Tetrasodium 4,4'-bis[[4-morpholino-6-(p-sulphonatoanilino)-1,3,5-triazin-2-yl]amino]stilbene-2,2'-disulphonate

Endpoint summary

Description of key information

In-vivo Skin Irritation:

The primary skin irritation potential of test item was investigated according to OECD test guideline no. 404. The test item was applied by topical semi-occlusive application of 0.5 g to the intact left flank of each of three young adult New Zealand White rabbits. The duration of treatment was four hours. The scoring of skin reactions was performed 1,24, 48 and 72 hours after removal of the dressing. The mean score was calculated across 3 scoring times (24, 48 and 72 hours after patch removal) for each animal for erythema/eschar grades and for oedema grades, separately. The mean erythema/eschar score of the three animals was 0.33 for all animals and the mean oedema score was 0.00 for all animals. The application of test item to the skin resulted in very slight to well-defined erythema. This effect was reversible and was no longer evident 48 hours after treatment. The test item caused no staining of the treated skin. No corrosive effects were noted on the treated skin of any animal at any of the measuring intervals and no clinical signs were observed. Based on the study results, FAT 66042/A is not considered as irritating to rabbit skin.

In-vivo Eye Irritation:

The primary eye irritation potential of test item 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 after test item instillation. 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 0.67 for reddening for all animals and 0.00 for chemosis for all animals. The instillation of test item into the eye resulted in mild to moderate, early-onset and transient ocular changes, such as reddening of the conjunctivae and sclerae, discharge and chemosis. These effects were reversible and were no longer evident 24, 48 or 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. Yellow test item remnants were evident in the eye or conjunctival sac of the male animal 1 hour after treatment. No staining of the treated eyes by the test item was observed and no clinical signs were observed.

Based on the study results, FAT 66042/A is considered as not irritating to rabbit eye.

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:

23 April 2008 to 2 May 2008.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

adopted Apri124, 2002

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: 0582900
- Expiration date of the lot/batch: 25-FEB-2013

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature (range of 20 ± 5 °C), light protected.
- Stability under storage conditions: Stable under storage conditions.

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands BV Kreuzelweg 53 NL-596l NM Horst / The Netherlands Postbus 6174 NL-5960 AD Horst / The Netherlands
- Age at study initiation: 15 weeks (male) and 14 weeks (females).
- Allocation: Male No. 61, Female Nos. 62, 63.
- Diet: pelleted standard Provimi Kliba 3418 rabbit maintenance (batch no. 03/08) provided by Provimi Kliba AG, CH-4303 Kaiseraugst ad libitum
- Water: community tap water from Füllinsdorf ad libitum
- Acclimation period: under laboratory conditions after health examination. Only animals without any visual signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature: continuously monitored environment with ranges for room temperature 17-23 °C.
- Humidity: between 30-70 % (values above 70 % during cleaning process possible).
- Air changes: air-conditioned with 10-15 air changes per hour.
- Photoperiod: automatically controlled light cycle of 12 hours light and 12 hours dark.
- Other: music during the daytime light period.

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

water

Controls:

not specified

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.5 g (per animal) of test item was weighed as delivered by the Sponsor and then moistened with approximately 0.5 ml of purified water before application.
- Concentration: a formulation of a 1 % (w/w) solution in purified water was prepared. The pH was found to be 7-8.

Duration of treatment / exposure:

4 hours

Observation period:

The skin reaction was assessed at approximately 1, 24, 48 and 72 hours, after exposure (removal of the dressing, gauze patch and test item).

Number of animals:

Three (one male and two females)

Details on study design:

TEST SITE
- Area of exposure: four days before treatment, the left flank was clipped with an electric clipper, exposing an area of approximately 100 cm2 (10 cm x 10 cm). The skin of the animals was examined one day before treatment, and regrown fur of all animals was clipped again. 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.
- Application: on the day of treatment, 0.5 g of test item was placed on a surgical gauze patch (ca. 2.5 cm x 2.5 cm). This gauze patch was applied to the intact skin of the clipped area. The patch was covered with a semi-occlusive dressing.
- Type of wrap if used: the dressing was wrapped around the abdomen and anchored with tape.

REMOVAL OF TEST SUBSTANCE
- Washing: the dressing was removed after 4 hours and the skin was flushed with lukewarm tap water to clean the application site.
Necropsy
No necropsy was performed on the animals sacrificed at termination of observation.
All rabbits were sacrificed by an intravenous injection of Pentobarbitone into the ear vein at a dose of at least 1 ml/kg body weight and discarded.

SCORING SYSTEM
The skin reaction was assessed according to the numerical scoring system listed in the Commission Directive 2004173/EC, April 29, 2004.

Irritation parameter:

erythema score

Basis:

mean

Remarks:

3 animal

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 h

Irritation parameter:

edema score

Basis:

mean

Remarks:

3 animal

Time point:

24/48/72 h

Score:

0 - 2.3

Max. score:

4

Irritant / corrosive response data:

A slight to well-defined erythema was observed in all animals 1-hour after test item exposure and persisted as slight up to the 24-hour reading. No abnormal findings were observed on the treated skin of any animal 48 hours after treatment.
Coloration: no staining produced by the test item of the treated skin was observed.
Corrosion: neither alterations of the treated skin were observed nor were corrosive effects evident on the skin.

Other effects:

No clinical signs of systemic toxicity were observed in the animals during the study and no mortality occurred.
The body weights of all rabbits were considered to be within the normal range of variability.

Individual animal skin irritation scores

Animal N.	Sex	Parameter	1 h	24 h	48 h	72 h	Mean*
61	M	Erythema	1	1	0	0	0.33
		Oedema	0	0	0	0	0.00
62	F	Erythema	2	1	0	0	0.33
		Oedema	0	0	0	0	0.00
63	F	Erythema	2	1	0	0	0.33
		Oedema	0	0	0	0	0.00

* Time point: 24, 48 and 72 hrs after patch removal.

Interpretation of results:

GHS criteria not met

Conclusions:

FAT 66042/A is not considered as irritating to rabbit skin.

Executive summary:

The primary skin irritation potential of test item was investigated according to OECD test guideline no. 404. The test item was applied by topical semi-occlusive application of 0.5 g to the intact left flank of each of three young adult New Zealand White rabbits. The duration of treatment was four hours. The scoring of skin reactions was performed 1,24, 48 and 72 hours after removal of the dressing. The mean score was calculated across 3 scoring times (24, 48 and 72 hours after patch removal) for each animal for erythema/eschar grades and for oedema grades, separately.

The mean erythema/eschar score of the three animals was 0.33 for all animals and the mean oedema score was 0.00 for all animals. The application of test item to the skin resulted in very slight to well-defined erythema. This effect was reversible and was no longer evident 48 hours after treatment. The test item caused no staining of the treated skin. No corrosive effects were noted on the treated skin of any animal at any of the measuring intervals and no clinical signs were observed. Based on the study results, FAT 66042/A is considered as 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:

7 May 7 2008 to 16 May 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

adopted April 24, 2002

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: 0582900
- Expiration date of the lot/batch: 25-FEB-2013
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature (range of 20 ± 5 °C), light protected.
- Stability under storage conditions: Stable under storage conditions.

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands BV.
- Age at study initiation: 16 weeks (male) and 14-15 weeks (females).
- Weight at study initiation: 2515 g - 3155 g.
- Allocation: Male No. 64 and Female Nos. 65, 66.
- Accommodation: individually in stainless steel cages equipped with feed hoppers and drinking water bowls. Wood blocks (RCC Ltd, Füllinsdorf) and haysticks 4642 (batch no. 36/07, Provimi Kliba AG) were provided for gnawing.
- Diet: ad libitum, pelleted standard Provimi Kliba 3418 rabbit maintenance diet (batch no. 03/08) provided by Provimi Kliba AG, CH-4303 Kaiseraugst.
- Water: ad libitum, community tap water from Füllinsdorf.
- Acclimation period: under laboratory conditions after health examination. Only animals without any visual signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature: continuously monitored environment with ranges for room temperature 17-23 °C.
- Humidity: between 30-70 % (values above 70 % during cleaning process possible).
- Air changes: air-conditioned with 10-15 air changes per hour.
- Photoperiod: automatically controlled light cycle of 12 hours light and 12 hours dark.
- Other: music during the daytime light period.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.1 g (per animal) of test item was weighed and applied undiluted as it was delivered by the sponsor.
- pH solution: the pH of a 1 % (w/w) solution was measured and was found to be 7-8.

Duration of treatment / exposure:

FAT 66042/A TE 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.

Observation period (in vivo):

The eyes of each animal were examined approximately 1, 24, 48, 72 hours after administration.

Number of animals or in vitro replicates:

Three (one male and two females)

Details on study design:

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 test item 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.

Necropsy:
No necropsy was performed on the animals sacrificed at termination of observation. The animals were killed by intravenous injection of Pentobarbitone into the ear vein at a dose of at least 1 ml/kg body weight (162 mg sodium pentobarbitone/kg body weight) and discarded.

SCORING SYSTEM
The eye reactions were assessed according to the numerical scoring system listed in the Commission Directive 2004173/EC, April 29, 2004 (see page 24). Scleral reddening and ocular discharge were also assessed.

TOOL USED TO ASSESS SCORE: Varta Cliptrix diagnostic-lamp (Roth AG, CH-4153 Reinachi Switzerland); the eyes of each animal were examined approximately 1, 24, 48, 72 hours after administration.

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

3 animal

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 animal

Time point:

24/48/72 h

Score:

0

Max. score:

2

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

Redness

Basis:

mean

Remarks:

3 animal

Time point:

24/48/72 h

Score:

0.67

Max. score:

3

Reversibility:

fully reversible within: 72 h

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

3 animal

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

No abnormal findings were observed in the cornea or iris of any animal at any of the measurement intervals. Slight to moderate reddening of the conjunctivae was noted in all animals 1 hour after treatment and persisted as slight up to the 48-hour reading. A slight to moderate reddening of the sclerae was present in all animals 1 hour after treatment and persisted as slight up to the 24-hour observation. A Slight swelling (chemosis) of the conjunctivae was observed 1 hour after instillation. Slight to moderate ocular discharge was recorded in all three animals 1 hour after treatment. No abnormal findings were observed in the treated eye of any animal 24, 48 or 72 hours after treatment, the end of the observation period for all animals.

Coloration: no staining produced by the test item of the treated eye was observed.
Test Item Remnants: yellow test item remnants were evident in the eye or conjunctival sac of the male animal 1 hour after treatment.
Corrosion: no corrosion of the cornea was observed at any of the reading times.

Other effects:

No clinical signs of systemic toxicity were observed in the animals during the study and no mortality occurred.

Eye irritation scores

Animal N.	Sex	Parameter	1 hr	24 hrs	48 hrs	72 hrs	Mean*
64	M	Corneal Opacity	0	0	0	0	0.00
		Iritis	0	0	0	0	0.00
		Conjunctival redness	2	1	1	0	0.67
		Conjunctival chemosis	1	0	0	0	0.00
65	F	Corneal Opacity	0	0	0	0	0.00
		Iritis	0	0	0	0	0.00
		Conjunctival redness	1	1	1	0	0.67
		Conjunctival chemosis	1	0	0	0	0.00
66	F	Corneal Opacity	0	0	0	0	0.00
		Iritis	0	0	0	0	0.00
		Conjunctival redness	1	1	1	0	0.67
		Conjunctival chemosis	1	0	0	0	0.00

* mean calculated across 24, 48 and 72 hours after instillation

Interpretation of results:

GHS criteria not met

Conclusions:

FAT 66042/A is considered as not irritating to rabbit eye.

Executive summary:

The primary eye irritation potential of test item 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 after test item instillation. 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 0.67 for reddening for all animals and 0.00 for chemosis for all animals. The instillation of test item into the eye resulted in mild to moderate, early-onset and transient ocular changes, such as reddening of the conjunctivae and sclerae, discharge and chemosis. These effects were reversible and were no longer evident 24, 48 or 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. Yellow test item remnants were evident in the eye or conjunctival sac of the male animal 1 hour after treatment. No staining of the treated eyes by the test item was observed and no clinical signs were observed. Based on the study results, FAT 66042/A is considered as not irritating to rabbit eye.

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

Based on the findings in the skin and eye irritation study, the test substance does not considered to be classified according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 to be irritating to the skin as well as eyes.