Fatty acids, C18-unsatd., phosphates

Endpoint summary

Description of key information

In vitro (OECD 442D) (WoE): Inconclusive

GPMT (WoE): Skin sensitisation was studied using The Guinea Pig Maximization Test (GPMT) for the test substance. In this study the test substance was not shown to have sensitisation potential.

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

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)

GLP compliance:

yes

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

Identification: Fatty acids, C18-unsatd., phosphates.
Appearance: Yellow liquid
Batch: 0101891886
Purity/Composition: 100% UVCB
Test item storage: At room temperature
Stable under storage conditions until: 02 November 2017 (retest date)
Test Facility test item number: 208073/A
Chemical name (IUPAC, synonym or trade name: Phosphorylated fatty acid
CAS number: 68604-99-9
pH (1% in water, indicative range): 2.28 – 2.21 (determined by Charles RiverDen Bosch)
Specific gravity / density: 1.04
Solubility and stability of the test substance in the solvent/vehicle:
The test item was dissolved in DMSO to a final concentration of 40 mg/ml. The 100-fold dilution in DMEM of 40 mg/ml formed a non-homogeneous solution and was therefore not suitable to test. The 100-fold dilution of the 20 mg/ml DMSO stock formed a homogeneous solution (slight precipitation). This concentration was selected as highest concentration for the main assay.
Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not indicated

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
In the first and second experiment, the test item was dissolved in DMSO to a final concentration of 20 mg/ml. The test item formed a clear colourless solution at 20 mg/ml. 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 (final concentration DMSO of 4%). These solutions were diluted 4-fold in the assay resulting in final test concentrations of 200, 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39, 0.20 and 0.098 μg/ml (final concentration DMSO of 1%). All concentrations of the test item were tested in triplicate. No precipitate was observed at the start or end of the treatment period.

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:
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). 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 (i.e. 25) and are employed for routine testing using the appropriate maintenance medium. Cell Culture

Basic medium
Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.

Maintenance medium
Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum and geneticin (500 μg/ml).

Exposure medium
Dulbecco’s minimal supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.

Environmental conditions
All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 -100% (actual range 51–90%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0±1.0°C (actual range 36.2–37.5°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.

Subculturing
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.

Experimental Design

Plating of Cells
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 overnight in the incubator. The passage numbers used were 21, 24 and 20 in experiment 1, 2 and 3, respectively.

Treatment of Cells
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 3 experiments were performed.

Luciferase 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 200μL 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 luminometer to assess the quantity of luciferase (integration time one second).

Cytotoxicity Assessment
For the KeratinoSensTM cell viability assay, medium was replaced after the 48 hour exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1) 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 with the TECAN Infinite® M200 Pro Plate Reader.

Positive control results:

Experiment 1: The positive control EDMG caused a dose related induction of the luciferase activity.The Imax was 2.49 and the EC1.5 93.8 μM.
Experiment 2: The positive control EDMG caused a dose related induction of the luciferase activity.The Imax was 4.72 and the EC1.5 14.2 μM.
Experiment 3: The positive control EDMG caused a dose related induction of the luciferase activity.The Imax was 4.09 and the EC1.5 23.5 μM.

Key result

Run / experiment:

run/experiment 3

Parameter:

EC 1.5 [442D]

Value:

38.5 µg/mL

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

run/experiment 3

Parameter:

Imax [442D]

Value:

1.9 µg/mL

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

run/experiment 3

Parameter:

IC30 [442D]

Value:

41 µg/mL

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

run/experiment 3

Parameter:

IC50 [442D]

Value:

54.1 µg/mL

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no

ACCEPTANCE OF RESULTS:
The KeratinoSensTM test is considered acceptable if it meets the following criteria:
a)The luciferase activity induction obtained with the positive control, EDMG, should be above the threshold of 1.5 in at least one of the tested concentrations (from 7.81 to 250 μM).
b)The EC 1.5 should be between 5 and 125 μM. Moreover, the induction for EDMGat 250 μM should be higher than 2-fold. If the latter criterion is not fulfilled, the dose-response of EDMG 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.All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

Concentration (ug/ml)	0.098	0.20	0 .39	0.78	1.56	3.13	6.25	12.5	25	50	100	200
Exp1 luminescence	0,90	0,79	0,95	0,98	0,98	0,99	0,95	0,83	0,82	1,26	1,06	0,001
Exp 1 viability (%)	98,2	101,1	98,9	97,2	97,7	96,1	92,7	87,0	85,6	84,2	54,2	0,1
Exp 2 luminescence	0,96	1,02	1,02	1,17	1,05	1,09	1,00	1,12	1,32	4,01	0,20	0,00
Exp 2 viability (%)	94,2	106,0	115,9	133,4	126,1	123,3	113,4	120,9	105,0	112,8	4,8	0,1
Concentration (ug/ml)	17,2	21,5	26,8	33,6	41,9	52,4	65,5	81,9	102,4	128	160	200
Exp 3 luminescence	1,02	0,87	1,00	1,21	1,70	1,90	0,99	0,08	0,00	0,00	0,00	0,00
Exp 3 viability (%)	85,4	81,2	81,2	73,1	69,9	55,0	16,3	0,4	0,1	0,0	0,0	0,0

of the luciferase activity (no EC1.5 value) was measured. The maximum luciferase activity induction (Imax) was 1.26-fold.  

In the second experiment, a statistically significant induction of the luciferase activity (EC1.5 value 26.7μg/ml; p<0.001 Student’s t test) was measured.  The maximum luciferase activity induction (Imax) was 4.01-fold at 50 μg/ml.  The test item induced the luciferase activity very close to cytotoxic dose levels. Therefore, the test item was retested with more narrow dose-response analysis using a lower dilution factor (1.25 fold dilution between wells), to determine if induction has occurred at cytotoxic levels or not.

In the third experiment, a statistically significant induction of the luciferase activity (EC1.5 value 38.5 μg/ml; p<0.001 Student’s t test) was measured.The luciferase activity inductions were 1.70-and 1.90 fold at 41.9 μg/ml and 52.4 μg/ml, respectively.  However, these inductions are observed at cytotoxic dose levels with a cellular viability < 70% (69.6% and 55.0% at 41.9 μg/ml and 52.4 μg/ml, respectively). Therefore, no real positive effect was measured at any of the tested concentrations. Based on the EC 1.5(38.5 μg/ml) and IC30(41.0μg/ml) values, the test item is positive in the KeratinoSensTM assay, although the values are very close together. Overall, the results of the third experiment are not clearly positive or negative and therefore inconclusive.

Three experiments were performed all with different conclusions. Due to the fact that induction of the luciferase activity was very close to the cytotoxic levels, it is not possible to make a final conclusion.

Interpretation of results:

study cannot be used for classification

Conclusions:

In conclusion, Fatty acids, C18-unsatd., phosphates. the result of the test is inconclusive under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate the ability of Fatty acids, C18-unsatd., phosphates.to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSensTM assay.

The study procedures described in this report were based on the most recent OECD guideline.

Batch 0101891886 of Fatty acids, C18-unsatd., phosphates. was a yellow liquid.  The test item was dissolved in dimethyl sulfoxide at 20 mg/ml. 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 0.098–200μg/ml(2-fold dilution series)in the first and second experiment and 17.2 –200 μg/ml(1.25-fold dilution series)in the third experiment.  

Both experiments 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 (93.8μM, 14.2μM and 23.5μM in experiment 1, 2 and 3, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (2.49-fold, 4.72-fold and 4.09-fold in experiment 1, 2 and 3,respectively).

- Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (17.5%,9.8% and 8.2% in experiment 1, 2 and 3,respectively).

Overall it is concluded that the test conditions were adequate and that the test system functioned properly.  

The test item showed toxicity (IC30 values of 73.7, 69.8 and 41.0 in experiment 1, 2 and 3,respectively, and IC50 values of 107.8, 79.1and 54.1 in experiment 1, 2 and 3,respectively).  

In the first experiment, no induction of the luciferase activity (no EC1.5 value) was measured. The maximum luciferase activity induction (Imax) was 1.26-fold.  

In the second experiment, a statistically significant induction of the luciferase activity (EC1.5 value 26.7μg/ml; p<0.001 Student’s t test) was measured.  The maximum luciferase activity induction (Imax) was 4.01-fold at 50 μg/ml. The test item induced the luciferase activityvery close to cytotoxic dose levels. Therefore, the test item was retested with more narrow dose-response analysis using a lower dilution factor (1.25 fold dilution between wells), to determine if induction has occurred at cytotoxic levels or not.  

In the third experiment, a statistically significant induction of the luciferase activity (EC1.5 value 38.5μg/ml; p<0.001 Student’s t test) was measured.The luciferase activity inductions were 1.70-and 1.90 fold at 41.9 μg/ml and 52.4 μg/ml, respectively.  However, these inductions are observed at cytotoxic dose levels with a cellular viability < 70% (69.6% and 55.0% at 41.9 μg/ml and 52.4 μg/ml, respectively).Therefore, no real positive effect was measured at any of the tested concentrations. Based on the EC1.5(38.5 μg/ml)and IC30(41.0μg/ml) values, the test item is positive in the KeratinoSensTM assay, although the values are very close together. Overall, the results of the third experiment are not clearly positive or negative and therefore inconclusive.

Three experiments were performed all with different conclusions. Due to the fact that induction of the luciferase activity was very close to the cytotoxic levels, it is not possible to make a final conclusion.

Reference 2

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

23 June 2017 - 22 Sep 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The guinea pig Maximization test was selected since the test item is a fatty acid and the Local Lymph Node Assay as preferred alternative has shown to provide false positive results for fatty acids.
Ref. Comparison of the skin sensitizing potential of unsaturated compounds as assessed by the murine local lymph node assay (LLNA) and the guinea pig maximization test (GPMT), Food and chemical Toxicology 46 (2008) 1896–1904

Specific details on test material used for the study:

Identification: Fatty acids, C18-unsatd., phosphates.
Appearance: Yellow liquid
Batch: 0101891886
Purity/Composition: 100% UVCB
Test item storage: At room temperature
Stable under storage conditions until: 02 November 2017 (retest date)
Test Facility test item number: 208073/A
Chemical name (IUPAC, synonym or trade name: Phosphorylated fatty acid
CAS number: 68604-99-9
pH (1% in water, indicative range): 2.28 – 2.21 (determined by Charles RiverDen Bosch)
Specific gravity / density: 1.04

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River France, L’Arbresle, France
- Age at study initiation: Young adult animals (approximately 4 to 7 weeks old) were selected.
- Weight at the initiation of dosing: 279 to 482 g
- Housing: On arrival and following assignment to the study, animals were group housed (up to 5 animals of the same sex and same dosing group together) in labeled Noryl cages (Tecniplast;74 cm x 54 cm x 25 cm height) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles.
- Diet (e.g. ad libitum):Complete maintenance diet for guinea pigs (MS-H, SSNIFF® Spezialdiäten GmbH, Soest,Germany) was provided ad libitum
- Municipal tap-water was freely available to each animal via water bottles.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.0 - 23.0 °C
- Humidity (%): 49 - 70 %
- Air changes (per hr): over 10 air changes / hour
- Photoperiod (hrs dark / hrs light): 12/12

Route:

intradermal

Vehicle:

corn oil

Concentration / amount:

0.2%

Day(s)/duration:

7

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Route:

epicutaneous, occlusive

Vehicle:

corn oil

Concentration / amount:

2%

Day(s)/duration:

2

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

corn oil

Concentration / amount:

2% (0.1 ml)

Day(s)/duration:

1

No. of animals per dose:

RANGE FINDING TEST
Epidermal exposure: 8 females, 8 doses tested
Intradermal exposure : 4 females; 2 doses per animal; 8 doses tested

MAIN STUDY
Experimental group: 10 females
Control group : 5 females

Details on study design:

RANGE FINDING TESTS:
A preliminary irritation study was conducted in order to select test item concentrations to be used in the main study. The selection of concentrations was based on the following criteria:
• The concentrations are well-tolerated systemically by the animals.
• For the induction exposures: the highest possible concentration that produced mild to moderate irritation (grades 2 - 3).
• For challenge exposure: the maximum non-irritant concentration.

The starting- and subsequent concentrations were taken from the series: 100% (undiluted), 50%, 20%, 10%, 5%, 2%, 1%, 0.5%, 0,2%

Intradermal injections:
Initially, a series of four test item concentrations was tested; the highest concentration was the maximum concentration that could technically be injected. Each of two animals received two different concentrations in duplicate (0.1 mL/site) in the clipped scapular region. The resulting dermal reactions were assessed 24 and 48 hours after treatment. Based on the results in the initially treated animals, two additional animals were treated in a similar manner with four lower concentrations at a later stage.

Epidermal application:
A series of four test item concentrations was tested; the highest concentration being the maximum concentration that could technically be applied. Two different concentrations were applied (0.5 mL each) per animal to the clipped flank, using Metalline patches# (2x3 cm) mounted on Medical tape, which were held in place with Micropore tape and subsequently coban elastic bandage. The initially used animals receiving intradermal injections were treated with the lowest concentrations and two other animals with the highest concentrations. After 24 hours, the dressing was removed and the skin cleaned of residual test item using water.

Based on the results, the test item concentrations selected for the main study were a 0.2% concentration for the intradermal induction and a 2% concentration for the epidermal induction exposure.
A 2% test item concentration was selected for the challenge phase.

MAIN STUDY
Induction-experimental animals
The concentrations and induction method were selected based on the results of the preliminary irritation study.

Day 1 The scapular region was clipped and three pairs of intradermal injections (0.1 mL/site) were made in this area as follows:
A) A 1:1 w/w mixture of Freunds' Complete Adjuvant (Sigma-Aldrich, Steinheim, Germany) with water for injection (B.Braun Melsungen AG, Melsungen. Germany).
B) The test item at a 0.2% concentration.
C) A 1:1 w/w mixture of the test item, at twice the concentration used in (B) and Freunds' Complete Adjuvant.
Day 3 The dermal reactions caused by the intradermal injections were assessed for irritation.
Day 8 The scapular area between the injection sites was clipped and subsequently treated with 0.5 mL of a 2% test item concentration using a Metalline patch (2x3cm) mounted on Medical tape, which was held in place with Micropore tape and subsequently Coban elastic bandage. The dressing was removed after 48 hours exposure, the skin cleaned of residual test item using water and the dermal reactions caused by the epidermal exposure were assessed for irritation.

Induction - Control animals
The control animals were treated as described for the experimental animals except that, instead of the test item, the vehicle was administered.

Challenge - All animals
Day 21 One flank of all animals was clipped and treated by epidermal application of a 2% test item concentration and the vehicle (0.1 mL each), using Patch Test Plasters (Curatest F®, Lohmann, Almere, The Netherlands). The patches were held in place with Micropore tape and subsequently Coban elastic bandage. The dressing was removed after 24 hours exposure and the skin cleaned of residual test item and vehicle using water. The treated sites were assessed for challenge reactions 24 and 48 hours after removal of the dressing.

Challenge controls:

In the control animals no visible skin reactions were observed following challenge with test item at a concentration of 2 % (w/v) in Corn oil at the 24 and 48 hours examination

Positive control substance(s):

yes

Positive control results:

The skin reactions observed in three experimental animals in response to the 20% Alpha- Hexylcinnamaldehyde concentration in the challenge phase were considered indicative of sensitisation, based on the absence of any response in the control animals. These results lead to a sensitisation rate of 33 percent to the 20% concentration. From these results, it was concluded that the female guinea pig of the Dunkin Hartley strain is an appropriate animal model for the performance of studies designed to evaluate the sensitizing potential of a test item in a Maximization type of test.

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

2 % (w/v)

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

There were no overt signs of an adverse clinical response to treatment with the test item during the course of the study.

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

2 % (w/v)

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

There were no overt signs of an adverse clinical response to treatment with the test item during the course of the study.

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

2 % (w/v)

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

There were no overt signs of an adverse clinical response to treatment with the test item during the course of the study.

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

2 % (w/v)

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

There were no overt signs of an adverse clinical response to treatment with the test item during the course of the study.

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

20 % Alpha-hexylcinnamaldedyde

No. with + reactions:

3

Total no. in group:

9

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

20 % Alpha- Hexylcinnamaldehyde

No. with + reactions:

1

Total no. in group:

9

There were no deaths. No signs of systemic toxicity were noted in the animals during the test. There were no notable differences in bodyweight changes between the test animal group and the control group.

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: expert judgment

Conclusions:

Sensitisation potential of of test item was studied in the guinea pig using the Magnusson and Kligman Method. Challenge with test item Fatty acids, C18-unsatd., phosphates. evoked no positive responses in the test animals sensitised previously with the test item or with control item. Under the conditions of the present assay the test item was shown to have no sensitisation potential.

Executive summary:

The objective of this study was to evaluate whether Fatty acids, C18-unsatd., phosphates. induces contact hypersensitivity in guinea pigs after intradermal and epidermal exposure

Test item concentrations selected for the main study were based on the results of a preliminary study. In the main study, ten experimental animals were intradermally injected with a 0.2% concentration and epidermally exposed to a 2% concentration. Five control animals were similarly treated, but with vehicle alone (corn oil). Two weeks after the epidermal application all animals were epidermally challenged with a 2% test item concentration and the vehicle.

Test group.

After the challenge with the test item at a concentration of 2% (w/v) in corn oil, no positive response was observed in the treated animals. This result indicates a sensitization rate of 0 per cent according to the 24 and 48 -hours results.

Control group.

After the challenge with the test item at a concentration of 2% (w/v) in corn oil no visible changes were found at the 24 and 48 hours examinations.

There was no evidence that Fatty acids, C18-unsatd., phosphates. had caused skin hypersensitivity in the guinea pig, since no responses were observed in the experimental animals in response to a 2% test item concentration in the challenge phase. This result indicates a sensitization rate of 0 per cent. Based on these results Fatty acids, C18-unsatd., phosphates. does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments) and EC criteria for classification and labelling requirements for dangerous items and preparations (Council Directive 67/548/EEC) (including all amendments).

This study was regarded reliable without restrictions since the the study was conducted according to the OECD 406 guideline and in compliance with GLP.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Weight of evidence approach was selected to assess the sensitisation potential of the substance.

 

In vitro study

 

In vitro study was conducted according the OECD 442D guideline by Westerink, W.M.A. (2017). 

The objective of this study was to evaluate the ability of Fatty acids, C18-unsatd., phosphates.to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSensTM assay.

The test item was dissolved in dimethyl sulfoxide at 20 mg/ml. 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 0.098–200μg/ml(2-fold dilution series)in the first and second experiment and 17.2 –200 μg/ml(1.25-fold dilution series)in the third experiment. 

Both experiments 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 125μM (93.8μM, 14.2μM and 23.5μM in experiment 1, 2 and 3, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (2.49-fold, 4.72-fold and 4.09-fold in experiment 1, 2 and 3,respectively).

- Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (17.5%,9.8% and 8.2% in experiment 1, 2 and 3,respectively).

 

Overall it is concluded that the test conditions were adequate and that the test system functioned properly. 

The test item showed toxicity (IC30 values of 73.7, 69.8 and 41.0 in experiment 1, 2 and 3,respectively, and IC50 values of 107.8, 79.1and 54.1 in experiment 1, 2 and 3,respectively). 

 

In the first experiment, no induction of the luciferase activity (no EC1.5 value) was measured. The maximum luciferase activity induction (Imax) was 1.26-fold. 

 

In the second experiment, a statistically significant induction of the luciferase activity (EC1.5 value 26.7μg/ml; p<0.001 Student’s t test) was measured. The maximum luciferase activity induction (Imax) was 4.01-fold at 50 μg/ml. The test item induced the luciferase activityvery close to cytotoxic dose levels. Therefore, the test item was retested with more narrow dose-response analysis using a lower dilution factor (1.25 fold dilution between wells), to determine if induction has occurred at cytotoxic levels or not. 

 

In the third experiment, a statistically significant induction of the luciferase activity (EC1.5 value 38.5μg/ml; p<0.001 Student’s t test) was measured.The luciferase activity inductions were 1.70-and 1.90 fold at 41.9 μg/ml and 52.4 μg/ml, respectively. However, these inductions are observed at cytotoxic dose levels with a cellular viability < 70% (69.6% and 55.0% at 41.9 μg/ml and 52.4 μg/ml, respectively).Therefore, no real positive effect was measured at any of the tested concentrations. Based on the EC1.5(38.5 μg/ml)and IC30(41.0μg/ml) values, the test item is positive in the KeratinoSensTM assay, although the values are very close together. Overall, the results of the third experiment are not clearly positive or negative and therefore inconclusive.

 

Three experiments were performed all with different conclusions. Due to the fact that induction of the luciferase activity was very close to the cytotoxic levels, it is not possible to make a final conclusion.

 

GPMT

 

The skin sensitisation potential of the target substance was assessed using The guinea pig Maximization test (OECD 406) (van Sas, P.H.T., 2017). The guinea pig Maximization test was selected since the test item contains fatty acid and the Local Lymph Node Assay as preferred alternative has shown to provide false positive results for fatty acids.

Test item concentrations selected for the main study were based on the results of a preliminary study. In the main study, ten experimental animals were intradermally injected with a 0.2% concentration and epidermally exposed to a 2% concentration. Five control animals were similarly treated, but with vehicle alone (corn oil). Two weeks after the epidermal application all animals were epidermally challenged with a 2% test item concentration and the vehicle.

Test group.

After the challenge with the test item at a concentration of 2% (w/v) in corn oil, no positive response was observed in the treated animals. This result indicates a sensitization rate of 0 per cent according to the 24 and 48 -hours results.

Control group.

After the challenge with the test item at a concentration of 2% (w/v) in corn oil no visible changes were found at the 24 and 48 hours examinations.

There was no evidence that Fatty acids, C18-unsatd., phosphates. had caused skin hypersensitivity in the guinea pig, since no responses were observed in the experimental animals in response to a 2% test item concentration in the challenge phase. The guinea pig Maximization test result indicates a sensitization rate of 0 per cent.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The substance is not classified for skin sensitisation according to the CLP Regulation No. 1272/2008.