Butanoic acid, 4-amino-4-oxosulfo-, N-coco alkyl derivs., monosodium salts, compds. with triethanolamine

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08/2017 - 01/2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Butanoic acid, 4-amino-4-oxosulfo-, N-coco alkyl derivs., monosodium salts, compds. with triethanolamine

EC Number:

308-662-5

EC Name:

Butanoic acid, 4-amino-4-oxosulfo-, N-coco alkyl derivs., monosodium salts, compds. with triethanolamine

Cas Number:

98171-53-0

IUPAC Name:

Butanoic acid, 4-amino-4-oxosulfo-, N-coco alkyl derivs., monosodium salts, compds. with triethanolamine

Test material form:

solid

Details on test material:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 0015567151
- Expiration date of the lot/batch: 11 March 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 0015567151
- Expiration date of the lot/batch: 11 March 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor.
- Solubility and stability of the test substance in the solvent/vehicle: No analysis of test substance preparation was performed because the test substance was applied minimally moistened with deionized water or PBS.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final preparation of a solid: Due to the texture of the solid test substance a bulk volume of ca. 25 µL ground test material was formed to a flat disc that matched to the size of the tissue (ca. 8 mm diameter).

FORM AS APPLIED IN THE TEST Test material was formed to a flat disc.

In vitro test system

Test system:

human skin model

Remarks:

EpiDerm model (EPI-200)

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: MatTek, Bratislava, Slovakia; Unspecified

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm
- Tissue batch number: 25837
- Date of certificate of analysis from MatTek: 16 August 2017
- Date of initiation of testing: 15 August 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature for 25 minutes overall and 37°C for 35 minutes (Incubator)
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
The tissues were washed with sterile PBS to remove residual test material 1 hour after start of application. Rinsed tissues were blotted on sterile absorbent paper and transferred into new
6-well plates pre-filled with 0.9 mL fresh medium. When all tissues were rinsed the surface of each tissue was carefully dried with a sterile cotton swab.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL MTT
- Incubation time: 3 hours
- Spectrophotometer: Sunrise Absorbance Reader
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The supplier demonstrates that each batch of the model used passes a MTT QC assay (4 hours, n=3, OD= 540-570nm).
- Reproducibility: given by historical data from Jan 2016 - Jul 2017 via given test method
- Barrier function and Quality control: The supplier demonstrates that each batch of the model used meets the defined production release criteria. MatTek determines the ET50 value following exposure to Triton X-100 (1%) for each EpiDermTM batch. The ET50 must fall within a range established based on a historical database of results.
Lower acceptance limit: ET50 = 4.0 hours
Upper acceptance limit: ET50 = 8.7 hours
- Sterility: The supplier demonstrates that each batch of the model used shows no contamination.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- EPI-200 tissue that is killed by freezing at –20°C
- N. of replicates: 3
- Method of calculation used: In case of direct MTT reduction by the test substance, the OD570 values measured in the freeze-killed control tissues (KC) will be used to correct the mean OD570 of the tissues treated with the test substance (mean OD570 KC corrected). Since killed tissue might still have a residual enzyme activity that is able to produce some formazan net OD570 KC is calculated by subtracting the OD570 KC of the negative control from the OD570 KC of the test substance. In case the net OD570 KC is greater than zero it is subtracted from the respective mean OD570 to result in the mean OD570 KC corrected. The mean OD570 KC corrected represents the formazan production linked to the tissue viability and therefore indicates the cytotoxic potency of the test substance.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- Justification for the selection of the cut-off points if different than recommended in TG 431 and 439:

Mean tissue viability (% of negative control):
Step 1: Identification of corrosives
< 45% after 3 min exposure -> Corrosive
> 45% after 3 min exposure and < 10% after 1 h exposure -> Corrosive
45 – 55% after 3 min exposure -> Borderline (inconclusive)
> 55% after 3 min exposure and 10 – 20% after 1 h exposure -> Borderline (inconclusive)
> 55% after 3 min exposure and > 20% after 1 h exposure -> Non-corrosive

Step 2: Optional UN GHS subcategorization of corrosives identified in step 1
< 20% after 3 min exposure -> UN GHS Cat 1A
20 – 30% after 3 min exposure -> Borderline (inconclusive) for UN GHS subcategorization
> 30% after 3 min exposure -> UN GHS Cat 1B or 1C

Mean tissue viability (% of negative control):
< 45% Irritant
45 - 55% Borderline3
> 55% Non-irritant

The “borderline“ evaluation (50 ± 5%, 25 ± 5% and 15 ± 5%) was statistically determined by using historic BASF data and hence considers the variance of the test method. This evaluation is an amendment to the evaluation provided in OECD Guideline 431 and 439.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: bulk volume of ca. 25 µL ground test material (about 48 mg)

NEGATIVE CONTROL
- Amount applied: 30 µL

POSITIVE CONTROL
- Amount applied: 30 µL
- Concentration: 5% (w/v) SDS

Duration of treatment / exposure:

- under laminar flow hood at room temperature for 25 minutes overall and for 35 minutes in the incubator (37°C)

Duration of post-treatment incubation (if applicable):

Tissues were post-incubated for 24 ± 2 hours at 37°.

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Remarks:

Final mean viability of tissues after killed control (for direct MTT-reduction) correction as % of negative control.

Run / experiment:

Test substance treatment.

Value:

16.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: The results of the killed control (KC) tissues indicate an increased MTT reduction (mean viability 0.3% of negative control).
Thus for the test substance, the final mean viability is given after KC correction.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The laboratory has demonstrated proficiency.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The absolute OD570 of the negative control tissues in the MTT test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is acceptable if the mean OD570 of the negative comtrol is >= 0.8. The mean OD570 of the negative control should not exceed 2.8.
- Acceptance criteria met for positive control: 5% SDS is used as positive control and reflects the sensitivity of the tissues used in the test conditions. A viability of <= 20% is acceptable.
- Acceptance criteria met for variability between replicate measurements: For every treatment, three tissues are treated in parallel. The inter-tissue variability is considered to be acceptable if the SD of % viability is <= 18%.
- Acceptance criteria for the killed controls (KC): The OD570 of the tissues for the KC of the negative control should be <= 0.35.
The OD570 value for direct MTT reduction of a test substance should be <= 30% of the OD570 of the negative control.

Any other information on results incl. tables

Table 1: Individual and mean OD₅₇₀values, individual and mean viability values, standard deviations and coefficient of variation.

Test substance identification			tissue 1	tissue 2	tissue 2	mean	SD	CV [%]
NC	viable tissues	mean OD570	1.757	1.476	1.646	1.626
		viability [% of NC]	108.0	90.8	101.2	100.0	8.7	8.7
	KC tissues	mean OD570	0.045	0.041	0.045	0.043
		viability [% of NC]	2.7	2.5	2.8	2.7	0.2	5.7
test substance	viable tissues	mean OD570	0.254	0.263	0.302	0.273
		viability [% of NC]	15.6	16.2	18.6	16.8	1.6	9.3
	KC tissues	mean OD570 KC NC corrected	0.007	0.007	0.002	0.005
		viability [% of NC]	0.4	0.4	0.1	0.3	0.2	58.9
	Final mean viability of tissues after KC correction [% of NC]:					16.5
PC	viable tissues	mean OD570	0.040	0.042	0.051	0.044
		viability [% of NC]	2.5	2.6	3.1	2.7	0.4	13.2

NC:       negative control

PC:       positive control

KC:       killed control (for direct MTT-reduction)

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Based on the results observed and by applying the evaluation criteria, it was concluded that the test material shows a skin irritation potential in the EpiDerm in vitro skin irritation test.

Executive summary:

The objective was to assess the skin irritation and corrosion potential of the test material. By using the currently available methods a single in vitro assay is not sufficient to cover the full range of skin irritating/corrosion potential. Therefore, two in vitro assays were part of this in vitro skin irritation and corrosion test strategy: The Skin Corrosion Test (SCT) and Skin Irritation Test (SIT).

The potential of the test substance to cause dermal corrosion/irritation was assessed by a single topical application of ca. 25 µL bulk volume (about 48 mg) of the undiluted test substance to a reconstructed three-dimensional human epidermis model (EpiDerm).

Due to the texture of the solid test substance the application with a sharp spoon was not possible. Therefore, a bulk volume of ca. 25 µL ground test material was formed to a flat disc matched to the size of the tissue (ca. 8 mm diameter). The discs were applied directly to the tissue surface.

The irritation test was performed with three EpiDerm tissues which were incubated with the test substance for 1 hour followed by a 42-hour post-incubation period.

Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation by using a colorimetric test. The reduction of mitochondrial dehydrogenase activity measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the epidermal tissues treated with the test substance is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability.

The following results were obtained in the EpiDerm skin corrosion/irritation test:

The test substance is able to directly reduce MTT. Therefore, an additional MTT reduction control KC (freeze-killed control tissues) was introduced.

Results of the Irritation Test (SIT):

The final mean viability of the tissues treated with the test substance determined after an exposure period of 1 hour with an about 42-hour post-incubation was 16.5%.

Based on the results observed and by applying the evaluation criteria, it was concluded that the test material (solvent free) shows a skin irritation potential in the EpiDerm in vitro skin irritation and corrosion test strategy under the test conditions chosen.