Sodium p-[(4,6-dichloro-1,3,5-triazin-2-yl)amino]benzenesulphonate

Endpoint summary

Description of key information

The test substance was tested in a validated in vitro skin model (EPISKIN) and did not show irritant effects.
The test substance was tested in a validated in vitro eye corrosion model (BCOP) and did not show corrosive effects. However, when tested in vivo in the eye of one rabbit, the material caused irreversible effects and is therefore considered as corrosive to the eye.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: According to: OECD Guideline for the Testing of Chemicals 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method (Original Guideline adopted July 22, 2010).

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for the Testing of Chemicals 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method (Original Guideline adopted July 22, 2010).

GLP compliance:

yes (incl. QA statement)

Amount / concentration applied:

Approximately 10 mg of the neat test item were applied to each EPISKIN (Skinethic) tissues. Additionally, the tissues were wetted with 15 µL of deionised water.

Duration of treatment / exposure:

The skin equivalents were exposed to the test item for 15 minutes. After completion of the treatment the test item was rinsed off and the skin equivalents were incubated for further 42 hours.

Details on study design:

Approximately 10 mg of the neat test item and 10 µL of the negative control (deionised water) or the positive control (5% SLS) were applied to each three EPISKIN (Skinethic) tissues. Additionally, the test item treated tissues were wetted with 15 µL deionised water. The test item as well as the controls were rinsed off after 15 minutes treatment. After further 42 hours incubation the tissues were treated with the MTT solution for 3 hours following approximately 72 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at a wavelength of 570 nm.

Remarks on result:

other: See comments below

Other effects / acceptance of results:

After treatment with the test item Sodium p-[(4,6-dichloro-1,3,5-triazin-2-yl) amino]benzenesulphonate the relative absorbance value did not decrease (105.2%; threshold for irritancy: ≤ 50%). Therefore, the test item is not considered to possess an irritant potential.

Results after treatment with Sodium p-[(4,6-dichloro-1,3,5-triazin-2-yl) amino]benzenesulphonate and controls

 

Dose group	Treatment Interval	Absorbance 570 nm Tissue 1*	Absorbance 570 nm Tissue 2*	Absorbance 570 nm Tissue 3*	Mean Absorbance of 3 Tissues	Relative Absorbance [%] Tissue 1, 2 + 3**	Standard Deviation [%]	Rel. Absorbance [% of Negative Control]***
Negative Control	15 min	0.843	0.766	0.702	0.770	109.4 99.5 91.1	9.2	100.0
Positive Control	15 min	0.127	0.382	0.266	0.259	16.5 49.3 34.6	16.6	33.6
Test Item	15 min	0.826	0.769	0.836	0.810	107.3 99.8 108.5	4.7	105.2

*       Mean of two replicate wells after blank correction

**      relative absorbance per tissue [rounded values]:
***    relative absorbance per treatment group [rounded values]:

Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

The relative absorbance value of the test item, corresponding to the cell viability, did not decrease (105.2%; threshold for irritancy:≤50%), consequently the test item was non irritant to skin.

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: OECD GHS

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item Sodium p-[(4,6-dichloro-1,3,5-triazin-2-yl) amino]benzenesulphonate is non irritant to skin.

Executive summary:

This in vitro study was performed to assess the irritation potential of Sodium p-[(4,6-dichloro-1,3,5-triazin-2-yl) amino]benzenesulphonate by means of the Human Skin Model Test.

Three tissues of the human skin model EpiSkin™ were treated with the test item, the negative or the positive control for 15 minutes.

Approximately 10 mg of the test item were applied to each tissue, wetted with 15 µL of deionised water, and spread to match the tissue size.

The test item and the positive and negative controls were washed off the skin tissues after 15 minutes treatment. After further incubation for about 41.5 hours the tissues were treated with the MTT solution for 3 hours following about 72 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm.

10 µL of either the negative control (deionised water) or the positive control (5% Sodium lauryl sulfate) were applied to each tissue.

After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD greater or equal than 0.6 till less or equal than 1.5 for the15 minutes treatment interval thus showing the quality of the tissues.

Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 33.6% thus ensuring the validity of the test system.

The standard deviations between the % variabilities of the test item, the positive and negative controls were below 17% (threshold of the "OECD TG 439 Guideline for the Testing of Chemicals 439: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method”: 18%),thus ensuring the validity of the study.

After treatment with the test item Sodium p-[(4,6-dichloro-1,3,5-triazin-2-yl) amino]benzenesulphonate the relative absorbance value did not decrease (105.2%; threshold for irritancy:≤50%). Therefore, the test item is not considered to possess an irritant potential.

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:

Between 26 June 2012 and 17 July 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

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)

Remarks:

The frequency of these process based inspections was changed from a monthly to a three monthly cycle. This deviation is considered not to affect the purpose or integrity of the study

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

One New Zealand White (Hsdlf:NZW) strain rabbit was supplied by Harlan Laboratories UK Ltd., Leicestershire, UK. At the start of the study the animal weighed 2.60 kg and was twelve to twenty weeks old. After an acclimatisation period of at least five days the animal was given a number which was written with a black indelible marker-pen on the inner surface of the ear and on the cage label.The animal was housed in a suspended cage. Free access to mains drinking water and food (2930C Teklad Global Rabbit diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study. The diet and drinking water were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.The temperature and relative humidity were set to achieve limits of 17 to 23°C and 30 to 70% respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness.The animal was provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

A volume of 0.1 ml of the test item was placed into the conjunctival sac of the right eye

Duration of treatment / exposure:

1 hour

Observation period (in vivo):

21 days

Number of animals or in vitro replicates:

1

Details on study design:

Immediately before the start of the test, both eyes of the provisionally selected test rabbit were examined for evidence of ocular irritation or defect with the aid of a light source from a standard ophthalmoscope. An animal free of ocular damage was used.A volume of 0.1 ml of the test item was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were held together for about one second immediately after treatment, to prevent loss of the test item, and then released. The left eye remained untreated and was used for control purposes. Immediately after administration of the test item, an assessment of the initial pain reaction was made according to the six point scale shown in Appendix 1.After consideration of the ocular responses produced in this animal, no additional animals were treated. Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment, according to the numerical evaluation given in Appendix 2, (from Draize J H (1977) "Dermal and Eye Toxicity Tests" In: Principles and Procedures for Evaluating the Toxicity of Household Substances, National Academy of Sciences, Washington DC p.48 to 49).Any other ocular effects were also noted. Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope.Any clinical signs of toxicity, if present, were also recorded.Additional observations were made on Days 7, 14 and 21 to assess the reversibility of the ocular effects.In order to confirm the area of corneal opacity, the treated eye was examined under ultra violet light following treatment with Sodium Fluorescein BP (Minims: Bausch & Lomb, Surrey, UK) at the 7–Day observation. The cornea, conjunctivae and iris were also examined for lesions.The animal’s bodyweight was recorded on Day 0 (the day of dosing) and at the end of the observation period.

Irritation parameter:

conjunctivae score

Basis:

animal: 72171 male

Time point:

24 h

Score:

2

Max. score:

3

Reversibility:

not reversible

Irritation parameter:

conjunctivae score

Basis:

animal: 72171 male

Time point:

48 h

Score:

2

Max. score:

3

Reversibility:

not reversible

Irritation parameter:

conjunctivae score

Basis:

animal: 72171 male

Time point:

72 h

Score:

2

Max. score:

3

Reversibility:

not reversible

Irritation parameter:

chemosis score

Basis:

animal: 72171 male

Time point:

24 h

Score:

2

Max. score:

4

Reversibility:

not reversible

Irritation parameter:

chemosis score

Basis:

animal: 72171 male

Time point:

48 h

Score:

1

Max. score:

4

Reversibility:

not reversible

Irritation parameter:

chemosis score

Basis:

animal: 72171 male

Time point:

72 h

Score:

1

Max. score:

4

Reversibility:

not reversible

Irritation parameter:

cornea opacity score

Basis:

animal: 72171 male

Time point:

24 h

Score:

2

Max. score:

4

Reversibility:

not reversible

Irritation parameter:

cornea opacity score

Basis:

animal: 72171 male

Time point:

48 h

Score:

2

Max. score:

4

Reversibility:

not reversible

Irritation parameter:

cornea opacity score

Basis:

animal: 72171 male

Time point:

72 h

Score:

1

Max. score:

4

Reversibility:

not reversible

Irritation parameter:

iris score

Basis:

animal: 72171 male

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

not reversible

Irritation parameter:

other: Discharge

Basis:

animal: 72171 male

Time point:

other: Highest score at 1, 24, 48 and 72 hours, 7, 14 and 21 days

Score:

2

Max. score:

3

Reversibility:

not fully reversible within: 21 days

Irritant / corrosive response data:

Individual and total scores for ocular irritation are given in Table 1.Scattered or diffuse corneal opacity was noted in the treated eye one hour after treatment with translucent corneal opacity at the 24 and 48-Hour observations and scattered or diffuse corneal opacity at the 72-Hour and all subsequent observations.Iridial inflammation was noted in the treated eye one hour after treatment and at all subsequent observations.Moderate conjunctival irritation was noted in the treated eye one hour after treatment and at the 24 and 48 Hour observations with minimal conjunctival irritation noted at the 72 Hour, 7 and 14 Day observations and moderate conjunctival irritation at the 21 Day observation.The persistence of reactions in the treated eye at the 21 day observation was considered to be indicative of irreversible ocular damage.

Other effects:

BodyweightIndividual bodyweights and bodyweight change are given in Table 2.The animal showed expected gain in bodyweight during the study.

Interpretation of Results

Data was summarised in tabular form, showing the irritation scores for the designated observation time, a description of the degree and nature of irritation, the presence of serious lesions and non‑ocular effects. 

If evidence of irreversible ocular damage is noted, the test item will be classified as corrosive to the eye.

The results were interpreted according to the Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008, relating to the Classification, Labelling and Packaging of Dangerous Substances.

Table 1              Eye Irritation Scores

Individual Scores for Eye Irritation

Rabbit Number and Sex	IPR	Evaluation interval*	Corneal Opacity	Area of CorneaI Opacity	Iris	Conjunctivae
						Redness	Chemosis	Discharge
72171Male	2	1 Hour	1	2	1	2	2	2
		24 Hour	2	3	1	2	2	2
		48 Hour	2	3	1	2	1	1
		72 Hour	1	2	1	2	1	0
		7 Days	1	3Ä	1	2	1	0
		14 Days	1	1	1	1	1	0
		21 Days	1	2	1	2	1	1

IPR=  Initial pain reaction

*=      Examinations were performed at the specified times after instillation of the test item

Ä=     Examination under ultra‑violet light following treatment with Sodium Fluorescein BP to help confirm area of corneal opacity

Table 2              Individual Bodyweights and Bodyweight Change

Rabbit Number and Sex	Individual Bodyweight (kg)		Bodyweight Change (kg)
	Day 0	Day 21
72171Male	2.60	3.02	0.42

Interpretation of results:

Category 1 (irreversible effects on the eye)

Remarks:

Migrated informationCriteria used for interpretation of results: EU

Conclusions:

The test item was classified as Irreversible effects on the eye (Category 1) (based on one rabbit only). It is reasonable to assume that the Signal Word “Danger” and the Hazard Statement “H318: Causes serious eye damage” are therefore required.

Executive summary:

The primary eye irritation potential of Sodiump-[(4,6-dichloro- 1,3,5-triazin-2-yl)amino]benzenesulphonate) was investigated according to a method compatible with OECD test guideline no. 405 and Method B5. The test item was applied by instillation of 0.1 ml into the right eye of one young adult New Zealand White rabbit. Scoring of irritation effects was performed approximately 1, 24, 48 and 72 hours, 7, 14 and 21 days after test item instillation. 

The instillation of Sodiump-[(4,6-dichloro- 1,3,5-triazin-2-yl)amino]benzenesulphonate) into the eye resulted in moderate to severe, early‑onset ocular changes, such as scattered or diffuse corneal opacity, iridial inflammation, reddening of the conjunctivae, ocular discharge and chemosis. These effects were irreversible and still evident 21 days after treatment, the end of the observation period. No clinical signs were observed.

Thus, the test item induced significant damage to the rabbit eye and induced irreversible damage to the rabbit eye.

The test item was classified asIrreversible effects on the eye (Category 1)(based on one rabbit only). It is reasonable to assume that the Signal Word “Danger” and the Hazard Statement “H318: Causes serious eye damage” are therefore required.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The test substance was tested in a validated in vitro skin model (EPISKIN) and did not show irritant effects.

The test substance was tested in a validated in vitro eye corrosion model (BCOP) and did not show corrosive effects. However, when tested in vivo in the rabbit eye, the material caused irreversible effects and is therefore considered as corrosive to the eye. This consideration is also supported by the chemical mechanism, which is the basis for the use of this substance as a leather tanning agent.

Justification for selection of skin irritation / corrosion endpoint:
only study

Justification for selection of eye irritation endpoint:
recent in vivo study according to standard method

Effects on eye irritation: corrosive

Justification for classification or non-classification

The test substance was tested in a validated in vitro skin model (EPISKIN) and did not show irritant effects. it is therefore not classified for skin irritation.

The test substance was tested in vivo in the rabbit eye. The material caused irreversible effects and is therefore considered as corrosive to the eye.