N-(2-chloroethyl)-4-[(2,6-dichloro-4-nitrophenyl)azo]-N-ethyl-m-toluidine

Endpoint summary

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

The batch of Disperse Brown 27 (4852-1501) tested was a brown powder with a purity of 99.7 %

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 16-EKIN-036

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Details on test system:

This model is a three-dimensional human epidermis model, which consists of adult humanderived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

The skin was moistened with 5 μl Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test item to the tissue and the solid test item (10.3 to 12.7 mg) was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 μl PBS (negative control) and 3 tissues with 25 μl 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time.

Duration of treatment / exposure:

The test consists of topical application of Disperse Brown 27 on the skin tissue for 15 minutes. The tissues were washed with phosphate buffered saline to remove residual test item. After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.

Duration of post-treatment incubation (if applicable):

Post-treatment incubation peiod was 42 hours.

Number of replicates:

Three sets of replicates, one each for Disperse Brown 27, positive and negative control.

Irritation / corrosion parameter:

% tissue viability

Value:

ca. 113

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

GHS criteria not met

Conclusions:

it is concluded that this test is valid and that Disperse Brown 27 is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report and should not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.

Executive summary:

Disperse Brown 27 was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because no colour changes were observed it was concluded that Disperse Brown 27 did not interact with the MTT endpoint.

The mean tissue viability obtained after 15 ± 0.5 minutes treatment with Disperse Brown 27 compared to the negative control tissues is as follows. Skin irritation is expressed asvthe remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with Disperse Brown 27 compared to the negative control tissues was 113%. Since the mean relative tissue viability for Disperse Brown 27 was above 50% Disperse Brown 27 is considered to be non-irritant. The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 5.3%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was 11% or less, indicating that the test system functioned properly.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not 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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to 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)

Deviations:

yes

Remarks:

In the repeat test, one of the negative control eyes in repeat experiment was excluded from the analysis since the IVIS >3. The other 2 eyes met criteria and test item results were not influenced by this result, this does not affect the study outcome.

GLP compliance:

yes

Specific details on test material used for the study:

Sample of Disperse Brown 27 was a brown powder with a purity of 99%.

Species:

cattle

Strain:

other:

Remarks:

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.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Disperse Brown 27 was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (322.6 to 355.1 mg)

Duration of treatment / exposure:

The test consists of topical application of Disperse Brown 27 on the epithelium of the bovine cornea for 4 hours. The non-surfactant solid test item is applied neat by direct application to the surface of the cornea. After exposure the corneas were thoroughly rinsed.

Duration of post- treatment incubation (in vitro):

The opacity of the corneas was determined directly after treatment and the permeability of the corneas was determined after a 90 minutes incubation period with sodium fluorescein.

Number of animals or in vitro replicates:

Two experiments were ran, each consisted of three replicates each for Disperse Brown 27, positive and negative control items.

Details on study design:

The Bovine Corneal Opacity and Permeability Assay (BCOP) is an organic model that provides short-term maintenance of normal physiological and biological function of the bovine cornea in an isolated system. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability with an opacitymeter and an ultraviolet/visible spectrophotometer, respectively.

The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded. The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The
compartments of the corneal holder were filled with cMEM of 32 ± 1 °C. The corneas were incubated for the minimum of 1 hour at 32 ± 1 °C.

After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

The first repeat test was rejected due to inappropriate responses of the negative control (not reported). Therefore a second repeat test was performed. The medium from the anterior compartment was removed and 750 µl of the negative control and 20% (w/v) Imidazole solution (positive control) were introduced onto the epithelium of the cornea. Disperse Brown 27 was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (322.6 to 355.1 mg).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 ± 1 °C. After the incubation the solutions and the test compound were removed and the epithelium was washed at least three times with MEM with phenol red (Earle’s Minimum Essential Medium Life Technologies).
Possible pH effects of the test item on the corneas were recorded. Each cornea was inspected visually for dissimilar opacity patterns. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM and the opacity determinations were performed.

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

>= -0.7 - <= 14.8

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Irritation parameter:

in vitro irritation score

Run / experiment:

2

Value:

>= -1.6 - <= 14.3

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Irritation parameter:

cornea opacity score

Run / experiment:

1

Value:

>= -1.6 - <= 1

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Irritation parameter:

cornea opacity score

Run / experiment:

2

Value:

>= -1.7 - <= 2.3

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Other effects / acceptance of results:

In the first experiment, the individual in vitro irritancy scores for the negative controls ranged from -0.8 to 0.9. The individual positive control in vitro irritancy scores ranged from 140 to 187. The corneas treated with the positive control were turbid after the 240 minutes of treatment.
The corneas treated with Disperse Brown 27 showed opacity values ranging from -1.6 to 1.0 and permeability values ranging from 0.041 to 0.917. The corneas were clear after the 240 minutes of treatment with Disperse Brown 27. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from -0.7 to 14.8 after 240 minutes of treatment with Disperse Brown 27. Since the IVIS scores were spread over two categories, the experiment was repeated. In the repeat experiment, the individual in vitro irritancy scores for the negative controls ranged from 2.2 to 2.6. The individual positive control in vitro irritancy scores ranged from 119 to 147. The corneas treated with the positive control were turbid after the 240 minutes of treatment.
The corneas treated with Disperse Brown 27 showed opacity values ranging from -1.7 to 2.3 and permeability values ranging from 0.002 to 0.800. The corneas were clear after the 240 minutes of treatment with Disperse Brown 27. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from -1.6 to 14.3 after 240 minutes of treatment with Disperse Brown 27. Again the IVIS scores were spread over two categories, the experiment was inconclusive.

Interpretation of results:

study cannot be used for classification

Conclusions:

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) were 159 and 136 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. Since Disperse Brown 27 induced IVIS scores spread over two categories in two independent experiment, no conclusion about the classification can be made based on this test.

Executive summary:

The eye damage of Disperse Brown 27 was tested through topical application for approximately 240 minutes.

Since no workable suspension in physiological saline could be obtained, the test item was used as delivered and added pure 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 159 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Since Disperse Brown 27 induced IVIS scores spread over two categories (-0.7, -0.5 and 14.8 respectively), the experiment was repeated.

In the repeat experiment, 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 136 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Disperse Brown 27 induced IVIS scores spread over two categories (-1.6, 2.0 and 14.3 respectively), comparable with the first experiment. Therefore no conclusion for classification can be made.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Justification for classification or non-classification

It was concluded that the testing for skin irritation was valid and that Disperse Brown 27 is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report, therefore it should not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.

The corneas treated with Disperse Brown 27 showed opacity values ranging from -1.7 to 2.3 and permeability values ranging from 0.002 to 0.800. The corneas were clear after the 240 minutes of treatment with Disperse Brown 27. No pH effect of the test item was observed on the rinsing medium. In the repeat experiment, 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 136 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

 

Since Disperse Brown 27 induced IVIS scores spread over two categories (-0.7, -0.5 and 14.8 respectively), the experiment was repeated. Disperse Brown 27 induced IVIS scores spread over two categories (-1.6, 2.0 and 14.3 respectively), comparable with the first experiment. Since mean IVIS scores were not > 55 the criteria for Category 1 eye irritant classification was not met.