Reaction products of disodium 3,5-diamino-2-[(2-sulfonatophenyl)diazenyl]benzoate and  diazotised sodium 2-[(4-aminophenyl)sulfonyl]ethyl sulfate

Endpoint summary

Description of key information

FAT 40810/A showed no skin irritation and no significant eye irritation. However, due to staining of the eyes, which did not fully reverse within 21 days, the substance is considered to cause irreversible effects 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

Study period:

Experiment start date - 25 February 2003; Experiment end date - 13 March 2003; Study completion date - 08 April 2003.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identity: FAT 40810/A
Batch: WP 6/02
Purity: approx. 75 %
Appearance: Solid, dark brownish powder
Expiration date: 12 December 2010
Storage: At room temperature at about 20 °C

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Test system: New Zealand White Rabbit, SPF
Rationale: Recognized by the international guidelines as the recommended test system.
Source: Elevage Scientifique des Dombes, F-01400 Chatillon sur Chalaronne, France
Number of animals per test: 3 (Animals of both sexes were used)
Age at treatment: 10 weeks (male), 11 weeks (females) g
Identification By unique cage number and corresponding ear number.
Acclimatization: Under laboratory conditions after health examination. Only animals without any visual signs of illness were used for the study.
Allocation: Male No. 61, Female Nos. 62 and 63

HUSBANDRY
Room no: 106/ RCC Ltd, Füllinsdorf
Conditions: Standard Laboratory Conditions, Air-conditioned with target ranges for room temperature 17 - 23 °C, relative humidity 30-70 % and approximately 10-15 air changes per hour. Room temperature and humidity were monitored continuously and values outside of these ranges may have occasionally occurred, usually following room cleaning. These transient variations are considered not to have any influence on the study and, therefore, these data are not reported but are retained at RCC. The animals were provided with an automatically controlled light cycle of 12 hours light and 12 hours dark. Music was played during the daytime light period.
Accommodation: Individually in stainless steel cages equipped with feed hoppers and drinking water bowls. Wood blocks (RCC Ltd, Füllinsdorf) and haysticks 4646 (batch no. 0206, Provimi Kliba AG) were provided for gnawing.
Diet: Pelleted standard Provimi Kliba 3418 rabbit maintenance diet ad libitum (batch no. 64/02) provided by Provimi Kliba AG, CH-4303 Kaiseraugst. Results of analysis for contaminants are archived at RCC Ltd, Itingen.
Water: Community tap water from Füllinsdorf, ad libitum. Results of bacteriological, chemical and contaminant analyses are archived at RCC Ltd, Itingen.

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

other: The test substance was moistened with bidistilled water.

Controls:

yes, concurrent no treatment

Amount / concentration applied:

500 mg

Duration of treatment / exposure:

4 h

Observation period:

10 days

Number of animals:

3

Details on study design:

TEST ITEM PREPARATION: 0.5 g (per animal) of FAT 40810/A was weighed as delivered by the sponsor and then moistened with approximately 0.1 mL of purified water before application. The pH of the test item was measured before the study initiation date. A formulation of 1 % in water was prepared. The pH was found to be 5.19. According to Directive 92/69 EEC, B.4. and OECD Guidelines 404, a test item needs not to be tested if the pH-value is less than 2 or greater than 11.5, owing to its predictable corrosive properties.

TREATMENT: Four days before treatment, one flank was clipped with an electric clipper, exposing an area of approximately 100 sq.cm (10 cm x 10 cm). The skin of the animals was examined one day before treatment, and regrown fur of all animals was again clipped. Animals with overt signs of skin injury or marked irritation which may have interfered with the interpretation of the results were not used in the test. On the day of treatment, 0.5 g of FAT 40810/A was placed on a surgical gauze patch (ca. 4 cm x 4 cm). This gauze patch was applied to the intact skin of the clipped area. The patch was covered with a semi-occlusive dressing. The dressing was wrapped around the abdomen and anchored with tape. The duration of treatment was 4 hours. Then the dressing was removed and the skin was flushed with lukewarm tap water to clean the application site so that any reactions (erythema) were clearly visible at that time.

OBSERVATIONS: Viability/Mortality: Daily from delivery of the animals to the termination of test. Clinical signs: Daily from delivery of the animals to the termination of test. Body weights: At start of acclimatization, on the day of application and at termination of observation.

IRRITATION SCORES: The skin reaction was assessed according to the numerical scoring system listed in the EEC Commission Directive 92/69/EEC, July 31, 1992 approximately 1, 24, 48 and 72 hours, as well as 7, 10 and 14 days after the removal of the dressing, gauze patch and test item. To allow further examination of the test site animal No. 63 was re-clipped on completion of the 48-hour examination.

Grading of Skin Reactions
ERYTHEMA AND ESCHAR FORMATION
No erythema.............................................0
Very slight erythema..................................1
Well-defined erythema................................2
Moderate to severe erythema......................3
Severe erythema (beet redness) or eschar formation (injuries in depth preventing erythema) reading................4

OEDEMA FORMATION
No oedema....................................................................................................................0
Very slight oedema (barely perceptible)...........................................................................1
Slight oedema (edges of area well-defined by definite raising).............................................2
Moderate oedema (edges raised approximately 1 mm)......................................................3
Severe oedema (raised more than 1 mm and extending beyond the area of exposure)..........4

Irritation parameter:

erythema score

Basis:

mean

Remarks:

3 animals

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

mean

Remarks:

3 animals

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

Neither oedema nor erythema were observed at any point during the study and thus there was no corrosion/irritation response observed.

Other effects:

COLORATION: The test item caused red staining of the treated skin. This effect was reversible and was no longer evident 10 days after treatment.
MORTALITY/CLINICAL SIGNS: No mortality and no clinical signs observed.

Interpretation of results:

GHS criteria not met

Conclusions:

FAT40810/A is non-irritating to skin.

Executive summary:

The primary skin irritation potential of FAT 40810/A was investigated by topical semi­ occlusive application of 0.5 g to the intact left flank of each of three young adult New Zealand White rabbits. This study was conducted in accordance with OECD test guideline 404 in a GLP certified laboratory. The duration of treatment was four hours. The scoring of skin reactions was performed 1, 24, 48 and 72 hours, as well as 7 and 10 days after removal of the dressing. The test item did not elicit any skin reactions at the application site of any animal at any of the observation times (all scores 0). The individual mean score for erythema/eschar and oedema for each of the three animals was therefore 0.

 

The application of FAT 40'810/A to the skin resulted in no signs of irritation. The test item caused red staining of the treated skin. This effect was reversible and was no longer evident 10 days after treatment, the end of the observation period for all animals. No corrosive effects were noted on the treated skin of any animal at any of the measuring intervals.

Thus, the test item did not induce significant or irreversible damage to the skin. Based upon the referred classification criteria (Commission Directive 2001/59/EC of August 06, 2001), FAT 40'810/A is considered to be "not irritating" to rabbit skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experiment start date - 18 March 2003, Experiment end date - 14 April 2003; Study completion date - 20 May 2003.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identity: FAT 40810/A
Batch: WP 6/02
Purity: approx. 75 %
Appearance: Solid, dark brownish powder
Expiration date: 12 December 2010
Storage: At room temperature at about 20 °C

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

Test system: New Zealand White Rabbit, SPF
Rationale: Recognized by the international guidelines as the recommended test system.
Source: Elevage Scientifique des Dombes, F-01400 Chatillon sur Chalaronne, France.
Number of animals per test: 3 (Animals of both sexes were used)
Age at treatment: 13 weeks (male), 14 weeks (females)
Identification By unique cage number and corresponding ear number.
Acclimatization: Under laboratory conditions after health examination. Only animals without any visual signs of illness were used for the study.
Allocation: Male No. 61, Female Nos. 62 and 63

HUSBANDRY
Room no.: 106/ RCC Ltd, Füllinsdorf
Conditions: Standard Laboratory Conditions, Air-conditioned with target ranges for room temperature 17 - 23 °C, relative humidity 30-70 % and approximately 10-15 air changes per hour. Room temperature and humidity were monitored continuously and values outside of these ranges may have occasionally occurred, usually following room cleaning. These transient variations are considered not to have any influence on the study and, therefore, these data are not reported but are retained at RCC. The animals were provided with an automatically controlled light cycle of 12 hours light and 12 hours dark. Music was played during the daytime light period.
Accommodation: Individually in stainless steel cages equipped with feed hoppers and drinking water bowls. Wood blocks (RCC Ltd, Füllinsdorf) and haysticks 4646 (batch no. 0206, Provimi Kliba AG) were provided for gnawing.
Diet: Pelleted standard Provimi Kliba 3418 rabbit maintenance diet ad libitum (batch no. 64/02) provided by Provimi Kliba AG, CH-4303 Kaiseraugst. Results of analysis for contaminants are archived at RCC Ltd, Itingen.
Water: Community tap water from Füllinsdorf, ad libitum. Results of bacteriological, chemical and contaminant analyses are archived at RCC Ltd, Itingen.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

100 mg

Duration of treatment / exposure:

Single application, eye not rinsed

Observation period (in vivo):

21 days

Number of animals or in vitro replicates:

3

Details on study design:

TEST ITEM PREPARATION: 0.1 g (per animal) of FAT 40810/A was weighed and applied undiluted as it was delivered by the sponsor. The pH of the test item was measured for a previous study (RCC Study number 847220, skin irritation with FAT 40810/A in rabbits) and was found to be 5.19. According to Directive 92/69 EEC, B.5. and OECD Guidelines 405, a test item needs not to be tested if the pH-value is less than 2 or greater than 11.5, owing to its predictable corrosive properties.

TREATMENT: The eyes of the animals were examined one day prior to test item administration. Animals with overt signs of ocular injury or irritation which may have interfered with the interpretation of the results were not used in the test. On the day of treatment, 0.1 g of FAT 40810/A was placed in the conjunctival sac of the left eye of each animal after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second to prevent loss of test item. The right eye remained untreated and served as the reference control. The treated eyes were not rinsed after instillation.

OBSERVATIONS:
Viability/Mortality Daily from acclimatization of the animals to the termination of test.
Clinical signs: Daily from acclimatization of the animals to the termination of test.
Body weights: At start of acclimatization, on the day of application and at termination of observation.
IRRITATION SCORES: The ocular reaction was assessed according to the numerical scoring system listed in the EEC Commission Directive 92/69/EEC, July 31, 1992 at approximately 1, 24, 48 and 72 hours, as well as 7, 10, 14, 17 and 21 days after application. When present, corrosion and/or staining of sclera and cornea by the test item were recorded and reported. Eye examinations were made with a Varta Cliptrix diagnostic-lamp (Roth AG, CH-4153 Reinach/Switzerland).

SCORING SYSTEM: Scale for scoring Ocular Lesions.
1.Cornea
(A) Opacity-degree of density (area most dense taken for reading)
- No opacity: 0
- Scattered or diffuse area, details of iris clearly visible: 1
- Easily discernible translucent areas, details of iris slightly obscured: 2
- Opalescent areas, no details of iris visible, size of pupil barely discernible: 3
- Opaque, iris invisible: 4

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

2.Iris
(A) Values
- Normal: 0
- Folds above normal, congestion, swelling, circumcorneal injection (any or all of these or combination of any thereof) iris still reacting to light (sluggish reaction is positive): 1
- No reaction to light, haemorrhage, gross destruction (any or all of these): 2
Ax5 Total maximum=10

3.Conjunctivae
(A) Redness (refers to palpebral and bulbar conjunctivae excluding cornea and iris)
- Vessels normal: 0
- Vessels definitely injected above normal: 1
- More diffuse, deeper crimson red, individual vessels not easily discernible: 2
- Diffuse beefy red: 3

(B) Chemosis
- No swelling: 0
- Any swelling above normal (includes nictitating membrane): 1
- Obvious swelling with partial eversion of lids: 2
- Swelling with lids about half closed: 3
- Swelling with lids about half closed to completely closed: 4

(C) Discharge'
- No discharge: 0
- Any amount different from normal (does not include small amounts observed" in inner canthus of normal animals): 1
- Discharge with moistening of the lids and hairs just adjacent to lids: 2
- Discharge with moistening of the lids and hairs, and considerable area around the eye: 3

TOOL USED TO ASSESS SCORE: Slit lamp

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

3 animals

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

mean

Remarks:

3 animals

Time point:

24/48/72 h

Score:

0

Max. score:

2

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

24/48/72 h

Score:

0.44

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

Reversibility of any observed effect: Changes fully reversible within 1 respectively 2 days

Other effects:

COLORATION: Light red staining of the eyes was observed in all animals from the 1-hour up to 10-day examination. It was still evident in 2 animals up to the observation time (21 days after treatment).

Comments: All irritant effects (slight reddening of the conjunctivae and the sclerea, slight swelling) were fully reversible within 1 to 2 days. However, due to the irreversible coloration after 21 days, the substance needs to be classified as irritant and labelled with R41.

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

Due to discoloration of the eyes by FAT 40810/A the substance is considered causing irreversible effects to eyes (CLP Category 1, H318)

Executive summary:

The primary eye irritation potential of FAT 40810/A was investigated according to OECD test guideline no. 405. The test item was applied by instillation of 0.1 g into the left eye of each of three young adult New Zealand White rabbits. Scoring of irritation effects was performed approximately 1, 24, 48 and 72 hours, as well as 7, 10, 14, 17 and 21 days after test item application. The mean score was calculated across 3 scoring times (24, 48 and 72 hours after instillation) for each animal for corneal opacity, iris, redness and chemosis of the conjunctivae, separately. The individual mean scores for corneal opacity and iris were 0.00 for all three animals. The individual mean scores for the conjunctivae were 0.33, 0.67 and 0.33 for reddening and 0.0, 0.0 and 0.0 for chemosis, respectively. The instillation of FAT 40810/A into the eye resulted in mild, early-onset and transient ocular changes, such as reddening of the conjunctivae and sclerae and chemosis. These effects were reversible and were no longer evident 72 hours after treatment. No abnormal findings were observed in the cornea or iris of any animal at any of the examinations. No corrosion was observed at any of the measuring intervals. Red staining of the treated eyes by the test item was observed in all animals throughout 10 days after treatment and persisted in two animals up to 21 days after treatment, the end of the observation time for all animals. No other clinical signs of substance related effects were observed. Due to discoloration of the eyes by FAT 40810/A the substance is considered causing irreversible effects to eyes (CLP Category 1, H318).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

FAT 40810/A was investigated for skin and eye irritation/corrosion in vivo using rabbits (OECD 404 and 405). Based upon the referred classification criteria (Commission Directive 2001/59/EC of August 06, 2001), FAT 40810/A is considered to be "not irritating" to rabbit skin, as no signs of skin irritation were observed throughout the entire study. Based upon the referred classification criteria (Commission Directive 2001/59/EC of August 06, 2001), with the exception of the red staining, which was still evident at termination, FAT 40810/A is considered to be "not irritating" to the rabbit eye. However, due to the irreversible coloration after 21 days, the substance needs to be classified as irritant (Xi, R41 according to DSD) respectively as causing irreversible damage to eyes according to CLP (Category 1, H318). Data for respiratory irritation are not available.

Effects on eye irritation: irritating

Justification for classification or non-classification

Based on two in vivo studies using rabbits, investigating skin and eye irritation potential, FAT40810/A is not classifiable for skin irritation corrosion according to CLP (Regulation EC No 1272/2008) or DSD (Directive 67/548/EEC). Although no significant eye irritation was observed, the substance caused staining of the eyes which did not fully revers during the observation period and thus FAT40810/A is classified as causing irreversible effects to eyes according to CLP (i.e. Category 1, H318) and accordingly as irritant (Xi), R41 according to DSD. Data for respiratory irritation is not available.