Benzenesulfonic acid, 2,2'-(1,2-ethenediyl)bis[5-[[4-(diethylamino)-6-[(4-sulfophenyl)amino]-1,3,5-triazin-2-yl]amino]-, sodium salt (1:4)

Endpoint summary

Description of key information

Not skin irritating.

Not eye irritating.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

other: read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Test procedures cannot be subsumed under testing guideline, nevertheless are well documented and scientifically acceptable. Justification for Read Across is reported in the endpoint summary and in the Category Justification Report attached to Section 13 of this dossier.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

GLP compliance:

not specified

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Kleintierfarm Madoerin AG, CH-4414 Fuellinsdorf.
- Age at study initiation: approx. 12 -14 weeks.
- Weight at study initiation: 2220 g - 2770 g.
- Housing: individually.
- Diet: ad libitum.
- Water: ad libitum.
- Acclimation period: at least 5 days.

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

Type of coverage:

occlusive

Preparation of test site:

shaved

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.5 g

Duration of treatment / exposure:

4 hours

Observation period:

1, 24, 48, 72 hours.

Number of animals:

3 males

Details on study design:

TEST SITE
- Area of exposure: 12 - 16 square cm
- Type of wrap if used: aluminium foil.

SCORING SYSTEM: OECD Scoring System.

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

ca. 0.33

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

4

Reversibility:

fully reversible

Irritant / corrosive response data:

Slighly erythema was robserved in one rabbit. The effect is reverseble within 72 hours.

Animal/sex	Reaction	1 hr	24 hrs	48 hrs	72 hrs	Mean 24, 48 and 72 hrs
1	Erythema	0	0	0	0	0.00
2	Erythema	2	1	0	0	0.33
3	Erythema	1	0	0	0	0.00
1	Oedema	0	0	0	0	0.00
2	Oedema	0	0	0	0	0.00
3	Oedema	0	0	0	0	0.00

Interpretation of results:

other: CLP criteria not met

Conclusions:

Non irritating.

Executive summary:

Method

The skin irritation potential was tested in New Zealand White rabbits. The procedure used are similar to those described in the OECD Guidelines 404.

Results

Slight erythema was observed in one rabbit. The effect is reversible within 72 hours.

Conclusion

Mean values from gradings at 24, 48 and 72 hours after patch removal were lower than 2.3 in all animals for both erythema/eschar and oedema reactions (in both intact and abraded skin).

In conclusion, the test item can be classified as NON irritating, according to the CLP (EC 1272/2008) Regulation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

other: read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Study period:

From April 14 to 18, 1988.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Test procedures cannot be subsumed under testing guideline, nevertheless are well documented and scientifically acceptable. Justification for Read Across is reported in the endpoint summary and in the Category Justification Report attached to Section 13 of this dossier.

Qualifier:

according to guideline

Guideline:

other: Bovine Eye and the Chicken Chorioallantoic Membrane (BECAM ASSAY).

GLP compliance:

yes

Details on test animals or tissues and environmental conditions:

Calf eyes were obtained from a local slaughterhouse (Viguurs, 's-Hertogenbosch). The eyes were excised by standard procedures within 15 minutes after killing the animal. They were transported to the laboratory and examined for injuries. This was done by holding the eyes submersed in saline; eyes with corneal damage or other abnormalities were discarded. The selected eyes were preserved at ambient temperature (approximately 20 °C) under saline not longer than 4 hours.
White eggs were obtained from a chicken breeding centre.
Date of arrival: April 5, 1988.
They were transported at a temperature between 15 and 20 °C.
After approximately 6 hours of rest, the incubation was started by placing the eggs in the incubator with the air chamber upwards.
The temperature was maintained at 37-38 °C and the relative humidity between 50 and 70 %. The eggs were turned once every hour.
They were candled at the seventh day of incubation; unfertilized eggs or eggs containing a dead embryo were removed.

Amount / concentration applied:

Calf eyes: 0.1 ml
eggs: 0.2 ml

Duration of treatment / exposure:

calf eyes: 30 sec
eggs: 20 sec

Observation period (in vivo):

calf eyes: 10 min
eggs: 30 sec, 1.5 and 4.5 min

Number of animals or in vitro replicates:

5 calf eyes

Irritation parameter:

other: all paramteres

Remarks on result:

other: See details on method and results

BE ASSAY

Treatment	Opacity	Detachment	Fluorescence	Total
Neg. control	0	0	0	0
Toluene	0	0	1	1
Acetone	2	0	1.5	3.5
Eye1	0	0	0	0
Eye2	0	0	0	0
Eye3	0	0	0	0
Eye4	0	0	0	0
Eye5	0	0	0	0
			Mean	0

CAM assay

		30 sec			2 min			5 min			Total
Time	Egg	I	H	C	I	H	C	I	H	C
09:37	1	+	-	-	+	-	-	+	+ (a)	-	3
09:40	2	+	-	-	+	-	-	+	-	-	2
09:43	3	+	-	-	+	-	-	+	+ (a)	-	3
09:46	4	+	-	-	+	-	-	+	+ (a)	-	3
										Mean:	2.75

^{a: Bleeding by 2 min}

The total scores are not consistent with the above mentioned scoring system (table 2). According to this scoring in table 2 the total score for egg 1 -4 would be: 4.5, 3.5, 4.5, 4.5, respectively, which would lead to a mean score of 4.25. The score of 4.25 would lead to moderate irritation.

REFERENCE SUBSTANCES

For toluene, a score of 1.0 was obtained in the BE assay whereas the average score in the CAM assay was 1.25.

Acetone treatment resulted scores of 3.5 in the BE assay and an average score of 2.5 in the CAM assay.

As the effects, produced by acetone and toluene, fell within the historical range and the untreated eyes and eggs showed no signs of irritation, the results were considered to be valid.

To evaluate the effects observed in both assays the documented in vivo effects of the two reference substances toluene and acetone were taken into consideration. Toluene produces slight transient conjunctival irritation with negligible to slight corneal epithelial damage without opacity in the rabbit eye, while no serious effects have been reported in case of accidental exposure of human eyes.

Acetone causes temporary edema and moderate corneal opacity with epithelial damage to exposed rabbit eyes while in man reversible corneal opacity has been observed.

Interpretation of results:

other: BE assay: not irritating (according to the CLP Regulation). CAM assay: inconclusive (according to the CLP Regulation).

Conclusions:

Today a validated methods and procedures protocol about the Bovine Eye assay is available. The differences between the present test and the validated protocol are not suspected of impairing the results. Therefore, it can be concluded that the substance is not irritating, based on the BE assay.
In the case of the CAM assay the protocol is not validated yet and the re-evaluation of the result obtained is not appicable. Therefore, the CAM assay can be considered as inconclusive.

Executive summary:

Method

The eye irritation potential was assessed according to the in vitro Bovine Eye and the Chicken Chorioallantoic Membrane (BECAM ASSAY) method.

Results

The test substance induced no effects in the bovine eye.

The effects on the CAM consisted of vascular injection almost instantly after application. In three eggs (no. 1, 3 and 4) haemorrhages were scored 2 minutes after application. On account of these results it is expected that eye contact with the test substance may lead to slight irritation. Acute corneal effects may be negligible.

Conclusion

Today a validated methods and procedures protocol about the Bovine Eye assay is available. The differences between the present test and the validated protocol are not suspected of impairing the results. Therefore, it can be concluded that the substance is not irritating, based on the BE assay.

In the case of the CAM assay the protocol is not validated yet and the re-evaluation of the result obtained is not appicable. Therefore, the CAM assay can be considered as inconclusive.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

No specific tests on the substance under registration was been performed; nevertheless the substance belongs to the Stilbene Fluorescent Whitening Agent category, group 1, in which the members share the common organic functional group diethylamino derivative, with different sulphonation degrees: tetrasulphonated (OB 1 -MSA) and hexasulphonated (OB 1 -DSA). The hexasulphonated is the best conservative representative in the group because of the structural similarity and the lower water solubility.

Read across within the same group is well justified in this case, also taking into account the impurities of the considered substances, since the identified organic impurities can have different substitution on the molecule, but the functional reactive groups are potentially the same, and molecules are of the same molecular size and polarity of the main component. As a consequence the systemic absorption and reactivity is practically the same than the main constituent and Read Across is justified.

The studies performed on OB 3a-MSA, the analogue dihydroxyethylamino derivative tetrasulphonated,, are reported here as supporting reference. The diethylamino substituent is less polar, less reactive and makes the molecule slightly less soluble compared to the dihydroxyethylamino derivative. Thus, the latter can be considered as a representative conservative analogous for the irritation endpoints.

Within the whole category, eleven over fourteen registered substances were tested and none of the existing tests arisen any concern for skin and eye irritation.

All substances of the category were modelled using the OECD Toolbox and the provisional results about skin and eye irritation were calculated for all members: no alerts were reported (see category Justification Report attached to the section 13) for inclusion for any substance.

Based on all those considerations, the available studies on the analogous substances are representative for the substance under registration; thus it can be considered not irritant for skin and eye.

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008) substances that have the potential to induce reversible damage to the skin following the application of a test substance for up to 4 hours are classified in Category 2 (irritating to the skin) if:

(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 the skin irritation test, mean values from gradings at 24, 48 and 72 hours after patch removal were lower than 2.3 in all animals for both erythema/eschar and oedema reactions (in both intact and abraded skin).

According to the CLP Regulation (EC 1272/2008) substances that have the potential to induce reversible eye irritation are classified in Category 2 (irritating to eyes) 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.

Today a validated methods and procedures protocol about the Bovine Eye assay is available. The differences between the present test and the validated protocol are not suspected of impairing the results. Therefore, it can be concluded that the substance is not irritating, based on the BE assay. This result is confirmed by the supporting study in which the mean values from gradings at 24, 48 and 72 hours for both rinsed and unrinsed eyes were lower than 1 for corneal opacity, lower than 1 for iritis, lower than 2 for both conjunctival redness and oedema, in all animals.

The available experimental data are adequate for classification and labelling and the results show that the substance is not classified for eye and skin irritation, according to the CLP Regulation (EC 1272/2008).