Registration Dossier

Administrative data

Description of key information

Skin and eye irritation of Sepisol Fast Yellow MG-F

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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From 28 November 2012 to 30 November 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
This read-across is based on the hypothesis that the source and the target substance have similar structural formula, with a similar toxicity profile amongst the raw materials used to synthetized the Sepisol Fast Yellow MG-F and MG-DPG. The target substance is a UCVB characterized qualitatively but not quantitatively, i.e. exact distribution of the various species is not well known. The UVCB is composed of a common core which is identical to the mono-constituent source substance. The source and the target substances share structural similarities with common functional groups varying in their substitutions. Adequate, reliable and available scientific information indicates that the source and target substances would have similar toxicity profiles. Both substances are manufactured according the same manufacturing process, with the same facilities, and will be handled and use in the same ways. The exposure to these 2 substances will be also identical. The source and target substances are also classified in the same way, with the same labelling requirements. Therefore, read-across from the existing skin irritation test on the source substance is considered as an appropriate adaptation to the standard information requirements of Annex VII, 8.1 of the REACH regulation for the target substance, in accordance with the provisions of Annex XI, 1.5 of the REACH regulation.
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The basic structures of the target and the source substances are the same: the target substance is the source substance which is substituted on the phenylamine cycles by 2 methyl groups or 1 ethyl group (see Figure I in the document attached).
Remark: the target and the source substances are manufactured by the same manufacturer, within the same facilities, and the anionic starting raw material used for the synthesis is identical.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The source substance (Sepisol fast yellow MG-DPG) and the target substance (Sepisol fast yellow MG-F) share the same impurities and a very close physicochemical properties profile (see table 3 of the attached document)


3. ANALOGUE APPROACH JUSTIFICATION
All the physicochemical properties were determined thank to same method for the 2 sources, and their profile is similar (see attached document). The structural differences in the substitution of the diphenylamine cycles do not significantly influence the physicochemical properties. However, some trends in the physicochemical parameters due to the substitution of the cycles can be observed : for example, while a clear and sharp melting point is observed at 160°C for the source substance (mono-constituent), a softening point with no clear melting is observed at 130°C for the target substance (UVCB). It is consistent with the fact that when substances with close melting point are mixed together, the mixture has a lowered melting point and a broadened melting range.
The solubility of the target substance is lower than the solubility of the source substance, which is consistent with the increase of its molecular weight. The partition coefficient has been calculated from the measured solubilities of the test item in octanol and in water separately: the one of the target substance is higher than the one of the source substance (higher molecular weight so lower solubility and high solubility in octanol) but it remains under the cut-off value of 4.

Comparison of the results of the acute toxicity test performed on the source and the target substance are detailled in the attached document: both have a LD50 between 300 mg/kg and 2000 mg/kg and are classification acute toxicity oral cat. 4.

4. DATA MATRIX
See the attached document.
Qualifier:
according to
Guideline:
other: OECD guideline 439 (In-vitro skin irritation)
Qualifier:
according to
Guideline:
other: EU method B.46 (In vitro skin irritation)
GLP compliance:
yes (incl. certificate)
Species:
human
Strain:
other: reconstructed epidermis of normal human keratinocytes.
Details on test animals and environmental conditions:
TEST SYSTEM
- Source: Skinethic Laboratories - 4 rue Alexander Fleming - 69366 Lyon CEDEX 07.
- Cell system used: reconstructed epidermis of normal human keratinocytes. Cells are grown on inert polycarbonate filter on chemically defined medium, arilifted for 17 days.
- RhE model : SkinEthic RHE/S/17
- Lot/Batch No.: 12 022A 1103
Type of coverage:
open
Preparation of test site:
other:
Vehicle:
unchanged (no vehicle)
Controls:
other: not applicable
Amount / concentration applied:
16 mg applied as such to the epidermal surface
Duration of treatment / exposure:
not applicable
Observation period:
not applicable
Number of animals:
not applicable
Details on study design:
I- TEST SYSTEM

Units of 0.50 cm² reconstituted epidermis (SkinEthic) were received on the day before the experiment. The insert (filter + epidermis) was gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter.

II- CHECK OF THE COMPATIBILITY OF THE TEST ITEM (direct MTT reduction, coloring potential, ability to stain tissues) WITH MTT SOLUTION

Prior to experiment, 16 mg of the test item was put in contact with 300 µL of MTT solution (1 mg/mL). After a 3 hrs incubation period at 37°C, 5% CO2, no blue or purple coloration was noted.

Therefore, there is no direct interaction between the test item and MTT.

III- APPLICATION OF THE TEST AND CONTROL CHEMICALS

* For all steps of the protocol, one 24 wells plate (3 columns of 4 wells) was used for each item (the test item, the negative control and the positive control).

* The insert were placed in a 24 wells culture plated which has been previously filled with 300 µL of maintenance medium (Batch No. 12 011J-M 037).

* 3 units of reconstructed human epidermis were used for the test item, the negative and positive controls.

* For the test item: 16 mg was applied as such to the epidermal surface of the models. For the positive control: unspecified quantity of 5% SDS (CAS N° 151-21-3, Sigma Batch N° BCBF8969V) were applied to uniformly cover the epidermis surface. For the negative control: unspecified quantity of PBS (Pan Biotech GmbH – Batch N° 2020312) were applied to uniformly cover the epidermis surface.

* At the end of an exposure period of 42 min at room temperature, the human epidermis was washed with 25 x 1 mL of PSB (PAN BIOTECH GmbH, Batch n° 2020312) except for the three epidermises treated with the test item wich were rinces with 35 x 1 mL of PBS. Even after this rinse, there was remaining test item on the surface of the three epidermises.

* Rinsed epidermis were incubated in fresh medium for a 42 hours 10 min post-treatment incubation period at 37°C, 5% CO2.

* Then, the epidermis were put in contact with the MTT solution.

IV-CELL VIABILITY MEASUREMENTS

* After the 42h10min incubation period, the cell viability was quantified by measurement of the cell succinate dehydrogenase activity. The skin sample was placed in 300 µL of a MTT solution of 1 mg/mL concentration for 3 hours at 37°C, 5% CO2. The precipitated blue formazan product is then extracted using isopropanol during 2 hours under agitation in the dark, and the concentration of formazan was measured by determining the Optical Density (OD) at 570 nm, just after dilution of the extractions in isopropanol (1:2).

The absorbance was measured in triplicate of MTT extract.

The optical density was performed usgin the ELx800 absorbance microplate reader and the validated software Gen5ELISA V1.05.11

V- INTERPRETATION OF RESULTS

* The results were expressed as a viability percentage compared with the negative control :

Viability %= [OD (test item)/OD (negative control)] ×100

The optical density (OD) values obtained for each test sample were used to calculate a percentage of viability relative to the negative control, which was arbitrarily set at 100%.
Irritation / corrosion parameter:
other: other: cell viability percentage
Value:
ca. 86.3
Remarks on result:
other:
Remarks:
Basis: mean. (migrated information)

Individual and average values of OD after 42 minutes of exposure:

 

Skin

OD

Mean OD/disc

(Mean of the 3 OD measurements)

Mean OD/ product

Viability %

Mean Viability %

SD

Conclusion

Negative Control

1

1.041

1.100

1.138

1.093

1.031*

106.0

100.0

5.9

 

2

0.961

1.039

1.092

1.030

99.9

3

0.908

0.976

1.029

0.971

94.1

Positive Control

4

0.012

0.014

0.013

0.013

0.013

1.3

1.2

0.1

Irritant

5

0.011

0.013

0.012

0.012

1.2

6

0.013

0.012

0.014

0.013

1.3

Test Item

7

0.216

0.243

0.250

0.236 – Aberrant values

 

 

 

 

 

8

0.763

0.761

0826

0.783

0.890

76.0

86.3*

14.6*

Non irritant

9

0.918

1.030

1.040

0.996

96.6

* The result is based on two tissues replicates (n° 8 and 9) instead of three as initially scheduled in the study plan. The rinsing of the epidermises in view to eliminate the test item after treatment was difficult and required more rinsing with PBS than usually. The first treated epidermis was compromised during this rinsing step and was not taken into account for the classification of the test item.

                 

Negative control and positive control are in the expected range

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The results obtained under these experimental conditions, enable to conclude that the test item must not be classified, according to the criteria for classification, packaging and labelling of dangerous substances and preparations in compliance with the E.E.C. Directives 67/548, 2001/59 and 99/45. No symbol or risk phrase is required.
In accordance with the Regulation EC No. 1272/2008, the test item must not be classified. No signal word or hazard statement is required.
Executive summary:

The aim of the study was to evaluate the possible irritating effects of SEPISOL FAST YELLOW MG-DPG after topical administration on in vitro human reconstructed epidermis (RHE® model). The test item SEPISOL FAST YELLOW MG-DPG was applied, as supplied, at the dose of 16 mg, to 3 Reconstructed Human epidermis small during 42 minutes, followed by a 42 hours post-incubation period at 37°C, 5% CO2.

The elimination of the test item on the epidermis after treatment was difficult. Even after a rinse with 35 mL of PBS, there was remaining test item. The first epidermis was compromised during rinsing and was not taken into account for the classification of the test item.

The experimental protocol was established in accordance with OECD guideline No. 439 adopted 22 July 2010 and the Test method B.46 of Council regulation No. 761/2009 dated 23 July 2009 (EU Journal L220)-ATP Council regulation No. 4401/2008 of 30 May 2008 (E. U. Journal L142).

The mean viability ofthe epidermis skins was 86.3%, versus 1.2% in the positive control (5% Sodium Dodecyl Sulfate ).

The results obtained, under these experimental conditions, enable to conclude that the test item SEPISOL FAST YELLOW MG-DPG must not be classified, according to the criteria for classification, packaging and labelling of dangerous substances and preparations in compliance with the E.E.C. Directives 67/548, 2001/59 and 99/45. No symbol or risk phrase is required.

In accordance with the Regulation EC No. 1272/2008, the test item must not be classified. No signal word or hazard statement is required.

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
Study period:
2012-11-05
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see "justification for type of information"
Remarks:
This read-across is based on the hypothesis that the source and the target substance have similar structural formula, with a similar toxicity profile amongst the raw materials used to synthetized the Sepisol Fast Yellow MG-F and MG-DPG. The target substance is a UCVB characterized qualitatively but not quantitatively, i.e. exact distribution of the various species is not well known. The UVCB is composed of a common core which is identical to the mono-constituent source substance. The source and the target substances share structural similarities with common functional groups varying in their substitutions. Adequate, reliable and available scientific information indicates that the source and target substances would have similar toxicity profiles. Both substances are manufactured according the same manufacturing process, with the same facilities, and will be handled and use in the same ways. The exposure to these 2 substances will be also identical. The source and target substances are also classified in the same way, with the same labelling requirements. Therefore, read-across from the existing eye irritation test on the source substance is considered as an appropriate adaptation to the standard information requirements of Annex VII, 8.2 of the REACH regulation for the target substance, in accordance with the provisions of Annex XI, 1.5 of the REACH regulation.
Reason / purpose:
reference to other study
Remarks:
Read-accross with a study conduct with the Sepisol fast yellow MG-DPG
Qualifier:
according to
Guideline:
EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)
Qualifier:
according to
Guideline:
OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)
GLP compliance:
yes (incl. certificate)
Species:
other: eyes from chickens freshly killed
Details on test animals or tissues and environmental conditions:
TEST EYES
- Source: Slaughterhouses Etablissement Brun, 33820 Etaulier, France
- Age of the donors animals: approximately 7 weeks old
- Weight of the donors animals: between 1.5 to 2.5 kg
- Transport of eyes: The intact heads were transported from the slaughterhouse at ambient temperature in polystrene boxes humidified with towels moistened with isotonic saline. The eyes are enucleated in the laboratory within less than 1h30 after the the heads are removed
- Time between collection and use of corneas:The corneas were transported to the laboratory straight after removal on animals and used in a maximum delay of 24 hours after reception at the laboratory.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied : 30 mg
Duration of treatment / exposure:
Treatment of 10 secondes
Observation period (in vivo):
4 hours post-treatment
Number of animals or in vitro replicates:
3 chicken eyes for the treatment group
3 chicken eyes for the positive control group
1 chicken eye for the negative control group
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes, with 40 mL of physiological saline (4 times for the treatement group and 2 times for the control groups)
- Time after start of exposure: after 10 secondes

SCORING SYSTEM: Determination of ICE classes for 3 endpoints (1- corneal thickness, 2- corneal opacitiy, 3- fluorescein retention) according to a predefine range. Each ICE classes are then combined to generate an Irritancy Classification

TOOL USED TO ASSESS SCORE:
- Corneal thickness : quantitative determination by optical pachymeter on a slit-lamp microscope.
- Corneal opacity: qualitative assessment based on the area of the cornea opacified
- Fluorescein retention : qualitative assessment based on the staining of the cornea
Irritation parameter:
other: ICE class
Remarks:
average values of the evaluation of corneal lesions
Remarks on result:
other: ICE class II
Irritant / corrosive response data:
Maximal mean corneal swelling score: +10% corresponding to the ICE class II
Maximal mean corneal opacity score: 0.2, corresponding to the ICE class I
Mean score of fluorescein retention: 0.8, corresponding to the ICE class II
Other effects:
no morphological effects were noted, whatever the examination time

Study acceptance criteria:

The concurrent negative and positive controls give an Irritancy Classification that falls within non irritant and severe irritant/corrosive classes, respectively.

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The results obtained, under these experimental conditions, enable to conclude that the test item SEPISOL FAST YELLOW MG-DPG must not be classified R41 "Risk of serious damage to eyes", according to the criteria for the classification, packaging and labelling of dangerous substances in compliance with the E.E.C. Directives 67/548, 2001/59 and 99/45. The symbol "Xi" and the danger label "irritant" are not required.

In accordance with the Regulation (EC) No. 1272/2008, the test item must not be classified in category l "irreversible effects on the eye". The signal word "Danger" and hazard statement H318 "Causes serious eye damage" are not required.
Executive summary:

The aim of the study was to evaluate the possible ocular corrosive or severe irritating effects of the test item after administration on enucleated chickeh eyes.

The test item SEPISOL FAST YELLOW MG-DPG was applied, as supplied, at the dose of 30 mg, to 3 enucleated chicken eyes, during 10 seconds. Then the eyes were rinsed twice with 10 mL of physiological saline. As there was remaining test item, the eyes were rinsed again twice with 10 mL of physiological saline. Test item remained on the cornea of one eye during all the study and on another eye up to the observation time 2 hours.

Three eyes were treated in the same manner (except for a rinse twice with 10 mL of physiological saline) with a positive control and one eye with a negative control. Damages by the test substance were assessed by determination of corneal swelling, opacity, and fluorescein retention at 30, 75, 120, 180 and 240 minutes post-dose.

The experimental protocol was established in accordance with the O.E.C.D. guideline No 438 adopted 07 September 2009 and the test method B.48 - Commission Regulation (EU) No. 1152/2010 dated 08 December 2010 (EU Journal L324) - ATP Council regulation No 440/2008 of 30 May 2008 (E. U Journal L142).

The ocular reactions observed in eyes treated with the test item were slight to moderate:

- maximal mean score of corneal opacity: 0.2, corresponding to the ICE class I;

- mean score of fluorescein retention: 0.8, corresponding to the ICE class II;

- maximal mean corneal swelling: +10%, corresponding to the ICE class II.

The combination of the three endpoints for the positive control, 10 % sodium hydroxide, was 3 x IV. Therefore, the positive control is classified as corrosive/severe irritant, as expected.

The combination of the three endpoints for the negative control, physiological saline solution, was 3 x I. Therefore, the negative control is classified as non corrosive/severe irritant, as expected.

The results obtained, under these experimental conditions, enable to conclude that the test item SEPISOL FAST YELLOW MG-DPG must not be classified R41 "Risk of serious damage to eyes", according to the criteria for the classification, packaging and labelling of dangerous substances in compliance with the E.E.C. Directives 67/548, 2001/59 and 99/45. The symbol "Xi" and the danger label "irritant" are not required.

In accordance with the Regulation (EC) No. 1272/2008, the test item must not be classified in category l "irreversible effects on the eye". The signal word "Danger" and hazard statement H318 "Causes serious eye damage" are not required.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Skin irritation / corrosion:

An EpiSkin study was conducted on a close analogue according to guidelines OECD 439 and EC B.46.

The results obtained (mean viability ofthe epidermis skins was 86.3%, versus 1.2% in the positive control) enable to conclude the substance must not be classified as "irritating".

In the sensitization study performed on the same analogue, it was not considered as an irritant (erythema score < 3).

Eye irritation:

An Isolated Chicken Eye Test was conducted with SEPISOL FAST YELLOW MG-DPG (a close analogue) according to guidelines OECD 438 and EC B.48.

While the results (slight to moderate ocular reactions) enable to conclude that SEPISOL FAST YELLOW MG-DPG must not be classified R41 "Risk of serious damage to eyes" or in category 1 "irreversible effects on the eye", it was not possible to conclude that the substance does not require classification for eye irritation or serious eye damage.

Above all, there are some uncertainties on the issue of the test and on its application domain.

First, the ICE test method does not consider conjunctival and iridal injuries, but it addresses only corneal effects. Based on in vivo test performed on other organic dye of the same group, the main corrosives effects are observed on the conjunctive and the iris, with moderated effects on the cornea.

Moreover, the applicability domain for this category of substance cannot be assessed as no comparison with an appropriate database was performed.

In addition, Sepisol Fast Yellow MG-DPG has surfactive properties, and one of the identified limitations for the ICE test method is a high false negative rate for surfactants. But, this limitation is not considered to be critical since “all test chemicals that come out negative would be subsequently tested with other adequately validated in vitro test(s) or as a last option in rabbits”.

Consequently, the results are doubtful and very likely under-estimated and further tests are needed to evaluate this endpoint.

In the past, the Bovine Corneal Opacity and Permeability Test Method (OECD 439) – BCOP – has been performed on another organic dye of the same category. Because of the coloring strength of the dye and the uncertainty of the applicability domain of the BCOP test method the result was inconclusive.

To conclude, no in vitro tests seems to be appropriate to evaluate the eye irritancy and/or corrosivity of the SEPISOL FAST YELLOW MG-DPG (coloring strength and uncertainties due to the applicability domain of the tests), and to avoid unnecessary in vivo test on rabbit, the SEPISOL FAST YELLOW MG-DPG will be classified corrosive for the eye.

By read-across, the SEPISOL FAST YELLOW MG-F will be classified corrosive for the eye.