Octan-1-ol, ethoxylated

Endpoint summary

Description of key information

Skin sensitisation (in vitro, h-CLAT, OECD 442E): negative

Skin sensitisation (in vitro, KeratinoSens, OECD 442D): positive for keratinocyte activation

The data generated with these in vitro methods is not sufficient to conclude on the skin sensitisation potential of octan-1-ol, ethoxylated (CAS No. 27252-75-1, EC No. 500-058-1) and should be considered in the context of an integrated approach to testing and assessment (IATA). However, according to the REACH Regulation (EC) No. 1907/2006, Annex VII, Section 8.3, Column 2, no further testing is warranted as the substance is classified for skin corrosion (Category 1).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

03 - 20 Dec 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 442E (in vitro Skin Sensitisation: human Cell Line Activation Test)

Version / remarks:

adopted 20 Jul 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Medicines and Healthcare Products Regulatory Agency, Department of Health, London, United Kingdom

Type of study:

activation of dendritic cells

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Details on the study design:

TEST METHOD:
The in vitro human Cell Line Activation Test (h-CLAT) is an alternative testing method for the evaluation of the skin sensitization potential of a test compound. It quantifies phenotypic changes, such as cell surface marker expression in cell lines following 24 h treatment with chemicals. The human leukemia cell line THP-1 is used as surrogate for human myeloic dendritic cells, which show enhanced CD86 and CD54 surface protein expression when treated with sensitiziers.
The dose for the h-CLAT assay was determined in two preliminary cytotoxicity test, yielding 75% cell viability (CV75). For the main assay, THP-1 cells were incubated for 24 ± 1 hours at 37 °C with the test compound, as well as the negative and positive controls. Changes of CD86 and CD54 expression were analyzed by flow cytometry, using fluorescently labelled antibodies against the two surface proteins. Relative fluorescence intensities compared to solvent controls are calculated and used in a prediction model to discriminate between sensitizing and non-sensitizing compounds.

TESTS SUBSTANCE PREPARATION:
The test item was dissolved in culture medium.

CONCENTRATIONS:
Pre-experimental dose-finding study: 5000, 2500, 1250, 625, 312.5, 156.25, 78.13, 39.06 µg/mL
Main experiment (h-CLAT): based on the results obtained in the pre-experimental dose-finding study: 595.25, 496.04, 413.37, 344.47, 287.06, 239.22, 199.35, 166.12 µg/mL.

VEHICLE CONTROL: Complete Roswell Park Memorial Institute (RPMI) culture medium containing 10% Human Serum and 0.05 mM 2-mercaptoethanol
POSITIVE CONTROL CV75: 2,4-Dinitrochlorobenzene (DNCB) prepared as 8 µg/mL in DMSO
POSITIVE CONTROL CD54 and CD86 expression: Nickel Sulphate prepared as 100 µg/mL in RPMI medium

TEST CELL LINE: THP-1 cells
- Source: ATCC, #TIB-202

CELL CULTURE CONDITIONS:
- Type and identity of media: RPMI supplemented with 10% Human Serum and 0.05 mM 2-mercaptoethanol

EXPOSURE CONDITIONS:
- Method of application: in medium
- Exposure duration: 24 ± 0.5 h

NUMBER OF REPLICATES: Each concentration was tested in two independent runs

DETERMINATION OF CYTOTOXICITY:
- Method: Propidium iodide, 24 ± 0.5 h exposure with test item, two independent experiments
- Determination of cell viability (= relative aborbance) for calculation of the CV75, which corresponds to the concentration needed to reduce the relative absorbance to 75% of the solvent control.

DETERMINATION OF FLUORESCENCE:
- Flow cytometry
- Antibodies: fluorochrome-tagged CD86 and CD54

Positive control results:

Relative fluorescence intensities first experiment:
100 µg/mL: CD54 = 212% (93.69% viability), CD86 = 152% (93.04% viability)

Relative fluorescence intensities, second experiment:
100 µg/mL: CD54 = 377% (88.95% viability), CD86 = 210% (86.58% viability)

Run / experiment:

other: 24 h incubation

Parameter:

other: RFI in % for CD54 in µg/mL

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: in 2/2 independent experiment data

Run / experiment:

other: 24 h incubation

Parameter:

other: RFI in % for CD86 in µg/mL

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: in 2/2 independent experiment data

Other effects / acceptance of results:

- Acceptance criteria met for CV75 determination: Yes, cell viability is ≥ 75% at the lowest dose and the highest test item concentration produces cytotoxicity (< 90% cell viability)
- Acceptance criteria met for negative control: yes, medium and solvent control RFI values do not exceed the positive criteria CD86 ≥ 150% and CD54 ≥ 200% and cell viability is > 90%
- Acceptance criteria met for positive control: yes, RFI values CD86 ≥ 150% and CD54 ≥ 200% and cell viability is > 50%

Table 1: Results of the h-CLAT test, first experiment:

Test Item Dose [µg/mL)	Cell Viability (%)			Average cell viability	CD54 RFI	CD86 RFI
	Isotype	CD54	CD86
595.25	55.95	60..92	58.46	58.44	-335	-871
496.04	51.28	53.04	53.36	52.56	-239	-625
413.37	57.3	52.62	53.8	54.57	-145	-425
344.47	60.74	55.92	56.7	57.79	135	120
287.06	71.13	65.12	65.53	67.26	124	126
239.22	78.66	75.32	75.64	76.54	114	105
199.35	86.92	83.85	82.52	84.43	87	87
166.12	85.81	85.22	85.14	85.39	82	65

Table 2: Results of the h-CLAT test, second experiment:

Test Item Dose [µg/mL)	Cell Viability (%)			Average cell viability	CD54 RFI	CD86 RFI
	Isotype	CD54	CD86
595.25	66.59	62.05	61.5	63.38	131	141
496.04	25.18	24.39	25.36	24.98	180	3
413.37	23.39	24.96	25.42	24.59	81	-236
344.47	81.07	84.13	85.74	83.64	2073*	2085*
287.06	42.93	45.66	38.86	42.48	191	173
239.22	64.97	63.79	61.91	63.56	140	81
199.35	81.3	80.15	79.64	80.36	116	89
166.12	89.72	88.53	87.3	88.51	67	90
* artefact, large amount of cell debris (and not live cells) that were picked up by the flow cytometer

Interpretation of results:

other: negative in the hCLAT

Conclusions:

The data generated with this method may not be sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of an integrated approach such as integrated approaches to testing and assessment (IATA).

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

10 - 17 Dec 2018

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:

adopted 05 Feb 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Medicines and Healthcare Products Regulatory Agency, Department of Health, London, United Kingdom

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Details on the study design:

TEST CELL LINE
- Cell type: HaCaT cells (human keratinocytes)
- Source: Givaudan, Switzerland
- Passage number: 20

TEST METHOD
The in vitro KeratinoSensTM assay enables the detection of the skin sensitizing potential of a test item by analyzing the activation of keratinocytes. This activation step represents the second molecular key event of the adverse outcome pathway, which is the induction of cyto-protective signaling pathways in keratinocytes in response to electrophiles and oxidative stress. The KeratinoSense assay addresses the effect on the antioxidant response element (ARE) Nrf2-dependent pathway in the transgenic KeratinoSens cell line, which stably expresses the ARE-Nrf2-dependet luciferase gene. The Nrf2-dependent induction of this reporter gene is analysed upon exposure to test chemicals. Luminescence detection in the cell lysate after 48 hours of exposure at 37 °C indicates luciferase induction and allows the discrimination between skin sensitisers and non-sensitisers.

TEST SUBSTANCE PREPARATION
The test item was dissolved in 1% DMSO in cell culture medium. Per plate, a single application of 12 concentrations of the test item was applied in cell culture medium (dilution factor 2) with a final concentration of DMSO of 1%.

CONCENTRATIONS:
0.195, 0.391, 0.781, 1.563, 3.125, 6.25, 12.5, 25, 50, 100, 200 and 44 µg/mL

CONTROLS:
NEGATIVE CONTROL: Dimethyl sulfoxide (DMSO), Fisher Scientific; Lot no. 1743585, purity 99.97% at a final concentration of 1% (v/v) in test item exposure medium. The negative control is actually a vehicle control.
POSITIVE CONTROL: Cinnamic aldehyde, Sigma; Lot no. STBG0250V, purity ≥ 99% used at a final concentration range of 8 - 128 µM. The final DMSO concentration was 1% (v/v).

CELL CULTURE CONDITIONS: CULTIVATION
- Temperature (°C): 37
- CO2 (%): 5

NUMBER OF REPLICATIONS: triplicates per individual run in 3 independent experiments
EXPERIMENTAL PROCEDURE:
Cells were seeded 10,000 cells / well in a 96-well format and grown for 24 h in assay medium. Thereafter, the test and control items were applied and cells exposed for 48 ± 2 h. After the exposure period, luciferase activity was evaluated by luminescence measurement and cell viability was determined using the MTT viability assay.

EXPOSURE: 48 ± 2 h at 37 °C

Positive control results:

Cinnamic aldehyde was tested as positive control in a concentration range of 8 - 128 µM. Cell viability was in the range of the required acceptability criterion of > 70% at all test item concentrations.
Luciferase activity increased dose-dependently and reached an > 1.5 fold induction at a concentration of 64 µM and 128 µM, which fits well in the required acceptability criterion of > 1.5 fold induction in at least one of the tested concentrations.

Run / experiment:

other: 48 h exposure / Experiment 1

Parameter:

other: EC1.50

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: 48 h exposure / Experiment 2

Parameter:

other: EC1.50

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: 48 h exposure / Experiment 3

Parameter:

other: EC1.50

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Technical proficiency of the assay was demonstrated by the testing facility using the 10 proficiency chemicals listed in OECD 442D and the 11 additional chemicals listed in the associated performance standards.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for positive control:
1. Yes, the luciferase activity induction of the positive control was > 1.5 in at least one of the tested concentrations and statistically significant compared to the solvent (negative) control (p < 0.05).
2. No, the average induction (Imax) in the 3 replicates for the positive control at a concentration of 32 µM was not within the historical range (between 1.6 and 3) but slightly below. Because all other acceptance criteria were met and because a dose-dependent increase of induction was observed with the positive control, the results were considered valid.
- Acceptance criteria met for variability between replicate measurements: Yes, the average coefficient of variation (CV) of the luciferase activity for the negative/blank control DMSO was < 20% in each repetition.

Table 1: Sensitisation potential of the test item: Experiment 1:

Rep 1	Test item concentration (µM)
	0.195	0.391	0.781	1.563	3.125	6.250	12.5	25	50	100	200	400
Mean fold induction	1.010	1.106	1.216	1.214	1.449	1.450	1.647	2.025	2.329	2.639	2.822	0.019
Viability %	123.398	128.114	125.049	133.376	129.084	136.833	139.741	146.626	201.962	194.370	184.020	1.387
T-test	9.18E-01	2.88E-01	3.90E-02	3.60E-02	2.82E-05	3.04E-05	7.07E-08	2.74E-14	1.91E-18	3.34E-20	2.80E-22	5.92E-14
SD	0.096	0.104	0.197	0.115	0.031	0.096	0.246	0.153	0.201	0.425	0.422	0.020
IMAX	2.822 at 200 µg/ml
EC1.5	7.831 µg/ml
IC30	324.862 µg/ml
IC50	346.764 µg/ml

Table 2: Sensitisation potential of the test item: Experiment 2:

Rep 2	Test item concentration (µM)
	0.195	0.391	0.781	1.563	3.125	6.250	12.5	25	50	100	200	400
Mean fold induction	0.904	0.924	1.002	1.058	1.144	1.291	1.516	2.225	2.152	2.482	2.695	-0.003
Viability %	111.219	113.219	118.417	126.476	128.868	127.370	145.925	178.329	206.597	199.354	185.482	1.408
T-test	3.32E-01	4.43E-01	9.84E-01	5.62E-01	1.58E-01	5.64E-03	7.12E-06	7.11E-10	8.28E-16	1.07E-20	2.05E-23	2.59E-14
SD	0.057	0.073	0.069	0.107	0.158	0.168	0.249	0.954	0.221	0.166	0.152	0.002
IMAX	2.695 at 200 µg/ml
EC1.5	12.052 µg/ml
IC30	325.474 µg/ml
IC50	347.204 µg/ml

Table 3: Sensitisation potential of the test item: Experiment 3:

Rep 3	Test item concentration (µM)
	0.195	0.391	0.781	1.563	3.125	6.250	12.5	25	50	100	200	400
Mean fold induction	0.888	1.008	1.174	1.418	1.409	1.606	1.920	2.230	2.710	2.883	3.399	0.000
Viability %	110.615	108.461	107.697	106.877	107.546	113.732	112.337	118.580	141.714	149.367	129.327	1.927
T-test	2.63E-01	9.37E-01	8.23E-02	9.34E-05	1.41E-04	1.41E-07	2.25E-12	2.17E-17	1.35E-22	1.02E-25	6.50E-29	2.90E-14
SD	0.094	0.072	0.019	0.103	0.131	0.152	0.212	0.144	0.280	0.123	0.344	0.008
IMAX	3.399 at 200 µg/ml
EC1.5	4.571 µg/ml
IC30	293.135 µg/ml
IC50	324.532 µg/ml

Interpretation of results:

other: positive for kerationcyte activation

Conclusions:

The data generated with this method may not be sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of an integrated approach such as integrated approaches to testing and assessment (IATA).

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

The skin sensitising potential of octan-1-ol, ethoxylated (CAS No. 27252-75-1, EC No. 500-058-1) was assessed using the in vitro human Cell Line Activation Test (h-CLAT) according to OECD guideline 442E under GLP conditions (Croda, 2019b). After 24 ± 0.5 h incubation with the test item, the expression of the cell surface markers CD54 and CD86 as indicators of dendritic cell activation was quantified by flow cytometry. The test substance dose that gave 75% cell viability was found to be 496.04 µg/mL. In either experiment at non-cytotoxic concentrations the threshold that determines a positive result for CD54 and CD86 expression was not reached, therefore the test item was considered negative for activation of dendritic cells under the experimental conditions of the test.

In a further in vitro investigation, the skin sensitising potential was assessed using the KeratinoSens in vitro assay according to OECD guideline 442D and observing GLP conditions (Croda, 2019c). After 48 h of exposure to test item concentrations of 0.195 to 400 µM, Luciferase measurements and MTT viability testing were performed. Under the experimental conditions of the study, the test item was found positive for keratinocyte activation in three independent experimental runs (Imax 2.82, 2.695 and 3.399 and EC1.5 7.831, 12.052 and 4.571).

 

Conclusion

The data generated with the in vitro methods is not sufficient to conclude on the skin sensitisation potential of octan-1-ol, ethoxylated (CAS No. 27252-75-1, EC No. 500-058-1) and should be considered in the context of an integrated approach to testing and assessment (IATA). Further testing would be necessary to unambiguously conclude on the potential to induce skin sensitisation in humans. However, due to the confirmation of skin corrosive properties in an appropriate in vitro study (Croda, 2019a), the substance is classified as Skin Corr. 1 and Eye Damage 1 (H314) and further testing for skin sensitisation is not required according to Annex VII, Section 8.3, Column 2, of Regulation (EC) No. 1907/2006 (REACH). Due to the corrosive properties it is considered inadequate to read-across data on skin sensitisation from the data pool of the Alcohol Ethoxylates (AE) category. For a detailed evaluation of the skin sensitisation potential of the substances in the AE category, please refer to the category justification attached to the category object.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The available data on skin sensitisation of octan-1-ol, ethoxylated (CAS No. 27252-75-1, EC No. 500-058-1) are not sufficient to conclude on the skin sensitisation potential of the substance. No further testing is warranted as the substance is classified for skin corrosion (Category 1). Since no further data are required for octan-1-ol, ethoxylated (CAS No. 27252-75-1, EC No. 500-058-1), the substance does not need to be classified according to the criteria of the CLP Regulation (EC) No. 1272/2008.