Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., compds. with 2-propanamine

Endpoint summary

Description of key information

The potential of LAS-IPA (target substance) to induce skin irritation/corrosion was evaluated in two suitable in vitro test methods (OECD 439 and OECD 431). The results show that the substance is not corrosive to the skin, but it does have irritating properties. Based on the results, the target substance can be considered as irritant to the skin. Therefore, classification as Skin Irrit. 2, H315 is warranted in accordance with the CLP criteria.

The potential of the target substance to induce eye irritation was assessed using data from a suitable in vitro eye irritation test. Based on the results, the target substance can be considered as irritant to the eye. Therefore, classification as Eye Irrit.2, H319 is warranted in accordance with the CLP criteria.

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:

Unavailable - study report dated 2012

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:

no

Qualifier:

according to guideline

Guideline:

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

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)

Details on test system:

Three-dimensional reconstructed human epidermis- EPISKIN model kit purchased from SkinEthic Laboratories (Lyon, France).

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 10 µL

NEGATIVE CONTROL
- Amount applied: 10 µL DPBS

POSITIVE CONTROL
- Amount applied: 10 µL 5% w/v SDS

Duration of treatment / exposure:

15 min

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

not specified

Details on study design:

Test MTT reduction by the test material: 10 µL test material + 2 mL 0.3 mg/mL MTT solution incubated for 3 h. The blue colour indicates reduced MTT.

MAIN TEST
DAY 1-Application of test item
Exposure: 15 min
Amount applied: 10 µL
Negative control: 10 µL DPBS
Positive control: 10 µL 5% w/v SDS
Washing: DPBS with Ca++ and Mg++
Post-exposure incubation: 42 h

DAY 3-MTT Loading/Formazan Extraction
Plate shaker: 15 min
MTT solution: 2 mL of 0.3 mg/mL per well
Incubation: 3 h
Biopsy of the epidermis was performed. The tissue was then transfered in micro tubes with 500 µL acidified isopropanol and refrigerated till day 6.

DAY 6-Absorbance/Optical Density
at 540 nm with Anthos 2001 microplate reader

Irritation / corrosion parameter:

% tissue viability

Remarks:

Relative Mean Viability

Value:

6.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

See below for detailed results.

Other effects / acceptance of results:

- OTHER EFFECTS:
The MTT solution conatining the test item did not turn blue, suggesting that the test material does not reduce directly MTT.

- ACCEPTANCE OF RESULTS:
Acceptance criteria met for negative and positive controls.

Table 1: Mean OD540 values and percentage viabilities for the negative control Item, positive control Item and test item

Item	OD540 of tissues	Mean OD540 of triplicate tissues	± SD of OD540	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	0.786	0.753	0.029	104.4	100	3.8
	0.737			97.9
	0.735			97.6
Positive Control Item	0.129	0.110	0.039	17.1	14.6	5.2
	0.065			8.6
	0.136			18.1
Test Item	0.032	0.051	0.017	4.2	6.8	2.3
	0.055			7.3
	0.066			8.8

 

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Under the conditions of this in vitro test benzenesulfonic acid, 4-C10-13-sec-alkyl derivs.-, compd. with 2-propanamine (1:1) is irritating to the skin.

Executive summary:

In a primary dermal irritation study conducted according to OECD Guideline for the Testing of Chemicals No. 439 “In Vitro Skin Irritation” (adopted 22 July 2010), EPISKIN™ reconstructed human epidermis model was topically exposed to 10 µL of undiluted benzenesulfonic acid, 4-C10-13-sec-alkyl derivs.-, compd. with 2 -propanamine (97% purity) for 15 min and 42 h post incubation period. The principle of the assay was based on the measurement of cytotoxicity in the human epidermal cultures following topical exposure to the test item, with the MTT reduction assay. The negative and positive controls confirmed the validity of the study.

The test item is irritating to the skin. The relative mean tissue viability was ≤ 50% (6.8%) after 15 min treatment and 42 h post-incubation. Based on the results, benzenesulfonic acid, 4-C10-13-sec-alkyl derivs.-, compd. with 2-propanamine is classified to be irritating to the skin in accordance with UN GHS "Category 2".

The study is considered acceptable based on the quality criteria.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Unavailable - study report dated 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

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: 50 µL

NEGATIVE CONTROL
- Amount applied: 50 µL of 0.9% w/v sodium chloride

POSITIVE CONTROL
- Amount applied: 50 µL glacial acetic acid

Duration of treatment / exposure:

3, 60 and 240 min

Number of replicates:

At least 2 per exposure time

Details on study design:

Pre-test: Test MTT reduction by the test material: 50 µL test material + 2.2 ml 0.3 mg/mL MTT solution incubated for 3 h. The blue colour indicates reduced MTT.

MAIN TEST
DAY 1-Application of test item
Exposure: 3, 60 and 240 min
Amount applied: 50 µL
Negative control: 50 µL of 0.9% w/v sodium chloride
Positive control: 50 µL glacial acetic acid
Washing: PBS with Ca++ and Mg++
MTT solution was applied: 2.2 mL of 0.3 mg/mL per well, incubation followed for 3 h ± 5 min
Biopsy of the epidermis was performed. The tissue was then transfered in micro tubes with 850 µL acidified isopropanol.

DAY 2-Absorbance/Optical Density
at 540 nm with Anthos 2001 microplate reader

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min

Value:

77.5

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

Value:

90.5

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:

240 min

Value:

85.2

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:
The MTT solution containing the test item did not turn blue, suggesting that the test material does not reduce directly MTT

- ACCEPTANCE OF RESULTS:
Acceptance criteria met for negative and positive controls.

Table 1: Mean OD540 values and viabilities for the negative control item, positive control item and test item

Item	Exposure period	Mean OD540 of duplicate tissues	Relative mean viability (%)
Negative control	240 min	0.169	100
Positive control	240 min	0.024	14.2
Test item	240 min	0.144	85.2
	60 min	0.153	90.5
	3 min	0.131	77.5

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of this in vitro test benzenesulfonic acid, 4-C10-13-sec-alkyl derivs.-, compd. with 2-propanamine (1:1), is not corosive to the skin.

Executive summary:

In a primary skin corrosion study conducted according to the OECD Guideline for the Testing of Chemicals No. 431 “In VitroSkin Corrosion” (adopted April 2004), the EPISKIN™ reconstructed human epidermis model was exposed to 50 µL of undiluted benzenesulfonic acid, 4-C10-13-sec-alkyl derivs.-, compd. with 2-propanamine (97% purity) for 3, 60, and 240 minutes. The principle of the assay was based on the measurement of cytotoxicity in the human epidermal cultures following topical exposure to the test item, with the MTT reduction assay. The negative and positive controls confirmed the validity of the study.

The test item showed no corrosive effects. The relative mean tissue viability was greater than 15% (90.5%) after 60 min treatment and greater than 50% (77.5%) after 3 min treatment. In addition, the relative mean viability of the test item treated tissues was 85.2% after 240 min treatment. Based on the results, benzenesulfonic acid, 4-C10-13-sec-alkyl derivs.-, compd. with 2-propanamine is classified as “non-corrosive“ in accordance with the UN GHS classification system.

The study is considered acceptable based on the quality criteria.

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:

weight of evidence

Study period:

2012-10-16 to 2013-01-15

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

The test is based on the hypothesis that irritant chemicals are able to penetrate the corneal epithelial tissue and are sufficiently cytotoxic to cause cell death. Cytotoxicity is measured with the MTT reduction assay.

GLP compliance:

yes (incl. QA statement)

Details on test animals or tissues and environmental conditions:

SkinEthic reconstructed Human Corneal Epithelium model (HCE, SkinEthic Laboratories, Lyon, France)

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 30 µL

Duration of treatment / exposure:

10 min

Number of animals or in vitro replicates:

2

Details on study design:

Pre-test: Test MTT reduction by the test material: 30 µL test material + 1 mL 0.5 mg/mL MTT solution incubated for 3 h. The blue colour indicates reduced MTT.

MAIN TEST
7-day old tissues were transferred into wells and treated with 30 µL test item
Exposure period: 10 min
Negative control: 30 µL solution A
Positive control: 30 µL of 2% w/v SDS
Washing: DPBS without Ca++ and Mg++
Placement of each tissue into a 24-well plate with 300 µl maintenance medium
MTT loading plates: 300 µL of a 0.5 mg/mL MTT solution
Incubation: 3 hours
Washing: phosphate buffered saline
Isopropanol was applied for the formation of formazan crystals
Optical density (OD) was measured at 540 nm with Anthos 2001 microplate reader

Tissue histopathology: one tissue/treatment group was kept for histopathological examination

Irritation parameter:

other: OD at 540 nm

Run / experiment:

1

Value:

ca. 0.141

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

other: OD at 540 nm

Run / experiment:

2

Value:

ca. 0.167

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

other: mean percent tissue viability  (migrated information)

Run / experiment:

mean of 2 tissues

Value:

ca. 16.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Table 1: Assessment of Eye Irritation Potential- Viability of HCE tissues

Item	OD540of Individual Tissue	Mean OD540	Relative Mean Viability (%)
Negative Control	0.965	0.926	100
	0.887
Positive Control	0.022	0.022	2.4
	0.021
Test item	0.141	0.154	16.6
	0.167

 

Tissue histopathological examination was not performed because it was not considered necessary.

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

Based on the results of the study, BIO-SOFT 411-E and hence, benzenesulfonic acid, 4-C10-13-sec-alkyl derivs.-, compd. with 2-propanamine (1:1), are both eye irritants.

Executive summary:

In anin vitro eye irritation study, 30 µL of BIO-SOFT 411-E and hence, benzenesulfonic acid, 4-C10-13-sec-alkyl derivs.-, compd. with 2-propanamine (1:1) (97% purity) was applied in the SkinEthic reconstructed Human Corneal Epithelium model (HCE, SkinEthic Laboratories, Lyon, France) for a treatment period of 10 minutes. The test is based on the hypothesis that irritant chemicals are able to penetrate the corneal epithelial tissue and are sufficiently cytotoxic to cause cell death. Cytotoxicity was measured with the MTT reduction assay.

Triplicate tissues were exposed to the test material and thereafter, to MTT. The optical density was measured at 540 nm. Data were presented in the form of percentage viability (MTT conversion relative to negative controls). The controls confirmed the validity of the study.

The test item showed irritant effects. The mean relative tissue viability of the three replicates was < 60% (16.6% ). Therefore, the test item is considered to be irritating to the eyes in accordance with UN GHS “Category 2”.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The potential skin effects of benzenesulfonic acid, 4-C10-13-sec-alkyl derivs.-, compd. with 2-propanamine (97%) were tested in two in vitro studies with the use of reconstructed human epidermis models (EPISKIN^TM). The principle of the assays is based on the measurement of cytotoxicity in the human epidermal cultures following topical exposure to the test material, with the MTT reduction assay. The in vitro test for skin corrosion gave a negative result, while the in vitro test for skin irritation indicates that the substance has skin irritating properties.

An in vitro eye irritation test was performed with the use of the SkinEthic Human Corneal Epithelium model in order to examine the eye irritation potential of benzenesulfonic acid, 4-C10-13-sec-alkyl derivs.-, compd. with 2-propanamine (97%). The results showed a relative mean viability of the test item treated tissues after a 10 min exposure period of 16.6 %, i.e. the test item shall be considered an eye irritant.

Justification for classification or non-classification

Based on the classification criteria laid down in Regulation (EC) No 1272/2008 benzenesulfonic acid, 4-C10-13-sec-alkyl derivs.-, compd. with 2-propanamine (LAS IPA) shall be classified and labelled as irritating to the skin (Skin Irrit. 2, H315) and eye (Eye Irrit. 2, H319).