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

Endpoint summary

Description of key information

The potential of the test item to induce skin irritation (OECD 439, 431) and eye irritation (OECD 437) was tested in suitable in vitro test methods. Based on the results, N-[2-[bis(carboxymethyl)amino]ethyl]-N-(1-oxononyl)Glycine can be considered as non-irritant to the skin and eye.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2022-04-05 to 2022-04-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

14 June 2021

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

31 July 2019

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation Protocol for: In Vitro EpiDermTM Skin Irritation Test (EPI-200-SIT) For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm (EPI-200-SIT)

Version / remarks:

12 April 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

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

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Firstly, 25 μL of sterile DPBS was applied to the epidermal surface in order to improve the contact between the powder and the epidermis. Afterwards, 25 mg (39 mg/cm2) of the test item was applied directly atop the EpiDerm tissue using an application spoon avoiding compression of the test item. The test item was spread to match size of the tissue by using a bulb-headed Pasteur pipette.

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e., the epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™-Standard Model (EPI-200-SIT, MatTek)
The EpiDerm tissues were provided as kits (e.g., EPI-200-SIT, MatTek), consisting of the following components relevant for this study:
1x sealed 24-well plate containing e.g., 24 reconstructed epidermis units (area: 0.63 cm2); each
reconstructed epidermis is attached to a cell culture insert and maintained on nutritive agar for
transport (Lot No.: 36132)
2x 24-well plates
8x 6-well plates
1x bottle of assay medium (DMEM-based medium, Lot No.: 033122KMC)
1x bottle of DPBS Rinse Solution (Lot No.: 021522MSA)
1x 1 vial 5% SDS Solution (TC-SDS-5%)
25 pieces Nylon Mesh circles (8 mm diameter, 200 μm pore)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C
- Temperature of post-treatment incubation (if applicable): 37 ± 1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: The tissues were washed by filling and emptying the inserts 15 times with DPBS using a constant stream in about 1.5 cm distance from the tissue surface, this process also occurred sequentially, e.g., in one-minute intervals. Subsequently, the inserts were completely submerged three times in 150 mL DPBS and shaken to remove rests of the test item. Finally, the inserts were rinsed once from the inside and the outside with sterile DPBS. Excess DPBS was removed by blotting the bottom with blotting paper.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h ± 5 min at 37 °C
- Wavelength: 570 nm
- Filter bandwidth: ± 30 nm

NUMBER OF REPLICATE TISSUES: 3

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the tissue viability after exposure and post-incubation is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability after exposure and post-incubation is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg + 25 μL DPBS

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL DPBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): 5% SDS solution

Duration of treatment / exposure:

60 min

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of triplicates

Value:

108

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction & colour interference with MTT: The test item has no non-specific MTT-reducing or colouring potential, therefore no additional controls were necessary.

ACCEPTANCE OF RESULTS:
The controls confirmed the validity of the study. The mean absolute OD570 of the three negative control tissues was ≥ 0.8 and ≤ 2.8 (1.652). The mean relative tissue viability (% negative control) of the positive control was < 20% (2.8%). Standard deviation of viability of replicate tissues of all dose groups was ≤ 18% (0.2% - 7.7%).

Pre-Experiments:

The mixture of 25 mg test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%.

The mixture of 25 mg of the test item per 300 µl aqua dest. and per 300 µL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSC equalled 0%.

The test item showed no non-specific reduction of MTT and no relevant colouring potential after mixture with aqua dest. and with isopropanol. Therefore, no additional controls for correction of possible false-negative results were necessary.

 

Table 1: Result of the Test Item N-[2-[bis(carboxymethyl)amino]ethyl]-N-(1-oxononyl)Glycine

Name	Negative Control			Positive Control			Test Item
Replicate Tissue	1	2	3	1	2	3	1	2	3
Absolute OD570	1.805	1.628	1.535	0.088	0.092	0.086	1.770	1.794	1.960
	1.767	1.630	1.549	0.089	0.094	0.086	1.754	1.649	1.762
Mean Absolute OD570	1.652****			0.089			1.782
OD570 (Blank Corrected)	1.761	1.583	1.490	0.044	0.047	0.042	1.726	1.750	1.916
	1.723	1.586	1.505	0.045	0.050	0.042	1.710	1.605	1.717
Mean OD570 of the Duplicates (Blank Corrected)	1.742	1.585	1.497	0.045	0.049	0.042	1.718	1.677	1.817
Total Mean OD570 of the 3 Replicate Tissues (Blank Corrected)	1.608*			0.045			1.737
SD of Mean OD570 of the 3 Replicate Tissues (Blank Corrected)	0.124			0.003			0.072
Relative Tissue Viability [%]	108.3	98.6	93.1	2.8	3.0	2.6	106.8	104.3	113.0
Mean Relative Tissue Viability [%]	100.0			2.8**			108.0
SD of Relative Tissue Viability [%]***	7.7			0.2			4.5
CV [% Viabilities]	7.7			7.4			4.1

^*           Blank-corrected mean OD₅₇₀ of the negative control corresponds to 100% absolute tissue viability.

^**          Mean relative tissue viability of the three positive control tissues is < 20%.

^***        Standard deviation (SD) obtained from the three concurrently tested tissues is ≤ 18%.

^****       The mean absolute OD₅₇₀ of the negative control is ≥ 0.8 and ≤ 2.8.

 

Table 2: Quality Criteria

	Value	Cut off	pass/fail
Mean Absolute OD570 nm NC	1.652	0.8 ≤ NC ≤ 2.8	pass
Mean Relative Viability [%] PC	2.8	< 20%	pass
SD Viability [%]	0.2 – 7.7	≤ 18%	pass

Interpretation of results:

GHS criteria not met

Conclusions:

In this in vitro skin irritation study (OECD 439), the test item is not considered to be irritant to the skin.

Executive summary:

In an in vitro dermal irritation study conducted according to OECD testing guideline 439, the EpiDerm™-Model (EPI-200) was topically exposed to 25 mg (39 mg/cm²) of N-[2-[bis(carboxymethyl)amino]ethyl]-N-(1-oxononyl)Glycine (≥ 99.7% purity) for 60 min and 42 h post-incubation. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 60 min exposure and 42 h post-incubation period and compared to those of the concurrent negative controls. The test item had no non-specific MTT-reducing or colouring potential, therefore no additional controls were necessary.

The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 50% (108.0%) after 60 min treatment and 42 h post-incubation. The controls confirmed the validity of the study.

Based on the results from this study, the test item can be classified as non-irritant.

The study is acceptable and satisfies the guideline requirements for an in vitro skin irritation study (OECD 439).

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2021-12-10 to 2022-03-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

18 June 2019

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation Protocol for: In Vitro EpiDermTM Skin Corrosion Test (EPI-200-SCT) For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm™

Version / remarks:

07/11/2014

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

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

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
25 mg of the test item were applied directly atop the EpiDerm tissue using an application spoon avoiding compression of the test item. To ensure good contact with the skin the test item was moistened with 25 µL H2O. The volume of H2O was increased (in the 60 min main experiment) and the test item was spread to match size of the tissue.

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The EpiDerm Skin Model is a well-established organotypic, three-dimensional model of the human epidermis and is used for in vitro experiments since many years. It is known for its similarity to human skin.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Reconstructed Human Epidermal Model EpiDerm™ (EPI-200, MatTek)
The EpiDerm tissues were provided as kits (e.g. EPI-200; MatTek), consisting of the following components relevant for this study:
1x sealed plate containing e.g. 24 reconstructed epidermis units (area: 0.63 cm2); each reconstructed epidermis is attached to a cell culture insert and maintained on nutritive agar for transport (Lot No.: 36115 [main exp. 60 min], 36121 [main exp. 3 min])
2x 24-well plates
4x 6-well plates
1x bottle of assay medium (DMEM-based medium; Lot: 121621LHB [main exp. 60 min], 020322LHB [main exp. 3 min])
1x bottle of DPBS Rinse Solution (Lot: 120721MSA)
25 pieces Nylon Mesh circles (8 mm diameter, 200 μm pore)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C
- Temperature of post-treatment incubation (if applicable): 37 ± 1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps:
- 3 min experiment: After 3 min of application, the first insert was removed from the 6-well plate with forceps. Using a wash bottle, the tissue was gently rinsed about 20 times with PBS (phosphate buffered saline) to remove any residual test item. Excess PBS was removed by gently shaking the insert and blotting bottom with blotting paper.
- 60 min experiment: After 60 min application, the first insert was removed from the 6-well plate with forceps. Using a wash bottle, the tissue was gently rinsed about 20 times with PBS to remove any residual test item. Excess PBS was removed by gently shaking the insert and blotting bottom with blotting paper.
- 3 min and 60 min experiment: after the 3 h MTT incubation period the MTT solution was aspirated. The wells were refilled with PBS and the PBS was aspirated. The rinsing was repeated twice and the tissues were dried.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2

PREDICTION MODEL / DECISION CRITERIA
See Table 1 in box "Any other information on materials and methods incl. tables".

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg + 50 μL H2O [main exp. 60 min]; 25 mg + 25 μL H2O [main exp. 3 min]

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
- Concentration (if solution): 8 N

Duration of treatment / exposure:

3 min and 60 min

Duration of post-treatment incubation (if applicable):

3 h

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min exposure, mean of replicates

Value:

88.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 min exposure, mean of replicates

Value:

32.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction & colour interference with MTT: The test item has no non-specific MTT-reducing or colouring potential, therefore no additional controls were necessary.

ACCEPTANCE OF RESULTS:
The controls confirmed the validity of the study. The mean OD570nm of the two negative control tissues was ≥ 0.8 and ≤ 2.8 for each exposure period (1.566, 1.432). The mean relative tissue viability (% negative control) of the positive control was ≤ 15% (5.9%) after 60 min treatment. The coefficient of variation (CV) (in the range of 20 – 100% viability) of replicate tissues of all dose groups was ≤ 30% (2.7% - 12.9%).

For details of the results please refer to Tables 2 and 3 in section "Any other information on results incl. tables".

Table 2:   Results of 3 min Experiment

Name	Negative Control		Positive Control		Test Item
Replicate Tissue	1	2	1	2	1	2
Absolute OD570	1.523	1.442	0.166	0.181	1.375	1.248
	1.643	1.565	0.176	0.195	1.499	1.368
	1.648	1.576	0.178	0.197	1.496	1.381
Mean Absolute OD570	1.566****		0.182		1.394
OD570 - Blank Corrected	1.476	1.395	0.119	0.134	1.328	1.201
	1.596	1.518	0.129	0.148	1.452	1.321
	1.601	1.529	0.131	0.151	1.449	1.334
Mean OD570 of 3 Aliquots (Blank Corrected)	1.558	1.481	0.126	0.144	1.410	1.286
SD OD570  of 3 Aliquots	0.071	0.074	0.006	0.009	0.071	0.073
Total Mean OD570 of 2 Replicate Tissues (Blank Corrected)	1.519*		0.135		1.348
SD OD570 of 2 Replicate Tissues	0.054		0.013		0.088
Mean Relative Tissue Viability [%]	100.0		8.9		88.7
Coefficient Of Variation [%]^***	3.6		9.4		6.5

^*               corrected mean OD₅₇₀ of the negative control corresponds to 100% absolute tissue viability

^***             coefficient of variation (CV) (in the range of 20 – 100% viability) between two tissues treated identically is ≤ 30%

^****            The mean absolute OD₅₇₀ of the negative control is ≥ 0.8 and ≤ 2.8

Table 3:   Results of 60 min Experiment

Name	Negative Control		Positive Control		Test Item
Replicate Tissue	1	2	1	2	1	2
Absolute OD570	1.312	1.569	0.124	0.133	0.494	0.512
	1.309	1.548	0.123	0.136	0.489	0.515
	1.297	1.557	0.122	0.132	0.488	0.497
Mean Absolute OD570	1.432****		0.128		0.499
OD570 - Blank Corrected	1.266	1.523	0.078	0.087	0.448	0.466
	1.262	1.502	0.077	0.090	0.443	0.469
	1.250	1.511	0.075	0.085	0.442	0.451
Mean OD570 of 3 Aliquots (Blank Corrected)	1.260	1.512	0.077	0.087	0.444	0.462
SD OD570  of 3 Aliquots	0.008	0.011	0.001	0.002	0.003	0.010
Total Mean OD570 of 2 Replicate Tissues (Blank Corrected)	1.386*		0.082		0.453
SD OD570 of 2 Replicate Tissues	0.178		0.008		0.012
Mean Relative Tissue Viability [%]	100.0		5.9**		32.7
Coefficient Of Variation [%]^***	12.9		9.2		2.7

^*               corrected mean OD₅₇₀ of the negative control corresponds to 100% absolute tissue viability

^**              mean relative tissue viability of the 60 min positive control is ≤ 15%

^***             coefficient of variation (CV) (in the range of 20 – 100% viability) between two tissues treated identically is ≤ 30%

^****            The mean absolute OD₅₇₀ of the negative control is ≥ 0.8 and ≤ 2.8

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item showed no corrosive effects. The mean relative tissue viability (% negative control) was above 15% after 60 min treatment and above 50% after 3 min treatment. Therefore, the test item is classified as “non-corrosive“.

Executive summary:

In an in vitro skin corrosion study conducted according to OECD testing guideline 431, two EpiDerm tissues per dose group were exposed to 25 mg of N-[2-[bis(carboxymethyl)amino]ethyl]-N-(1-oxononyl)Glycine (≥ 99.7% purity) for 60 min and 3 min. Cytotoxicity was measured in comparison to the concurrent negative controls (distilled water) via the MTT reduction assay and irritation was scored by the method of mean relative tissue viability. The test item showed no corrosive effects. The mean relative tissue viability (% negative control) was ≥50% (88.7%) after 3 min treatment and ≥15% (32.7%) after 60 min treatment. The controls confirmed the validity of the study and all acceptance criteria were fulfilled. Based on the results from this study, the test item can be classified as non-corrosive. To specify the classification in accordance with CLP regulation 1272/2008 the results from an OECD 439 study must be additionally consulted.

The study is acceptable and satisfies the guideline requirements for an in vitro skin corrosion study (OECD 431).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2022-04-04 to 2022-06-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

26 June 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

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

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): The test item was used as provided by the sponsor and moistened wit ha drop of physiological saline 0.9% NaCl.

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Abattoir Vion Beef B.V., Buchloe, Germany

On the test day, fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories. Immediately after arrival of the eyes, cornea preparation was initiated.
The eyes were carefully examined for defects and any defective eyes were discarded.
The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS. Before the corneas were mounted in corneal holders (Duratec GmbH) with the endothelial side against the O-ring of the posterior chamber, they had been visually examined for defects and any defective cornea had been discarded. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with RPMI 1640 medium (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI 1640 medium). The posterior chamber was always filled first. The corneas were incubated for one hour at 32 ± 1 °C to allow equilibration.

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): enough test item to completely cover the cornea (due to the low density of the test material less than 750 mg sufficed for completely covering the cornea which is conformity to OECD guideline 437)
- Concentration (if solution): The test item was moistened with a drop of physiological saline

VEHICLE
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): 0.9%
- Lot/batch no. (if required): 21412415

Duration of treatment / exposure:

4 hours ± 5 minutes

Duration of post- treatment incubation (in vitro):

The optical density at 490 nm was measured upon 90 minutes of incubation with fluorescein after exposure to the test item by using a spectrophotometer.

Number of animals or in vitro replicates:

3 corneas each for the test item, solvent control and positive control

Details on study design:

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: physiological saline 0.9% NaCl

POSITIVE CONTROL USED: 20% imidazole

APPLICATION DOSE AND EXPOSURE TIME: enough test item to completely cover the cornea was applied; 4 hours ± 5 minutes incubation

TREATMENT METHOD: open chamber

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
After incubation the test substance was removed and the epithelium washed at least three times with MEM (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI 1640 medium (without phenol red). The anterior chamber was refilled with complete RPMI 1640 medium (without phenol red) and an illuminance measurement was performed.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacitometer (BASF-OP3.0, Duratec GmbH). Calibration was performed before the test and is documented in the raw data. The filter holder was placed into the opacitometer and the readout was adjusted to 1000 ± 10 lux using the “Calibrate”-turning knob. For calibration the glass filter F2 was introduced into the filter holder. The readout lay in the range between 540-560 lux.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry (OD490)
- Others: Following treatment and washing steps, each cornea was observed visually and pertinent observations were recorded.

SCORING SYSTEM:
In Vitro Irritancy Score (IVIS) = mean opacity value + (15x mean permeability OD490 value)

DECISION CRITERIA: As indicated in the OECD TG 437:
- IVIS ≤ 3: No Category
- 3- >55: Category 1

Irritation parameter:

in vitro irritation score

Run / experiment:

mean of triplicates

Value:

0.34

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: not specified

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control responses resulted in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control (opacity 2.07; permeability 0.043).
- Acceptance criteria met for positive control: Due to the fact that the IVIS of the positive control did not fall within two standard deviations of the current historical mean in the first experiment, it was not considered to be valid. Therefore, the experiment was repeated. The results of both experiments are reported, noting that data from the first experiment is invalid and is not considered for experimental outcome in the present study. All data presented refers to the 2nd experiment. In the second experiment the in vitro irritation score obtained with the positive control fell within two standard deviations of the current historical mean and therefore this assay is considered to be valid (Range of historical mean IVIS Score of Positive Control: 83.91 - 155.02).

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, based on the mean in vitro irritation score of 0.34 obtained in the bovine corneal opacity and permeability assay (BCOP, OECD 437), the test item can be considered to be non-irritant and no classification for eye irritation/serious eye damage is warranted.

Executive summary:

The eye irritation potential of the test item was investigated in the bovine corneal opacity and permeability assay (BCOP, OECD 437). The test item N-[2-[bis(carboxymethyl)amino]ethyl]-N-(1-oxononyl)Glycine (purity: 99%) was directly applied on the cornea and moistened with a drop of 0.9% NaCl. A mean in vitro irritation score of 0.34 was determined. The positive and negative controls induced the appropriate responses, indicating the validity of the assay. Based on the results obtained, no classification for eye irritation/serious eye damage is warranted.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Irritant and corrosive effects of the test material to the skin were investigated in vitro with the EpiDerm™-Model according to OECD TG 431, which allows the discrimination between corrosive and non-corrosive substances, and OECD TG 439, which can identify chemicals requiring classification and labelling according to UN GHS Category 2 or Category 1.

In the in vitro skin corrosion study conducted according to OECD testing guideline 431, two EpiDerm tissues per dose group were exposed to 25 mg of N-[2-[bis(carboxymethyl)amino]ethyl]-N-(1-oxononyl)Glycine (≥ 99.7% purity) for 60 min and 3 min.

The test item showed no corrosive effects. The mean relative tissue viability (% negative control) was ≥50% (88.7%) after 3 min treatment and ≥15% (32.7%) after 60 min treatment. The controls confirmed the validity of the study and all acceptance criteria were fulfilled. Based on the results from this study, the test item was classified as non-corrosive.

In the in vitro dermal irritation study conducted according to OECD testing guideline 439, the EpiDerm™-Model (EPI-200) was topically exposed to 25 mg (39 mg/cm²) of N-[2-[bis(carboxymethyl)amino]ethyl]-N-(1-oxononyl)Glycine (≥ 99.7% purity) for 60 min and 42 h post-incubation. The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 50% (108.0%) after 60 min treatment and 42 h post-incubation. The controls confirmed the validity of the study.

Based on the results from this study, the test item can be classified as non-irritant.

If the results from both assays are taken together, no indications for skin irritating or corrosive effects of the test item were observed.

The potential of the test item to cause eye irritation or serious eye damage was assessed in vitro in the bovine corneal opacity and permeability test according to OECD TG 437. This assay is capable of identifying chemicals causing both serious eye damage according to UN GHS Category 1 or not requiring classification for eye irritation or serious eye damage according to the UN GHS criteria.

In this assay, the test item N-[2-[bis(carboxymethyl)amino]ethyl]-N-(1-oxononyl)Glycine (purity: 99%) was directly applied on the cornea and moistened with a drop of 0.9% NaCl. A mean in vitro irritation score of 0.34 was determined. The positive and negative controls induced the appropriate responses, indicating the validity of the assay. Based on the results obtained, no classification for eye irritation/serious eye damage is warranted.

Justification for classification or non-classification

Based on the results obtained from suitable in vitro studies conducted according to OECD test guidelines, no classification for skin and eye irritation is warranted according to CLP Regulation 1272/2008.