Disodium 2,4-diamino-3-{[4-(ethenylsulfonyl)phenyl]diazenyl}-5-{[4-(ethenylsulfonyl)-2-sulfonatophenyl]diazenyl}benzenesulfonate

Endpoint summary

Description of key information

FAT 40866/A TE was found to be non-irritant and non-corrosive to skin and eyes in an in vitro study.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Guidelines followed

GLP compliance:

yes

Species:

other: skin tissue

Strain:

not specified

Details on test animals or test system and environmental conditions:

Test system:

EpiDerm Skin Model (EPI-200, Lot no.: 20532 kit N).

The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.

Type of coverage:

other: EpiDerm Skin Model

Preparation of test site:

not specified

Vehicle:

not specified

Controls:

not required

Amount / concentration applied:

None

Duration of treatment / exposure:

None

Observation period:

Acceptability of the assay:
The in vitro skin corrosion test is considered acceptable if it meets the following criteria:
a) The absolute mean OD540 of the two tissues of the negative control should reasonably be within the laboratory historical control data range.
b) The mean relative tissue viability following 3-minute exposure to the positive control should be <= 30%.
c) The maximum inter-tissue variability (in viability) is 30% between two tissues treated identically.
d) The maximum difference in percentage between the mean viability of two tissues and one of the
two tissues is 15%.

Number of animals:

None

Details on study design:

None

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1-hour application viability (percentage of control)

Value:

92

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3-minute application viability (percentage of control)

Value:

88

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

Finally, it is concluded that this test is valid and that FAT 40866/A TE is not corrosive in the in vitro skin corrosion test under the experimental conditions.

Other effects:

None

FAT 40866/A TE was checked for possible direct MTT reduction by adding the test substance to MTT medium. Because no colour change was observed it was concluded that FAT 40866/A TE did not interact with MTT.

The mean absorption at 540 nm measured after treatment with FAT 40866/A TE.

The mean tissue viability obtained after 3-minute and 1-hour treatments with FAT 40866/A TE compared to the negative control tissues. Skin corrosion is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with FAT 40866/A TE compared to the negative control tissues was

88% and 92% respectively. Because the mean relative tissue viability for FAT 40866/A TE was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment FAT 40866/A TE is considered to be not corrosive.

The absolute mean OD540 (optical density at 540 nm) of the negative control tissues was within the laboratory historical control data range. The mean relative tissue viability following 3-minute exposure to the positive control was 9%. The maximum inter-tissue variability in viability between two tissues treated identically was less than 20% and the maximum difference in percentage

between the mean viability of two tissues and one of the two tissues was less than 11%. It was therefore concluded that the test system functioned properly.

Interpretation of results:

other: not corrosive

Remarks:

Criteria used for interpretation of results: OECD GHS

Conclusions:

Finally, it is concluded that this test is valid and that FAT 40866/A TE is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

Executive summary:

In vitro skin corrosion test with FAT 40866/A TE using a human skin model. This report describes the ability of FAT 40866/A TE to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)). The possible corrosive potential of FAT 40866/A TE was tested through topical application for 3 minutes and 1 hour.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

Batch BOP 04-13 (Lot: BS-MIF 975) of FAT 40866/A TE was a red-brown powder. Skin tissue was moistened with 25 μl of Milli-Q water and 25 mg of FAT 40866/A TE was applied directly on top of the skin tissue.

The positive control had a mean relative tissue viability of 9% after 3 minutes exposure. The absolute mean OD540 (optical density at 540 nm) of the negative control tissues was within the laboratory historical control data range. The maximum inter-tissue variability in viability between two tissues treated identically was less than 20% and the maximum difference in percentage between the mean

viability of two tissues and one of the two tissues was less than 11%, indicating that the test system functioned properly.

Skin corrosion is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with FAT 40866/A TE compared to the negative control tissues was 88% and 92%, respectively. Because the mean relative tissue viability for FAT 40866/A TE was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment FAT 40866/A TE is considered to be not corrosive.

Finally, it is concluded that this test is valid and that FAT 40866/A TE is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

Principles of method if other than guideline:

Guidelines followed

GLP compliance:

yes

Species:

other: cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco,'s Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.

Transport: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.

Vehicle:

water

Controls:

not specified

Amount / concentration applied:

No correction was made for the purity/composition of the test substance.

A 20% (w/w) solution of FAT 40866/A TE was prepared in physiological saline (Merck, Darmstadt, Germany). The stock solution was treated with ultrasonic waves until the test substance had completely dissolved.

Duration of treatment / exposure:

The medium from the anterior compartment was removed and 750 µl of either the negative control, positive control (20% (w/v) Imidazole solution) or 20% (w/w) test substance was introduced onto the epithelium of the cornea. The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 240 ± 10 minutes at 32 ± 1ºC. After the incubation the solutions were removed and the epithelium was washed at least three times with MEM with phenol red (Eagle’s Minimum Essential Medium, Invitrogen Corporation). Possible pH effects of the test substance on the corneas were recorded. The anterior and the posterior compartment were refilled with fresh cMEM and an opacity determination was performed without any further incubation. After the completion of the incubation period each cornea were inspected visually for dissimilar opacity patterns and the opacity determination was performed. To verify the results a repeat experiment was performed (Experiment 2).

Observation period (in vivo):

None

Number of animals or in vitro replicates:

None

Details on study design:

Acceptability of the assay:

The assay is considered acceptable if:
- The positive control gives an in vitro irritancy score that falls within the laboratory historical mean value.
- The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean In vitro Irritation Score

Value:

3.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Run / experiment:

Mean In vitro Irritation Score

Value:

0.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

None

Other effects:

None

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas.

The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 115 in experiment 1 and 128 in experiment 2 and within the historical positive control data range.

It was therefore concluded that the test conditions were adequate and that the test system functioned properly. FAT 40866/A TE showed in vitro irritancy scores of 0.6, 0.5 and 10.8 after 240 minutes of treatment. Since one out of three corneas showed an IVIS above 3, the test was repeated to confirm the results, resulting in IVIS scores of -0.2 to 1.0. Therefore the value of 10.8 observed in the first experiment can be considered an outlier.

Finally, it is concluded that this test is valid and that FAT 40866/A TE is not irritant or corrosive in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.

Since FAT 40866/A TE induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: OECD GHS

Conclusions:

It is concluded that this test is valid and that FAT 40866/A TE is not irritant or corrosive in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.

Executive summary:

Screening for the eye irritancy potential of FAT 40866/A TE using the Bovine Corneal Opacity and Permeability test (BCOP test).

This report describes the ocular irritation properties of FAT 40866/A TE on an isolated bovine cornea.

The possible ocular irritancy of FAT 40866/A TE was tested through topical application for approximately 240 minutes.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

Batch BOP 04-13 (Lot: BS-MIF 975) of FAT 40866/A TE was a red-brown powder. The test substance was applied as a 20% (w/w) solution (750 μl) directly on top of the corneas.

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas.

The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 115 (experiment 1) and 128 (experiment 2) and within the historical positive control data range in two independent experiments. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

FAT 40866/A TE showed in vitro irritancy scores of 0.6, 0.5 and 10.8 after 240 minutes of treatment. Since one out of three corneas showed an IVIS above 3, the test was repeated to confirm the results, resulting in IVIS scores of -0.2 to 1.0. Therefore the value of 10.8 observed in the first experiment can be considered an outlier.

Finally, it is concluded that this test is valid and that FAT 40866/A TE is not irritant or corrosive in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.

Since FAT 40866/A TE induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Two in vitro studies were carried out to evaluate the skin irritation/corrosion and eye irritation potency of the test substance FAT 40866/A following the OECD guidelines.

An in vitro skin corrosion test was conducted with FAT 40866/A TE using a three dimensional epidermal model test, EpiDerm (EPI-200) test. FAT 40866/A TE was tested through topical application for 3 minutes and 1 hour.

The relative mean tissue viability obtained after 3-minute and 1-hour treatments with FAT 40866/A TE compared to the negative control tissues was 88% and 92%, respectively. Because the mean relative tissue viability for FAT 40866/A TE was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment FAT 40866/A TE is considered to be not corrosive.

Another key study conducted in an in vitro skin irritation test with FAT 40866/A TE following a standard three dimensional epidermal model, EPISKIN Standard model (EPISKIN-SMTM) method. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with FAT 40866/A TE compared to the negative control tissues was 113%. Since the mean relative tissue viability for FAT 40866/A TE was above 50% after 15 minutes treatment FAT 40866/A TE is considered to be non-irritant. The positive control had a mean cell viability of 40% after 15 minutes exposure. Based on results, it is concluded that the FAT 40866/A TE is non-irritant in the in vitro skin irritation test under the described experimental conditions.

The possible ocular irritation potential of FAT 40866/A TE was assessed using the Bovine Corneal Opacity and Permeability test (BCOP test) followed by OECD and EC guidelines.

The meanin vitroirritancy score of the positive control (20% (w/v) Imidazole) was 115 (experiment 1) and 128 (experiment 2) and within the historical positive control data range in two independent experiments. FAT 40866/A TE showedin vitroirritancy scores of 0.6, 0.5 and 10.8 after 240 minutes of treatment. Since one out of three corneas showed an IVIS above 3, the test was repeated to confirm the results, resulting in IVIS scores of -0.2 to 1.0. Therefore the value of 10.8 observed in the first experiment can be considered an outlier.

The FAT 40866/A TE is not irritant or corrosive in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.

Since FAT 40866/A TE induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Justification for selection of skin irritation / corrosion endpoint:
The study was conducted according to OECD and EC guidelines and in accordance with GLP.

Justification for selection of eye irritation endpoint:
The study was conducted according to OECD and EC guidelines and in accordance with GLP.

Justification for classification or non-classification

Test item FAT 40866 did not show any irritation/ corrosive effects in in vitro tests conducted to determine its potency to cause skin irritation/ corrosion and eye irritation.