Propane-1,3-diyl bis(4-aminobenzoate)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Justification for type of information:

A skin sensitiser refers to a substance that will lead to an allergic response following skin contact as defined by the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS) [6]. The potential to induce skin sensitisation is an important consideration included in procedures for the safe handling, packaging and transports of chemicals.
The assessment of skin sensitisation typically involves the use of laboratory animals. Classical methods comprise the Magnusson Kligman Guinea Pig Maximisation Test, the Buehler Test (TG 406) [7] as well as the local lymph node assay, in its radioactive and non-radioactive form (TG 429, TG 442A/B [8], [9], [10]. In order to replace in vivo experiments validation studies on alternative, mechanistically based in chemico and in vitro test methods on skin sensitisation were conducted under the auspices of ECVAM and have been considered scientifically valid for the evaluation of the skin sensitisation hazard of chemicals [4], [11]. It was concluded that the KeratinoSens™ assay showed evidence of being a reliable and relevant method to support the discrimination between sensitisers and non-sensitisers for the purpose of hazard classification and labelling for skin sensitisation testing [4], [12], [13], [14], [15]. However, only combinations of several non-animal testing methods within an Integrated Approach to Testing and Assessment (IATA) will be able to fully substitute for the animal test currently in use [4], [11].
The KeratinoSens™ assay is supposed to address the second key event of the skin sensitisation process as defined by the adverse outcome pathway (AOP) [4], [16], the induction of cyto-protective signalling pathways in keratinocytes in response to electrophiles and oxidative stress. The KeratinoSens™ assay addresses the effect on the antioxidant response element (ARE)-dependent pathway in the KeratinoSens™ cell line by measuring the induction of an ARE dependent gene product, the luciferase gene. The luciferase gene induction following exposure to test chemicals is measured in cell lysates by luminescence detection, allowing the discrimination between sensitisers and non-sensitisers [4], [11], [12].
This test may be used for supporting the discrimination between skin sensitisers and non-sensitisers in accordance with UN GHS “Category 1”. It does not allow the classification of chemicals to the subcategories 1A and 1B as defined by UN GHS nor predict potency for safety assessment decisions. Therefore, all substances giving a positive result in the KeratinoSens™ will be classified into UN GHS “Category 1” [4], [11].
15. References
15.1. Guidelines
[1] Chemikaliengesetz (“Chemicals Act”) of the Federal Republic of Germany, Appendix 1 to § 19a as amended and promulgated on July 18, 2017 (BGBl. I S. 2774)
[2] Konsens-Dokument der Bund-Länder-Arbeitsgruppe Gute Laborpraxis (“Consensus Document of the National and Länder Working Party on Good Laboratory Practice“) on the archiving and storage of records and materials, 5 May 1998
[3] OECD Principles of Good Laboratory Practice (as revised in 1997); OECD Environmental Health and Safety Publications; Series on Principles of Good Laboratory Practice and Compliance Monitoring - Number 1. Environment Directorate, Organisation for Economic Co-operation and Development, Paris 1998
[4] OECD Guidelines for Testing of Chemicals, number 442d “In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method” (adopted: February 04, 2015). Available at: http://www.oecd.org/env/testguidelines
[5] KeratinoSens™, EURL ECVAM DB-ALM Protocol n°155, July 1st, 2015. Available at: https://ecvam-dbalm.jrc.ec.europa.eu/beta/index.cfm/methodsAndProtocols/index?idmm=6&idsm=7
15.2. Literature
[6] UN (2015), United Nations Globally Harmonized System of Classification and Labelling of Chemicals (GHS), Sixth revised edition, UN New York and Geneva. Available at: http://www.unece.org/trans/danger/publi/ghs/ghs_rev06/06files_e.html
[7] OECD Guidelines for Testing of Chemicals, number 406 “Skin Sensitisation” (adopted July 07, 1992). Organisation for Economic Cooperation and Development, Paris. Available at: http://www.oecd.org/env/testguidelines
[8] OECD Guidelines for Testing of Chemicals, number 429 “The Local Lymph Node Assay” (adopted July 22, 2010). Organisation for Economic Cooperation and Development, Paris. Available at: http://www.oecd.org/env/testguidelines
[9] Guidelines for Testing of Chemicals, number 442A “The Local Lymph Node Assay: DA” (adopted July 22, 2010). Organisation for Economic Cooperation and Development, Paris. Available at: http://www.oecd.org/env/testguidelines
[10] Guidelines for Testing of Chemicals, number 442B “Skin Sensitisation: BrdU-ELISA: DA” (adopted July 22, 2010). Organisation for Economic Cooperation and Development, Paris. Available at: http://www.oecd.org/env/testguidelines
[11] EURL-ECVAM (2014). Recommendation on the KeratinoSens™ assay for skin sensitization testing, 42 pp. Available at: https://eurl-ecvam.jrc.ec.europa.eu/eurl-ecvam-recommendations/recommendation-keratinosens-skin-sensitisation
[12] Emter R., Ellis G., Natsch A.(2010). Performance of a novel keratinocyte-based reporter cell lineto screen skin sensitizers in vitro. Toxicology and Applied Pharmacology 245, 281-290.
[13] Natsch A., Bauch C., Foertsch L., Gerberick F., Normann K., Hilberer A., Inglis H., Landsiedel R., Onken S., Reuter H., Schepky A., Emter R. (2011). The intra- and inter-laboratory reproducibility and predictivity of the KeratinoSens assay to predict skin sensitizers in vitro:results of a ring-study in five laboratories. Toxicol. In Vitro 25, 733-744.
[14] Natsch A., Ryan C.A., Foertsch L., Emter R., Jaworska J., Gerberick G.F., Kern P. (2013). A dataset on 145 chemicals tested in alternative assays for skin sensitization undergoing prevalidation. Journal of Applied Toxicology, 33, 1337-1352.
[15] Natsch A. (2010). The Nrf2-Keap1-ARE Toxicity Pathway as a Cellular Sensor for Skin Sensitizers-Functional Relevance and Hypothesis on Innate Reactions to Skin Sensitizers. Toxicological Sciences 113, 284-292.
[16] OECD Series on Testing and Assessment No. 168. The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins. Part 1, (2012).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

methods comprise the Magnusson Kligman Guinea Pig Maximisation Test, the Buehler Test (TG 406) [7] as well as the local lymph node assay, in its radioactive and non-radioactive form (TG 429, TG 442A/B [8], [9], [10]. In order to replace in vivo experiments validation studies on alternative, mechanistically based in chemico and in vitro test methods on skin sensitisation were conducted under the auspices of ECVAM and have been considered scientifically valid for the evaluation of the skin sensitisation hazard of chemicals [4], [11]. It was concluded that the KeratinoSens™ assay showed evidence of being a reliable and relevant method to support the discrimination between sensitisers and non-sensitisers for the purpose of hazard classification and labelling for skin sensitisation testing [4], [12], [13], [14], [15]. However, only combinations of several non-animal testing methods within an Integrated Approach to Testing and Assessment (IATA) will be able to fully substitute for the animal test currently in use [4], [11].

Principles of method if other than guideline:

The KeratinoSens™ assay is supposed to address the second key event of the skin sensitisation process as defined by the adverse outcome pathway (AOP) [4], [16], the induction of cyto-protective signalling pathways in keratinocytes in response to electrophiles and oxidative stress. The KeratinoSens™ assay addresses the effect on the antioxidant response element (ARE)-dependent pathway in the KeratinoSens™ cell line by measuring the induction of an ARE dependent gene product, the luciferase gene. The luciferase gene induction following exposure to test chemicals is measured in cell lysates by luminescence detection, allowing the discrimination between sensitisers and non-sensitisers [4], [11], [12].

GLP compliance:

yes

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

The induction of the Keap1-Nrf2-ARE signalling pathway by small electrophilic substances such as skin sensitizers was reported by several studies [12], [13], [14], [15] and represents the second key event of the skin sensitisation process as described by the AOP [4], [16]. Therefore the KeratinoSens™ assay is considered relevant for the assessment of the skin sensitisation potential of chemicals [4].
This test method is able to detect chemicals that cause skin sensitisation and allows for hazard identification in accordance with UN GHS “Category 1”. Data generated with this method may be not 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 an IATA, combining them with other complementary information e.g., derived from in chemico or in vitro assays addressing other key events of the AOP [4], [11].

Test material

Test material information

Constituent 1

Reference substance name:

Propane-1,3-diyl bis(4-aminobenzoate)

EC Number:

260-847-9

EC Name:

Propane-1,3-diyl bis(4-aminobenzoate)

Cas Number:

57609-64-0

Molecular formula:

C17H18N2O4

IUPAC Name:

3-(4-aminobenzoyloxy)propyl 4-aminobenzoate

In vitro test system

Details on the study design:

10.2. Preparation of the Test Item
All test item solutions were freshly prepared immediately prior to use.
The test item was dissolved in dimethyl sulfoxide (DMSO, CAS No.: 67-68-5, purity ≥99%; AppliChem; Lot No.: 0001055932, 00001179895). A stock solution of 200 mM was prepared for experiment 1, 2 and 3. During experiment 4 a stock solution of 25 mM was prepared.
Based on the stock solution a set of twelve master solutions in 100% solvent was prepared. The stock solution of the test item was diluted eleven times using a constant dilution factor of 1:2 for experiment 1, 2 and 3 and a constant dilution factor of 1:1.333 for experiment 4. Then the 100x concentrated master solutions were further diluted 1:25 in cell culture medium resulting in a 4% share of the solvent.
These 4x concentrated test item solutions were finally diluted 1:4 when incubated with the cells. Based on this procedure the final concentration of the solvent was 1% (v/v) in all test item concentrations and controls
10.3. Controls
A blank, a negative control and a positive control were set up in parallel in order to confirm the validity of the test.
Blank
A blank well with no seeded cells was included in every plate to determine the background. The well was incubated with the negative control.
Negative Control
DMSO (AppliChem; Lot No.: 0001055932, 00001179895) at a final concentration of 1% (v/v) in test item exposure medium was used as negative control. Six wells were included in every testing plate. The preparation of the negative control was carried out analogous to the test item.
Positive Control
Cinnamic aldehyde (CA, (2E)-3-phenylprop-2-enal; CAS 104-55-2; >98%; Alfa Aesar; Lot No.: 10176010) was used as positive control. CA was dissolved in DMSO (AppliChem; Lot No.: 0001055932, 00001179895) at a concentration of 6.4 mM and was further diluted four times with a constant dilution factor of 1:2 resulting in a concentration range of 0.4 mM – 6.4 mM. The following preparation of the positive control was carried out analogous to the preparation of the test item, resulting in a final concentration range of 4 μM – 64 μM. The final concentration of the solvent DMSO was 1% (v/v) for all wells.
10.4. Cell line
The test was carried out using the transgenic cell line KeratinoSens™ (Givaudan, Switzerland), a cell line derived from human keratinocytes (HaCaT) transfected with a stable insertion of the Luciferase construct. Cells from frozen stock cultures, tested routinely for mycoplasma, were seeded in culture medium at an appropriate density and were used for routine testing. Only cells at a low passage number <25 (P 11 in experiment 1; P 03 in experiment 2; P 6 in experiment 3, P 7 in experiment 4) were used.
Cells were cultured in 75 cm2 culture flasks (Greiner) in maintenance medium at 37 ± 1°C and 5% CO2. For test item exposure, cells were cultured in medium for test item exposure.
10.5. Composition of Media
Maintenance Medium
Dulbecco’s Modified Eagle Medium (GlutaMAX™) (Gibco Life Science, Cat. No.: 21885-025, Lot No.: 1896185, 1896184) with 1.0 g/L D-glucose and Na-Pyruvate. The medium was supplemented with the following components:
- 10% fetal bovine calf serum (Biochrom, Cat. No.: S 0615, Lot No.: 0879F, 1000F)
- 1% geneticin (final concentration: 500 μg/mL; Gibco Life Science, Cat. No. 10131-027, Lot No.: 1881779; 1894716)
Assay Medium
Dulbecco’s Modified Eagle Medium (GlutaMAX™) (Gibco Life Science, Cat. No.: 21885-025, Lot No.: 1896185) with 1.0 g/L D-glucose and Na-Pyruvate. The medium was supplemented with the following components:
- 10% fetal bovine calf serum (Biochrom, Cat. No.: S 0615, Lot No.: 0879F, 1000F)
Test Item Exposure Medium
Dulbecco’s Modified Eagle Medium (GlutaMAX™) (Gibco Life Science, Cat. No.: 21885-025, Lot No.: 1896185, 1906036) with 1.0 g/L D-glucose and Na-Pyruvate. The medium was supplemented with the following components:
- 1% fetal bovine calf serum (Biochrom, Cat. No.: S 0615, Lot No.: 1000F, 0879F)
10.6. Luciferase Assay System
The luciferase activity was determined using the following products purchased from Promega. All components were used according to the instructions of the manufacture manual.
Luciferase Assay System 10-Pack
The kit (Promega, Cat. No.: E1501, Lot No.: 0000277734) consisted of the following components relevant for this study:
- 10 vials Luciferase Assay Substrate (lyophilized)
- 10 x 10 mL Luciferase Assay Buffer
If freshly prepared, Luciferase Assay Substrate was dissolved in Luciferase Assay Buffer.
If thawed from -80 °C, Luciferase Assay Reagent was allowed to equilibrate to room temperature prior to use.
Luciferase Cell Culture Lysis 5x Reagent
The kit (Promega, Cat. No.: E1531, Lot No.: 0000246522, 000254353, 00002584055) consisted of the following components relevant for this study:
- 30 mL Luciferase Cell Culture Lysis 5x Reagent
Prior to use lysis buffer was diluted 1:5 with dist. water (Sigma; Lot No.: RNBG3519, RNBG3520)
10.7. Further Reagents
MTT solution
- MTT (VWR, CAS No.: 298-93-1, Lot No.: 1017C385, 0977C002) stock solution: 5 mg/mL MTT in DPBS (Gibco Life Science; Lot No.: 1909266)
SDS solution:
- 10% (w/v) sodium dodecyl sulfate (SDS; AppliChem, CAS No.: 151-21-3, Lot No.: 7J011684, 40015277) in dist. water (Sigma; Lot No.: RNBG3519, RNBG3520)
DPBS:
DPBS solution (without Ca2+/Mg2+) (Gibco Life Science; Lot No.: 1909266, 1943446)
10.8. Dose Groups
1. Negative Control: 1% (v/v) DMSO in test item exposure medium
2. Positive Control: CA: 4 μM, 8 μM, 16 μM; 32 μM; 64 μM
3. Test Item: 12 concentrations of the test item
Experiment 1, 2 and 3:
2000, 1000, 500.0, 250.0, 125.0, 62.50, 31.25, 15.63, 7.81, 3.91, 1.95 and 0.98 μM
Experiment 4:
250.00, 187.55, 140.70, 105.55, 79.18, 59.40, 44.56, 33.43, 25.08, 18.81, 14.11, 10.59 μM
Each concentration step of the test item and the positive control was assessed in three replicates in every independent run. The negative control was assessed using six replicates per plate in every independent run.
10.9. Experimental Procedure
A cell suspension of 8 × 104 cells/mL in assay medium was prepared. 125 μL of the cell suspension corresponding to 1 × 104 cells were dispensed in each well, except for the blank. To determine the luciferase activity cells were seeded in white 96-well plates (flat bottom). In parallel cells were seeded in a transparent 96-well plate (flat bottom) for the determination of the cell viability.
After seeding cells were grown for 24 h ± 1 h in assay medium at 37 °C ± 1 °C and 5% CO2. Thereafter, the assay medium was discarded and replaced by 150 μL test item exposure medium. 50 μL of the shortly before prepared 4x master concentrations were transferred to the luciferase and cell viability plates, resulting in an additional 1:4 dilution of the test item.
All plates were sealed using a sealing tape to avoid evaporation of volatile compounds and cross-contamination between wells by the test items. Treated plates were incubated for 48 h ± 1 h at 37 °C ± 1 °C and 5% CO2.
Luciferase activity
After 48 h ± 1 h of exposure, the supernatant was aspirated from the white assay plates and discarded. Cells were washed once with DPBS (Gibco Life Science; Lot No.: 1909266). Subsequently 20 μL of passive lysis buffer were added into each well and the plate was incubated for 20 min at room temperature in the absence of light.
Plates with the cell lysate were placed in the plate reader for luminescence measurement. Per well 50 μL of the luciferase substrate were injected by the injector of the plate reader. The plate reader waited for 1.000 ms before assessing the luciferase activity for 2.000 ms. This procedure was repeated for each individual well.
Cell viability
For the cell viability plate the medium was replaced with 200 μL test item exposure medium. 27 μL MTT solution were added directly to each individual well. The plate was covered with a sealing tape and incubated for 4 h at 37 °C ± 1 °C and 5% CO2. Afterwards the medium was removed and replaced by 200 μL 10% SDS solution per well. The plate was covered with sealing tape and incubated in the incubator at 37 °C ± 1 °C and 5% CO2 overnight (experiment 1 and 3) and over the weekend (experiment 2). After the incubation period the plate was shaken for 10 min and the OD was measured at λ = 600 nm.

Results and discussion

Positive control results:

Under the condition of this study the test item is therefore considered as non-sensitiser.
The controls confirmed the validity of the study.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: first experiment, 125 µM, max luciferase activity (l max)

Parameter:

other: l max

Value:

1.57

Vehicle controls validity:

valid

Remarks:

solvent

Positive controls validity:

valid

Results 2

Key result

Run / experiment:

other: second experiment, 250 µM, max luciferase activity (l max)

Parameter:

other: l max

Value:

2.1

Vehicle controls validity:

valid

Remarks:

solvent

Positive controls validity:

valid

Results 3

Key result

Run / experiment:

other: third experiment, 1000 µM, max liciferase activity (l max)

Parameter:

other: l max

Value:

2.21

Vehicle controls validity:

valid

Remarks:

solvent

Positive controls validity:

valid

Results 4

Key result

Run / experiment:

other: fourth experiment, 79.18 µM, max luciferase activity (l max)

Parameter:

other: l max

Value:

1.54

Vehicle controls validity:

valid

Remarks:

solvent

Positive controls validity:

valid

Other effects / acceptance of results:

10.12. Acceptance Criteria
The test meets acceptance criteria if:
- the luciferase activity induction of the positive control is statistically significant above the threshold of 1.5 (using a t-test) in at least one of the tested concentrations
- the average induction in the three technical replicates for the positive control at a concentration of 64 μM is between 2 and 8
- the EC1.5 value of the positive control is within two standard deviations of the historical mean
- the average coefficient of variation (CV; consisting of 6 wells) of the luminescence reading for the negative (solvent) control DMSO is <20% in each repetition.

Any other information on results incl. tables

The in vitro KeratinoSens™ assay enables detection of the sensitising potential of a test item by addressing the second molecular key event of the adverse outcome pathway (AOP), namely activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line KeratinoSens™. The luciferase activity, assessed by luminescence measurement, compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers.

In the present study PU-2017-775 was dissolved in DMSO. Based on a molecular weight of 314.34 g/mol a stock solution of 200 mM was prepared for experiment 1, 2 and 3. In order to verify these experiments, a fourth experiment with adapted concentrations was performed. For experiment 4 a stock solution of 25 mM was prepared.

Based on the stock solution a set of twelve master solutions in 100% solvent was prepared by serial dilution using a constant dilution factor of 1:2 for experiment 1, 2 and 3 and a constant dilution factor of 1:1.333 for experiment 4. These master solutions were diluted 1:100 in cell culture medium. The following concentration range was tested in the assay:

Experiment 1, 2 and 3

2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 μM

Experiment 4

250.00, 187.55, 140.70, 105.55, 79.18, 59.40, 44.56, 33.43, 25.08, 18.81, 14.11, 10.59 μM

Cells were incubated with the test item for 48 h at 37°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement.

In the first experiment, a max luciferase activity (Imax) induction of 1.57 was determined at a test item concentration of 125 μM. The corresponding cell viability was >70% (89.2%). No further significant luciferase induction >1.5 was found in the tested concentration range. The calculated EC1.5 was <1000 μM (70.24 μM). A cytotoxic effect was detected starting from a concentration of 250 μM onwards.

In the second experiment, a max luciferase activity (Imax) induction of 2.10 was determined at a test item concentration of 250 μM, but the corresponding cell viability was <70% (32.1%). The lowest tested concentration with a significant luciferase induction >1.5 (1.74) was found to be 62.50 μM, but the corresponding cell viability was <70% (69.8%). The calculated EC1.5 was <1000 μM (41.66 μM). A cytotoxic effect was detected starting from a concentration of 62.5 μM onwards.

In the third experiment, a max luciferase activity (Imax) induction of 2.21 was determined at a test item concentration of 1000 μM, but the corresponding cell viability was <70% (25.8%). The lowest tested concentration with a significant luciferase induction >1.5 (1.67) was found to be 125 μM. The corresponding cell viability was >70% (103.3%). The calculated EC1.5 was <1000 μM (73.42 μM). A cytotoxic effect was detected starting from a concentration of 250 μM onwards.

Since the test item induced the gene activity very close to the cytotoxic levels, a fourth experiment with an adapted concentration range and more narrow dose-response analysis with dilution of 1.333-fold between wells instead of two-fold dilutions was performed to decide if induction is at cytotoxic levels or not.

In the fourth experiment, a max luciferase activity (Imax) induction of 1.54 was determined at a test item concentration of 79.18 μM. The corresponding cell viability was >70% (95.9%). No further significant luciferase induction >1.5 was found in the tested concentration range. The calculated EC1.5 was <1000 μM (75.54 μM). A cytotoxic effect was at the highest concentration of 250 μM.

A dose response for luciferase activity induction was observed for experiments 1, 2 and 3.

Within all four experiments the induced the gene activity was very close to the cytotoxic levels.

In the first experiment the increase of luciferase activity was detected only at one concentration near the cytotoxicity threshold. Furthermore, the significant induction was with 1.57 only slightly increased and showed a standard deviation of ± 0.25. Therefore, the test item could be considered as non-sensitiser in the first experiment.

Within the second experiment the test item showed no significant induction in the non-cytotoxic range and could therefore be considered as non-sensitiser.

Within the third experiment the test item showed a significant induction in the non-cytotoxic range with a dose-response dependency and could therefore be considered as sensitiser.

Within the fourth experiment the increase of luciferase activity was detected only at one concentration, but no dose-response curve was observed and no further significant luciferase induction >1.5 was found in the higher concentrations with a viability >70%. Therefore, the test item could be considered as non-sensitiser in the fourth experiment.

Under the condition of this study the test item is therefore considered as non-sensitiser.

The controls confirmed the validity of the study.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item might be considered as non sensitiser.
The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Executive summary:

Under the condition of this study the test item is therefore considered as non-sensitiser.