conclusion, Tetraethylene glycol dimethacrylate is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions.

Executive summary:

The objective of this study was to evaluate the ability of Tetraethylene glycol dimethacrylateto activate theantioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens assay.

Tetraethylene glycol dimethacrylate was a clear colourless liquid. Tetraethylene glycol dimethacrylate was dissolved in dimethyl sulfoxide at 200 mM. From this stock 11 spike solutions in DMSO were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.98 – 2000 µM (2-fold dilution series). The highest test concentration was thehighest dose required in the current guideline. No precipitate was observed at any dose level tested. Two independent experiments were performed.

Both experiments passed the acceptance criteria. Overall it is concluded that the test conditions were adequate and that the test system functioned properly. 

Tetraethylene glycol dimethacrylate showed toxicity in experiment 1 and 2. The IC30 value was 939 µMand IC50 value was 1313 µM in experiment 1. In experiment 2, the IC30 value was 1198 µM but no IC50 value was reached. A biologically relevant, dose-related induction of the luciferase activity (EC 1.5 values of 49 µM and 18 µM in experiment 1 and 2, respectively) was measured in both experiments. The maximum luciferase activity induction (Imax) was 23.17-fold and 19.77-fold in experiment 1 and 2 respectively. Tetraethylene glycol dimethacrylate is classified as positive in the KeratinoSens assay since positive results (>1.5-fold induction) were observed at test concentrations < 1000 µM with a cell viability of >70% compared to the vehicle control.

In conclusion, Tetraethylene glycol dimethacrylate is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Registered substance:Tetraethylene Glycol Diacrylate (CAS number 17831-71-9)

 

Reporting format for defined approaches to testing and assessment based on multiple information sources

 

1 Summary

No experimental data is available to evaluate the skin sensitisation potential of Tetraethylene Glycol Diacrylate.

A read-across is proposed with analogue substances: Tetraethylene Glycol Dimethacrylate and triethylene Glycol Trimethacrylate, which are considered to be moderate skin sensitizers based on the in vitro and in vivo skin sensitisation studies.

 

2 General information

2.1 Identifier:In order to evaluate the potential skin sensitizer properties of Tetraethylene Glycol Diacrylate and to not use new animal tests, a strategy of read-across was proposed.

 

2.2 Reference to main scientific papers:

-       ECHA.Guidance on Information Requirements and Chemical Safety Assessment.ChapterR.7a: Endpoint specificguidance. Version 6.0 – July 2017

-       ECHA.Guidance on Information Requirements and Chemical Safety Assessment.ChapterR.7c: Endpoint specificguidance. Version 2.0 – November 2014

-       OECD (2012) The Adverse Outcome Pathway for Skin Sensitization Initiated by Covalent Binding to Proteins, Part 1: Scientific Evidence, Series on Testing and Assessment No.168 (ENV/JM/MONO(2012)10/PART1), available at:http://www.oecd.org/officialdocuments/publicdisplaydocumentpdf/?cote=env/jm/mono(2012)10/part1&doclanguage=en

 

3 Endpoint addressed

Skin sensitization

 

4 Definition of the purpose

Meeting the REACH information requirement for skin sensitization (Annex VII, 8.3) and the relevant classification and/or risk assessment obligations.

 

5 Rationale underlying the construction of the defined approach

 “Due to the complexity of the skin sensitization endpoint, a combination of alternative test methods (e.g.in silico,in chemicoandin vitro) in a Weight-of-Evidence approach needs to be considered to increase confidence in the final assessment of skin sensitization, e.g. a combination of read-across and non-animal test methods can be useful in concluding on the assessment of skin sensitization. “ (ECHA Guidance R7a, 2017, page 302).

The rationale of the defined approach is based on the strategy described in the ECHA Guidance R7a (2017, Pages 304-307,Figure R.7.3–2 Testing and assessment strategy for evaluating the skin sensitization potential of substances).

 

Element	Information	Conclusion for the registered substance
Existing data on physico-chemical properties
1	Is the substance a strong acid (pH= 2.0) or base (pH= 11.5), corrosive to the skin or (spontaneously) flammable in air or in contact with water or moisture at room temperature?	NO
Existing human data
2	Are there adequate existing human data, which provide evidence that the substance is a skin sensitizer?	NO
Existing animal data from sensitisation studies
3	Are there data from existing studieson skin sensitisation in laboratory animals (LLNA, GPMT, or Buehler test, EU B.42, B.50, B.51 and B.6/OECD TGs 429, 442A, 442B and 406), which provide sound conclusive evidence that the substance is a sensitizer, or non-sensitizer?	NO
Existing (Q)SAR data and read-across
4	Do “read-across” from structurally and mechanistically related substances and/or do suitable (Q)SAR predictions reliably indicate skin sensitization potential or the absence thereof of the substance?	YES
Existing in chemico and in vitro data
5a	Is there evidence/hypothesis of dermal bioavailability based on physico-chemical, in silico, in vitro or in vivo data?	YES, based on physical-chemical properties
5b	Has the substance demonstrated peptide/protein binding properties in an EU/OECD adopted in chemico test (e.g. OECD TG 442c)? (Key event 1 of the AOP), and/or Has the substance demonstrated activation of the Nrf2-Keap1-ARE toxicity pathway in an EU/OECD adopted in vitro test (e.g. OECD TG 442d)?(Key event 2 of the AOP), and/or Has the substance demonstrated induction of the cell surface markers(CD54 and/or CD86) on monocytic cells in a validated in vitro test, e.g. h-CLAT?(Key event 3 of the AOP).	NO
5c	Are there data from (a) non-validated in vitro test(s), which provide evidence that the substance may be a skin sensitizer?	NO
Weight-of-Evidence analysis
6	The “elements” described above may be arranged as appropriate. Taking all existing and relevant data (elements 1-5) into account, is there sufficient information to meet the information requirement of Section 8.3 of Annex VII and to make a decision on whether classification and labelling are warranted?	YES

 

 

6 Description of the individual information sources used within the approach

 

·        Dermal bioavailability :

No experimental toxicokinetic study is available on Tetraethylene glycol diacrylate.

However, as per REACH guidance document R7.c (2014, pages 172-174), information on dermal absorption may be deduced from the physicochemical properties, including: mean molecular weight (302.32 g/mol), water solubility (0.956 g/L) and the partition coefficient (Log Kow of 0.814).

Tetraethylene glycol diacrylate is a liquid having low molecular weight (< 500 g/mol), favourable to the dermal absorption. With a water solubility of 0.956 g/L, the substance is considered to be moderately soluble. However Log Kow value below 1 could limit penetration into the stratum corneum.To sum up, the physical-chemical properties of Tetraethylene glycol diacrylate concluded that the substance is absorbed after dermal exposure.

 

·        READ-ACROSS with Tetraethylene glycol dimethacrylate : In vitro skin sensitization tests:

First of all,an in Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA) was performed according to the OECD test guideline (N°442c).The objective of this study was to determine the reactivity of Tetraethylene glycol dimethacrylate towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL). After incubation of the test item with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and photodiode array (PDA) detection at 220 nm and 258 nm. SPCC and SPCL Percent Depletion Values were calculated. In the cysteine reactivity assay, the test item showed 66.3% SPCC depletion while in the lysine reactivity assay the test item showed 12.2% SPCL depletion.The mean of the SPCC and SPCL depletion was 39.3% and as a result Tetraethylene glycol dimethacrylate was considered to be positive in the DPRA.

 

Then, anIn Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method was performed according to the OECD test guideline (N°442D).The objective of this study was to evaluate the ability of Tetraethylene glycol dimethacrylate to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSensÔassay.

Tetraethylene glycol dimethacrylate showed toxicity in experiment 1 and 2. The IC30 value was 939µM and IC50 value was 1313 µM in experiment 1. In experiment 2,the IC30 value was 1198 µM but no IC50 value was reached. A biologically relevant, dose-related induction of the luciferase activity (EC1.5 values of 49 µM and 18 µM in experiment 1 and 2, respectively) was measured in both experiments. The maximum luciferase activity induction (I max) was 23.17-fold and 19.77-fold in experiment 1 and 2 respectively.Tetraethylene glycol dimethacrylate is classified as positive in the KeratinoSens assay since positive results (>1.5-fold induction) were observed at test concentrations < 1000 µM with a cell viability of >70% compared to the vehicle control.

 

·        READ-ACROSS with Triethylene glycol dimethacrylate : In vivo skin sensitization study:

In this valid dermal sensitisation study according to OECD Guideline 429 (2010), Triethylene glycol dimethacrylate (purity: 99.68%) in acetone:olive oil (4+1, v/v), groups of 5 female CBA/CaOlaHsd mice were tested using the LLNA method with the individual approach. No mortality or systemic findings were observed during the study period. Only the highest dose (100%) induced slight erythema on the ear skin on days 3 to 6 (Score 1).

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.

Stimulation indices (S.I.) of 1.40, 1.51 and 3.30 were determined with the test substance at concentrations of 25%, 50% and 100% (w/v) in vehicle, respectively. The positive control substance wasa-Hexylcinnamaldehyde, which gave an EC3 at 9.3 % (w/v). A result is regarded as positive when the S.I. is =3. Based on these criteria, the test substance was found to be a skin sensitiser. The EC3 was calculated to be 91.6 %. According to the Regulation EC n°1272 (CLP), Triethylene glycol dimethacrylate is classified as skin sensitizer, subcategory 1B (EC3 value > 2%).

 

7 Data interpretation procedure applied

The data interpretation procedure (DIP) is currently not designed to provide information on the potency of a sensitiser. The combination of test methods used covers the first three key events (KEs) of the adverse outcome pathway (AOP) leading to skin sensitisation as formally described by the OECD: KE 1: protein binding (e.g. via the direct peptide reactivity assay (DPRA); OECD TG 442C); KE 2: keratinocyte activation (e.g. via the KeratinoSensTM or LuSens assay; OECD TG 442D); and dendritic cell activation [e.g. via the human cell line activation test (h-CLAT); OECD TG 442E or the modified Myeloid U937 Skin Sensitisation Test (mMUSST)]. The prediction model entails that two concordant results obtained from methods addressing different steps of first three KEs of the AOP, determine the final classification.

 

8 Substances used to develop and test the DIP

The DIP is described in the OECD document (2012):http://www.oecd.org/officialdocuments/publicdisplaydocumentpdf/?cote=env/jm/mono(2012)10/part1&doclanguage=en

Performance and classifications derived from the “2 out of 3 - Sens ITS” of 213 substances were compared to both high quality animal and human data. Depending on the combination of tests used, the “2 out of 3 - Sens ITS” prediction model generally achieved accuracies slightly exceeding those of the murine local lymph node assay (LLNA) when compared to human data. These results compellingly verify the applicability of this easy to understand integrated testing approach (ITS) for a wide range of chemicals.

 

9 Predictive capacity of the approach

The DIP can be applicable to Tetraethylene glycol dimethacrylate.

The training set used to define the DIP includes both methacrylates: methyl methacrylate and ethylene glycol dimethacrylate. Both substances are similar to Tetraethylene glycol dimethacrylate.

 

10 Discussion and conclusion

The physical-chemical properties of Tetraethylene glycol diacrylate concluded that the substance is absorbed after dermal exposure. So, the skin sensitization potential of this substance should be evaluated as required by REACh regulation.

In absence of reliable skin sensitization data on Tetraethylene glycol diacrylate,an in Chemico Skin Sensitization: Direct Peptide Reactivity Assay (DPRA) performed according to the OECD test guideline (N°442c) onTetraethylene glycol dimethacrylate is used.The mean of the cysteine and lysine depletion was 39.3% and as a result Tetraethylene glycol dimethacrylate was considered to be positive in the DPRA.

Then, anIn Vitro Skin Sensitization: ARE-Nrf2 Luciferase Test Method performed onTetraethylene glycol dimethacrylate showed positive in the KeratinoSens assay since positive results (>1.5-fold induction) were observed at test concentrations < 1000µMwith a cell viability of >70% compared to the vehicle control.

In summary, Tetraethylene glycol dimethacrylate showed positive results for the Key Events 1 and 2 of the skin sensitization AOP developed by OECD. So, the substance is considered to be a skin sensitizer according to the “2 out of 3 - Sens ITS” rules.

However, no information is available on the potency of skin sensitization allowing to choose a sub-category of classification according to the Regulation EC n°1272/2008. As the Category 1A cannot be excluded based on in vitro/in chemico testing, additional information would need to be generated to strengthen the weight of evidence (ECHA Guidance R7a, 2017, page 277).

In the present case, an in vivo skin sensitization study is needed to evaluate the potency of skin sensitization.However, in order to avoid to perform a new animal study, the specific rules of adaptation (column 2 of Annexes VII to X) is used, specially the use of read-across.

ALocal Lymph Node Assay (LLNA) in mouse was performed on a structural analogue of Tetraethylene glycol diacrylate: triethylene glycol dimethacrylate.The test substance was found to be a weak skin sensitizer (category 1B) with an EC3 value of 91.6 %.

Based on the weight-of-evidence approach, Tetraethylene glycol diacrylate is classified as skin sensitizer, subcategory 1B according to the Regulation EC n°1272 (CLP).