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

Endpoint summary

Description of key information

An OECD 439 skin irritation study was performed and showed a skin irritation potential in the EpiDerm model.

An OECD 431 skin corrosion study was performed and showed no skin corrosion potential in the EpiDerm model.

An OECD 437 study was performed and showed serious eye damage in the BCOP model.

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:

08/2017 - 01/2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

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)

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.

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

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.

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)

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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:

July 2017 - January 2018

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 July 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test-substance preparation was produced on a weight per volume (w/v) basis shortly before application by stirring with a high-speed homogenizer (Ultra-Turrax) and a magnetic stirrer.
- Final dilution of a dissolved solid, stock liquid or gel: 20% (w/v)

FORM AS APPLIED IN THE TEST: suspension

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Schlachthof (slaughterhouse) Mannheim, Schlachthofstr. 21, 68165 Mannheim, Germany
- Characteristics of donor animals: age minimum 12 months, maximum 60 months
- indication of any existing defects or lesions in ocular tissue samples: none

Vehicle:

water

Remarks:

deionized

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 750µL
- Concentration: 20% (w/v)

Duration of treatment / exposure:

4 hours

Duration of post- treatment incubation (in vitro):

The negative control and the positive control were removed from the anterior chamber by using a syringe and the epithelium was washed at least 3 times with Eagle’s MEM (containing phenol red) and once with Eagle’s MEM (without phenol red). Both chambers were then refilled with fresh Eagle’s MEM (without phenol red).
Because the test substance could not be removed by using a syringe the epithelium was rinsed with the open chamber method.

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION, PREPARATION AND QUALITY CHECK OF CORNEAS
Corneas free of defects (opacity, scratches, pigmentation etc.) were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in cornea holders that consists of anterior and posterior chambers. Both chambers were filled to excess with pre-warmed Eagle’s MEM (without phenol red) and then equilibrated in a vertical position at about 32°C for at least 1 hour.

TREATMENT METHOD: [closed chamber / open chamber]
The test-substance preparation (non-surfactant) was applied directly to the epithelial surface of the cornea by using a pipette (open chamber method).

POST-INCUBATION PERIOD: no

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity readings were taken for each cornea with an opacitometer.
- Corneal permeability: The amount of sodium fluorescein that permeated through the corneas into the posterior chamber was spectrophotometrically measured. Three aliquots per cornea were transferred to a 96 well microtiter plate (Sunrise Absorbance Reader) and the optical density (OD490) was determined. An aliquot was diluted 1:5 in Eagle’s MEM (without phenol red) and analogously measured (positive control and test substance).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: The decision criteria were modified and amended with a borderline evaluation based on additional internal validation data: The borderline evaluation was statistically determined by using historic BASF data and hence considers the variance of the test method.

IVIS Prediction
< 1.5 No classification for eye irritation
1.5 – 4.5 Borderline
> 4.5; < 45 No prediction can be made for eye irritation, further testing with another suitable method is required
45 - 65 Borderline
> 65 Ocular corrosive or severe irritant

The “borderline“ evaluation (IVIS 3.0 ± 1.5 and 55.0 ± 10.0) was statistically determined by using historic BASF data and takes the test facility specific variance of the test method into account. This evaluation is an amendment to the evaluation provided in OECD Guideline 437. There is no relevance for the outcome of this study since the IVIS is greater than 65.

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean value from three replicates.

Value:

84.6

Vehicle controls validity:

valid

Negative controls validity:

other: The mean OD value of the negative control of the present study lies out of the range of the data given above. However, as all other quality criteria of the test were met, this deviation is not considered to have any influence on the validity of the study.

Positive controls validity:

valid

Table 1: In Vitro Irritancy score (IVIS) of the test substance, the negative control and the positive control

Test substance identification	Cornea-No.	Opacity per cornea	Permeability per cornea	IVIS
				per cornea	per group
					mean	SD
16/0407-1	13	0.0	5.271	79.1	84.6	5.5
	14	6.0	5.231	84.5
	15	3.4	5.785	90.2
NC	1	14.2	0.001	14.2	16.2	8.4
	2	9.0	0.002	9.0
	3	25.4	0.003	25.5
PC	4	85.6	2.283	119.8	110.6	8.0
	5	77.0	1.926	105.9
	6	88.7	1.153	106.0

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

Based on the results observed for the BCOP test alone and by applying the evaluation criteria, it was concluded that the test material causes ocular corrosion or severe irritation in the in vitro eye irritation test strategy under the test conditions chosen.

Executive summary:

The objective was to assess the eye irritating potential of the test material. By using the methods currently available a single in vitro assay is not sufficient to cover the full range of eye irritating potential. Therefore, two in vitro assays were part of this in vitro eye irritation test strategy: The Bovine Corneal Opacity and Permeability Test (BCOP Test) and EpiOcular Eye Irritation Test.

However, in the current case the results derived with BCOP alone were sufficient for a final assessment. Therefore, further testing in EpiOcular Eye Irritation Test was waived.

BCOP

The potential of the test substance to cause ocular irritation or serious damage to the eyes was assessed by a single topical application of 750 µL 20% test-substance preparation to the epithelial surface of isolated bovine corneas.

Three corneas were treated with the test-substance preparation for an exposure period of 4 hours.

In addition to the test substance, a negative control (NC; deionized water) and a positive control (PC; 20% imidazole in deionized water) were applied to three corneas each.

Corneal opacity was quantitatively measured as the amount of light transmitted through the cornea. Permeability was quantitatively measured as the amount of sodium fluorescein dye that passes across the full thickness of the cornea. Both measurements were used to calculate an In Vitro Irritancy Score of the test substance.

Based on the results observed (refer to table 1) in the BCOP test alone and by applying the evaluation criteria described, it was concluded that the test material causes ocular corrosion or severe irritation in the in vitro eye irritation test strategy under the test conditions chosen.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Irritation/Corrosion

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 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.

For the corrosion test, two EpiDerm tissues were incubated with the test substance for 3 minutes and 1 hour, each.

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.

As a result of 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 Corrosion Test (SCT):

The final mean viability of the tissues treated with the test substance determined after an exposure period of 3 minutes was 101.9% and it was 71.9% after an exposure period of 1 hour.

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 shows a skin irritation potential in the EpiDerm in vitro skin irritation and corrosion test strategy under the test conditions chosen.

Eye irritation

By using the methods currently available a single in vitro assay is not sufficient to cover the full range of eye irritating potential. Therefore, two in vitro assays were part of this in vitro eye irritation test strategy: The Bovine Corneal Opacity and Permeability Test (BCOP Test) and EpiOcular Eye Irritation Test.

However, in the current case the results derived with BCOP alone were sufficient for a final assessment. Therefore, further testing in EpiOcular Eye Irritation Test was waived.

BCOP

The potential of the test substance to cause ocular irritation or serious damage to the eyes was assessed by a single topical application of the test-substance preparation to the epithelial surface of isolated bovine corneas.

Three corneas were treated with the test-substance preparation for an exposure period of 4 hours.

In addition to the test substance, a negative control (NC; deionized water) and a positive control (PC; 20% imidazole in deionized water) were applied to three corneas each.

Corneal opacity was quantitatively measured as the amount of light transmitted through the cornea. Permeability was quantitatively measured as the amount of sodium fluorescein dye that passes across the full thickness of the cornea. Both measurements were used to calculate an In Vitro Irritancy Score of the test substance.

Based on the results observed (refer to table 1) in the BCOP Test alone and by applying the evaluation criteria described, it was concluded that the test material causes ocular corrosion or severe irritation in the in vitro eye irritation test strategy under the test conditions chosen.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008.

As a result the substance is considered to be classified as skin irritant and causing serious eye damage under Regulation (EC) No. 1272/2008.