Disodium 6-hydroxy-5-[(2-methoxy-4-sulphonato-m-tolyl)azo]naphthalene-2-sulphonate

Endpoint summary

Description of key information

Skin Irritation

The test uses a single rabbit ear, to indicate the Comedogenicity and irritancy of group of skin care products that may contain follicular and surface epithelial irritating ingredients. The test chemical falls under Grade 2 (diffuse scaling and no erythema). Hence it can be concluded that the test chemical was not irritating to rabbit ears.

Eye Irritation

An ocular irritation study was carried out to assess the irritation potential of the test chemical.0.1 ml of 1.0% aqueous solution of the test chemical was instilled into the rabbit eyes and the effects were observed (duration not specified).0.1 ml of 1.0% aqueous solution of the test chemical did not any changes in rabbit eyes. Hence, the test chemical can be considered not irritating to rabbit eyes.

An in vitro assay was performed using Hen’s egg test on the chorioallantoic membrane (HET-CAM) with a 1 % aqueous dilution of the test chemical to determine its irritation potential. The 1% aqueous solution  of the test chemical did not cause any damaging effect on the CAM as the obtained evaluation resulted in score 0 Hence, the test chemical can be considered to be not irritating to eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

data is from peer reviewed journals

Qualifier:

according to guideline

Guideline:

other: as mentioned below

Principles of method if other than guideline:

To assess the comedogenicity and irritancy potential of the test chemical in rabbits

GLP compliance:

not specified

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Source: No data
Age at study initiation: No data
Weight at study initiation: 3 kg
Fasting period before study: No data
Housing: singly housed, in suspended cages
Diet (e.g. ad libitum): Purina Rabbit Chow, ad libitum
Water (e.g. ad libitum): water, ad libitum
Acclimation period: No data

ENVIRONMENTAL CONDITIONS
Temperature (°C): No data
Humidity (%):No data
Air changes (per hr): No data
Photoperiod (hrs dark / hrs light): 12 hour light and 12 hr dark cycle

IN-LIFE DATES: From: No data To: No data

Type of coverage:

other: Entire inner surface of one ear

Preparation of test site:

other: A colony of rabbits that had genetically good ears and which were free from mites were used for the assay

Vehicle:

other: The test chemical was mixed in propylene glycol at a 9 to 1 dilution for testing.

Controls:

yes, concurrent no treatment

Amount / concentration applied:

Dose of 1ml of the test material was applied once daily for five days per week for 2 weeks.

Duration of treatment / exposure:

five days per week for 2 weeks

Observation period:

2 weeks

Number of animals:

3 animals

Details on study design:

TEST SITE
Area of exposure: Ear
% coverage: A dose of 1ml of the test chemical is applied and spread once daily to the entire inner surface of one ear five days per week for 2 weeks.
Type of wrap if used: No data

REMOVAL OF TEST SUBSTANCE
Washing (if done): No data
Time after start of exposure: No data

SCORING SYSTEM: The irritancy produced by repeated application of a chemical or skin care product on the surface epidermis in the rabbit ear is evaluated on a scale of 0 to 5. The grades are summarized as follows:
0 = No irritation
1 = few scales, no Erythema
2 = diffuse scaling, no Erythema
3 = Generalized scaling with Erythema
4 = Scaling, Erythema and Edema
5 = Epidermal necrosis and slough

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

14 d

Score:

2

Max. score:

5

Reversibility:

no data

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

Grade 2 indicates diffuse scaling, no Erythema

 Table 1: Ingredients and their Comedogenicity and Irritancy

Test chemical	Comedogenicity* [Grade 0-5]	Irritancy** [Grade 0-5]
	2	2

 

*Comedogenicity or the ability of test substance to produce follicular hyperkeratosis

** Irritancy or ability of test substance to produce surface epithelial irritation

Interpretation of results:

other: not irritating

Conclusions:

The test chemical falls under Grade 2 (diffuse scaling and no erythema). Hence it can be concluded that the test chemical was not irritating to rabbit ears.

Executive summary:

The test uses a single rabbit ear, to indicate the Comedogenicity and irritancy of group of skin care products that may contain follicular and surface epithelial irritating ingredients. Ingredients are mixed in propylene glycol at a 9 to 1 dilution for testing unless otherwise indicated ( 10% concentration). A colony of New Zealand albino rabbits that have genetically good ears and is free from mites is used. Three rabbits, weighing two to three kilograms, are used for each assay. Animals are housed singly in suspended cages and fed Purina Rabbit Chow and water ad libitum. Animals are maintained on a 12-hour light and 12-hour dark cycle. A dose of 1 ml of test chemical was applied and spread once daily to the entire inner surface of once for five days per week for two weeks. The opposite untreated ear of each animal served as an untreated control. The irritancy produced by repeated application of a chemical or skin care product on the surface epidermis in the rabbit ear is evaluated on a scale of 0 to 5. The grades are summarized as follows:

 0 = No irritation, 1 = few scales, no Erythema, 2 = diffuse scaling, no Erythema, 3 = Generalized scaling with Erythema, 4 = Scaling, Erythema and Edema

and 5 = Epidermal necrosis and slough.

The test chemical falls under Grade 2 (diffuse scaling and no erythema). Hence it can be concluded that the test chemical was not irritating to rabbit ears.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Justification for type of information:

data is from safety assessment reports

Qualifier:

equivalent or similar to guideline

Guideline:

other: as mentioned below

Principles of method if other than guideline:

To evaluate the ocular irritation potential of the test chemical

GLP compliance:

not specified

Species:

rabbit

Strain:

not specified

Details on test animals or tissues and environmental conditions:

not specified

Vehicle:

water

Controls:

not specified

Amount / concentration applied:

0.1 ml of 1.0% aqueous solution

Duration of treatment / exposure:

no data available

Observation period (in vivo):

no data available

Duration of post- treatment incubation (in vitro):

no data available

Number of animals or in vitro replicates:

no data available

Details on study design:

no data available

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

other: not specified

Score:

0

Reversibility:

not specified

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

No changes were observed, hence the test chemical can be considered as not irritating

Interpretation of results:

other: not irritating

Conclusions:

0.1 ml of 1.0% aqueous solution of the test chemical had no cause any changes in rabbit eyes. Hence, the test chemical can be considered not irritating to rabbit eyes.

Executive summary:

An ocular irritation study was carried out to assess the irritation potential of the test chemical.0.1 ml of 1.0% aqueous solution of the test chemical was instilled into the rabbit eyes and the effects were observed (duration not specified).0.1 ml of 1.0% aqueous solution of the test chemical did not cause any changes in rabbit eyes. Hence, the test chemical can be considered not irritating to rabbit eyes.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Justification for type of information:

data is from safety assessment reports

Qualifier:

according to guideline

Guideline:

other: chorioallantoic membrane (HET-CAM)

Principles of method if other than guideline:

Assessment of the eye irritation potential of the test chemical in the Hen’s egg test on the chorioallantoic membrane (HET-CAM)

GLP compliance:

not specified

Species:

other: Hens egg

Strain:

not specified

Details on test animals or tissues and environmental conditions:

no data

Vehicle:

water

Controls:

not specified

Amount / concentration applied:

1 % aqueous dilution

Duration of treatment / exposure:

at day 9 of incubation for 30 sec

Number of animals or in vitro replicates:

6 eggs per groups

Details on study design:

TEST SITE
- Area of exposure: chorioallantoic membrane
- % coverage: no data
- Type of wrap if used: no data

REMOVAL OF TEST SUBSTANCE
- Washing (if done): 30 seconds
- Time after start of exposure: no data

SCORING SYSTEM:

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

no indication of irritation

Interpretation of results:

other: not irritating

Conclusions:

The 1% aqueous solution of test chemical did not cause any damaging effect on the CAM as the obtained evaluation resulted in score 0 . Hence, the test chemical can be considered to be not irritating to eyes.

Executive summary:

Hen’s egg test on the chorioallantoic membrane (HET-CAM) were performed with a 1 % aqueous dilution of the test chemical.The diluted test item was applied onto the CAM of fertilized chicken eggs at day 9 of incubation and was rinsed off 30 sec after application and evaluation of the irritation parameters was performed. The 1% aqueous solution  of the test chemical did not cause any damaging effect on the CAM as the obtained evaluation resulted in score 0

Hence, the test chemical can be considered to be not irritating to eyes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Irritation

Various studies have been summarized to evaluate the dermal irritation potential of the test chemical in living organisms. These include in vivo experimental studies performed on rabbits for the test chemical.

The test uses a single rabbit ear, to indicate the Comedogenicity and irritancy of group of skin care products that may contain follicular and surface epithelial irritating ingredients. Ingredients are mixed in propylene glycol at a 9 to 1 dilution for testing unless otherwise indicated ( 10% concentration). A colony of New Zealand albino rabbits that have genetically good ears and was free from mites was used. Three rabbits, weighing two to three kilograms, are used for each assay. Animals are housed singly in suspended cages and fed Purina Rabbit Chow and water ad libitum. Animals are maintained on a 12-hour light and 12-hour dark cycle. A dose of 1 ml of test chemical was applied and spread once daily to the entire inner surface of once for five days per week for two weeks. The opposite untreated ear of each animal served as an untreated control. The irritancy produced by repeated application of a chemical or skin care product on the surface epidermis in the rabbit ear was evaluated on a scale of 0 to 5. The grades are summarized as follows:

 0 = No irritation, 1 = few scales, no Erythema, 2 = diffuse scaling, no Erythema, 3 = Generalized scaling with Erythema, 4 = Scaling, Erythema and Edema and 5 = Epidermal necrosis and slough. The test chemical falls under Grade 2 (diffuse scaling and no erythema). Hence it can be concluded that the test chemical was not irritating to rabbit ears.

 

This is supported by the results of the tests for dermal irritations on intact and abraded skin were performed on rabbits. 0.316, 1.0, 3.16 and 10 g/kg bw of the test chemical was applied on the skin of rabbits and observed for effects. The test chemical showed no gross irritation at levels tested; also no skin staining occured. Hence, the test chemical was considered to be not irritating to skin.

The above results are further supported by a Prophetic patch test used for determination of cutaneous toxicity of a chemical compound.

The test chemical was applied either as a neat or as a 25% aqueous solution to the skin of 200 human subjects. The initial exposure to the compound was for 72 hours, and this was followed by a 24-hour application 10 to 14 days later. None of the subjects exhibited compound induced irritation. Hence, the test chemical was considered to be not irritating to skin.

These results are also supported by a study performed to determine the dermal irritation potential of the test chemical in rabbits.

The test chemical was applied daily at rates of 0.5 g/kg, five days a week for 15 applications on abraded and 65 applications on intact skin of rabbits, of 0.1 and 1% solutions in water or as a hydrophilic ointment and observed for effects.No dermal irritation was noted after daily application of the test chemical. Also, No changes in the body weight, clinical laboratory studies and gross and microscopic pathology were observed. Hence, the test chemical was considered to be not irritating to skin.

Based on the available results, the test chemical can be considered to lack the potential to cause irritation/corrosion to skin. Hence, the test chemical can be considered to be not irritating to skin. Comparing the above annotations with the criteria of CLP regulations, the test chemical can be classified under the category “Not Classified”.

Eye irritation

In different studies, the test chemical has been investigated for potential for ocular irritation in living organisms to a greater or lesser extent. These include in vivo experimental studies performed on rabbits as well as in vitro experimental studies for the test chemical. The results are summarized as follows:

An ocular irritation study was carried out to assess the irritation potential of the test chemical.0.1 ml of 1.0% aqueous solution of the test chemical was instilled into the rabbit eyes and the effects were observed (duration not specified).0.1 ml of 1.0% aqueous solution of the test chemical did not any changes in rabbit eyes. Hence, the test chemical can be considered not irritating to rabbit eyes.

This is supported by an in vitro assay performed using Hen’s egg test on the chorioallantoic membrane (HET-CAM) with a 1 % aqueous dilution of the test chemical to determine its irritation potential. The diluted test item was applied onto the CAM of fertilized chicken eggs at day 9 of incubation and was rinsed off 30 sec after application and evaluation of the irritation parameters was performed. The 1% aqueous solution  of the test chemical did not cause any damaging effect on the CAM as the obtained evaluation resulted in score 0

Hence, the test chemical can be considered to be not irritating to eyes.

The above in vitro result is supported by another in vitro screening assay using the measurement of cytotoxicity in human keratinocytes (NRU-assay) was performed to assess the ocular irritation potential of the test chemical. Monolayers of human keratinocytes (HaCat) were exposed in 96-well microtiter plates to various concentrations of the test chemical for 24 hours and cell viability was measured by neutral red uptake. The concentration causing a 50 % reduction in neutral red uptake compared to the concurrent control (NRU-50) is determined. This figure, as a measure for cytotoxicity of a test item in this cell culture, is compared to findings with known eye irritants under identical test conditions and allows a prediction of the eye irritation potential of a test item. No NRU-50 value could be determined as the viability was still 71 % in the first and 69 % in the second assay after treatment with the highest test concentration of 10000 μg/ml medium. Thus, the NRU-50 was > 10000 μg/ml. The NRU-50 values obtained with the positive control SLS (12.5 and 15.6 μg/ml) were in the range of the historical control data of the laboratory and demonstrate the validity and sensitivity of the assay.

According to the classification system of the performing laboratory discriminating between nonirritant, not-classified and severe, the test chemical was classified as non-irritant, as the cut-off for this classification is ≥750 μg/ml under the described test conditions.

The in vitro and the in vivo results are in agreement with each other, indicating a very strong possibility that the test chemical lacks the potential to cause any irritation to eyes. Hence, the test chemical can be considered to be not irritating to eyes. Comparing the above annotations with the criteria of CLP regulations, the test chemical can be classified under the category “Not Classified”.

Justification for classification or non-classification

Based on the available results, the test chemical can be considered to lack the potential to cause irritation/corrosion to skin. Hence, the test chemical can be considered to be not irritating to skin. Comparing the above annotations with the criteria of CLP regulations, the test chemical can be classified under the category “Not Classified”.The in vitro and the in vivo results are in agreement with each other, indicating a very strong possibility that the test chemical lacks the potential to cause any irritation to eyes. Hence, the test chemical can be considered to be not irritating to eyes. Comparing the above annotations with the criteria of CLP regulations, the test chemical can be classified under the category “Not Classified”.