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Endpoint summary

Description of key information

In the KeratinoSens^TM assay, the test substance gave inconclusive results. As other in vitro/in chemico methods are not suitable for this type of substances (UVCB), an in vivo test (LLNA) was conducted.

In a GLP-compliant OECD guideline 429 (LLNA) test, the test item elicited SI > 3 at 50% concentration in DMF. The EC3 was calculated to be 40.4%. Based on this, the test item is concluded to be sensitizing to skin.

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:

key study

Study period:

03 November 2017 - 01 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted February 2015

Deviations:

no

GLP compliance:

yes

Type of study:

activation of keratinocytes

Details on the study design:

MATERIALS AND METHODS
- Vehicle of the test item: Dimethyl sulfoxide (DMSO, Merck, Darmstadt, Germany).
- Negative control: vehicle of the test item
- Positive control: Ethylene dimethacrylate glycol, cas n 97-90-5
- blank: no cells and no treatment
- Item solutions preparations: the test substance was first dissolved in DMSO at 10 mg/mL concentration. From this stock 11 spike solutions in DMSO were prepared (2-fold dilution series). The stock and spike solution were diluted 25-fold with exposure medium. These solutions were diluted 4-fold in the assay
- tested concentrations: 100, 50, 25, 13, 6.3, 3.1, 1.6, 0.78, 0.39, 0.20, 0.098 and 0.049 µg/mL (final concentration DMSO of 1%). All formulations were clear soilutions
- number of tests: all concentrations were tested in triplicate

PARAMETERS
- Imax = The maximal average fold induction of luciferase activity value observed at any concentration of the tested chemical and positive control
- EC1.5 = the value representing the concentration for which induction of luciferase activity is above the 1.5 fold threshold
- IC30 = concentration values for 30% reduction of cellular viability.
- IC50 = concentration values for 50% reduction of cellular viability

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

SUBCULTURING
Cells were subcultured upon reaching 80-90% confluency.

ENVIROMENTAL CONDITIONS
- humid atmosphere: 80 - 100% (actual range 72 – 100 %)
- CO2: 5.0 ± 0.5% CO2 in air
- light conditions: dark
- temperature: 37.0 ± 1.0°C (actual range 36.0 – 37.0°C).
Temperature and humidity were continuously monitored throughout the experiment.
The CO2 percentage was monitored once on each working day.

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 number used was p+8 in experiment 1 and p+6 in experiment 2.
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.
- The treated plates were then incubated for about 48 hours at 37±1.0oC in the presence of 5% CO2.
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 TECAN Infinite® M200 Pro Plate Reader to assess the quantity of luciferase (integration time two seconds).
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 at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.

NUMBER OF EXPERIMENTS
- Initially, experiment 2 did not pass all the acceptability criteria and therefore this part of the study was repeated. In total 2 valid experiments were performed

DEVIATIONS
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

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

Positive control results:

The positive control was Ethylene dimethacrylate glycol.
Two indipendent experiments were performed.

- The luciferase activity induction obtained with the positive control 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 (43 µM and 28 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (3.18-fold and 4.41-fold in experiment 1 and 2, respectively).

Key result

Run / experiment:

other: first experiment

Parameter:

other: I max

Remarks:

Maximum Luciferase Activity Induction

Value:

1.48

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: inconclusive

Remarks:

Imax experiment 1 = 1.48 fold

Key result

Run / experiment:

other: In both independent experiments

Parameter:

other: induction of luciferase acticity

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: inconclusive

Remarks:

No EC1.5 value was measured at any of the concentrations in both experiments

Key result

Run / experiment:

other: first experiment

Parameter:

other: IC30

Remarks:

value in µg/mL

Value:

9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The test item showed toxicity

Key result

Run / experiment:

other: second experiment

Parameter:

other: IC30

Remarks:

value in µg/mL

Value:

10

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The test item showed toxicity

Key result

Run / experiment:

other: first experiment

Parameter:

other: IC50

Remarks:

value in µg/mL

Value:

10

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The test item showed toxicity

Key result

Run / experiment:

other: second experiment

Parameter:

other: IC50

Remarks:

value in µg/mL

Value:

11

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The test item showed toxicity

Key result

Run / experiment:

other: second experiment

Parameter:

other: Imax

Remarks:

Maximum Luciferase Activity Induction

Value:

1.35

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: inconclusive

Remarks:

Imax experiment 2 = 1.35 fold

Interpretation of results:

other: inconclusive

Conclusions:

Under the applied experimental conditions, the test substance is classified as inconclusive in the KeratinoSensTMassay since negative results (<1.5-fold induction) were observed at test concentrations < 200µg/mL.

Executive summary:

The ability of the test substance to induce skin sensitization was evaluated according to the OECD guideline 442D (In Vitro Skin Sensitization: ARE-Nrf2 Luciferase Test Method), adopted in February 2015

Two independent experiments were performed. In these experiments, the cells were incubated for 48 hours with the following concentrations of the test item: 0.049; 0.098; 0.20; 0.39, 0.78; 1.6; 3.1; 6.3; 13; 25; 50; 100 µg/mL. The esperiments were performed in triplicate

The activation of the ARE-dependent pathway was assessed by measuring the luminescence induction compared to the vehicle control. In addition, the viability was assessed with an MTT assay.

In both the experiments:

- the test item showed toxicity (IC₃₀values of 9.0 µg/mLand 10 µg/mL and IC₅₀values of 10 µg/mL and 11 µg/mL)

- No biologically relevant induction of the luciferase activity (no EC_1.5value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (I_max) was 1.48-fold and 1.35-fold

The test item is classified as inconclusive in the KeratinoSens^TMassay since negative results (<1.5-fold induction) were observed at tested concentrations under the applied experimental conditions.

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 January 2018- 20 February 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

2003

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Solubility and stability of the test substance in the solvent/vehicle: soluble in N,N-dimethylformamide (DMF)

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: SPF
- Age at study initiation: ca. 10 weeks
- Weight at study initiation: 17.4-23.5 g
- Housing: in groups of 5/sex of the same dosing group in polycarbonate Makrolon MIII type cages, with sterilized sawdust as bedding material. For psychological/environmental enrichment, animals were provided with paper and shelters, except when interrupted by study procedures/activities.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum, except during designated procedures
- Water: municipal tap water, ad libitum
- Acclimation period: at least 5 days
- Indication of any skin lesions: none

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 (actual mean)
- Humidity (%): 40-43
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From: 31 January 2018 To: 19 February 2018

Vehicle:

dimethylformamide

Concentration:

0% (vehicle controls), 25%, 50% and 100% w/w

No. of animals per dose:

5 females/dose

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: The vehicle was chosen from the vehicles specified in the test guideline (in order of preference): Acetone/Olive oil (4:1 v/v), N,N-dimethylformamide, methylethylketone, propylene glycol, dimethylsulfoxide and 1% Pluronic© L92 in Elix water (in case an aqueous vehicle is suitable). The vehicle was selected on the basis of maximizing the solubility based on trial preparations performed at the testing laboratory and on information provided by the sponsor. Trial preparations were not used for dosing and were discarded after the assessment is complete. There was no information available about the stability and solubility of the test item in vehicle.
- Irritation: A pre-screen test was conducted in order to select the highest test item concentration to be used in the main study. In principle, this highest concentration should cause no systemic toxicity, may give well-defined irritation as the most pronounced response (maximum grade 2 and/or an increase in ear thickness < 25%) and/or is the highest possible concentration that can technically be applied. Two test item concentrations were tested; a 50% and 100% concentration. The highest concentration was the maximum concentration as required in the test guidelines. At a 50 and 100% test item concentration, no signs of excessive irritation were observed and therefore the 100% concentration was selected as highest concentration for the main study.
- Systemic toxicity: in the pre-screen test, no signs of systemic toxicity were noted.
- Ear thickness measurements: in the pre-screen ear thickness was increased by max. 14% on Day 3 following application of 100% substance, compared to Day 1 pre-dose value. A 25% value is used as the threshold for selection for use in the main study.
- Erythema scores: max. 1 (Day 2 and 3 following application of 100% pure substance)

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: Stimulation Index (SI) >= 3; EC3 value ≤ 2%: sub-category 1A, EC3 value > 2%: sub-category 1B


TREATMENT PREPARATION AND ADMINISTRATION:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily and dosed within 4 hours after adding the vehicle to the test item. The dosing formulations were kept at room temperature until dosing and stirred until and during dosing.
No adjustment was made for specific gravity of the vehicle and no correction was made for the purity/composition of the test item, since the test method requires a logical concentration range rather than specific dose levels.
On days 1, 2 and 3 three groups of five animals were treated with one test item concentration per group. The highest test item concentration was selected from the pre-screen test. One group of five animals was treated with the vehicle. The dorsal surface of both ears was topically treated (25 μL/ear or an equivalent amount when dosed with a spatula) with the test item, at approximately the same time on each day. The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test item. On day 6, each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) containing 20 μCi of 3H-methyl thymidine. After five hours, all animals were killed by intraperitoneal injection of Euthasol® 20%, and the draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in PBS.

Positive control substance(s):

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

Statistics:

Not used.

Positive control results:

An extensive data base is available at the test laboratory with reliability checks performed each half year during at least the recent 9 years showing reproducible and consistent positive results.

Key result

Parameter:

SI

Value:

2.2

Variability:

± 0.4

Test group / Remarks:

25% test substance in DMF

Key result

Parameter:

SI

Value:

3.5

Variability:

± 0.3

Test group / Remarks:

50% test substance in DMF

Key result

Parameter:

SI

Value:

2.4

Variability:

± 0.5

Test group / Remarks:

100% test substance

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
Mean DPM/animal values for the experimental groups treated with test item concentrations 25, 50 and 100% were 1131, 1794 and 1236 DPM, respectively. The mean DPM/animal value for the vehicle control group was 513 DPM.
The majority of auricular lymph nodes were considered normal in size, except for the nodes in three animals treated at 50% which were considered enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

DETAILS ON STIMULATION INDEX CALCULATION
A Stimulation Index (SI) was calculated for each group using the individual SI values. The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean.

EC3 CALCULATION
The EC3 value (the estimated test item concentration that will give a SI =3) was determined, using linear interpolation.

CLINICAL OBSERVATIONS:
No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. No erythema was observed in any of the animals. Transparent test item remnants were present on the dorsal surface of the ears of all test item treated animals between Days 1 and 6, which did not hamper scoring of the skin reactions. Bald skin behind the ears was noted for all test item treated animals from Day 2 to 6. Bald skin in the neck was noted for all animals treated at 50% on Days 4, 5 and 6 and for all animals treated at 100% on Days 4 and 5.

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

The response of the 100% group did not follow the expected dose-response relationship which is more often seen in these kind of studies. The response might be less due to differences in skin penetration (no vehicle present) or viscosity.

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

In a GLP-compliant OECD guideline 429 study, the test item elicited SI ≥ 3 at concentration 50%. The EC3 was calculated to be 40.4%. Based on this, the test item is considered to be sensitizing to skin.

Executive summary:

In a GLP-compliant OECD guideline 429 study (LLNA), the test item elicited SI ≥ 3 at concentration 50% in DMF. The EC3 was calculated to be 40.4%. No mortality, clinical signs of toxicity or erythema were noted. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. Positive control hexylcinnamaldehyde, tested in a separate study not more than 6 months previously, showed satisfactory response, confirming the sensitivity of the test system. Based on this, the test item is considered to be sensitizing to skin. Based on the EC3 value of 40.4%, the test substance needs to be classified as Skin Sens. 1B, H317 according to Regulation (EC) 1272/2008 (CLP).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

The objective was the evaluation of the skin sensitization potential of the test substance, by in vitro test methods. In vitro skin sensitization studies according to EU COM regulation 2016/1688 for UVCB's and metals (effective 20 September 2016) were attempted. The DPRA test is not applicable for UVCB substances.

As first the KeratinoSens^TM assay was performed, according to the most recent OECD guideline. This study evaluated the ability of the test substance to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway. Under the applied experimental conditions, negative results (<1.5-fold induction) were observed at test concentrations < 200 µg/mL.

The EC1.5 could not be calculated and therefore the test substance is classified as inconclusive in the KeratinoSensTMassay.

 

The  MUSST/hCLAT was not executed because it would not contribute to obtain a conclusive result from the in vitro methods, neither in case of a negative nor in case of a positive result of this test.

Therefore, since in vitro studies remain inconclusive a Local Lymph Node Assay is required to be executed to be able to conclude for this endpoint.

In a GLP-compliant OECD guideline 429 study (LLNA), the test item elicited SI ≥ 3 at concentration 50% in DMF. The EC3 was calculated to be 40.4%. No mortality, clinical signs of toxicity or erythema were noted. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. Positive control hexylcinnamaldehyde, tested in a separate study not more than 6 months previously, showed satisfactory response, confirming the sensitivity of the test system. Based on this, the test item is considered to be sensitizing to skin.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In the OECD guideline 429 test, the test substance elicited SI > 3 at concentration 50% in DMF. The calculated EC3 was 40.4%. Based on these results, classification of the substance as Skin Sens. 1B, H317 is required according to Regulation (EC) 1272/2008.