N-[2-[bis(carboxymethyl)amino]ethyl]-N-(1-oxononyl)Glycine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2022-04-05 to 2022-10-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442E (In Vitro Skin Sensitisation assays addressing the key event on activation of dendritic cells on the Adverse Outcome Pathway for skin sensitisation)

Version / remarks:

30 June 2022

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

human Cell Line Activation Test (h-CLAT)

Test material

Test material information

Constituent 1

Reference substance name:

2-(N-{2-[bis(carboxymethyl)amino]ethyl}nonanamido)acetic acid

EC Number:

853-587-8

Cas Number:

199387-97-8

Molecular formula:

C17 H30 N2 O7

IUPAC Name:

2-(N-{2-[bis(carboxymethyl)amino]ethyl}nonanamido)acetic acid

Test material form:

solid: particulate/powder

Details on test material:

- Test material form: white to off-white crystalline powder
- Batch No.: LFND3B1003
- Storage conditions: 2 - 8 °C
- Expiry date: 01 August 2025

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- lot/batch number of test material: LFND3B1003
- Purity: 100% (HPLC)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Store in a cool place, protected from light

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): The test item was soluble in dimethyl sulfoxide at a maximal soluble concentration of 312.5 mg/mL. Vortex mixing, sonication and warming to 37 °C were used to aid solubilisation.

In vitro test system

Details of test system:

THP-1 cell line [442E]

Details on the study design:

442E

PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical stock solution: The test item was soluble in dimethyl sulfoxide (DMSO, CAS No.: 67-68-5, purity ≥ 99%) at a maximal soluble concentration of 312.5 mg/mL. From this initial solution, 8 stock solutions were prepared by 1:1.2 dilution.
- Preparation of the test chemical serial dilutions: The working stock solutions were prepared by diluting each stock solution 250 times with cell culture medium. The working stock solutions were applied to the cells by adding equal volumes of each working solution to prepared cells, resulting in a further 1:2 dilution of the working solutions.
- Preparation of the positive controls: 2,4-dinitrochlorobenzene at a final concentration of 4 µg/mL was tested concurrently with the test item. DNCB was dissolved in DMSO and diluted, resulting in a final DMSO concentration of 0.2% (v/v)
- Preparation of the solvent, vehicle and negative controls: The solvent controls were diluted (mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well plate), resulting in a final concentration of 0.2% (v/v) of DMSO.
- Stable dispersion obtained: yes, it was taken care that the test chemical was dissolved or stably dispersed in the chosen solvent and that it did not interfere with the test design.

DOSE RANGE FINDING ASSAY:
- Highest concentration used: 312.5 mg/mL (maximal soluble concentration in DMSO (solvent). From this starting solution, eight stock solutions (eight concentrations) were prepared by 2-fold serial dilutions using the corresponding solvent. These stock solutions were further diluted 250-fold (DMSO) into culture medium (working solutions). The working solutions were finally used for treatment by adding an equal volume of working solution to the volume of THP-1 cell suspension in a 96-well plate to achieve a further 2-fold dilution.
- Results of selecting appropriate concentration and determination of cytotoxicity e.g. CV75: The CV75 value, i.e. the concentration showing 75% cell survival, was calculated by log-linear interpolation. The CV75 value was used to calculate the concentration range of the test item for the main experiment. The dose finding assay was performed in one independent run. Since there was no cytotoxicity, no CV75 could be derived. The main experiment was performed covering a concentration range from 625– 4.88 µg/mL (312.50– 2.44 mg/mL stock solution) corresponding to the maximal soluble concentration of the test item.

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates: single replicates
- Number of repetitions: two independent experiments
- Test chemical concentrations: 625.00, 520.83, 434.03, 361.69, 301.41, 251.17, 209.31, 174.43 µg/mL
- Application procedure: For testing, THP-1 cells were pre-cultured for 48 h – 72 in culture flasks at a cell density of 0.1 – 0.2 x 10E6 cells/mL. Prior to test item application, cells were harvested from the cell culture flask by centrifugation (125 x g) and were re-suspended in fresh culture medium at a density of 2 x 10E6 cells/mL. Then, 500 µL of the cell suspension were seeded into a 24 well flat-bottom plate (1 x 10E6 cells/well).
The solvent controls, the positive control and the working solutions were mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well plate. Treated plates were incubated for 24 h ± 0.5 h at 37 °C ± 1 °C and 5% CO2.
- Exposure time: 24 h ± 0.5 h
- Study evaluation and decision criteria used: For test chemicals classified as sensitiser the effective concentration 150 for CD86 (EC150) and the effective concentration 200 for CD54 (EC200) can be calculated with the following equation:
EC150=B_dose+[((150-B_RFI)/((A_RFI-B_RFI ))*(A_dose-B_dose )]
EC200=B_dose+[((200-B_RFI ))/((A_RFI-B_RFI ) )*(A_dose-B_dose )]
If the RFI value of the lowest dose is above the positive criteria of CD86 and CD54, the EC150 and EC200 values can be calculated using the lowest dose by log linear extrapolation according to the following equation:
EC150=2^{〖log〗_2 (B_dose )+(150-B_RFI)/(A_RFI-B_RFI )*[(〖log〗_2 (A_dose )-〖log〗_2 (B_dose )]}
EC200=2^{〖log〗_2 (B_dose )+(200-B_RFI)/(A_RFI-B_RFI )*[(〖log〗_2 (A_dose )-〖log〗_2 (B_dose )]}
Adose is the lowest concentration in µg/mL with RFI > 150 (CD86) or 200 (CD54)
Bdose is the highest concentration in µg/mL with RFI < 150 (CD86) or 200 (CD54)
ARFI is the RFI at the lowest concentration with RFI > 150 (CD86) or 200 (CD54)
BRFI is the RFI at the highest concentration with RFI < 150 (CD86) or 200 (CD54)
For the purpose of more precisely deriving the EC150 and EC200 values, three independent runs should be performed for CD86/CD54 expression measurement. The EC150 and EC200 values are the median value of the ECs calculated from three independent runs. In order to obtain a median value, three independent runs are necessary. If only two of three independent runs meet the positive criteria, the higher EC150 or EC200 of the two calculated values is adopted. The EC values could potentially contribute to the assessment of sensitising potency when used in integrated approaches such as IATA.

- Description on study acceptance criteria:
The test meets acceptance criteria if:
• the cell viability of the medium and the solvent controls is >90%,
• the cell viability of at least four tested doses of the test item in each run is >50%,
• the RFI values of the positive control (DNCB) is ≥150% for CD86 and ≥200% for CD54 at a cell viability of >50%,
• the RFI values of the solvent control is not ≥150% for CD86 and not ≥200% for CD54,
• the MFI ratio of CD86 and CD54 to isotype IgG1 control for the medium and DMSO control, is >105%.

SEEDING AND INCUBATION
- Seeding conditions (passage number and seeding density): Cells at passage number (<30) were used. Cells are routinely passaged every 2-3 days at a density of 0.1 – 0.2 x 10E6 cells/mL. Cells were cultured in 175 cm2 culture flasks (Greiner) in Roswell Park Memorial Institute medium (RPMI-1640) supplemented with 10% fetal bovine serum, 25 mM HEPES, L-glutamine, 0.05 mM 2-mercaptoethanol and 100 U/ml penicillin / 100 µg/mL streptomycin in a humidified incubator at 37 ± 1°C and 5% CO2.
- Incubation conditions: The solvent controls, the positive control and the working solutions were mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well plate. Treated plates were incubated for 24 h ± 0.5 h at 37 °C ± 1 °C and 5% CO2.
- Washing conditions: After 24 h ± 0.5 h of exposure, cells were transferred into sample tubes and collected by centrifugation (approx. 250 x g). The following steps were carried out on ice with pre-cooled buffers and solutions. The supernatant was discarded and the remaining cells were washed twice with FACS buffer.

MEASUREMENT OF CELL SURFACE EXPRESSION/LUCIFERASE ACTIVITY:
After washing, cells were blocked using 600 µL of a FcR blocking buffer (FACS buffer containing 0.01% (w/v) Globulin Cohn Fraction) and incubated at 4 °C for 15 min. After blocking, cells were split in three aliquots into a 96-well V-bottom plate. After centrifugation (approx. 250 x g), cells were stained with 50 µL of FITC-labelled anti-CD86, anti-CD54, or mouse IgG1 (isotype) antibodies in the dark for 30 min. All antibodies were diluted in FACS buffer at an appropriate manner. After washing with FACS buffer two times, cells were re-suspended in FACS buffer and PI solution was added. PI staining was done immediately prior to the measurement by adding PI solutions to each sample (final concentration of PI was 0.625 µg/mL).
The expression levels of CD86 and CD54 as well as cell viability were analysed by flow cytometry using an excitation wavelength of λ = 488 nm and an emission wavelength of λ = 530 nm ± 15 nm for FITC and λ > 650 nm for PI. Based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of CD86 and CD54 and the cell viability was calculated.

DATA EVALUATION
- Cytotoxicity assessment: PI staining
- Prediction model used:
For CD86/CD54 expression measurement, each test item was tested in at least two independent runs to derive a single prediction. Each independent run was performed on a different day or on the same day provided that for each run: independent fresh stock solutions and working solutions of the test chemicals and antibody solutions were prepared and independently harvested cells were used.
Sensitising potential of the test item was predicted from the mean percentage expression of CD86 and CD54. Any test chemical tested by the h-CLAT was considered positive if the RFI of CD86 was equal to or greater than 150% at any tested dose at a cell viability ≥ 50% in at least two independent runs or if the RFI of CD54 was equal to or greater than 200% at any tested dose at a cell viability ≥ 50% in at least two independent runs or if the RFIs of both the CD86 and CD54 were equal to or are greater than 150% and 200% respectively at any tested dose at a cell viability ≥ 50% in at least two independent runs. In case of not concordant results a third run should be conducted to make the final prediction. Otherwise the results were considered as inconclusive.
A negative test result of a test item was only accepted if the cell viability at a concentration of 1.2 x CV75 is <90%. In contrast, a positive test outcome was accepted irrespective of cell viabilities >90% at a concentration of 1.2 x CV75. If no CV75 could be derived negative test results can be accepted when the test item is tested at the highest soluble concentration (5000 µg/mL for 0.9% NaCl solution; 1000 µg/mL for DMSO or a different organic solvent) even if the cell viability is >90%.

Vehicle / solvent control:

DMSO

Negative control:

not applicable

Positive control:

dinitrochlorobenzene (DNCB) [442E]

Results and discussion

Positive control results:

The positive control led to an upregulation of CD54 and CD86 in all experiments. The threshold of 150% for CD86 (234% experiment 1; 316% experiment 2;) and 200% for CD54 (251% experiment 1; 447% experiment 2) were clearly exceeded.

In vitro / in chemico

Results

Results 1

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

RFI CD54>150 [442E]

Remarks:

maximum RFI determined

Value:

105

At concentration:

312.5 other: mg/ml stock (625 µg/ml)

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 2

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

RFI CD54>150 [442E]

Remarks:

maximum RFI determined

Value:

113

At concentration:

150.7 other: mg/ml stock (301.41 µg/ml)

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 3

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

RFI CD86>200 [442E]

Remarks:

maximum RFI determined

Value:

115

At concentration:

312.5 other: mg/ml stock (625 µg/ml)

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 4

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

RFI CD86>200 [442E]

Remarks:

maximum RFI determined

Value:

123

At concentration:

180.84 other: mg/ml stock (361.69 µg/ml)

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Outcome of the prediction model:

negative [in vitro/in chemico]

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
For individual results see Table 1 in "Any other information on results incl. tables".

Any other information on results incl. tables

Summary of results:

No cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 94.5% (CD86), 93.1% (CD54) and 95.3% (isotype IgG1 control) in the first experiment and to 96.6% (CD86), 96.2% (CD54) and 95.7% (isotype IgG1 control) in the second experiment. The expression of the cell surface marker CD86 was not upregulated above the threshold of 150% in any of the experiments. The expression of cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments.

Table 1: CD54 and CD86 Expression Experiment 1

Sample	Conc. (stock) [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			Corrected Mean Fluorescence Intensity		Relative Fluorescence Intensity (RFI)		Ratio Isotype IgG1 to [%]
		CD86	CD54	Isotype IgG1	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	CD86	CD54
Medium Control	-	93.4	94.0	94.1	1681	705	375	1306	330	93	82	448	188
Solvent Control	0.20%	93.9	93.9	95.4	1754	752	348	1406	404	100	100	504	216
DNCB	4.00	78.5	78.9	80.8	3752	1475	462	3290	1013	234	251	812	319
N-[2-[bis(carboxymethyl)amino]ethyl]-N- (1-oxononyl) Glycine	312.5	94.5	93.1	95.3	1964	766	341	1623	425	115	105	576	225
	260.42	94.8	93.8	95.6	1679	739	371	1308	368	93	91	453	199
	217.01	95.1	94.2	94.9	1785	690	382	1403	308	100	76	467	181
	180.84	94.1	94.4	94.1	1884	693	364	1520	329	108	81	518	190
	150.70	92.9	94.9	95.2	1743	716	361	1382	355	98	88	483	198
	125.59	94.6	92.9	94.0	1817	734	357	1460	377	104	93	509	206
	104.66	93.6	94.3	93.3	1702	706	350	1352	356	96	88	486	202
	87.21	91.4	93.6	94.3	1846	714	378	1468	336	104	83	488	189

Table 2: CD54 and CD86 Expression Experiment 2

Sample	Conc. (stock) [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			Corrected Mean Fluorescence Intensity		Relative Fluorescence Intensity (RFI)		Ratio Isotype IgG1 to [%]
		CD86	CD54	Isotype IgG1	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	CD86	CD54
Medium Control	-	95.9	97.0	96.5	1950	720	471	1479	249	94	87	414	153
Solvent Control	0.20%	94.6	96.7	95.8	2007	716	431	1576	285	100	100	466	166
DNCB	4.0	78.3	78.1	79.0	5379	1678	405	4974	1273	316	447	1328	414
N-[2-[bis(carboxymethyl)amino]ethyl]-N- (1-oxononyl) Glycine	312.50	96.6	96.2	95.7	2135	724	427	1708	297	108	104	500	170
	260.42	96.9	96.8	92.2	2125	704	413	1712	291	109	102	515	170
	217.01	97.2	96.7	96.7	2261	681	434	1827	247	116	87	521	157
	180.84	96.4	96.9	96.3	2354	739	423	1931	316	123	111	557	175
	150.70	96.4	96.6	95.7	2015	735	412	1603	323	102	113	489	178
	125.59	93.9	96.0	96.9	2270	704	398	1872	306	119	107	570	177
	104.66	96.4	96.0	95.4	1879	712	392	1487	320	94	112	479	182
	87.21	96.7	97.2	94.4	1919	675	440	1479	235	94	82	436	153

 

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 upregulate the cell surface markers CD54 and CD86 in two independent experiment runs. Based on these results, the test item is not considered to be a skin sensitiser.

Executive summary:

In a skin sensitisation study conducted according to OECD 442E with N-[2-[bis(carboxymethyl)amino]ethyl]-N-(1-oxononyl)Glycine (100 % purity), the sensitisation potential of the test item was assessed on the basis of the activation of dendritic cells using the in vitro human cell line activation test (h-CLAT). Cells were incubated with the test item in a concentration range of 174.43 to 625.00 µg/ml (based on stock concentrations of 87.21 to 312.5 mg/ml) for 24 h at 37 °C and later checked for cell viability and expression of CD86 and CD54 cell surface markers. Sensitisation was scored by measuring cell viability and checking the expression of both cell surface markers. CD54 and CD86 were not upregulated above the threshold of 200% (CD54) and 150% (CD86) in two experiments. Based on these results, the test item is not considered to be a skin sensitiser under the UN GHS criteria.