N,N-bis(carboxymethyl)glycine, compound with 2-aminoethanol (1:3)

Endpoint summary

Description of key information

A GLP-compliant study was performed to determine the skin corrosion potential of N,N’-bis(carboxymethyl)glycine, compound with 2-aminoethanol (1:3) in line with OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method). N,N’-bis(carboxymethyl)glycine, compound with 2-aminoethanol (1:3) was applied as a ~8 mm disc (50 mg) to reconstructed human epidermal tissue (Epiderm™) prior to wetting with 15 µl of water. Negative controls of tissue grade sterile water and positive controls of potassium hydroxide were prepared and applied at 50 µl. Exposure was permitted for a 3- and 60-minute period at 37 °C, 5 % CO2, 95 % RH. Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. The results are then used to make a prediction of the corrosivity potential of the test item. All controls were valid and demonstrated the reliability of the test system. Mean tissue viability (as a percentage of the negative control), was 82.7 and 22.6 % after 3 and 60 minutes of exposure, respectively. Therefore, N,N’-bis(carboxymethyl)glycine, compound with 2-aminoethanol (1:3) does not meet the criteria for classification according to CLP Regulation (EC) No. 1272/2008.

The skin irritation potential of N, N’-bis (carboxymethyl glycine) compound with 2-aminoethanol (1:3), was evaluated in accordance with OECD guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method). The test was GLP compliant.

Triplicate tissues were treated with Approximately 10 mg (26.3 mg/cm2) of N, N’-bis (carboxymethyl glycine) compound with 2-aminoethanol (1:3). Additional triplicate tissues individually treated with 5% Sodium dodecyl sulphate and 10µl of Dulbecco’s phosphate buffered saline served as positive and negative controls respectively.

All the tissues were exposed for 15 minutes followed by an incubation period of 42 hours at 37 °C, 5% C02 in air. Cell viability is measured by the enzymatic reduction of the yellow MTT tetrazolium salt to a blue/ purple formazan salt in the mitochondria of viable cells in the tissue treated with the test item compared to the tissues treated with negative controls. The results are used to predict the irritation potential of the test item.

The validity of both the positive and negative controls was established thus demonstrating the test system reliability. The relative mean tissue viabilities for N, N’-bis (carboxymethyl glycine) compound with 2-aminoethanol (1:3) were compared to the mean of the negative controlled tissues (100%).

The relative mean viability of tissues treated with N, N’-bis (carboxymethyl glycine) compound with 2-aminoethanol (1: 3) was 104.1%. According to the interpretation guidance provided in the test guideline OECD TG 439 N,N'-bis (carboxymethyl glycine) compound with 2- aminoethanol (1:3) will be considered as non-irritant. Therefore the substance does not meet the criteria for classification as irritant to the skin in accordance with Regulation (EC) No 1272/2008.

An in vitro GLP compliant test was performed in accordance with OECD TG 437 to assess the eye damage potential of N, N’- bis(carboxymethyl)glycine, compound with 2-aminoethanol (1:3). Quantitative measurements of changes in corneal opacity and permeability are used to assess damage by the test item. N, N’- bis(carboxymethyl)glycine, compound with 2-aminoethanol (1:3) was applied at a concentration of 20% w/v in sodium chloride 0.9% w/v for four hours. Both positive (Imidazole 20% w/v.) and negative (Sodium Chloride 0.9% w/v.) controls were tested simultaneously. Two endpoints namely opacity and permeability were used to generate an In Vitro Irritancy Score (IVIS). The IVIS for N, N’- bis(carboxymethyl)glycine, compound with 2-aminoethanol (1:3) was found to be 14.6 and for the positive and negative controls the scores were 133.2 and 1.0 respectively. In accordance with the decision criteria guidance table provided in OECD TG 437 as the IVIS score obtained for N, N’- bis(carboxymethyl)glycine, compound with 2-aminoethanol (1:3) was ≥3 and ≤ 55 no prediction could be made on the eye irritation potential of N, N’- bis(carboxymethyl)glycine, compound with 2-aminoethanol (1:3).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 2018

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: ~8mm disc (50 mg) prior to wetting of the tissue with 15 µl water

NEGATIVE CONTROL
- Amount applied: Amount applied: 50µl
- Lot/batch no.: RNBG4913

POSITIVE CONTROL
- Amount applied: 50µl
- Lot/batch no.: SLBD3295V

Duration of treatment / exposure:

3 and 60 minutes

Number of replicates:

Triplicate

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes

Value:

82.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes

Value:

22.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

GHS criteria not met

Conclusions:

In the reported study, mean tissue viability (as a percentage of the negative control) was 22.6 % after a 60-minute exposure and 82.7 % after a 3-minute exposure. N,N’-bis(carboxymethyl)glycine, compound with 2-aminoethanol (1:3) does not meet the criteria for classification according to CLP Regulation (EC) No. 1272/2008

Executive summary:

A GLP-compliant study was performed to determine the skin corrosion potential of N,N’-bis(carboxymethyl)glycine, compound with 2-aminoethanol (1:3) in line with OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method). N,N’-bis(carboxymethyl)glycine, compound with 2-aminoethanol (1:3) was applied as a ~8 mm disc (50 mg) to reconstructed human epidermal tissue (Epiderm™) prior to wetting with 15 µl of water. Negative controls of tissue grade sterile water and positive controls of potassium hydroxide were prepared and applied at 50 µl. Exposure was permitted for a 3- and 60-minute period at 37 °C, 5 % CO2, 95 % RH. Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. The results are then used to make a prediction of the corrosivity potential of the test item. All controls were valid and demonstrated the reliability of the test system. Mean tissue viability (as a percentage of the negative control), was 82.7 and 22.6 % after 3 and 60 minutes of exposure, respectively. Therefore, N,N’-bis(carboxymethyl)glycine, compound with 2-aminoethanol (1:3) does not meet the criteria for classification according to CLP Regulation (EC) No. 1272/2008.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 September 2018- 01 October 2018

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)

Deviations:

yes

Remarks:

see 'Principles of method if other than guideline'

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

see 'Principles of method if other than guideline'

Principles of method if other than guideline:

The following deviations from study plan occurred: Due to the nature of the test item a sharps spoon could not be used to weigh out approximately 10 mg required for dosing. It was established that weight would be used for this study.Using a dispersion pipette 8 µL weighing approximately 10.2 mg was used to achieve the approximate required does (10mg). The 5 uL of water to pre-wet the tissue was not required prior to application of the test item, this decision was supported by the Episkin supplier SOP. An assessment found the test item was able to directly reduce MTT. Therefore, an additional procedure using water killed tissues was performed. However, results showed negligible interference due to direct reduction of MTT. Therefore considered unnecessary to use the results of the water killed tissues for quantitative correction of results or for reporting purposes.These deviations were considered to have not affected the integrity or validity of the study.

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: human-derived epidermal keratinocytes

Justification for test system used:

Following a full validation study the EpiSkinTM reconstructed human epidermis model showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation when the endpoint is measured by MTT reduction and for being used as a replacement for the Draize Skin Irritation Test for the purpose of distinguishing between Irritating and Non-Irritating test items.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:EPISKIN Reconstructed Human Epidermis Model kit
- Tissue batch number(s):18-EKIN-039
- Delivery date: 25 September 2018
- Date of initiation of testing: 26 September 2018

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

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds
- Observable damage in the tissue due to washing: None reported
- Modifications to validated SOP: None reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/ml freshly prepared
- Incubation time: The solution was incubated in the dark at 37 °C, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control
- Spectrophotometer: Microplate reader: Labtech LT-4500
- Wavelength:570nm OD570
- Filter: without a reference filter
- Filter bandwidth: N/A
- Linear OD range of Microplate reader: Not reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Relative mean viability of the test item treated tissues was 104.1%, the positive control item was 5.6% and the negative control viability was set at 100%
- Barrier function: Not reported
- Morphology: Not reported
- Contamination: Not reported
- Reproducibility: Not reported

NUMBER OF REPLICATE TISSUES: Triplicate

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues
- Procedure used to prepare the killed tissues: Water-killed tissues prepared prior to study by inserting untreated EPISKIN tissues in a 12-well plate holding 2.0 mL of sterile distilled water in each well. Tissues were incubated at 37 °C, 5% CO2 in air for at least 48 hours. Following incubation, the water was discarded. Once killed the tissues were stored in a freezer (−14 to −30 °C) for up to 6 months. Prior to use each tissue was thawed by placing in 2.0 mL of maintenance medium for approximately 1 hour at room temperature
- N. of replicates: 3 treated 3 untreated
- Method of calculation used: Results obtained showed no interference owing to direct reduction of MTT arose. Thus, it was considered redundant to use the results of the water-killed tissues for quantitative correction of results or for reporting purposes.

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is predicted to be irritant to skin if after a 15-minute exposure followed by a post exposure incubation period of 42 hours the relative mean tissue viability is equal to or less than 50%.
- The test substance is predicted to be non-irritant to skin if after 15 minutes exposure followed by a post exposure incubation period of 42 hours the relative mean tissue viability is greater than 50%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: Single topical application of neat test item following which approximately 10 mg (26.3 mg/cm2) of the neat test item was applied to the epidermal surface.

NEGATIVE CONTROL
- Amount applied (volume): 10 μL of Dulbecco’s Phosphate Buffered Saline

POSITIVE CONTROL
- Amount applied (volume ): 10 μL
- Concentration:5% w/v

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

three

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

104.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: An assessment found the test item was able to directly reduce MTT. Therefore, an additional procedure using water-killed tissues was performed. However,the results obtained showed that no interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the water-killed tissues for quantitative correction of results or for reporting purposes. The solution containing the test item was colorless. It was therefore unnecessary to run color correction tissues.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes . Mean OD570 for negative control treated tissues was 0.726 and the standard deviation value of the viability was 2.2%.
- Acceptance criteria met for positive control: Yes. The relative mean tissue viability for the positive control treated tissues was 5.6% relative to the negative control treated tissues and the standard deviation value of the viability was 3.4%.
- Acceptance criteria met for variability between replicate measurements: Yes. The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 2.0%.

See attached background document

Interpretation of results:

GHS criteria not met

Conclusions:

The skin irritation potential of N,N’-bis(carboxymethylglycine) compound with 2-aminoethanol (1:3) was evaluated. The relative mean viability of the test item treated tissues was 104.1% after a 15-minute exposure and 42-hour post-exposure incubation period. The substance did not meet the critera for classification as irritant to the skin in accordance with Regulation (EC) No. 1272/2008.

Executive summary:

The skin irritation potential of N, N’-bis (carboxymethyl glycine) compound with 2-aminoethanol (1:3), was evaluated in accordance with OECD guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method). The test was GLP compliant.

Triplicate tissues were treated with Approximately 10 mg (26.3 mg/cm2) of N, N’-bis (carboxymethyl glycine) compound with 2-aminoethanol (1:3). Additional triplicate tissues individually treated with 5% Sodium dodecyl sulphate and 10µl of Dulbecco’s phosphate buffered saline served as positive and negative controls respectively.

All the tissues were exposed for 15 minutes followed by an incubation period of 42 hours at 37 °C, 5% C02 in air. Cell viability is measured by the enzymatic reduction of the yellow MTT tetrazolium salt to a blue/ purple formazan salt in the mitochondria of viable cells in the tissue treated with the test item compared to the tissues treated with negative controls. The results are used to predict the irritation potential of the test item.

The validity of both the positive and negative controls was established thus demonstrating the test system reliability. The relative mean tissue viabilities for N, N’-bis (carboxymethyl glycine) compound with 2-aminoethanol (1:3) were compared to the mean of the negative controlled tissues (100%).

The relative mean viability of tissues treated with N, N’-bis (carboxymethyl glycine) compound with 2-aminoethanol (1: 3) was 104.1%. According to the interpretation guidance provided in the test guideline OECD TG 439 N,N'-bis (carboxymethyl glycine) compound with 2- aminoethanol (1:3) will be considered as non-irritant. Therefore the substance does not meet the criteria for classification as irritant to the skin in accordance with Regulation (EC) No 1272/2008.

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:

key study

Study period:

25 September- 26 September

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)

Deviations:

yes

Remarks:

See 'Principles of method if other than guideline'

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

yes

Remarks:

See 'Principles of method if other than guideline'

Principles of method if other than guideline:

The following deviation from the study plan occured:

The positive control group had an overall IVIS of 133.2 which was slightly higher than the defined criteria range for the test acceptability. Due to the amount that it was exceeded by being so small ,the result was accepted on the basis that the positive control group still fulfilled its purpose of showing sensitivity of the test system to a known occular irritant.

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Cattle (12 to 60 months old)
- Number of animals: Not specified
- Characteristics of donor animals (e.g. age, sex, weight): Not specified
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Eyes excised from freshly slaughtered cattle were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100IU/ml and streptomycin at 100uh/ml) and transported to the test facility over ice packs on the same day of slaughter.
- Time interval prior to initiating testing: The corneas were refrigerated on arrival and used within 24 hours of receipt.
- indication of any existing defects or lesions in ocular tissue samples: None reported
- Indication of any antibiotics used: penicillin at 100IU/ml and streptomycin at 100µg/ml

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Concentration: The test item was prepared as a 20% w/v solution in Sodium chloride 0.9% w/v

VEHICLE
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Lot/batch no. (if required): 3012487
- Purity: 0.9%

Duration of treatment / exposure:

4 hours

Duration of post- treatment incubation (in vitro):

none

Number of animals or in vitro replicates:

Three

Details on study design:

SELECTION AND PREPARATION OF CORNEAS : Eyes from adult cattle typically 12 to 60-month-old were obtained from a local abattoir as a by-product from freshly slaughtered animals.The eyes were excised by an abattoir employee post slaughter, placed in Hanks' Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/ml and stretomycin at 100ug/ml). they were transported on the same day of slaughter to the test facility on ice packs.The corneas were refrigerated on arrival and used within 24 hours of receipt. All eyes were visibly inspected before and after dissection. Only corneas free from damage were used.

QUALITY CHECK OF THE ISOLATED CORNEAS : The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders The front and back chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 65 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

NUMBER OF REPLICATES : 3 for Test item, 3 for postive control, 3 for negative control.

NEGATIVE CONTROL USED : Sodium Chloride 0.9% w/v.

POSITIVE CONTROL USED : Imidazole 20% w/v.

APPLICATION DOSE AND EXPOSURE TIME : 0.75ml for 4 hours

TREATMENT METHOD: open chamber

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: Post exposure, the test and control items were removed from the front chamber and the cornea was rinsed 3 times with fresh EMEM containing phenol red prior to a final rinse with complete EMEM without phenol red

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Change in opacity for each cornea (including the negative control) calculated via substraction of the initial opacity reading from the final opacity reading. Subsequently these values were corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD492) using the Labtech LT-4500 microplate reader.
- Others histopathology: Corneas were retained after testing for possible conduct of histopathology.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS) : The formula used to determine the In Vitro Irritancy Score was as follows: In vitro Irritancy score= mean opacity value + (15 X mean permeability OD492 value).

DECISION CRITERIA:The decision criteria indicated in OECD TG437 was used.
IVIS= mean opacity value + (15 X mean permeability OD492 value).
Opacity and permeability values were evaluated individually to determine if the test item produced a response through only one of the two endpoints. Cornea condition post treatment was assessed macroscopically. Classification of the test item was made in accordance with the following prediction model:

IVIS Classification:
≤3: No Category. Not requiring classification to UN GHS or EU CLP;
≥3: ≤ 55 No prediction of eye irritation can be made;
> 55: Category 1. UN GHS or EU CLP Causes serious eye damage

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean

Value:

14.6

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Irritation parameter:

cornea opacity score

Run / experiment:

Mean

Value:

8

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: The corneas treated with the test item were slightly cloudy post treatment. The corneas treated with the negative control item were clear post treatment. The corneas treated with the positive control item were opaque post treatment.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control gave opacity of ≤2.3 and permeability ≤0.44. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The positive control In Vitro Irritancy Score was marginally outside the range of 71.2 to 132.9. The positive control acceptance criterion was therefore not strictly satisfied and is reported as a deviation. However, therefore the difference of a 0.3 IVIS value is considered negligible.

Interpretation of results:

study cannot be used for classification

Conclusions:

The eye damage potential of N, N’- bis(carboxymethyl)glycine, compound with 2-aminoethanol (1:3) was evaluated. Based on the results, no conclusion can be made regarding the capacity of the substance to induce eye damage or eye irritation.

Executive summary:

An in vitro GLP compliant test was performed in accordance with OECD TG 437 to assess the eye damage potential of N, N’- bis(carboxymethyl)glycine, compound with 2-aminoethanol (1:3). Quantitative measurements of changes in corneal opacity and permeability are used to assess damage by the test item. N, N’- bis(carboxymethyl)glycine, compound with 2-aminoethanol (1:3) was applied at a concentration of 20% w/v in sodium chloride 0.9% w/v for four hours. Both positive (Imidazole 20% w/v.) and negative (Sodium Chloride 0.9% w/v.) controls were tested simultaneously. Two endpoints namely opacity and permeability were used to generate an In Vitro Irritancy Score (IVIS). The IVIS for N, N’- bis(carboxymethyl)glycine, compound with 2-aminoethanol (1:3) was found to be 14.6 and for the positive and negative controls the scores were 133.2 and 1.0 respectively. In accordance with the decision criteria guidance table provided in OECD TG 437 as the IVIS score obtained for N, N’- bis(carboxymethyl)glycine, compound with 2-aminoethanol (1:3) was ≥3 and ≤ 55 no prediction could be made on the eye irritation potential of N, N’- bis(carboxymethyl)glycine, compound with 2-aminoethanol (1:3).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Justification for classification or non-classification

GLP-compliant studies were performed in order to investigate the skin corrosion and skin irritation of N,N’-bis(carboxymethylglycine) compound with 2-aminoethanol (1:3) in accordance with the OECD Testing Guidelines 431 and 439 respectively. The substance did not meet the criteria for classification as corrosive or irritant to the skin according to Regulation (EC) N° 1272/2008.

A GLP-compliant study was performed in order to investigate the eye irritation of N,N’-bis(carboxymethylglycine) compound with 2-aminoethanol (1:3) in accordance with the OECD Testing Guideline 437 (BCOP). The study results do not provide enough information to make a conclusion on the potential for the substance to induce eye irritation.

According to ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R.7a: Endpoint specific guidance (Version 6.0 - July 2017), the BCOP possesses a good predictivity to identify substances inducing serious eye damage and therefore meeting the criteria for classification as Eye Dam. 1 according to Regulation (EC) N° 1272/2008. Since N,N’-bis(carboxymethylglycine) compound with 2-aminoethanol (1:3) was not identified during the study as inducing serious eye damage, it can be concluded that the substance does not meet the criteria for classification as Eye Dam. 1 according to Regulation (EC) N° 1272/2008.

Therefore it is proposed to classify N,N’-bis(carboxymethylglycine) compound with 2-aminoethanol (1:3) as Eye Irrit 2 without performing additional in vitro eye irritation testing as it represents a worst-case scenario.