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

Rat oral LD50 > 2000 mg/kg bw

Rat inhalation LC50 > 1895 mg/m3

Rat dermal LD50 > 2000 mg/kg bw

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From September 29 to Ocober 15, 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Version / remarks:

adopted 17 December 2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.1 (Acute Toxicity (Oral))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SPF breeding, VELAZ s.r.o., Únětice, Czech Republic
- Age at study initiation: 8 - 10 weeks at the time application
- Fasting period before study: twenty hours before oral administration the animals were not fed.
- Housing: animal room with monitoring conditions – 3 animals of one sex in one plastic breeding cage Velaz T4.
- Bedding: sterilized shavings of soft wood.
- Diet: ad libitum, ST - 1, VELAS a.s., Hrabanov 535, 289 22 Lysá nad Labem, CZ 801080-01.
- Water: ad libitum, drinking tap water ad libitum (quality corresponding to Regulation No. 252/2004 Czech Coll. of Law).
- Acclimation period: 20 days
- Health condition: certificate of good health condition from breeding farm; no signs of diseases were observed at clinical check-in, during the acclimatisation period and before the start of study.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 – 70 %
- Photoperiod: light period 12-hour light/12 hour dark.

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Aqua pro injection

Details on oral exposure:

Preparation and application of the test substance: immediately before application the test substance was weighed and mixed in vehicle. The test substance was administered dissolved in water pro injection.

Dosing: the test substance was administered to the stomach by tube. The single volume of administered solution was 1 ml/100 g of animal body weight.

Doses:

2000 mg/kg of body weight

No. of animals per sex per dose:

6 females (3 per group)

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: immediately before application the animals were weighed, 8th day and at the 15th day, before euthanasia of animals.
- Necropsy of survivors performed: yes.
- Clinical examination: after application the animals were observed individually. The first day twice (30 minutes and 3 hours after application), the second day twice (in the morning and in the afternoon) and daily thereafter for 14 days. Observations included changes in skin and fur, eyes, visible mucous membranes, behaviour of animals, somatomotor activity, reactions to stimuli, and presence of lacrimation, salivation and discharge from nostrils, function of respiratory, digestive and urogenital system.
- Pathological examination: all test animals survived to the end of study were sacrificed on the 15th day by injection of veterinary preparation T61 (1 ml iv.) and gross necropsy was carried out. Nutritious status, body surface, body foramina, thoracic, abdominal and cranial cavity were evaluated.

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No death occured.

Clinical signs:

other: No clinical signs of intoxication were observed during the study.

Gross pathology:

No pathologic changes were recorded.

Interpretation of results:

other: CLP criteria not met

Conclusions:

The LD50 of the test substance for female rats is higher than 2000 mg/kg of body weight.

Executive summary:

Method

The aim of the study was to investigate acute toxic effects of the test substance after a single oral administration to Wistar CRL rats.

The testing was performed according Method B.1 tris: Acute Oral Toxicity - Acute Toxic Class Method, Council Regulation (EC) No.440/2008, published in O.J. L 142, 2008. The test substance was administered dissolved in water pro injection as a single dose, given orally via gavage to three groups of three female Wistar CRL rats. 

The dosing was performed sequentially in two groups of three females:

group No. 1 - first step using the starting dose of 2000 mg/kg of body weight

group No. 2 - second step using the same dose 2000 mg/kg.

Results

The test substance administered at the dose of 2000 mg/kg caused no death of animals. No clinical signs of intoxication were detected during whole study. No pathologic macroscopic changes were diagnosed during pathological examination.

According to the study results the value of LD50 of the test substance for female rats is higher than 2000 mg/kg of body weight.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

acute toxicity: inhalation

Type of information:

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

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Particle size not specified. The analogous substance, part of the Stilbene Fluorescent Withening Agents, group 3, is the acid form of the disulphonated derivative dihydroxyethyl derivative. 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 403 (Acute Inhalation Toxicity)

GLP compliance:

no

Test type:

standard acute method

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Winkelmann, Borchen, Germany.
- Weight at study initiation: 180 - 200 g.

Route of administration:

inhalation: dust

Type of inhalation exposure:

not specified

Vehicle:

air

Analytical verification of test atmosphere concentrations:

yes

Remarks:

gravimetric with membrane filter.

Duration of exposure:

1 - 4 h

Concentrations:

163.3, 375, 1225 and 1895 mg/m³ air at 4 hour exposure.
1820 mg/m³ air at 1 hour exposure.

No. of animals per sex per dose:

10

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days .
- Necropsy of survivors performed: yes.
- Other examinations performed: clinical signs.

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 1 895 mg/m³ air (nominal)

Based on:

test mat.

Exp. duration:

4 h

Sex:

male/female

Dose descriptor:

LC50

Effect level:

1 820 mg/m³ air (nominal)

Based on:

test mat.

Exp. duration:

1 h

Mortality:

No mortality occuerred.

Clinical signs:

other:

Gross pathology:

No abnormalities detected.

Interpretation of results:

study cannot be used for classification

Conclusions:

LC50 (4 h): > 1.895 mg/l air

Executive summary:

Method

Acute inhalation toxicity was assessed following the method described into the OECD guideline 403.

Results

LC50 (4 h): > 1.895 mg/l air

LC50 (1 h): 1.820 mg/l air

Conclusion

According to CLP Regulation (EC 1272/2008), 3.1 Acute toxicity section, substances can be allocated to one of four toxicity categories based on acute toxicity by the oral, dermal or inhalation route according to the numeric criteria. Acute toxicity values are expressed as (approximate) LD50 (oral, dermal) or LC50 (inhalation) values or as acute toxicity estimates (ATE).

The inhalation LD50 value classification limit for dust is 5.0 mg/l (category 4: 1.0 < ATE ≤ 5.0 mg/l). In the current test an LD50 was non identified; considering the fact that no mortality occurred, a classification category can not be assigned. Thus, a classification according to the CLP Regulation (EC 1272/2008) is not applicable.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Value:

1 895 mg/m³ air

Acute toxicity: via dermal route

Link to relevant study records

Reference

Endpoint:

acute toxicity: dermal

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted according to internationally accepted testing guidelines and performed according to GLP. 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:

OECD Guideline 402 (Acute Dermal Toxicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.3 (Acute Toxicity (Dermal))

Deviations:

no

GLP compliance:

yes

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd., Wblferstrasse 4, CH-4414 Fullinsdorf.
- Age at study initiation: 10 weeks (males), 12 weeks (females).
- Weight at study initiation: 225 - 247 g (males), 200 - 222 g (females).
- Housing: individually in Makrolon type-2 cages with standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz).
- Diet: pelleted standard Kliba 343, Batches 77/90 and 78/90 rat maintenance diet ("Kliba", Klingentalmuehle AG, CH-4303 Kaiseraugst); ad libitum.
- Water: community tap water from Itingen; ad libitum.
- Acclimation period: one week.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 40-70 %
- Air changes: 10 - 15 air changes per hr.
- Photoperiod: 12 / 12 hrs dark / hrs light.

Type of coverage:

semiocclusive

Vehicle:

unchanged (no vehicle)

Details on dermal exposure:

TEST SITE
- Area of exposure: backs of the animals.
- Preparation: clipping 24 h before application.
- % coverage: 10% of the total body surface.

REMOVAL OF TEST SUBSTANCE
- Washing: with lukewarm tap water.
- Time after start of exposure: 24 h

TEST MATERIAL
- Amount applied: 4 ml
- Concentration: 100%
- For solids, paste formed: yes

Duration of exposure:

24 h

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

5

Control animals:

no

Details on study design:

- Duration of observation period following administration: 15 days
- Frequency of observations and weighing: observations four times during test day 1, and daily during days 2 - 15; weighing on test days 1 (pre-administration), 8 and 15.
- Necropsy of survivors performed: yes
- Other examinations performed: gross pathology.

Statistics:

The LOGIT-Model could not be applied to the observed rate of death. The toxicity was estimated without use of a statistical model.

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No mortality.

Clinical signs:

other: No systemic signs were observed in the animals during the entire observation period. Skin observations: males/females: scales (back) (10/10); general erythema (back) (3/10); males: erythema focal (back) (3/5). All animals had recovered from the local sign

Gross pathology:

No findings noted.

Interpretation of results:

other: CLP criteria not met

Conclusions:

LD50 > 2000 mg/kg bw

Executive summary:

Method

Upon an acute oral single administration (2000 mg/kg bw) and a 15 day post-treatment observation period, the dermal LD50 was determined for the test substance, according to the OECD guideline 402.

Results

LD50 > 2000 mg/kg bw

No deaths accorred and no systemic signs were observed in the animals during the entire observation period. Skin reactions are all recovered after 7 observation days.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Additional information

The oral acute toxic effects of the test substance were investigated after a single oral administration at the dose of 2000 mg/kg bw to rats; the test was performed according to the OECD guideline 423 and EU Method B.1. No death of animals was recorded, as no clinical signs of intoxication and no pathologic macroscopic changes were observed (CETA, 2014).

No specific tests about inhalation and dermal acute toxicity on the substance under registration were performed, but it is part of the Stilbene Fluorescent Whitening Agents category. The substance OB 1 -MSA belongs to the group 1 of the category, in which the substitution on the triazino moiety is a diethylamine. The functional group is the least reactive between all the other functional groups of the category, both from a chemical and from a metabolic point of view. The most similar substances, the dihydroxyethylamino derivatives, di and tetrasulphonated (OB 3a-A(Na), OB 3a-A(NaK) and OB 3a-MSA) are respectively less and more water soluble; therefore, the result of acute toxicity can be interpolated on the basis of the water solubility, Kow and specific functional group reactivity, between the results of those substances.

Skin adsorption has been evaluated and calculated for all members of the category (see Category Justification Report attached to the Section 13).

As it can be noted, the influence of the variability in functional groups is very low, more related to the variability in the polarity of the substance than in the potential reactivity that can arise a concern. From a metabolic point of view, an estimation was performed in order to verify if breakdown products may be formed by dermal metabolism, using the OECD Toolbox. in the category.

Seven registered substances belonging to the category were tested for acute dermal toxicity and they represent several groups: the results indicated a LD50 > 2000 mg/kg bw (the highest tested dose) for all members.

A good GLP Klimish 1 study on OB 3a-MSA was chosen as representative and was here reported for acute dermal toxicity (Ciba-Geigy Ltd., 1991): it was performed at 2000 mg/Kg bw with no effect.

Because of the physical state and the trade forms, inhalation is not an appropriate route of exposure. Acute toxicity results for the other two exposure routes indicate no concern therefore no testing was performed. One test is available on an analogous substance, performed at the maximum allowed concentration of 1890 mg/m3: no effects were observed. The analogous substance, part of the Stilbene Fluorescent Withening Agents, group 3, is the acid form of the disulphonated derivative dihydroxyethyl derivative, therefore contains the same organic functional groups, but due to the sulphonation/salification degree is less soluble. This property makes the Read Across substance OB 3a-A(free acid) a conservative representative because of the potential higher bioavailability.

As a conclusion it can be stated that the substance is not acutely toxic for all the three exposure ways.

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), 3.1 Acute toxicity section, substances can be allocated to one of four toxicity categories based on acute toxicity by the oral, dermal or inhalation route according to the numeric criteria. Acute toxicity values are expressed as (approximate) LD50 (oral, dermal) or LC50 (inhalation) values or as acute toxicity estimates (ATE).

The oral LD50 value was established to be greater than 2000 mg/kg body weight, therefore the test substance is out of any classification limit for acute oral toxicity (oral acute toxicity category 4: 300 < ATE ≤ 2000 mg/kg bw).

The dermal LD50 value was established to exceed 2000 mg/kg body weight, which exceeded the highest CLP classification limit (dermal acute toxicity category 4: 1000 < ATE ≤ 2000 mg/kg bw).

The inhalation LC50 value was established to be greater than 1895 mg/m3. For powder the limit for classification is ATE > 5 mg/l i. e. 5000 mg/m3.

Since no effect was observed at the tested concentration and this was the maximum reachable concentration in the test condition, it is assumed that the substance is not classified for inhalation acute toxicity.

In conclusion, the available experimental data are adequate for classification and labelling and the test substance is non classified for oral and dermal acute toxicity, according to the CLP Regulation (EC 1272/2008).