Benzenesulfonic acid, 2,2'-(1,2-ethenediyl)bis[5-nitro-, disodium salt, reaction products with 4-[(4-aminophenyl)azo]benzenesulfonic acid monosodium salt

Endpoint summary

Description of key information

 Appraisal of the safety of chemicals in foods drugs and cosmetics of 1959 by AFDO, the substance is not skin irritant in rabbits.

 OECD 405, the substance is not eye irritation in rabbits.

 

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro skin irritation study does not need to be conducted because adequate data from an in vivo skin irritation study are available

Reason / purpose for cross-reference:

data waiving: supporting information

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

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

Adequacy of study:

key study

Study period:

1973

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Remarks:

Source study has reliability 2. Details on the read across are attached in section 13.

Principles of method if other than guideline:

Appraisal of the safety of chemicals in foods drugs and cosmetics (1959) by AFDO.

GLP compliance:

no

Species:

rabbit

Strain:

other: Russian breed

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 1.5 to 1.6 kg
- Housing: Singly in metal cages, overall dimensions 47×32×34 cm
- Diet: Nafag, ad libitum
- Water: ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature: 18 ± 1 °C
- Humidity: 55 ± 5 %
- Photoperiod: 14 hrs light day

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

water

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 g

Duration of treatment / exposure:

24 hours

Observation period:

8 days

Number of animals:

6 rabbits, 3 males and 3 females

Details on study design:

The shaved skin area on the left side was slightly scarified just before treatment. A gauze patch of 2.5 × 2.5 cm with test substance was applied immediatly to the prepared skin. The patch was covered with an impermeable foil of 5 × 5 cm, which was fixed to the body of the animals with adhesive tape.
Test substance was applied to each side in quantities of 0.5 g. Before application, test material was wetted with tap water.
The gauze patches were removed 24 hours after application. Skin reaction was appraised upon removal during an observation period of 8 days on the basis of erythema and edema formation.

Irritation parameter:

erythema score

Basis:

animal #1

Remarks:

026/M

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

edema score

Basis:

animal #1

Remarks:

026/M

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

erythema score

Basis:

animal #2

Remarks:

028/M

Time point:

24/48/72 h

Score:

0.66

Max. score:

4

Reversibility:

fully reversible within: 8 days

Irritation parameter:

edema score

Basis:

animal #2

Remarks:

028/M

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

erythema score

Basis:

animal #3

Remarks:

030/M

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

edema score

Basis:

animal #3

Remarks:

030/M

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible within: 8 days

Irritation parameter:

erythema score

Basis:

animal #4

Remarks:

007/F

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 8 days

Irritation parameter:

edema score

Basis:

animal #4

Remarks:

007/F

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

erythema score

Basis:

animal #5

Remarks:

009/F

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

edema score

Basis:

animal #5

Remarks:

009/F

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

erythema score

Basis:

animal #6

Remarks:

011/F

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

animal #6

Remarks:

011/F

Time point:

24/48/72 h

Score:

0

Max. score:

4

Results tabulated

animal no.	erythema			oedema			mean erythema	mean oedema
	24 h	48 h	72 h	24 h	48 h	72 h
1	1	0	0	0	0	0	0.33	0
2	1	0	1	0	0	0	0.66	0
3	0	1	0	0	0	0	0.33	0
4	1	1	1	0	0	0	1	0
5	1	0	0	0	0	0	0.33	0
6	0	0	0	0	0	0	0	0

 

Erythema and Eschar Formation

0       No erythema

1       Very slight erythema (barely perceptible)

2       Well defined erythema

3       Moderate to severe erythema

4       Severe erythema (beet redness) to slight eschar formation (injuries in depth)

Total possible erythema score 4

 

Edema formation

0       No edema

1       Very slight edema (barely perceptible)

2       Slight edema (edges of area well defined by definite raising)

3       Moderate edema (raised appoximately 1mm)

4       Severe edema (raised more than 1mm and extending beyond area of exposure)

Total possible edema score 4

 

 

Interpretation of results:

other: not classified, according to the CLP Regulation (EC 1272/2008)

Conclusions:

The substance was tested as described in Appraisal of the safety of chemicals in foods drugs and cosmetics of 1959 by AFDO.
Under the conditions of the experiments the substance is not irritant when applied to intact skin of rabbits.

Executive summary:

Method

6 rabbits (3 males and 3 females) of the Russian breed were used. Test substance was applied on both intact and abraded skin in amount of 0.5 g. The gauze patches were removed 24 hours after application. The reaction of the skin was appraised upon removal during an observation period of 8 days.

Results

On intact skin, slight signs of erythema and oedema were noted in 5/6 rabbits. Such signs were found to be reversible within 8 days.

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

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro eye irritation study does not need to be conducted because adequate data from an in vivo eye irritation study are available

Reason / purpose for cross-reference:

data waiving: supporting information

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Source study has reliability 2. Details on the read across are attached in section 13.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Dr. K. Thomae GMBH; Chemisch-pharmazeutische Fabrik D-7950 Biberach/Riss
- Weight at study initiation: 2230 to 2620 g
- Housing: individually
- Diet: ad libitum standard rabbit pellet
- Water: fresh water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 ± 3°C
- Humidity: 30 - 70 %
- Photoperiod (hrs dark / hrs light): 12 hours light

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 ml (32 mg) of test substance were applied into the conjunctival sac of the left eye of each animal.
- The right eye remained untreated and served as control

Duration of treatment / exposure:

After instillation the lids were held together for about one second to prevent loss of test article.

Observation period (in vivo):

Ocular reactions were evaluated after 1, 24, 48 and 72 hours after instillation
Reaction was observed after 7, 10 and 14 days of instillation

Number of animals or in vitro replicates:

3

Details on study design:

SCORING SYSTEM:
Cornea
A. Opacity-degree of density
0 No opacitiy
1 Scaterred or difussed area, details of iris clearly visible
2 Easily discernible translucent areas, details of iris slightly obscured
3 Opalescent areas, no details of iris visible
4 Opaque, iris invisible

B. Area of cornea involved
1 One quarter (or less) but not zero
2 Greater than one quarter, but less than half
3 Greater than half, but less than three quarters
4 Greater than three quarters, up to whole area

A×B×5 Total maximum = 80

Iris
A. Values
0 Normal
1 Folds above normal, congestion, swelling, circumcorneal injection
2 No reaction to light, hemorrhage, gross destruction

A×5 Total maximum=10

Conjuctivae
A. Redness
0 Vessels normal
1 Vessels definitely injected above normal
2 More diffuse, deeper crimson red, individual vessels not easily discernible
3 Diffuse beefy red

B. Chemosis
0 No swelling
1 Any swelling above normal
2 Obvious swelling with partial eversion of lids
3 Swelling with lids about half closed
4 Swelling with lids about half closed to completely closed

C. Discharge
0 No discharge
1 Any amount different from normal
2 Discharge with moistening of the lids and hairs just adjacent to lids
3 Discharge with moistening of the lids and hairs, and considerable area around the eye

(A+B+C)×2 Total maximum=20

TOOL USED TO ASSESS SCORE: slit-lamp was used to facilitate the evaluation

Irritation parameter:

cornea opacity score

Basis:

animal #1

Remarks:

658/F

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 14 days

Irritation parameter:

iris score

Basis:

animal #1

Remarks:

658/F

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

fully reversible within: 14 days

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #1

Remarks:

658/F

Time point:

24/48/72 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 14 days

Irritation parameter:

chemosis score

Basis:

animal #1

Remarks:

658/F

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 14 days

Irritation parameter:

cornea opacity score

Basis:

animal #2

Remarks:

110/F

Time point:

24/48/72 h

Score:

0.67

Max. score:

4

Reversibility:

fully reversible within: 14 days

Irritation parameter:

iris score

Basis:

animal #2

Remarks:

110/F

Time point:

24/48/72 h

Score:

0.33

Max. score:

2

Reversibility:

fully reversible within: 14 days

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #2

Remarks:

110/F

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

fully reversible within: 14 days

Irritation parameter:

chemosis score

Basis:

animal #2

Remarks:

110/F

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 14 days

Irritation parameter:

cornea opacity score

Basis:

animal #3

Remarks:

098/F

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 14 days

Irritation parameter:

iris score

Basis:

animal #3

Remarks:

098/F

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

fully reversible within: 14 days

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #3

Remarks:

098/F

Time point:

24/48/72 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 14 days

Irritation parameter:

chemosis score

Basis:

animal #3

Remarks:

098/F

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible within: 14 days

Results tabulated

 

3 animals or less	mean score animal (1 / 2 / 3)			maximum value	maximum duration of any effect	maximum value at the end of the observation period
conjunctiva/redness	1	2	1	2	10 days	0
conjunctiva/chemosis	0.33	0.33	0	1	24 hours	0
cornea	0.33	0.67	0.33	1	48 hours	0
iris	0	0.33	0	1	24 hours	0

Interpretation of results:

other: not classified, according to the CLP Regulation (EC 1272/2008)

Conclusions:

The substance was tested according to the guidelines OECD 405.
Based on scores at 24 to 72 hours after instillation of the substance, the substance resulted as not irritant to rabbit eye. Changes were fully reversible within 14 days.

Executive summary:

Method

Eye irritation potential was assessed following OECD guideline 405. Test substance was applied to one eye of 3 rabbits; the other eye served as control. No indication of eye rinse was reported. Observations were done at 1, 24, 48 and 72 h and 7d, 10 d and 14 after application. Effects on cornea, iris and conjunctivae were noted.

Results

Test material induced reactions of cornea, iris and conjunctiva when instilled into the conjunctival sac of rabbits eyes.

The following mean scores over the period from 24 to 72 h were calculated:

animal no.	658	110	98
conj. redness	1	2	1
conj. chemosis	0.33	0.33	0
cornea	0.33	0.67	0.33
iris	0	0.33	0

Effects were fully reversible within the 14 days observation period.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

No data on target substance was available, thus a read across approach was followed using available data on Similar Substance 01. Details on the read across are reported in section 13.

 

A study on Similar Substance 01 was carried out as described in Appraisal of the safety of chemicals in foods drugs and cosmetics of 1959 by AFDO. A test sample containing 66 % of active substance was used. The substance was applied to intact and abraded skin of 6 rabbits. Effects on intact skin were evaluated to the purpose of classification and were found to be reversible within 8 days.

Eye irritation was tested according OECD guideline 405, using Similar Substance 01. A test sample containing 91 % of active substance was used. Slight effects on cornea, iris and conjunctivae were noted, which were fully reversible within 14 days.

Justification for classification or non-classification

Skin Irritation

According to the CLP Regulation (EC 1272/2008), skin corrosion means the production of irreversible damage to the skin, and skin irritation means the production of reversible damage to the skin following the application of a test substance for up to 4 hours.

A substance has to be classified as category 1 (corrosive) if it produces destruction of skin tissue, namely, visible necrosis through the epidermis and into the dermis, in at least 1 tested animal after exposure up to a 4 hour duration.

 

A substance has to be classified as category 2 (irritant) if shows:

(1) mean value of ≥ 2,3 - ≤ 4,0 for erythema/ eschar or for oedema in at least 2 of 3 tested animals from gradings at 24, 48 and 72 hours after patch removal or, if reactions are delayed, from grades on 3 consecutive days after the onset of skin reactions; or

(2) inflammation that persists to the end of the observation period normally 14 days in at least 2 animals, particularly taking into account alopecia (limited area), hyperkeratosis, hyperplasia, and scaling; or

(3) in some cases where there is pronounced variability of response among animals, with very definite positive effects related to chemical exposure in a single animal but less than the criteria above.

In case 6 rabbits are used, a response above the threshold of 2.3 in 4 of 6 rabbits implies a classification in category 2.

As irritation scores were below the threshold set by the CLP Regulation (EC 1272/2008) and were reversible within 8 days, no classification applied.

 

Eye Irritation

According to the CLP Regulation (EC 1272/2008), serious eye damage means the production of tissue damage in the eye, or serious physical decay of vision, following application of a test substance to the anterior surface of the eye, which is not fully reversible within 21 days of application .

Eye irritation means the production of changes in the eye following the application of test substance to the anterior surface of the eye, which are fully reversible within 21 days of application.

 

A substance has to be classified as category 1 (irreversible effects on the eye), If, when applied to the eye of an animal, a substance produces:

- at least in one animal effects on the cornea, iris or conjunctiva that are not expected to reverse or have not fully reversed within an observation period of normally 21 days; and/ or

- at least in 2 of 3 tested animals, a positive response of:

- corneal opacity ≥ 3 and/or

- iritis > 1,5 calculated as the mean scores following grading at 24, 48 and 72 hours after installation of the test material.

 

A substance has to be classified as category 2 (irritating to eye) if, when applied to the eye of an animal, a substance produces:

at least in 2 of 3 tested animals, a positive response of:

- corneal opacity ≥ 1 and/or

- iritis ≥ 1, and/or

- conjunctival redness ≥ 2 and/or

- conjunctival oedema (chemosis) ≥ 2

calculated as the mean scores following grading at 24, 48 and 72 hours after installation of the test material, and which fully reverses within an observation period of 21 days.

As irritation scores of treated eyes were all below the thresholds set by the CLP Regulation (EC 1272/2008), no classification applied.