1,3-bis(2-methylphenyl)guanidinium m-[[p-anilinophenyl]azo]benzenesulfonate

Endpoint summary

Description of key information

Skin and eye irritation of the substance

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:

key study

Study period:

From 28 November 2012 to 30 November 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)

Version / remarks:

2010

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

other: reconstructed epidermis of normal human keratinocytes.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic RHE/S/17
- Tissue batch number(s): 12 022A 1103
- Certificate of analysis of reconstructed human epidermis date: 2012-11-26
- Delivery date: recevied on 2012-11-27
- Expiration date : 2012-12-03
- Date of initiation of testing: 2012-11-28


TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 25 x 1 mL with PBS
- Observable damage in the tissue due to washing: 1 of the replicate was compromised.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hr

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the tissue viability after 42 minutes exposure and 42 hrs of post-treatment incubation is < than 50%.
- The test substance is considered to be non-corrosive to skin if the tissue viability after 42 minutes exposure and 42 hrs of post-treatment incubation is > than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 mg

NEGATIVE CONTROL : PBS - Pan BIOTECH GmbH - in the same experimental conditions

POSITIVE CONTROL : 5% SDS CAS 151-21-3 (0.5 g SDS in a 10 mL volumetric flask qsp 10 mL distilled water), in the same experimental conditions.

Duration of treatment / exposure:

42 min

Duration of post-treatment incubation (if applicable):

42 hrs

Number of replicates:

3 replicates

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

replicate 1 (skin #7)

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: aberrants values

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

replicate 2 (skin #8)

Value:

ca. 76

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

replicate 3 (skin #9)

Value:

ca. 96.6

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: mean valibility = 100 %
- Acceptance criteria met for positive control: mean viability = 1.2 %

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:

GHS criteria not met

Conclusions:

The test item must not be classified, in accordance with the Regulation EC No. 1272/2008.

Executive summary:

The aim of the study was to evaluate the possible irritating effects of the test item after topical administration on in vitro human reconstructed epidermis (RHE® model). The test item was applied, as supplied, at the dose of 16 mg, to 3 Reconstructed Human epidermis small during 42 minutes, followed by a 42 hours postincubation period at 37°C, 5% C02.

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

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the structural similarity between the source and the target substances. Their skin toxicity are expected to be similar because of this structural similarity (see document attached).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target and the source substances are structurally related, and have the same impurity profile. Therefore, it is concluded that the impurities will not affect the read-across (see document attached).


3. ANALOGUE APPROACH JUSTIFICATION
In the absence of a skin irritation study on the target substance, a read-across using experimental study performed on source substance was used. Based on structural similarity and a similar impurity profile, no significant difference on their skin irritation toxicity potential is anticipated between both the target and the source substances (see document attached). Therefore, the skin irritation study conducted with the source substance is highly likely to predict the properties of the target substance and is considered as adequate to fulfil the information requirement.


4. DATA MATRIX
See document attached

Reason / purpose for cross-reference:

read-across source

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

-

Value:

>= 76 - <= 96.6

Vehicle controls validity:

not applicable

Remarks:

read-across

Negative controls validity:

not applicable

Remarks:

read-across

Positive controls validity:

not applicable

Remarks:

read-across

Remarks on result:

no indication of irritation

Remarks:

by read-across

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the result of the source subtance, the target substance must not be classified, in accordance with the Regulation EC No. 1272/2008.

Executive summary:

The irritating potency of the target substance has been determined according to the 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), performed on the source substance.

The test item was applied, as supplied, at the dose of 16 mg, to 3 Reconstructed Human epidermis small during 42 minutes, followed by a 42 hours postincubation period at 37°C, 5% C02.

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

Based on the result, the source and target substances must not be classified, in accordance with the Regulation EC No. 1272/2008.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-11-05

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)

GLP compliance:

yes (incl. QA statement)

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:

cornea opacity score

Remarks:

maximal mean score

Value:

ca. 0.2

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class I

Irritation parameter:

fluorescein retention score

Remarks:

mean score

Value:

ca. 0.8

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class II

Irritation parameter:

corneal swelling 

Remarks:

maximal mean (+ in %)

Value:

ca. 10

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class II

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no morphological effects were noted, whatever the examination time

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: the combinaison of the 3 endpoints was 3 x class I: classified as non corrosive/severe irritant.
- Acceptance criteria met for positive control: the combinaison of the 3 endpoints was 3 x class IV: classified as corrosive/severe irritant.
- Range of historical values if different from the ones specified in the test guideline:

Interpretation of results:

not irritating

Conclusions:

The results obtained, under these experimental conditions, enable to conclude that the test item must not be classified the test item must not be classified in category l "irreversible effects on the eye", in accordance with the Regulation (EC) No. 1272/2008, 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 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 must not be classified in category l "irreversible effects on the eye", in accordance with the Regulation (EC) No. 1272/2008.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the structural similarity between the source and the target substances. Their eye toxicity are expected to be similar because of this structural similarity (see document attached).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target and the source substances are structurally related, and have the same impurity profile. Therefore, it is concluded that the impurities will not affect the read-across (see document attached).


3. ANALOGUE APPROACH JUSTIFICATION
In the absence of a eye irritation study on the target substance, a read-across using experimental study performed on source substance was used. Based on structural similarity and a similar impurity profile, no significant difference on their eye irritation toxicity potential is anticipated between both the target and the source substances (see document attached). Therefore, the eye irritation study conducted with the source substance is highly likely to predict the properties of the target substance and is considered as adequate to fulfil the information requirement.


4. DATA MATRIX
See document attached

Reason / purpose for cross-reference:

read-across source

Irritation parameter:

cornea opacity score

Remarks:

maximal mean score

Value:

ca. 0.2

Remarks on result:

other: ICE class I

Irritation parameter:

fluorescein retention score

Remarks:

mean score

Value:

ca. 0.8

Remarks on result:

other: ICE class II

Irritation parameter:

corneal swelling 

Remarks:

maximal mean (+ in %)

Value:

ca. 10

Remarks on result:

other: ICE class II

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the result of the source subtance, the target substance must not be classified, in accordance with the Regulation EC No. 1272/2008.

Executive summary:

The irritating potency of the target substance has been determined according to the OECD guideline No. 438 adopted 07 September 2009 and the Test method B.48 of Council regulation No. 1152/2010 dated 08 December 2010 (EU Journal L324)-ATP Council regulation No. 4401/2008 of 30 May 2008 (E. U. Journal L142), performed on the source substance.

The test item 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 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.

Based on the result of the source subtance, the target substance must not be classified, in accordance with the Regulation EC No. 1272/2008.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Skin irritation / corrosion:

An EpiSkin study was conducted on the source substance 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 source substance, it was not considered as an irritant (erythema score < 3).

Eye irritation:

An Isolated Chicken Eye Test was conducted with the source substance according to guidelines OECD 438 and EC B.48.

While the results (slight to moderate ocular reactions) enable to conclude that the source substance 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.

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 source substance (coloring strength and uncertainties due to the applicability domain of the tests), and to avoid unnecessary in vivo test on rabbit, the source substance has been classified corrosive for the eye.

By read-across, the target substance is classified corrosive for the eye.