N,N'-(2-(4-(2-aminobenzamido)butyl)pentane-1,5-diyl)bis(2-aminobenzamide)

Endpoint summary

Description of key information

Sensitisation data summary.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Phase:16 December 2020 to 19 January 2021.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Female CBA/CaCrl strain mice were supplied by Charles River (UK) Ltd, Margate, UK.).
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 9 to 10 weeks
- Weight at study initiation: 17 to 20g. Individual body weights were within ±20% of the mean body weight for mice on the study.
- Housing: Animals were housed in groups of up to five during acclimatisation and in pairs. From day 1 the preliminary study animal was individually housed from and the main study animals were housed in pairs. Bedding was provided on a weekly basis to each cage by use of clean European softwood bedding (Datesand Ltd., Manchester, UK).
. - Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 - 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 °C
- Humidity (%): 40 to 70%
- Air changes (per hr): At least fifteen per hour
Photoperiod (hrs dark / hrs light): 12 hours in light, 12 hours in darkness

Vehicle:

dimethyl sulphoxide

Remarks:

The vehicle was chosen because it produced the highest suitable concentration (25% w/v) out of the vehicles listed in the protocol.

Concentration:

Control = vehicle only, low dose = 5 % w/v test substance, mid-dose = 10% w/v test substance, high dose = 25% w/v test substance

No. of animals per dose:

Four per dose group.

Positive control substance(s):

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

Positive control results:

The positive control article produced a Stimulation Index of 4.48, demonstrating adequate performance of the assay.

Key result

Parameter:

SI

Value:

ca. 1.29

Test group / Remarks:

5 % w/v

Key result

Parameter:

SI

Value:

ca. 1.72

Test group / Remarks:

10 % w/v

Key result

Parameter:

SI

Value:

ca. 2.112

Test group / Remarks:

25% w/v

Mortality

There were no deaths on the study.

Clinical Signs

There were no clinical signs indicative of a systemic effect of treatment among mice treated with the vehicle or with 5, 10 or 25% w/v formulations of the test article.

 

The vehicle and test formulation application sites remained free of irritation.

 

Greasy fur to the back of the ears and neck were observed in all Group 5 animals from Day 1 to Day 5 indicative of the positive control vehicle being acetone/olive oil (4:1 (v/v)).

Body Weights

There was no indication of a treatment related effect on body weight.

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the result from the Local Lymph Node Assay, the test substance was concluded not have the potential to cause skin sensitisation.

Executive summary:

Introduction

This study was conducted to assess the potential of the test article, N,N'-(2-(4-(2-aminobenzamido)butyl)pentane-1,5-diyl)bis(2-aminobenzamide), to cause skin sensitisation in the mouse. The method used was deigned to meet the requirements of the following guidelines:

• OECD Guidelines for Testing of Chemicals Method 429 (adopted 22 July 2010).


• Method B42 of Council Regulation (EC) No 440/2008

 

Method

Following a preliminary screening test using a 25% w/v formulation, the test article was prepared for administration at 5, 10 or 25% w/v in dimethyl sulphoxide (DMSO).

Groups of four female CBA / CaCrl mice were subjected to topical applications of vehicle control, positive control or of one of the test formulations to the outer aspect of the auditory pinnae once daily on Days 1, 2 and 3. On Day 6 a 20 μCi dose of tritiated ³H-methyl thymidine was injected intravenously into each mouse. Approximately five hours later the auricular lymph nodes were recovered from each animal. The pairs of nodes from each animal were pooled and suspensions of the cellular components of the lymph nodes were prepared in 5% w/v trichloroacetic acid and processed through a scintillation counter.

Test results are expressed in terms of Stimulation Indices and the threshold level for the Stimulation Index to be considered a positive indicator of the potential to cause skin sensitisation is 3.0.

Results

	Concentration of Test Article in Applied Formulation (% w/v)
	5%	10%	25%
Stimulation Index	1.29	1.72	2.12

 

The positive control article produced a Stimulation Index of 4.48, demonstrating adequate performance of the assay.

Conclusion

The test substance, N,N'-(2-(4-(2-aminobenzamido)butyl)pentane-1,5-diyl)bis(2-aminobenzamide) did not induce skin sensitisation in the Local Lymph Node Assay and is considered not to be a skin sensitser.

 

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Phase: 28 September 2020 to 27 October 2020.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: of OECD Guidelines for Testing of Chemicals Method 442E: In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT), adopted June 2018.

Deviations:

yes

Remarks:

An adjustment to the guideline serial dilutions when preparing the stock dose solutions had to be made due to the viscous nature of the test article introducing air bubbles. This did not affect interpretation or compromise the integrity of the study.

GLP compliance:

yes

Type of study:

other: Expression of cell surface markers CD86 and CD54.

Justification for non-LLNA method:

The h-CLAT assay was conducted as part of a stepwise approach to the assessment of skin sensitisation of the test substance, as required by REACH from 11 October 2016.

Details on the study design:

TEST ITEM CONCENTRATIONS
7.81, 15.63, 31.25, 62.50, 125, 250, 500 and 1000 µg/mL

The test item concentrations for use in the main test were determined from a dose range-finder test.

CONTROLS
Positive control: This was performed using DNCB (CAS no. 97-00-7, ≥99% purity), nickel sulphate (CAS no. 10101-97-0, ≥99% purity)
Negative Control: lactic acid (CAS no. 50-21-5, ≥85% purity)

THP-1 CELL CULTURE
THP-1 cells were cultured in a humidified incubator set to 37ºC, 5% CO2, in RPMI 1640 medium supplemented with 10% heat inactivated (HI) foetal bovine serum (FBS), 0.05 mM 2-mercaptoethanol, 100 units/mL penicillin and 100 µg/mL streptomycin. The cells were passaged every 2-3 days at a density of 0.1 to 0.2 x 106 cells/mL and maintained at a density from 0.1 x 106 to 0.8 x 106 cells/mL. Cell density did not exceed 1 x 106 cells/mL, and cells did not exceed 30 passages.

PLATE PREPARATION
THP-1 cells were pre-cultured in culture flasks either at a density of 0.2 x 106 cells/mL for 48 hours or at a density of 0.1 x 106 cells/mL for 72 hours. On the days of testing, cells were harvested from the flasks and were resuspended with fresh culture medium at 2 x 106 cells/mL. The cells were then distributed into a 24 well flat-bottom plate (500 µL/well) (expression assay) or a 96-well flat-bottomed plate (80 µL/1.6 x 105 cells per well) (DRF assay(s)).

CD86/CD54 EXPRESSION MEASUREMENT
Three independent runs (experiments) were needed to drive a prediction. Each independent run was performed on a different day.

In the initial Experiment 2, the RFI ratio of CD54 for the DNCB positive control did not meet the relevant positive threshold criteria (RFI of 187% was obtained vs protocol criteria of 200%). These data were therefore invalidated and are not reported. The Experiment 2 treatments were repeated in order to provide the data presented in this report.

On the days of testing, cells harvested from the flasks were resuspended with fresh culture medium at 2 x 106 cells/mL. The cells were then distributed into a 24-well plate (500 µL/1 x 106 cells per well).

The test article working solution or solvent control was mixed 1:1 (v/v) with the cell suspensions in the 24-well plates. The plates were sealed and then incubated for 24 hours (incubator set to 37ºC, 5% CO2).

After the 24-hour incubation period, the cells were transferred into sample tubes, collected by centrifugation (approximately 250 g, 5 minutes) and washed twice with 1 mL of FACS buffer. After washing, the cells were blocked with 600 µL of blocking solution (FACS buffer containing 0.01% (w/v) globulin) on ice for 15 minutes.

After blocking, the cells were split into three aliquots of 180 µL into a 96-well plate and centrifuged (approximately 250 g, 3 minutes). After centrifugation, the cells were stained with 50 µL of FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 antibodies on ice for 30 minutes. The stained cells were washed three times with an excess of FACS buffer, re-suspended in FACS buffer and 12.5 µg/mL PI solution was added (to give a final PI concentration of 0.625 µg/mL).

The expression levels of CD86 and CD54 and cell viability were analysed using flow cytometry.

Positive control results:

This was performed using DNCB (CAS no. 97-00-7, ≥99% purity) and nickel sulphate (CAS no. 10101-97-0, ≥99% purity). RFI values were ≥150% for CD86 and ≥200% for CD54, and cell viability was >50% in each independent run.

Key result

Run / experiment:

mean

Parameter:

EC150, CD86 [442E]

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: No EC150 value was calculated for CD86 as this marker was negative in all experiments.

Key result

Run / experiment:

mean

Parameter:

EC200, CD54 [442E]

Value:

13.3 µg/mL

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

All assay acceptance criteria were met as highlighted below:

The cell viabilities of medium and solvent/vehicle control were higher than 90% in each independent run.

In the solvent control, RFI values of both CD86 and CD54 did not exceed the positive criteria (CD86 RFI ≥150% and CD54 RFI ≥200%).

For both medium and solvent/vehicle controls, the MFI ratio of both CD86 and CD54 to isotype control was >105% on all occasions.

For the positive control, RFI values were ≥150% for CD86 and ≥200% for CD54, and cell viability was >50% in each independent run.

For the test article, the cell viability was more than 50% in all tested concentrations in each independent run.

The relative fluorescence intensity (RFI) values for the test article were calculated as follows:

 

 

Concentration (µg/mL)	RFI (CD86)			RFI (CD54)
	Exp 1	Exp 2	Exp 3	Exp 1	Exp 2	Exp 3
7.81	57	100	88	119	220	154
15.63	70	94	69	152	243	220
31.25	53	82	89	166	255	289
62.50	44	47	71	176	238	305
125	35	59	54	171	205	311
250	31	41	48	191	227	333
500	49	52	51	188	258	254
1000	43	87	71	30	88	51
Solvent/ vehicle Control (DMSO)	147	85	94	149	1 04	76
Positive Control (DNCB)	374	327	354	421	416	845

In Experiment 1, the RFI values for CD86 were <150% and the RFI values for CD54 were <200% at all concentrations.

In Experiment 2, the RFI values for CD86 were <150% at all concentrations, and the RFI values for CD54 were >200% (with cell viability >50%) at concentrations of 7.81 to 500 μg/mL. A positive prediction was therefore obtained for the CD54 marker.

In Experiment 3, the RFI values for CD86 were <150% at all concentrations and the RFI values for CD54 were >200% (with cell viability >50%) at concentrations of 15.63 to 500 μg/mL.

Therefore, based on the majority result of the three independent runs, the test article gave a positive prediction in the assay.

The EC200 value for CD54 calculated by linear regression of endpoint assay data was 13.3 μg/mL. No EC150 value was calculated for CD86 as this marker was negative in all experiments.

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Remarks:

This result will not be interpreted as a stand alone result, but incorporated in a battery of test results to provide an overall conclusion as to the skin sensitising status of the test substance.

Conclusions:

The test item was considered to be positive in the human Cell Line Activation Test.
 

Executive summary:

Introduction

The study was conducted to investigate the potential of N,N'-(2-(4-(2-aminobenzamido)butyl)pentane-1,5‑diyl)bis(2-aminobenzamide) to activate monocytes and dendritic cells in the human monocytic leukemia cell line THP-1, by quantifying changes in the expression of cell surface markers (CD86 and CD54). The procedure was designed to meet the requirements of the following guideline:

OECD Guidelines for Testing of Chemicals Method 442E:In VitroSkin Sensitisation: human Cell Line Activation Test (h-CLAT), adopted June 2018.

The data may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.

Method

For the dose finding assay, the test article was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 500 mg/mL giving a maximum test concentration of 1000 µg/mL. No reduction in viability was observed.

For the expression measurements, test concentrations in a range from 7.81 to 1000 μg/mL (after dilution in medium) were used.

Aliquots of 500 µL of each of the working solutionswere mixed 1:1 with cell suspensions at 1 x 10⁶cells per well. After blocking, the cells were stained with FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 antibodies on ice for 30 minutes. The stained cells were washed, re-suspended in FACS buffer and propidium iodide solution was added. The expression levels of CD86 and CD54 and cell viability were analysed using flow cytometry.

Results

For CD86, the RFI values were <150% in all three experiments. For CD54, the RFI values for CD54 were <200% at all concentrations in Experiment 1, however in Experiments 2 and 3 the RFI values for CD54 were >200% (with cell viability >50%) at most test concentrations Therefore, based on the majority result of the three independent runs, the test article gave a positive prediction in the assay.

The EC200 value for CD54 was calculated to be 13.3 μg/mL.

All acceptance criteria of the h-CLAT assay parameters were met in each experiment.

Conclusion

The test article, N,N'-(2-(4-(2-aminobenzamido)butyl)pentane-1,5‑diyl)bis(2-aminobenzamide), was considered to be positive in the human Cell Line Activation Test.

 

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental phase: 06 October 2020 to 20 November 2020.

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 KeratinoSens™ test method)

Deviations:

no

GLP compliance:

yes

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

The ARE-Nrf2 Luciferase test was conducted as part of a stepwise approach to the assessment of skin sensitisation of the test substance as required by REACH from 11 October 2016

Details on the study design:

EXPERIMENTAL DESIGN

Test System
The cells used in the assay were the KeratinoSens™ cell line supplied by Givaudan Schweiz, Zurich, Switzerland. A fresh vial of cells was used for each experimental occasion and cultured using Dulbecco’s modified Eagle medium (DMEM) containing serum and Geneticin. The cells (passage 12) were 80 - 90% confluent. On the day prior to treatment, cells were harvested and distributed into 96-well plates (10000 cells/well) and incubated for 24±1 hours an incubator set to 37°C, 5% CO2.

Replicates
For each experiment three replicates were used for the luciferase activity measurements and one parallel replicate used for the cell viability assay.

Preparation of Test Item
Test formulations were prepared using DMSO. This was the first of the listed vehicles that produced a visually clear solution / stable dispersion at a concentration of 200 mM. Serial dilutions were made (from the 200 mM stock) using the vehicle to obtain 12 master concentrations ranging from 0.098 to 200 mM. The master concentrations were then further diluted 25-fold into culture medium containing serum and finally used for treatment with a further 4-fold dilution factor in the treatment plates, so that the final concentrations range from 0.98 to 2000 µM.

Treatment
At the end of the 24-hour incubation period (see test system above), the medium was removed and replaced with 150 µL fresh culture medium (containing serum but without Geneticin) to which test article and control formulations were added. One well per plate was left empty (no cells and no treatment) to assess background values. Aliquots of 50 µL of each of the 25-fold test article or control dilutions (see Section 3.1) were transferred to give three luciferase replicates on a white walled plate and a single viability replicate on a clear walled plate. Each plate was sealed and incubated for 48±1 hours in a humidified incubator set to 37ºC, 5% CO2.

For each test article and positive control, one experiment, consisting of at least two independent repetitions each containing three replicates of each concentration are needed to derive a prediction (positive or negative).

Cytotoxicity Assessment
After the 48-hour exposure period, the medium was replaced with fresh medium containing MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). The plate was sealed and placed in an incubator set to 37°C, 5% CO2 for 4 hours. The MTT medium was removed and SDS (at 10% w/v) added per well. The plate was sealed and placed into an incubator set to 37°C, 5% CO2, and left overnight. After the overnight incubation, the plate was shaken to ensure homogeneity of the solution in the wells and then absorption read at 600 nm.

Luciferase Activity Measurements
After the 48-hour exposure period, the cells were washed with phosphate buffered saline and lysis buffer for luminescence readings was added to each well. The plate was then incubated for 20 minutes in an incubator set to 25°C, loaded into the luminescence plate reader and read using the following parameters: 100 µL injection (Luciferase assay substrate), 15 second delay, 7 second luminescence integration time.

Positive control results:

The positive and negative controls responded as expected and thus confirmed assay validity.

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: Luciferase activity induction (Imax)

Value:

1.09

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

The test did not meet the criteria for acceptance of results as either positive or negative.

Key result

Run / experiment:

other: Experiment 2

Parameter:

other: Luciferase activity induction (Imax)

Value:

1.18

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

The test did not meet the criteria for acceptance of results as either positive or negative.

Key result

Run / experiment:

other: Experiment 3

Parameter:

other: Luciferase activity induction (Imax)

Value:

1.09

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

The test did not meet the criteria for acceptance of results as either positive or negative.

Key result

Run / experiment:

other: Mean (of all 3 experiments)

Parameter:

other: Luciferase activity induction (Imax)

Value:

1.12

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

The test did not meet the criteria for acceptance of results as either positive or negative.

RESULTS

At the end of the 48-hour treatment period, precipitation was observed at concentrations of 125 – 2000µM in Experiment 1; the maximum soluble test concentration was therefore considered to be 62.5 μM. Cytotoxicity (<70% viability) was not reached at the maximum soluble concentration.

For Experiment 2, an unusual data recording of precipitation and toxicity was noted, which was not consistent with Experiment 1. A third experiment (Experiment 3) was therefore performed for clarification.

In Experiment 3, precipitation was noted at concentrations of 250 - 2000 μM and the maximum soluble test concentration was therefore considered to be 125 μM. Cytotoxicity (<70% viability) was not reached at the maximum soluble concentration.

Experiment 3 therefore confirmed the same toxicity and precipitation observations as Experiment 1, and thereby clarified the unusual data recording mentioned above for Experiment 2, in that the maximum soluble test concentration in Experiment 2 was below 1000 μM, and cytotoxicity (<70% viability) was not reached at the maximum soluble concentration.

There were no EC_1.5 values for Experiments 1, 2 or 3 as there were no statistically significant increases in induction.

 

Viability

 

The cell viability measurement was not applicable as there were no EC_1.5 determining concentrations in Experiments 1, 2 or 3. It may however be noted thatcytotoxicity (<70% viability) was not reached at the maximum soluble concentration in any experiment. The IC₅₀ value could not be calculated for Experiment 3 as the minimum viability did not drop below 50%.

 

The four conditions required for a positive prediction were not met in any experiment, however as a negative result was obtained in a test with maximal soluble concentrations below 1000 µM, and cytotoxcity (<70%) was not reached at the maximum soluble concentration, the criteria for negativity cannot be applied. In accordance with the test guideline the result is therefore considered inconclusive.

Interpretation of results:

study cannot be used for classification

Remarks:

The results were considered inconclusive and no prediction could be made.

Conclusions:

The four conditions required for a positive prediction were not met in any experiment. In accordance with the test guideline. As a negative result was obtained in a test with maximal soluble concentrations below 1000 µM, and cytotoxicity (<70%) was not reached at the maximum soluble concentration, the criteria for negativity cannot be applied and the result of the ARE-Nrf2 Luciferase Test of N,N'-(2-(4-(2-aminobenzamido)butyl)pentane-1,5-diyl)bis(2-aminobenzamide) is considered to be inconclusive.

Executive summary:

Introduction

The study was conducted to investigate the potential of N,N'-(2-(4-(2-aminobenzamido)butyl)pentane-1,5-diyl)bis(2-aminobenzamide) to induce genes that are regulated by the antioxidant response element (ARE). The study was conducted to meet the requirements of the following guideline:

 

• OECD Guidelines for Testing of Chemicals Method 442D (adopted June 2018).

 

 

Method

The test article was dissolved in dimethyl sulfoxide (DMSO) and diluted to give a final test concentration ranging from 0.98 to 2000 µM. Each plate was sealed using a plate sealer and then incubated for 48±1 hours in a humidified incubator set to 37ºC, 5% CO₂. There were three luciferase replicates on a white walled plate and a single viability replicate on a clear walled plate.

 

After the 48-hour exposure period, the medium in the viability plate was replaced with fresh medium containing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The plate was sealed and incubated for 4 hours in an incubator set to 37°C, 5% CO₂. The MTT medium was then removed and SDS (at 10% w/v) added per well. The plate was sealed and placed into an incubator set to 37°C, 5% CO₂, and left overnight. After the overnight incubation absorption was read at 600 nm.

 

After the 48-hour exposure period, the cells in the luciferase plate were washed with phosphate buffered saline and lysis buffer for luminescence readings was added to each well. The plate was then incubated for 20 minutes in an incubator set to 25°C, loaded into the luminescence plate reader and read.

 

Results

The I_max value in all three experiments was less than 1.5.

 

The positive and negative controls responded as expected and thus confirmed assay validity.

 

Precipitation of the test article was observed in each experiment, such that the maximum soluble test concentration was below 1000 μM. Cytotoxicity (<70% viability) was not reached at the maximum soluble concentration.

 

 

Conclusion

The four conditions required for a positive prediction were not met in any experiment. In accordance with the test guideline. As a negative result was obtained in a test with maximal soluble concentrations below 1000 µM, and cytotoxicity (<70%) was not reached at the maximum soluble concentration, the criteria for negativity cannot be applied and the result of the ARE-Nrf2 Luciferase Test of N,N'-(2-(4-(2-aminobenzamido)butyl)pentane-1,5-diyl)bis(2-aminobenzamide) is considered to be inconclusive.

Reference 4

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Phase: 02 November 2020 to 06 November 2020.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Deviations:

yes

Remarks:

The Test Item could not be solubilised at the target concentration of 100 mM in a solvent compatible with the assay. A reduced concentration solution (10 mM) in DMF/propan-2-ol 10/90 v/v was prepared for use in the assay.

GLP compliance:

yes

Type of study:

other: Direct Peptide Reactivity Assay (DPRA)

Justification for non-LLNA method:

The direct peptide binding assay is a recognised assay for conducting as part of a battery of tests for the assessment of skin sensitisation for registration under REACH.

Details of test system:

other: DPRA (OECD 442c)

Details on the study design:

Experimental Procedure

Assessment of Test Item Solubility

The solubility of the test substance was assessed at a concentration of 100 mM and 10 mM in a selection of solvents and solvent mixtures.

Preparation of Peptide Stock Solutions
Stock solutions of each peptide at concentrations of 0.667 mM were prepared by dissolution of pre-weighed aliquots of the appropriate peptide in ca 20 mL aliquots of the appropriate buffer solution (Cysteine in 100 mM phosphate buffer pH 7.5, Lysine in 100 mM Ammonium acetate buffer pH 10.2).

Preparation of Peptide Calibration Standards
Calibration standards of both peptides were prepared by diluting the requisite stock solution in the appropriate buffer and acetonitrile and contained each peptide at concentrations of 0.0167 mM, 0.0334 mM, 0.0667 mM, 0.133 mM, 0.267 mM and 0.534 mM. A buffer blank was also prepared.

Preparation of Stability Controls and Precision Controls
Precision controls (Reference Control A), stability controls (Reference Control B) and (Reference Control C), of both peptides were prepared at a peptide concentration of 0.5 mM. Reference Control A and Reference Control B were the appropriate peptide buffer and acetonitrile, Reference Control C were the appropriate peptide buffer and DMF/Propan-2-ol 10/90 v/v.

Preparation of Positive Control Solution and Test Item Stock Solution
The positive control chemical (Cinnamic Aldehyde) was prepared at a concentration of 100 mM in acetonitrile. A 10 mM stock solution of the test substance was prepared in DMF/Propan-2-ol 10/90 v/v.

Preparation of Positive Control and Cysteine Peptide Depletion Samples and Co-elution Controls
Accurate volume aliquots of the test substance stock solution and the positive control solution were diluted with the Cysteine peptide stock solution to prepare solutions containing 0.5 mM Cysteine and either 0.5 mM of test substance or 5mM positive control. For the co-elution control, the appropriate peptide buffer solution was used in place of the Cysteine stock solution.

Preparation of Positive Control and Lysine Peptide Depletion Samples and Co elution Controls
Accurate volume aliquots of test substance stock solution and the positive control solution were diluted with the Lysine peptide stock solution to prepare solutions containing 0.5 mM Lysine and either 2.5 mM test substance or 25 mM positive control. For the co-elution control, the appropriate peptide buffer solution was used in place of the Lysine stock solution.

Incubation
The appearance of the test substance and positive control samples in the HPLC vials was documented after preparation and then the vials placed into the autosampler of the HPLC set at 25°C for a minimum of 22 hours incubation prior to injection of the samples as part of analytical run. Before initiation of the run the appearance of the samples in the vials was assessed and documented again.

Analysis
The concentration of both the Cysteine and Lysine peptides in the presence of the test substance and the associated positive controls was quantified by HPLC using UV detection as detailed in the chromatographic section.

Instrument
Instrument: Waters Alliance 2695 separation module and 2487 dual wavelength detector
Column: Agilent Zorbax SB C18, 3.5µm, 100 x 2.1 mm
Column temperature: 30°C
Sample temperature: 25°C
Detector wavelength: UV, 220 nm

Positive control:

other: Cinnamic Aldehyde

Positive control results:

The positve control substance Cinnamic Aldehyde, responded as expected demonstrating that the asay was working as expected.

Key result

Run / experiment:

other: Cysteine

Parameter:

other: Mean peptide depletion

Value:

3.2

Vehicle controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

not determinable because of methodological limitations

Remarks:

Due to solubility constraints the test item was applied to the assay system at a concentration below 100 mM. As per OECD TG 442C, a negative prediction cannot be confirmed with regards to the test item sensitising potential, and the assay outcome is inconclusive.

Key result

Run / experiment:

other: Lysine

Parameter:

other: Mean peptide depletion

Value:

0

Vehicle controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

not determinable because of methodological limitations

Remarks:

Due to solubility constraints the test item was applied to the assay system at a concentration below 100 mM. As per OECD TG 442C, a negative prediction cannot be confirmed with regards to the test item sensitising potential, and the assay outcome is inconclusive.

Solubility Assessment

The solubility of the test substance was tested in the following solvents at a nominal concentration of 100 mM, and was not achieved: acetonitrile, propan-2-ol and methanol. Solubility at 100 mM was achieved in N,N-dimethylformamide (DMF), however neat DMF is not a suitable solvent for the assay. Further attempts were made to solubilise the test item in DMF/acetonitrile 10/90 v/v and DMF/propan-2-ol 10/90 v/v mixtures, but solubility was not achieved at 100 mM.

 

A solution of the test item in DMF at 100 mM was added to propan-2-ol to give a final solution of 10 mM concentration in DMF/propan-2-ol 10/90 v/v, and a clear solution was obtained. This preparation was used for the depletion samples.

 

Observation of assay samples for both Cysteine and Lysine peptides indicated that the test item was not fully soluble in the assay media.

 

In Lysine assay samples and the coelution control test item in assay media (no peptide), there was immediate evidence of test item precipitation, with the presence of a beige precipitate. The dissolution control (test item in DMF/propan-2-ol 10/90 v/v) was a clear colourless solution.

 

In Cysteine assay samples, a clear solution was formed initially, however upon observation after overnight incubation, precipitate was noted. In contrast, the dissolution and coelution controls were clear colourless solutions.

 

Reactivity Assessment

 

The DPRA prediction and the reactivity of the test substance wasbased on theoverall mean and the individual depletion values in the Cysteine peptide and the Lysine peptide.  The depletion of peptide in the presence of the test substance is presented in the following table:

 

 

Peptide	Mean peak area of reference control (µV.sec)	Mean peak area of peptide with test item(µV.sec)	Mean peptide depletion by the test substance (%)
Cysteine	Control B:839090 (n=6) Control C:832020 (n=3)	805570 (n=3)	3.2
Lysine	Control B:728810 (n=6) Control C:708484 (n=3)	708720 (n=3)	0.0

Interpretation of results:

study cannot be used for classification

Conclusions:

Solutions of N,N’-(2-(4-(2-aminobenzamido)butyl)pentane1,5-diyl)bis(2-aminobenzamide) were analysed by a DPRA analytical method in both Cysteine and Lysine containing synthetic peptides.

There was minimal reactivity in both peptides and therefore the test substance is classified into the reactivity class of “no or minimal”.

However, due to solubility constraints the test item was applied to the assay system at a concentration below 100 mM. As per OECD TG 442C, in this situation a negative prediction cannot be confirmed with regards to the test item sensitising potential, and the assay outcome is inconclusive.

Executive summary:

Introduction

The purpose of this study was to assess the reactivity and sensitizing potential of N,N’-(2-(4-(2-aminobenzamido)butyl)pentane1,5-diyl)bis(2-aminobenzamide in the Direct Peptide Reactivity Assay (DPRA). The method was designed to be meet the requirements of the following guideline:

 

OECD Guideline 442C: In Chemico Skin Sensitisation

 

Method

Solutions of test substance were analysed by the validated DPRA analytical method in both Cysteine and Lysine containing synthetic peptides. 

 

Results

There was minimal reactivity in both peptides therefore the test item was classified as reactivity class “no or minimal”.

 

Conclusion

Solutions of N,N’-(2-(4-(2-aminobenzamido)butyl)pentane1,5-diyl)bis(2-aminobenzamide) were analysed by a DPRA analytical method in both Cysteine and Lysine containing synthetic peptides.

 

There was minimal reactivity in both peptides and therefore the test substanceis classified intothe reactivity class of “no or minimal”.

 

However, due to solubility constraints the test item was applied to the assay system at a concentration below 100 mM. As per OECD TG 442C, in this situation a negative prediction cannot be confirmed with regards to the test item sensitising potential, and the assay outcome is inconclusive. 

Reference 5

Endpoint:

skin sensitisation, other

Remarks:

QSAR Assessment

Type of information:

(Q)SAR

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Remarks:

The QSAR assessment was made in accorndance with current ECHA guidelines.

Justification for type of information:

According to Annex VII (Regulation (EC) No 1907/2006) the information needed for the classification or risk assessment of a substance should be obtained through non-animal methods as a first step.

Data from valid (Q)SAR is one of the first steps in assessing whether a substance may be a skin sensitiser.

Qualifier:

no guideline available

Principles of method if other than guideline:

QSAR assessment is part of the initial steps in a stepwise approach to assessing the skin sensitising potential of a substance.

GLP compliance:

no

Justification for non-LLNA method:

QSAR assessment is recognised as a key first step in a battery of assessments of a test substance for skin sensitisation potential for registration under REACH. Depending upon the outcome further assessment may, or may not be required.

Key result

Parameter:

other: QSAR

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

not determinable because of methodological limitations

Remarks:

The exclusion criteria for testing according to ECHA are not met and the QSAR predictions are not considered conclusive for a conclusion based on weight of evidence, in vitro testing of the assesed substance should therefore be performed.

Appraisal of (Q)SAR Modelling

The prediction results are presented in the following table:

Prediction Results

Model	Prediction Result	Reliability
TOPKAT	Not sensitising	Not reliable
CAESAR (VEGA)	Not sensitising	Not reliable
IRFMN/JRC ( VEGA)	Sensitising	Moderately reliable
OECD Toolbox	Not sensitising	Reliable
DEREK	Skin sensitisation in mammal is PLAUSIBLE Alert matched: 427 Aromatic primary or secondary amine	Inconclusive
TOXTREE	No alert for skin sensitisation reactivity domain triggered	Not applicable

TOPKAT predicted N,N'-(2-(4-(2-aminobenzamido)butyl)pentane-1,5-diyl)bis(2-aminobenzamide) not to be sensitising. However, considerations on structural similar compounds in the training set and prediction statistics indicate no confidence in the prediction.

CAESAR predicted N,N'-(2-(4-(2-aminobenzamido)butyl)pentane-1,5-diyl)bis(2-aminobenzamide) not to be sensitising. The query compound is however out of the applicability domain of the model and the prediction not considered reliable.

The query compound was predicted to be sensitising by the IRFMN/JRC model. However, uncertainties such as low structural similarity between the compounds in the training set and the query compound and study results of these compounds not corresponding with the prediction result indicate uncertainties and the prediction is therefore considered moderately reliable.

Profiling with OECD Toolbox indicates the query compound to be subject of biotic metabolic transformation. While no protein binding alert for skin sensitisation was triggered for the compound itself, the Toolbox simulated 18 potential metabolites of which 12 triggered an alert for nitroso protein binding. These metabolites are similar in structure and size to their parent and only differ in the position of the nitroso moiety. Using analogues search accounting for biotic metabolism (and excluding abiotic metabolism) and matching nitroso protein binding alerts, not sensitising pigment red 177 (CAS 4051-63-2) was obtained.

The formation of a nitroso compound as protein reactive species is also proposed by DEREK though as a result of non-enzymatic autoxidation. A second proposed mechanism suggests the formation of a reactive nitrenium ion. As test/example compound triggering the same alert DEREK provided sensitising 4-choloraniline. The test item is considered structurally more similar to pigment red 177 than to 4-chloroaniline. In addition, water solubility and log K_ow of pigment red 177 and the test item suggest only limited or low dermal uptake, while the log K_ow and water solubility of 4-chloroaniline favour absorption.

However, with only one prediction considered reliable there are not enough arguments for a weight of evidence conclusion and therefore additional in vitro testing is recommended.

Interpretation of results:

study cannot be used for classification

Remarks:

The QSAR assessment was not conclusive as to whether the test substance could be skin sensitiser and further investigations are recommended.

Conclusions:

As the exclusion criteria for testing according to ECHA are not met and the QSAR predictions are not considered conclusive for a weight of evidence, in chemico/in vitro testing of the test item should be performed.

Results of the TIMES metabolism simulator and the absence of protein binding alerts in OECD Toolbox and Toxtree indicate that the test item may act exclusively as pro hapten. However, abiotic transformation cannot fully be excluded as suggested by DEREK.

Executive summary:

Introduction

The prediction of the skin sensitising potential of N,N'-(2-(4-(2-aminobenzamido)butyl)pentane-1,5-diyl)bis(2-aminobenzamide) was performed with BIOVIA Discovery Studio (TOPKAT) 2020, VEGA NIC 1.1.5 (CAESAR, IRFMN/JRC), OECD QSAR Toolbox 4.5, Toxtree 3.1.0 and DEREK Nexus 6.0.1. The TOPKAT model for skin sensitisation was extended by including 70 compounds from an internal company database.

 

Results

The prediction results are presented in the following table:

Prediction Results

Model	Prediction Result	Reliability
TOPKAT	Not sensitising	Not reliable
CAESAR (VEGA)	Not sensitising	Not reliable
IRFMN/JRC ( VEGA)	Sensitising	Moderately reliable
OECD Toolbox	Not sensitising	Reliable
DEREK	Skin sensitisation in mammal is PLAUSIBLE Alert matched: 427 Aromatic primary or secondary amine	Inconclusive
TOXTREE	No alert for skin sensitisation reactivity domain triggered	Not applicable

   

Conclusion

As the exclusion criteria for testing according to ECHA are not met and the QSAR predictions are not considered conclusive for a weight of evidence, in chemico/in vitro testing of the test item should be performed.

Results of the TIMES metabolism simulator and the absence of protein binding alerts in OECD Toolbox and Toxtree indicate that the test item may act exclusively as pro hapten. However, abiotic transformation cannot fully be excluded as suggested by DEREK.

 

Reference 6

Endpoint:

skin sensitisation: in chemico

Type of information:

(Q)SAR

Adequacy of study:

key study

Study period:

Report issue: 02 December 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

According to Annex VII (Regulation (EC) No 1907/2006) the information needed for the classification or risk assessment of a substance should be obtained through non-animal methods as a first step.

Before starting any new testing an assessment of all available in vitro data, in vivo data, historical human data, data from valid (Q)SARs and data from structurally related substances (read-across approach) is required.

Qualifier:

no guideline followed

Principles of method if other than guideline:

The first step in the assessment is to undertake a literature search for existing data on skin sensitisation potential.

As no relevant existing data on skin sensitisation potential was found following searches a QSAR assessment was undertaken to meet the requirements of conducting skin sensitisation assessment for the test substance according to Annex VII section 8.3 of the REACH Regulation (Regulation (EC) No 1907/2006 and Commission Regulation (EU) 2016/1688).

The QSAR assessment is the first step in a stepwise approach to assessing the skin sensitising potential of a substance by consideration of the structural properties of the test substance and any available existing data.

GLP compliance:

no

Justification for non-LLNA method:

Review of existing data and QSAR assessment is recognised as a key first step in a battery of assessments of a test substance for skin sensitisation potential for registration under REACH. Depending upon the outcome further assessment may, or may not be required.

Key result

Parameter:

other: QSAR and expert assessment of skin sensitisation potential.

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

other: The exclusion criteria for testing according to ECHA are not met and the QSAR predictions are not considered conclusive for a conclusion based on weight of evidence, in vitro testing of the assessed substance should therefore be performed.

The exclusion criteria for testing according to ECHA are not met and the QSAR predictions are not considered conclusive for a weight of evidence assessment.

Only one prediction was considered reliable there are not enough arguments for a weight of evidence conclusion. Results of the TIMES metabolism simulator and the absence of protein binding alerts in OECD Toolbox and Toxtree indicate that the test item may act exclusively as pro hapten.

Abiotic transformation cannot fully be excluded as suggested by DEREK.

Interpretation of results:

study cannot be used for classification

Remarks:

The expert /QSAR assessment was not conclusive as to whether the test substance could be skin sensitiser and further investigations are recommended.

Conclusions:

As the exclusion criteria for testing according to ECHA are not met and the QSAR predictions are not considered conclusive for a weight of evidence, in vitro testing of N,N'-(2-(4-(2-aminobenzamido)butyl)pentane-1,5-diyl)bis(2-aminobenzamide) should be performed.

Executive summary:

Introduction

An assessment of the skin sensitisation potential of N,N'-(2-(4-(2-aminobenzamido)butyl)pentane-1,5-diyl)bis(2-aminobenzamide was conducted based on a review of existing data, QSAR and expert assessment to addresses the requirements of conducting skin sensitisation assessment according to Annex VII section 8.3 of the REACH Regulation (Regulation (EC) No 1907/2006.

The assessment(s) form the first part of stepwise approach to assessing skin sensitisation potential. Depending on the results further assessments and / or in vivo in vitro testing may be required.

Method

Screening of databases such as ECHA, ChemIDplus, ChemSpider, PubChem, PubMed, US EPA was undertaken to as part of an existing data assessment.

The prediction of the skin sensitising potential was performed with BIOVIA Discovery Studio (TOPKAT) 2020, VEGA NIC 1.1.5 (CAESAR & IRFMN/JRC), OECD QSAR Toolbox 4.4, Toxtree 3.1.0 and DEREK Nexus 6.0.1.

Results

The databases examined provided no information on the skin sensitising properties of the assessed substance. Only one QSAR prediction was considered reliable there are not enough arguments for a weight of evidence conclusion.

Conclusion

As the exclusion criteria for testing according to ECHA are not met and the QSAR predictions are not considered conclusive for a weight of evidence, in vitro testing of N,N'-(2-(4-(2-aminobenzamido)butyl)pentane-1,5-diyl)bis(2-aminobenzamide) should be performed.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Justification for classification or non-classification

A QSAR assessment concluded that there was insufficient information to draw conclusions about the sensitising potential of the substance. 

 

The in vitro Luciferase and Direct Peptide Reactivity Assays were inconclusive while the h-CLAT assay gave a positive result.  Overall, the in vitro tests were considered inconclusive with regards to skin sensitisation.

 

As the results of the QSAR and in vitro assessments being inconclusive, an in vivo LLNA test was conducted which was negative.

 

Based on the results of the LLNA and an overall weight of evidence approach the substances is concluded as negative for skin sensitisation and no classification is required.