Sodium 4-[(C16-18-(even numbered)-alkylamino)carbonyl]benzoate

Endpoint summary

Description of key information

The skin irritation and skin corrosion endpoints have been completed using GLP-compliant studies following OECD 439 and OECD 431 guidelines. The substance showed a cell viability of 96.4%, which is above the threshold for irritancy (≤50%) and consequently the test item was not irritant to skin. The test item was considered to be non-corrosive to skin as the viability of the cells was 97.7% after 3 minutes of exposure and 99.4% after 60 minutes exposure. The test substance did not meet the criteria for classification for skin irritation or corrosion.

No studies have been conducted for the eye irritation or eye corrosion endpoints. No data were identified which would indicate that the substance would meet the criteria for classification for eye irritation or corrosion and experimental data on a structural analogue support that the substance would not meet the criteria for classification.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 April 2017 - 02 June 2017

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:

(Original Guideline adopted July 28, 2015)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch: A026/99 and A026/50-01
- Purity: 100% (UVCB)
- Appearance: White solid
- Expiration date: 01 July 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

Test system:

other: EPISKIN™ Reconstructed Human Epidermis Model

Source species:

other: EPISKIN™ Reconstructed Human Epidermis Model

Cell type:

non-transformed keratinocytes

Cell source:

other: EPISKIN™ Reconstructed Human Epidermis Model

Source strain:

other: EPISKIN™ Reconstructed Human Epidermis Model

Details on animal used as source of test system:

Source: EpiSkin™ model kit.
Supplier: EpiSkin™ kits are purchased from SkinEthic Laboratories (69007 Lyon, France).
EpiSkin™ Kit Lot No.: 17-EKIN-022

Justification for test system used:

In an international validation study performed by ECVAM, the in vitro skin irritation test using the human skin models EpiSkin™ and EpiDerm™ and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for skin irritancy potential.

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): 17-EKIN-022
- Delivery date: 30 May 2017
- Date of initiation of testing: 30 May 2017

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 using a soft stream of PBS to gently remove any residual test item
- Observable damage in the tissue due to washing: Not specified
- Modifications to validated SOP: None

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 2 mL assay medium containing 0.3 mg/mL MTT per well
- Incubation time: 3 hours
- Spectrophotometer: Microplate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, version 4.7.1)
- Filter: 570 nm
- Filter bandwidth: Not applicable
- Linear OD range of spectrophotometer: Not specified

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- The results for the positive control are within the historical data (means, rel. standard deviation, and ranges) of Envigo CRS GmbH
- The results for the negative control are within the historical data (means, rel. standard deviation and ranges) of Envigo CRS GmbH (Please note: the study was performed by using PBS, the Historical Control Data Base annexed to the report shows the Data for deionized water; data for PBS are in process and the number of studies is still too low for presenting Historical Data). Comparing the results of PBS to the Historical Control Data Base generated with deionized water is legitimate since both controls are aqueous from the chemical point of view. In other toxicological studies (e.g. OECD 487 or OECD 473) Historical Data Bases distinguish only between aqueous or organic solvents.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Since the colour did not turn blue/purple, the test item was not considered to be a MTT reducer. Therefore, it was not necessary to perform an additional test with freeze-killed tissues to determine a correction factor for calculating the true viability in the main experiment.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: Three
- Test for Direct MTT Reduction
- Test for Direct Colour Interference
- Main test

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be an irritant to skin if the relative mean tissue viability after a 15 minute exposure period followed by the 42-Hour post-exposure incubation period is less than or equal to 50%.
- The test substance is considered to be non-corrosive to skin if the viability after 15-Minute exposure period followed by the 42-Hour post-exposure incubation period is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 5 μL of deionised water followed by 10 ± 2 mg of the test item were topically applied to the epidermal surface
- Concentration (if solution): Test material is a solid

VEHICLE
- Not applicable

NEGATIVE CONTROL
- Name: PBS (produced in-house: 40 g NaCl, 1 g KCl, 1 g KH2PO4, 0.94 g Na2HPO4 + 2 H2O filled to 5000 mL with deionised water)
- Amount(s) applied: 10 μL
- Concentration: Not specified.

POSITIVE CONTROL
- Name: 5% SLS (Fluka, Sigma-Aldrich) solution in deionised water
- Amount(s) applied: 10 μL
- Concentration (if solution): 5% w/v aqueous solution

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

The tissues were incubated for approximately 41.5 hours at 37 ± 1.5°C, 5 ± 0.5% CO2

Number of replicates:

3 test replicates

Species:

other: reconstituted human epidermis model

Strain:

other: reconstituted human epidermis model

Details on test animals or test system and environmental conditions:

Not applicable

Type of coverage:

other: Topical

Preparation of test site:

other: Not applicable

Vehicle:

other: No vehicle used

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 5 μL of deionised water followed by 10 ± 2 mg of the test item were topically applied to the epidermal surface
- Concentration (if solution): Test material is a solid

VEHICLE
- Not applicable

NEGATIVE CONTROL
- Name: PBS (produced in-house: 40 g NaCl, 1 g KCl, 1 g KH2PO4, 0.94 g Na2HPO4 + 2 H2O filled to 5000 mL with deionised water)
- Amount(s) applied: 10 μL
- Concentration: Not specified.

POSITIVE CONTROL
- Name: 5% SLS (Fluka, Sigma-Aldrich) solution in deionised water
- Amount(s) applied: 10 μL
- Concentration (if solution): 5% w/v aqueous solution

Duration of treatment / exposure:

Treatment: The test item tissues were soaked in 5 μL of deionised water. The controls and test item was added into the insert above the EpiSkin™ triplicate tissues for 15 minutes. The post exposure incubation period was 42 hours.

Observation period:

Not applicable

Number of animals:

Not applicable

Details on study design:

TEST SITE
- Area of exposure: The test material was applied to a human reconstructed epidermis model. The epidermis surface had previously been moistened with 5 μL of distilled water to improve contact between the solid test material and the epidermis.
- % coverage: The test material was applied topically to the corresponding tissues ensuring uniform covering.
- Type of wrap if used: None used

REMOVAL OF TEST SUBSTANCE
- Washing: After the end of the treatment interval the inserts were immediately removed from the 12-well plate. The tissues were gently rinsed with PBS to remove any residual test material. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper.
- Time after start of exposure: 15 Minutes

SCORING SYSTEM:
- Quantitative MTT Assessment (percentage tissue viability): For the test material, the relative mean tissue viabilities obtained after the 15 minute treatment followed by the 42-hour post-exposure incubation period were compared to the mean of the negative control treated tissues (n=3). The relative mean viabilities were calculated in the following way:
Relative mean tissue viability (percentage of negative control) = (mean OD570 of test material / mean OD570 of negative control) x 100

Classification of irritation potential is based upon relative tissue viability following the 15-minute exposure period followed by the 42-hour post-exposure incubation period according to the following:
Mean tissue viability is ≤50%: Irritant (I) H315 Category 2
Mean tissue viability is >50%: Non-Irritant (NI) Not classified. (Category 3 cannot be determined)

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Relative mean variability

Value:

96.4

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:

The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not lead to a change in colour.
Optical evaluation of the MTT-reducing capacity of the test item after 3 hour incubation with MTT-reagent did not show blue colour.
The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 96.4% (threshold for irritancy: ≤ 50%), consequently the test item was not irritant to skin.

Quality Criteria

The acceptance criteria were met:

• the mean OD of the three negative control exposed tissues is ≥ 0.6 till ≤ 1.5 (range: 0.856 to 0.972).

• the rel. standard deviations between tissues of the same treatment group was ≤ 18% (range: 6.6 to 12.7%).

• the mean relative tissue viability of the positive control was ≤ 40% (5.3%).

• the acceptance limit of the IC50 of the respective EpiSkin™ lot was between 1.5 and 3.0 mg/mL after 18 hours treatment with SLS (1.7 mg/mL).

• the results for the positive control are within the historical data (means, rel. standard deviation, and ranges) of Envigo CRS GmbH

• the results for the negative control are within the historical data (means, rel. standard deviation and ranges) of Envigo CRS GmbH (Please note: the study was performed by using PBS, the Historical Control Data Base annexed to the report shows the Data for deionized water; data for PBS are in process and the number of studies is still too low for presenting Historical Data). Comparing the results of PBS to the Historical Control Data Base generated with deionized water is legitimate since both controls are aqueous from the chemical point of view. In other toxicological studies (e.g. OECD 487 or OECD 473) Historical Data Bases distinguish only between aqueous or organic solvents.

RESULTS

The individual and mean absorbance values, standard deviations and tissue viabilities for the test material, negative control material and positive control material are given in Table 1. The relative mean viability of the test material treated tissues was 96.4% after a 15 minutes exposure.

Table 1: Mean absorbance values and percentages for the blank control, negative control material, positive control material and test material.

Results after treatment with test item and controls

Test Group	Absor-bance 570 nm Tissue Well 1	Absor-bance 570 nm Tissue Well 2	Mean Absor-bance 570 nm	Mean Absor-bance 570 nm *	Mean Absor-bance of 3 Tissues	Relative Absor-bance [%] Tissue 1, 2, 3**	Relative Standard Deviation [%]	Rel. Absor-bance [% of Negative Control]***
Blank	0.038	0.038	0.038
Negative Control	0.945	0.905	0.925	0.887	0.880	100.8	6.6	100.0
	0.983	0.960	0.972	0.934		106.2
	0.872	0.840	0.856	0.818		93.0
Positive Control	0.073	0.083	0.078	0.040	0.046	4.5	12.7	5.3
	0.091	0.080	0.086	0.048		5.4
	0.087	0.091	0.089	0.051		5.8
Test Item	0.986	0.961	0.974	0.936	0.848	106.4	9.6	96.4
	0.848	0.890	0.869	0.831		94.5
	0.832	0.794	0.813	0.775		88.1

Interpretation of results:

GHS criteria not met

Conclusions:

The test material was considered to be a non-irritant to skin using the EPISKIN™ Reconstructed Human Epidermis Model.

Executive summary:

The study is assigned a reliability score of 1 (reliable without restrictions) as it followed OECD Guideline 439: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method, is compliant with GLP and met the acceptance criteria. The substance, under test conditions, had a mean relative absorbance value, corresponding to cell viability, of 96.4%, which is above the threshold for irritancy (≤50%) and consequently the test item was not irritant to skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The skin irritation and skin corrosion endpoints have been completed using GLP-compliant studies following OECD 439 and OECD 431 guidelines. The test substance did not meet the criteria for classification for skin irritation or corrosion.

No studies have been conducted for the eye irritation or eye corrosion endpoints. A literature search and the available data for the substance did not identify any hazards and no data were identified which would lead to predictions that the substance would meet the criteria for classification for human or environmental effects under the CLP regulation. Therefore, the substance is not expected to meet the criteria for classification for eye irritation or corrosion and as the substance is being registered with reduced information requirements, as set out under REACH Annex III, no studies for eye irritation or corrosion were conducted.

Furthermore, the reaction mass of methyl N-octadecylterephthalamate and N,N'‑dioctadecylterephthalamide (with methyl N-octadecylterephthalamate, a structural analogue for sodium 4‑[(octadecylamino)carbonyl]benzoate) has been registered under REACH and does not meet the criteria for classification for human health or the environment. The replacement of the methyl functional group present in the source substance with the sodium ion present in the target is not expected to lead to a change in classification and therefore it is expected that the target substance would also not meet the criteria for classification.

Therefore, using experimental and read across data, it is concluded that Sodium 4-[(C16-18-(even numbered)-alkylamino)carbonyl]benzoate does not meet the criteria for classification for irritation or corrosion.

Justification for classification or non-classification

Experimental data are available to show that the substance would not meet the criteria for classification for skin irritation or corrosion. No data were identified which would indicate that the substance would meet the criteria for classification for eye irritation or corrosion. Furthermore, experimental data on a structural analogue support that the substance would not meet the criteria for classification. Therefore, it is concluded that Sodium 4-[(C16-18-(even numbered)-alkylamino)carbonyl]benzoate does not meet the criteria for classification for irritation or corrosion.