Phosphoric acid, mono- and di-C12-18-(even numbered)-alkyl esters, sodium salts

Endpoint summary

Description of key information

Based on the results of read across guinea pig maximisation tests, the test substance is not considered to be a skin sensitiser.

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:

supporting study

Study period:

From April 20, 2017 to May 16, 2017

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for the Testing of Chemicals No. 442E: In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)

Version / remarks:

OECD (2016)

Deviations:

not specified

GLP compliance:

no

Type of study:

other: Changes in CD86 cell surface marker expression on a human histiocytic lymphoma cell line, U937 cells

Details on the study design:

PRINCIPLE OF THE STUDY: The h-CLAT assay measures markers of dendritic cell (DC) activation in the human leukaemia cell line THP-1. The ability of test substance to induce activation of CD86 and CD54 in THP-1 cells is used as an assessment of the chemical’s skin sensitisation potential. Activation of CD54 and CD86 protein markers was measured by flow cytometry following 24 h exposure to the test substance.

DOSE FINDING ASSAY
A solubility assessment and two independent dose finding assay (determining CV75 value i.e. the estimated concentration yielding 75% cell viability) were performed, using doses of test substance between 0.003 mg/mL and 0.3 mg/mL in saline.

TEST SYSTEM: The test system was THP-1 human monocytic leukaemia cells (ATCC® catalogue no. TIB 202, batch no. 62996831).

Positive control results:

The study was terminated prior to its completion

Key result

Run / experiment:

other: Mean of two run

Parameter:

other: CV75 (mg/mL)

Remarks:

A concentration showing 75% of THP-1 cell survival (25% cytotoxicity)

Value:

0.067

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not examined

Remarks on result:

not determinable

Remarks:

The study was terminated prior to its completion, due to technical difficulties related to Test facility

Other effects / acceptance of results:

The study was terminated prior to its completion

Dose Finding Assay

• Test substance was soluble at a concentration of 30 mg/mL in saline.
• The mean CV₇₅ value calculated was 0.067 mg/mL.

Results of h-CLAT Assay:

• The study was terminated prior to its completion, due to technical difficulties with the h‑CLAT assay. These technical difficulties were not due to incompatibility of the test substance with the test system, but instead related to the Test Facility being no longer able to perform the assay, independent of the test substance.
• A second acceptable h‑CLAT assay was not conducted, therefore no prediction of the sensitizing potential of test substance was made.

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the study conditions, h-CLAT assay was not completed due to technical difficulties related to test facility, therefore no prediction can be made regarding skin sensitisation potential of test substance.

Executive summary:

A study was conducted to determine the skin sensitisation potential of test substance, using the in vitro human Cell Line Activation Test (h-CLAT) method according to OECD Guideline 442E. The study was terminated prior to its completion. One h‑CLAT assay was performed but a second assay could not be performed. Under the study conditions, h‑CLAT assay was not completed due to technical difficulties related to the test facility, therefore no prediction can be made regarding skin sensitisation potential of the test substance (Vinall, 2017).

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From May 02, 2017 to September 29, 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:

February, 2015

Deviations:

no

GLP compliance:

yes

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin sensitization tests is the KeratinoSensTM assay, which is recommended in international guidelines (e.g. OECD).

Details on the study design:

PRINCIPLE OF THE TEST:
The test method makes use of an immortalised adherent cell line derived from HaCaT human keratinocytes stably transfected with a selectable plasmid. The cell line contains the luciferase gene under the transcriptional control of a constitutive promoter fused with an Antioxidant 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 (transcription factor involved in the antioxidant response pathway) 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. Test chemicals are considered positive if they induce a statistically significant induction of the luciferase activity above a given threshold (i.e. > 1.5 fold or 50% increase), below a defined concentration which does not significantly affect cell viability (i.e. below 1000 µM and at a concentration at which the cellular viability is above 70%).

EXPERIMENTAL MATERIALS
- TEST SYSTEM SOURCE AND MAINTENANCE:
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 (i.e. 25) and are employed for routine testing using the appropriate maintenance medium.

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 from the frozen stock.

CELL CULTURE MEDIUM:
- 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.

TEST SUBSTANCE, CONTROLS AND WORKING SOLUTIONS:
- A correction factor was used to correct for the purity of of the test substance
- Vehicle: The vehicle of the Test substance was ethanol (Extra pure, Merck, Darmstadt, Germany). The vehicle of the positive control was dimethyl sulfoxide (DMSO, Merck, Darmstadt, Germany).
- Negative Control: The negative control is the vehicle of the test substance.
- Positive Control (RS582): Ethylene dimethacrylate glycol (Batch# SHBG0572V)
- Solvent control: The solvent control of the positive control was 1% DMSO in exposure medium. Eighteen wells were tested per plate. The solvent control of the test substance was 0.25% ethanol in exposure medium.
- On each plate three blank wells were tested (no cells and no treatment).

TEST SUBSTANCE SOLUBILITY TEST: A solubility test was performed. No solution or homogeneous suspension could be prepared in DMSO and milli-Q at 40 and 20 mg/mL. Therefore ethanol was tested as solvent. A concentration of 50 µg/mL was determined to be the limit of solubility. a stock of 20 mg/mL was prepared which was 400-fold diluted in the assay resulting in a top concentration in the assay of 50 µg/mL

DOSE LEVELS:
In the main experiments the stock solution of test substance followed series with ethanol and exposure medium to get following test concentrations in Experiment 1 and 2: 50, 25, 13, 6.3, 3.1, 1.6, 0.78, 0.39, 0.20, 0.098, 0.049 and 0.024 µg/mL (final concentration ethanol of 0.25%).

NUMBER OF RUN: All concentrations of the test substance and positive controls were tested in triplicate.

EXPERIMENTAL DESIGN
TREATMENT OF CELLS: 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 cells were incubated overnight in the incubator. The passage number used was 20 in Experiment 1 and 22 in Experiment 2. 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 substance 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 h at 37±1.0ºC in the presence of 5% CO2.

Initially, experiment 1 did not pass all the acceptability criteria and therefore this part of the study was repeated.

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 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 luminometer to assess the quantity of luciferase (integration time two seconds).

CELL VIABILITY ASSAY: medium was replaced after the 48 h 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 h 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.

- ENVIRONMENTAL CONDITION: Temporary deviations from the temperature and humidity occurred due to opening and closing of the incubator door. Based on laboratory historical data these deviations were not considered to affect the study integrity.

Positive control results:

The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity.
Experiment 1: The Imax was 2.40 and the EC1.5 85 µM.
Experiment 2: The Imax was 3.08 and the EC1.5 38 µM.
Both Experiment passed the acceptance criteria:

Key result

Run / experiment:

other: Experiment 1 and 2

Parameter:

other: Imax value (overall maximal fold luciferase activity induction)

Remarks:

The maximum luciferase activity

Value:

0.87

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments (Imax <1.5)

Other effects / acceptance of results:

1. 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.
2. The EC1.5 of the positive control was between 5 and 125 µM (85 µM and 38 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (2.40-fold and 3.08-fold in experiment 1 and 2, respectively).
3. The average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (8.3% and 6.3% in experiment 1 and 2, respectively).
4. Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control Ethanol was below 20% (7.2% and 4.3% in experiment 1 and 2, respectively).
5. Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

No precipitation was observed at the start and end of the incubation period in the 96-well plates in either of Experiment 1 or 2

Experiment 1

·          The test substance showed toxicity. The calculated IC₃₀ was 0.30 µg/mL and the calculated IC₅₀ was 1.04 µg/mL.  

·          No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with the test substance. The I_max was 0.87 and therefore no EC_1.5 could be calculated. 

 

Experiment 2

·          The test substance showed toxicity. The calculated IC₃₀ was 1.54 µg/mL and the calculated IC₅₀ was 2.87 µg/mL. 

·          No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with the test substance. The I_max was 1.37 and therefore no EC_1.5 could be calculated.  

The test substance showed toxicity (IC₃₀ values of 0.30 µg/mL and 1.54 µg/mL and IC₅₀ values of 1.04 µg/mL and 2.87 µg/mL in experiment 1 and 2, respectively). No biologically relevant induction of the luciferase activity (no EC_1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (I_max) was 0.87-fold and 1.37-fold in experiment 1 and 2 respectively. The test substance is classified as inconclusive in the KeratinoSens^TM assay since negative results (<1.5-fold induction) were observed at test concentrations < 200 µg/mL.

Interpretation of results:

study cannot be used for classification

Conclusions:

Under study conditions, the test substance was determined to be inconclusive for skin sensitisation potential

Executive summary:

A study was conducted to determine the skin sensitisation potential of the test substance using the ARE-Nrf2 Luciferase test method according to the OECD Guideline 442D, in compliance with GLP. Two independent experiments were performed. In both experiments, cells were incubated with the test substance at 12 different concentrations in a range of 0.024 – 50 µM. The activation of the ARE-dependent pathway was assessed by measuring the luminescence induction compared to the vehicle control to determine test substance concentration needed for a statistically significant induction of luciferase activity above the threshold (i.e. EC1.5 value). In addition, the viability was assessed with an MTT assay. The EC1.5 value observed at test concentrations with a cell viability of >70% compared to the vehicle control was considered positive for skin sensitisation. The test substance showed toxicity (IC30 values of 0.30 µg/mL and 1.54 µg/mL and IC50 values of 1.04 µg/mL and 2.87 µg/mL in experiment 1 and 2, respectively). No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (IMAX) was 0.87 fold and 1.37 fold in experiment 1 and 2 respectively. The test substance was classified as inconclusive in the KeratinoSensTM assay since negative results (<1.5 -fold luciferase induction) were observed at all test concentrations. Under study conditions, the results were determined to be inconclusive (Westerink, 2017).

Reference 3

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Dec. 11, 2017 to Mar. 06, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EC No 440/2008, part B. “Skin Sensitization: Guinea-Pig Maximization Test (GPMT)”

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

2003

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 substance is a surfactant and the Local Lymph Node Assay as preferred alternative has shown to provide false positive results for surfactants

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River France, L’Arbresle, France; Charles River Deutschland, Sulzfeld, Germany
- Age at the Initiation of Dosing: Young adult animals (approximately 4 weeks old).
- Weight at the Initiation of Dosing: 239 to 288 g.
- Acclimation: 5 d before the commencement of dosing.
- Housing: Up to 5 animals of the same sex and same dosing group together in labeled Noryl cages containing sterilized sawdust as bedding material
- Diet: Complete maintenance diet for guinea pigs (MS-H, SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum
- Water: Municipal tap-water was freely available to each animal via water bottles.
- Other: Nulliparous and non-pregnant animals were included in the study

ENVIRONMENTAL CONDITIONS
- Temperature: 18 to 24°C
- Humidity: 41 to 71%
- Air changes: Ten or greater air changes per hour with 100% fresh air
- Photoperiod: 12 hour light/12 hour dark cycle

ANIMAL ENRICHMENT: For psychological/environmental enrichment, animals were provided with shelters, Play tunnels, Datesand, except when interrupted by study procedures/activities.

No. of animals per dose:

Experimental group: 10 females
Control group: 5 females

Details on study design:

VEHICLE: Water (Elix, Millipore S.A.S., Molsheim, France).

PREPARATION OF TEST SUBSTANCE
Test substance dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements.
The dosing formulations were kept at room temperature until dosing. The dosing formulations were stirred until and during dosing.
No adjustment was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test substance.
Any residual volumes were discarded.
Analysis of test substance in vehicle for concentration, stability, homogeneity was not performed.

ANIMAL SELECTION, IDENTIFICATION AND ASSIGNMENT TO EXPERIMENTAL GROUPS
- Animal Identification: At study assignment, each animal was identified using an ear tattoo.
- Assignment to experimental groups: Animals were assigned to the study at the discretion of the coordinating biotechnician according to body weights, with all animals within ± 20% of the sex mean. Animals in poor health or at extremes of body weight range were not assigned to the study.
- Before the initiation of dosing, a health inspection was performed and any assigned animal considered unsuitable for use in the study were replaced by alternate animals obtained from the same shipment and maintained under the same environmental conditions.

RANGE FINDING TESTS:
A preliminary irritation study was conducted in order to select test substance 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.
Series of test substance concentrations were tested. Practical feasibility of administration determined the highest starting-concentration for each route. The starting- and subsequent concentrations were taken from the series: 50%, 20%, 10%, 5%, 2%, 1% and if needed, further lower concentrations using the same steps.
The test system and procedures were identical to those used during the main study, unless otherwise specified. The six animals selected were between 4 and 9 weeks old. No body weights were determined.

- Intradermal injections:
Initially, a series of four test substance 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 h 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 substance concentrations was tested; the highest concentration being the maximum concentration that could technically be applied. Two different concentrations were applied (0.5 mL each or an equivalent amount when dosed with a spatula) 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 h, the dressing was removed and the skin cleaned of residual test substance using water.
The resulting dermal reactions were assessed for irritation 24 and 48 h after removal of the dressings.

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

A. INDUCTION EXPOSURE (Experimental animals)
- 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 (Fresenius AG, Bad Homburg, Germany).
b) The test substance at a 0.5% concentration.
c) A 1:1 w/w mixture of the test substance, at twice the concentration used in (b) and Freunds' Complete Adjuvant.
Note: One of each pair was on each side of the midline and from cranial a) to caudal c).

- Day 3: The dermal reactions caused by the intradermal injections were assessed for irritation.

- Day 7: The scapular area between the injection sites was clipped and subsequently rubbed with 10% sodium-dodecyl-sulfate (SDS, Boom, Meppel, The Netherlands) in vaseline using a spatula. This concentration of SDS provokes a mild inflammatory reaction.

- Day 8: The 10% SDS treated area between the injection sites was treated with 0.5 mL or an equivalent amount when dosed with a spatula of a 50% test substance concentration using a Metalline patch (2x3 cm) mounted on Medical tape, which was held in place with Micropore tape and subsequently Coban elastic bandage.

- CONTROL ANIMALS: The control animals were treated as described for the experimental animals except that, instead of the test substance, the vehicle was administered.

B. CHALLENGE EXPOSURE (Experimental and Control animals)
- Day 21: One flank of all animals was clipped and treated by epidermal application of a 20% test substance 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 h exposure and the skin cleaned of residual test substance and vehicle using water. The treated sites were assessed for challenge reactions 24 and 48 h after removal of the dressing.

TERMINATION
- After termination, animals were sacrificed using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and an intra-peritoneal injection of Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands).

Challenge controls:

Control animals were treated similar to test animals in 'CHALLENGE EXPOSURE'. For details refer to the section 'Details on study design'

Positive control substance(s):

no

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

20% test substance concentration

No. with + reactions:

5

Total no. in group:

10

Clinical observations:

Five experimental animals showed skin reactions of grade 1; A skin reaction of grade 1 was seen in one experimental animal in response to the vehicle

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

20% test substance concentration

No. with + reactions:

3

Total no. in group:

6

Clinical observations:

Skin reactions of grade 1 were observed

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

20% test substance concentration

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No skin reactions were noted for any animal 48 h after exposure

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Remarks on result:

not measured/tested

PRELIMINARY IRRITATION STUDY

Based on the results, the test substance concentrations selected for the main study were a 0.5% concentration for the intradermal induction and a 50% concentration for the epidermal induction exposure.

Only very slight erythema was observed to the highest test substance concentration epidermally tested. Therefore, the test site of all animals of the main study were treated with 10% SDS approximately 24 h before the epidermal induction, to provoke a mild inflammatory reaction. A 20% test substance concentration was selected for the challenge phase.

MAIN STUDY

INDUCTION PHASE: The reactions noted in the experimental and control animals after the epidermal induction exposure were considered to be enhanced by the SDS treatment.

CHALLENGE PHASE: Skin reactions of grade 1 were observed in five out of ten experimental and 3 out of six control animals in response to the 20% test substance concentration 24 h after exposure. No skin reactions were noted for any animal 48 h after exposure. A skin reaction of grade 1 was seen in one experimental animal in response to the vehicle 24 h after exposure.

TOXICITY / MORTALITY: No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

BODY WEIGHTS: Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the study conditions, test substance was determined to be non-sensitising.

Executive summary:

A study was conducted to determine the skin sensitisation potential of the test substance using the Guinea-Pig Maximisation Test method (GPMT) according to OECD Guideline 406, in compliance with GLP. Test substance concentrations selected for the main study were based on the results of a preliminary study. In the main study, 10 experimental animals were intradermally injected with a 0.5% concentration and epidermally exposed to a 50% concentration in vehicle (water). Five control animals were similarly treated, but with vehicle alone. Approximately 24 h before the epidermal induction exposure all animals were treated with 10% SDS. Two weeks after the epidermal application all animals were epidermally challenged with a 20% test substance concentration and the vehicle. Skin reactions of grade 1 were observed in five out of ten experimental and 3 out of six control animals in response to the challenge application, 24 h after exposure. No skin reactions were noted for any animal 48 h after exposure.  A skin reaction of grade 1 was seen in one experimental animal in response to the vehicle 24 h after exposure. No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study. Since skin reactions of the same severity and of a comparable frequency were noted for the experimental and control animals and the same response was noted against the vehicle in one experimental animal it was considered that these findings were not indicative of sensitization. Under the study conditions, test substance was determined to be non-sensitising (Van Sas, 2018).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

Skin sensitization in vitro (Keratinosens):

A study was conducted to determine the skin sensitisation potential of the test substance using the ARE-Nrf2 Luciferase test method according to the OECD Guideline 442D, in compliance with GLP. Two independent experiments were performed. In both experiments, cells were incubated with the test substance at 12 different concentrations in a range of 0.024 – 50 µM. The activation of the ARE-dependent pathway was assessed by measuring the luminescence induction compared to the vehicle control to determine test substance concentration needed for a statistically significant induction of luciferase activity above the threshold (i.e. EC1.5 value). In addition, the viability was assessed with an MTT assay. The EC1.5 value observed at test concentrations with a cell viability of >70% compared to the vehicle control was considered positive for skin sensitisation. The test substance showed toxicity (IC30 values of 0.30 µg/mL and 1.54 µg/mL and IC50 values of 1.04 µg/mL and 2.87 µg/mL in experiment 1 and 2, respectively). No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (IMAX) was 0.87 fold and 1.37 fold in experiment 1 and 2 respectively. The test substance was classified as inconclusive in the KeratinoSensTM assay since negative results (<1.5 -fold luciferase induction) were observed at all test concentrations. Under study conditions, the results were determined to be inconclusive (Westerink, 2017).

Skin sensitization in vitro (h-CLAT):

A study was conducted to determine the skin sensitisation potential of test substance, using the in vitro human Cell Line Activation Test (h-CLAT) method according to OECD Guideline 442E. The study was terminated prior to its completion. One h‑CLAT assay was performed but a second assay could not be performed. Under the study conditions, h‑CLAT assay was not completed due to technical difficulties related to the test facility, therefore no prediction can be made regarding skin sensitisation potential of the test substance (Vinall, 2017).

Skin sensitization in vivo (GPMT):

A study was conducted to determine the skin sensitisation potential of the test substance using the Guinea-Pig Maximisation Test method (GPMT) according to OECD Guideline 406, in compliance with GLP. Test substance concentrations selected for the main study were based on the results of a preliminary study. In the main study, 10 experimental animals were intradermally injected with a 0.5% concentration and epidermally exposed to a 50% concentration in vehicle (water). Five control animals were similarly treated, but with vehicle alone. Approximately 24 h before the epidermal induction exposure all animals were treated with 10% SDS. Two weeks after the epidermal application all animals were epidermally challenged with a 20% test substance concentration and the vehicle. Skin reactions of grade 1 were observed in five out of ten experimental and 3 out of six control animals in response to the challenge application, 24 h after exposure. No skin reactions were noted for any animal 48 h after exposure.  A skin reaction of grade 1 was seen in one experimental animal in response to the vehicle 24 h after exposure. No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study. Since skin reactions of the same severity and of a comparable frequency were noted for the experimental and control animals and the same response was noted against the vehicle in one experimental animal it was considered that these findings were not indicative of sensitization. Under the study conditions, test substance was determined to be non-sensitising (Van Sas, 2018).

Justification for classification or non-classification

Based on the results of in vitro studies, the substance does not meet the criteria for classification for skin sensitisation according to CLP criteria (Regulation 1272/2008).​