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Administrative data

Description of key information

QSAR (OECD QSAR toolbox) profiling for skin sensitisation: no alert

In chemico Direct Peptide Reactivity Assay (OECD 442 C): negative

In vitro ARE-Nrf2 Luciferase assay (OECD 442 D): negative

In vivo Buehler (Read-across, similar to OECD 406): not sensitising

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
13 Dec 2016 - 11 Jan 2017
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Remarks:
Limited documentation
Qualifier:
according to guideline
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
adopted in 2015
Deviations:
yes
Remarks:
Limited data on materials and methods available (such as information on general materials, suppliers etc. are documented solely in the study data and not in the report);
GLP compliance:
yes
Type of study:
direct peptide reactivity assay (DPRA)
Details on the study design:
The test item was incubated for 24 h (± 2 h) at 25 ± 2.5 °C in solution at 100 mM in combination with either cysteine (96% purity) or lysine (98% purity) containing peptides and then run on an HPLC system (20-minute run-time) using gradient elution and UV detection at 220 nm to measure peptide concentration. The test item was compared to reference controls containing the test item solvent in combination with either cysteine or lysine peptide in order to determine the relative percent peptide depletion. Relative percent peptide depletion values were used in a prediction model to assign the test item to one of four reactivity classes.

Statistical method: Data analysis for this study was performed using the validated EURL-ECVAM analysis template (DPRA validated study template) available from the EURL-ECVAM website.
The template is a Microsoft Excel workbook containing formulae to process the raw data as per OECD TG 442C, it has been validated initially for internal use at XCellR8 during the DPRA proficiency study 16XC014. The final data output is a percentage peptide depletion value for the cysteine and lysine peptides after exposure to the test items. The validated template also assesses adherence to the acceptance criteria.



Positive control results:
Cinnamic aldehyde, CAS 104-55-2, 100 mM in HPLC Grade acetonitrile (CAS 75-05-8), freshly prepared on Day 1 of main testing
Key result
Run / experiment:
other: 1/2
Parameter:
other: Mean % peptide depletion (cysteine + lysine)
Value:
5.286
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 1/2
Parameter:
other: % cysteine peptide depletion
Value:
9.732
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 1/2
Parameter:
other: % lysine peptide depletion
Value:
0.84
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 3
Parameter:
other: % cysteine peptide depletion
Value:
12.541
Vehicle controls validity:
not valid
Negative controls validity:
not specified
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: not specified

DEMONSTRATION OF TECHNICAL PROFICIENCY: due to limited reporting details not specifiable

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for vehicle control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: due to limited reporting details not specifiable
- Range of historical values if different from the ones specified in the test guideline: due to limited reporting details not specifiable

ACCEPTANCE CRITERIA

Acceptance criteria for all controls and the test item were met in both runs with the exception of the RefC controls highlighted in bold. The cysteine peptide in the RefC controls was deemed to have been partially depleted by the acetone solvent used. However, as the test items were also solubilised in acetone the control remained valid for use as a reference for the test item depletion values. RefA and RefB controls in acetonitrile met all the acceptance criteria and therefore the cysteine runs were deemed valid. As acetone partially depleted the cysteine peptide, the positive control was compared to RefB controls in order to calculate percent depletion, as both the positive control and RefB were prepared in acetonitrile.

Criterion

Run 1 (Cysteine)

Run 2 (Lysine)

Run 3 (Cysteine Additional)

Outcome

Std Curve r2>0.99

0.993

0.997

0.991

PASSED

PC 60.8% to 100% depletion Cysteine

66.249

-

68.930

PASS

PC 40.2% to 69.0% depletion Lysine

-

68.210

-

PASS

SD Cysteine Depletion PC < 14.9 %

4.949

-

9.731

PASS

SD Lysine Depletion PC < 11.6 %

-

1.477

-

PASS

RefA Mean Conc 0.50 ±0.05 mM

0.536

0.508

0.504

PASS

Peak Area CV RefB < 15.0 %

8.615

3.008

12.934

PASS

Peak Area CV RefC < 15.0 %

3.520

0.773

19.876

PASS/PASS/FAILED

SD Cysteine Depletion Test ltem < 14.9 %

9.046

-

10.861

PASS

SD Lysine Depletion Test ltem < 11.6 %

-

0.734

-

PASS

RefC Mean Conc 0.50 ±0.05 mM

0.326

0.499

0.333

FAIL/PASS/FAIL

PC = positive control, SD = standard deviation, CV = coefficient of variation

RESULTS SUMMARY

The test item produced 5.286 % mean cysteine and lysine peptide depletion, therefore, using the cysteine 1:10 / lysine 1:50 prediction model, the test item was classified as a Non­ Sensitizer with No or Minimal Activity. A single HPLC analysis for the lysine peptide was considered sufficient for the test item as the result was unequivocal with this peptide. However, for the cysteine peptide an additional run was carried out as the first result generated was borderline. The second result was assessed using the cysteine 1:10 prediction model. This also produced a borderline result with the same outcome.

Run

Test item ID

% Cysteine Peptide Depletion

% Lysine Peptide Depletion

Mean % Peptide Depletion (Cys + Lys)

DPRA Prediction

DPRA

Reactivity Class

1/2

TEN0001

9.732

0.840

5.286

Non-Sensitizer

No or Minimal Reactivity

3

TEN0001

12.541

N/A

N/A

Non-Sensitizer

No or Minimal Reactivity

 N/A = not applicable

Interpretation of results:
other: The result of the DPRA performed with Reaction mass of 2-hydroxyethyl laurate and ethylene dilaurate was negative.
Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
05 - 13 Dec 2016
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Remarks:
Limited data on materials and methods (materials, cell culture media, purity of control substances, suppliers, expiry dates, passage number of cells are documented not in the report); Result values shown only as mean of triplicates, not for each repetition; no coefficient of variation for negative control. Additional bibliographic data source indicates that cell culture medium contained human serum to allow completely animal-product-free testing.
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
adopted in 2015
Deviations:
yes
Remarks:
Limited data in the study report; result values shown only as mean of triplicates, not for each repetition; no coefficient of variation for negative control.
Principles of method if other than guideline:
As mentioned in the free available bibliographic source, the testing laboratory obtained clarification from the European Chemicals Agency (ECHA) that data using the adapted method (animal product-free conditions) may be used in REACH submissions, provided that the Performance Standards data, demonstrating equivalence with the validated reference method, is included in the dossier.
GLP compliance:
yes
Type of study:
activation of keratinocytes
Details on the study design:
Skin sensitisation (In vitro test system) - Details on study design:

EXPERIMENTAL DESIGN
Day 1: Seeding cells (3 x 96-well plates for Luminescence; 1 x 96-well plate for MTT)
Day 2: 24h after seeding, application of test items and controls for 48 ± 2 h
Day 4: Evaluation of luciferase activity by luminescence (3 plates) and cell viability by MTT testing (1 plate)

TEST METHOD
The in vitro KeratinoSens™ assay enables detection of the sensitising potential of a test substance by addressing the second molecular key event of the Adverse Outcome Pathway (AOP), namely activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line KeratinoSens™. The luciferase activity, assessed by luminescence measurement, compared to the respective solvent controls is used to support discrimination between skin sensitizer and non-sensitizers.

TEST CELL LINE
- Type: KeratinoSens™
- Source: Givaudan, Switzerland

CELL CULTURE CONDITIONS
- Type and identity of media, culture conditions: Not stated in study report, according to the publication of testing laboratory, human serum was used (animal product-free conditions).

TEST CONCENTRATIONS
0.488, 0.977, 1.953, 3.906, 7.813, 15.625, 31.25, 62.5, 125, 250, 500, and 1000 μg/mL.
The highest non-cytotoxic concentration of the test item to human dermal fibroblasts was tested in a pre-test in the laboratory and determined at 250 µg/mL. A top test concentration of 1000 µg/mL was selected for this study, to ensure detection of sensitising potential at the maximum possible concentration.

CONTROLS
Vehicle control
- Substance: dimethyl sulfoxide (DMSO)
- Final concentration in cell culture medium: 1%
Positive control
- Substance: cinnamic aldehyde
- Tested concentrations in 1% DMSO containing cell culture medium: 8, 16, 32, 64, and 128 μM
Negative control


EXPOSURE CONDITIONS
- Exposure duration: 48 ± 2 h

NUMBER OF REPLICATIONS: 3 repetitions; each repetition included 3x96 well plates for luminescence and 1x96 well plate for MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide)) assay

DETERMINATION OF CELL VIABILITY
- Method: MTT assay
DETERMINATION OF LUMINESCENCE
- Luciferase activity

The KeratinoSens™ cell line (test system) is an immortalized adherent cell line derived from HaCaT human keratinocytes, stably transfected with a selectable plasmid containing the luciferase gene under the transcriptional control of the Anti-oxidant Response Element (ARE) from a gene that is known to be up-regulated by contact sensitisers. The luciferase signal reflects the activation by sensitisers of endogenous Nrf2 dependent genes, and the dependence of the luciferase signal in the recombinant cell line on Nrf2 has been demonstrated. This allows
quantitative measurement (by luminescence detection) of luciferase gene induction,using well established light producing luciferase substrates, as an indicator of the activity of the Nrf2 transcription factor in cells following exposure to electrophilic test substances.


Positive control results:
Cinnamic aldehyde was tested at concentrations of 8, 16, 32, 64 and 128 µM. Viability % was ≥ 97.99 % at all tested concentrations. Luciferase gene induction of ≥ 1.5 fold was seen at all tested concentrations ≥ 32 µM.
lmax was 7.026 at 128 µM and EC (1.5) was at 31.65 µM. One acceptance criterion was not met, as the average induction of cinnamic aldehyde at 32 µM was at 1.508 and therefore not in the historical range of 1.6 to 3 of the laboratory. However cinnamic aldehyde showed the expected dose-response curve of induction and therefore the test was considered acceptable.
Key result
Run / experiment:
other: Mean of 3 repetitions
Parameter:
other: EC(1.5) in µg/mL
Value:
21.01
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: Mean of 3 repetitions
Parameter:
other: lmax at 65.5 µg/mL
Value:
2.705
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
At concentrations of 31.25 and 62.5 µg/mL the test item caused luciferase induction ≥ 1.5. The EC1.5 value was calculated to be 21.01 µg/mL. The test item induced luciferase activity in a dose dependent manner. However, at the lowest concentration showing an induction value above 1.5 (31.25 µg/mL) viability was below 70 % so the test item was classified as a non-sensitizer in this test. Maximum induction (lmax) was observed at a concentration of 62.5 µg/mL, with an lmaxvalue of 2.705. For reference, during test validation sensitizing proficiency chemicals produced lmax values of up to 36-fold over untreated controls.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: CV% of blank values was 12.1% and therefore below 20%
- Acceptance criteria met for positive control: Yes, cinnamic aldehyde induced a greater than 1.5 fold induction in at least one concentration in a dose-dependent manner
- Range of historical values if different from the ones specified in the test guideline: see acceptence criteria under methods.

The used test item concentrations were selected on the basis of compatibility tests carried out prior to the study. The top non-cytotoxic concentration of Reaction mass of 2-Hydroxyethyl laurate
and ethylene dilaurate to human dermal fibroblasts was 250 μg/mL. A top test concentration of 1000 μg/mL was selected to ensure detection of sensitising potential at the maximum possible concentration.

Table 1: Induction values at different test item concentrations:

 Test item concentration (µg/mL]

0.488

0.977

1.953

3.906

7.813

15.625

31.25

62.5

125

250

500

1000

Mean of fold induction

0.848

0.982

0.999

0.933

1.125

1.291

1.897

2.705

0.735

0.015

0.001

-0.001

Standard deviation

0.156

0.185

0.169

0.160

0.171

0.206

0.312

0.601

0.771

0.017

0.004

0.003

Viability%

86.94

79.53

82.70

68.49

60.91

65.60

50.69

84.12

22.75

3.92

3.58

2.78

 

lmax : 2.705 at 65.5 µg/m  

EC1.5: 21.01 µg/mL

Induction values above 1.5 and viability results below 70% highlighted in bold

Interpretation of results:
other: The result of the Keratinosens test performed with Reaction mass of 2-hydroxyethyl laurate and ethylene dilaurate was negative.
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
Refer to analogue justification provided in IUCLID section 13.
Reason / purpose for cross-reference:
read-across source
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
100% moistened with few drops of distilled water
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
100% moistened with few drops of distilled water
No. with + reactions:
0
Total no. in group:
20
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
100% moistened with few drops of distilled water
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
100% moistened with few drops of distilled water
No. with + reactions:
0
Total no. in group:
20
Key result
Reading:
other:
Group:
positive control
Remarks on result:
not measured/tested
Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.
Conclusions:
The read across approach is justified in the analogue justification. The target and source substances are considered unlikely to differ in their skin sensitisation potential. In a Buehler test with the source substance ethylene distearate (CAS 627-83-8) no skin sensitising potential was found. Therefore, no skin sensitisation potential is expected for target substance Reaction mass of 2-hydroxyethyl laurate and ethylene dilaurate (EC 908-917-6).
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

In silico, in chemico and in vitro data on the skin sensitising potential of Reaction mass of 2-hydroxyethyl laurate and ethylene dilaurate were available. The assessment was further supported with data from an in vivo study conducted with a source substance as part of a read across approach, which is in accordance with Regulation (EC) No. 1907/2006, Annex XI, 1.5. Read-across is justified based on different compounds having the same type of effect(s) as described in scenario 2 of the Read-Across Assessment Framework (Read-Across Assessment Framework (RAAF), European Chemicals Agency, Helsinki, Finland, 2017. A detailed justification for the analogue read-across approach is provided in the technical dossier (see IUCLID Section 13).

In silico - QSAR

The two main components of Reaction mass of 2-hydroxyethyl laurate and ethylene dilaurate were profiled in the OECD QSAR Toolbox v4.1 in Nov 2017. Profiling for general mechanistic and endpoint specific protein binding alerts for skin sensitization did not trigger any alert for the two components regarding protein binding by OASIS, protein binding potency, protein binding by OECD and endpoint specific protein binding alerts for skin sensitization according to GHS or by OASIS. These data were not specified in a robust study summary, as only a short profiling check was done.

In chemico

A Direct Peptide Reactivity Assay (DPRA) was conducted according to OECD guideline 442C under GLP conditions (WoE, 2017). Reaction mass of 2-hydroxyethyl laurate and ethylene dilaurate was solved in acetone and tested at a concentration of 100 mM. After 24 h of incubation with both cysteine and lysine containing peptides, the percent peptide depletion was measured by High Performance Liquid Chromatography (HPLC). The test item was compared to reference controls containing the solvent in combination with either cysteine or lysine peptide in order to determine the relative percent peptide depletion. The cysteine peptide in one reference control was deemed to have been partially depleted by the acetone solvent used. However, as the test items were also solubilised in acetone, the control remained valid for use as a reference for the test item depletion values. The % cysteine peptide depletion was 9.732 and % lysine peptide depletion was 0.840. The mean % peptide depletion (cysteine + lysine) observed was 5.286% (a borderline result as per OECD guideline 442C). As the cysteine peptide led to the borderline result, an additional cysteine run was carried out. The % cysteine peptide depletion was found to be 12.541% in the additional run and therefore produced a borderline result. As the mean of cysteine and lysine % depletion was found to be < 6.38, Reaction mass of 2-hydroxyethyl laurate and ethylene dilaurate was judged to be a non-sensitizer with no or minimal reactivity as per the prediction model. The mean percent depletion value of the positive control substance cinnamic aldehyde was within the expected acceptance criteria. The result of the DPRA performed with Reaction mass of 2-hydroxyethyl laurate and ethylene dilaurate did was negative.

In vitro

An ARE-Nrf2 Luciferase assay was conducted according to OECD TG 442D under GLP conditions using an adapted method, which was accepted from the European Chemicals Agency (ECHA) to be used in REACH submissions (WoE, 2017). Reaction mass of 2 -hydroxyethyl laurate and ethylene dilaurate was dissolved in cell culture medium with 1% DMSO and tested at different concentrations. The highest non-cytotoxic concentration of the test item to human dermal fibroblasts was tested in a pre-test in the laboratory and determined to be 250 µg/mL. A top test concentration of 1000 µg/mL was selected to ensure detection of sensitising potential at the maximum possible concentration. At concentrations of 31.25 and 62.5 µg/mL the test item caused luciferase induction ≥ 1.5. The EC1.5 value was calculated to be 21.01 µg/mL. The test item induced luciferase activity in a dose dependent manner. However, at the lowest concentration showing an induction value above 1.5 (31.25 µg/mL) viability was below 70 %, so the test item was classified as a non-sensitizer in this test. Maximum induction (lmax) was observed at a concentration of 62.5 µg/mL, with an lmax value of 2.705. For reference, during test validation sensitizing proficiency chemicals produced lmax values of up to 36-fold over untreated controls, indicating validity of the test system. The negative vehicle control (DMSO at 1% in cell culture medium) was found to be valid. The result for Reaction mass of 2-hydroxyethyl laurate and ethylene dilaurate was negative (no indication for skin sensitisation potential) in this assay.

In vivo

CAS 627-83-8

The skin sensitising potential of the source substance ethylene distearate (CAS No. 627-83-8) was investigated. The study was performed according to a Buehler test protocol similar to OECD guideline 406 in guinea pigs (WoE, 1983). The solid test material was mixed with a few drops of water and applied at a concentration of 100% for epidermal induction and challenge under occlusive conditions in 20 animals. The negative control group (n=10) was treated with the vehicle only. No positive control data was included in the study report for reliability check. At 24, 48, and 72 hours after challenge, the neat test substance induced no skin effects in the test and negative control group. No skin reactions after induction and challenge were observed. Under the conditions of this study ethylene distearate was not skin sensitising.

Overall conclusion for skin sensitisation

No sensitising potential was indicated in experimental studies performed in vitro and in chemico. No skin sensitising potential was seen in an in vivo study performed with a source substance. Based on the available information, Reaction mass of 2-hydroxyethyl laurate and ethylene dilaurate is not expected to be skin sensitising.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

According to Article 13 of Regulation (EC) No. 1907/2006 "General Requirements for Generation of Information on Intrinsic Properties of substances", information on intrinsic properties of substances may be generated by means other than tests e.g. from information from structurally related substances (grouping or read-across), provided that conditions set out in Annex XI are met. Annex XI, "General rules for adaptation of this standard testing regime set out in Annexes VII to X” states that “substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group, or ‘category’ of substances. This avoids the need to test every substance for every endpoint". Since the analogue concept is applied to Reaction mass of 2-hydroxyethyl laurate and ethylene dilaurate data will be generated from data for reference source substance(s) to avoid unnecessary animal testing. Additionally, once the analogue read-across concept is applied, substances will be classified and labelled on this basis.

Therefore, the available data on skin sensitisation do not meet the classification criteria according to Regulation (EC) No. 1272/2008, and are therefore conclusive but not sufficient for classification.