Benzenesulfonic acid, 2,2'-(1,2-ethenediyl)bis[5-nitro-, disodium salt, reaction products with 4-[(4-aminophenyl)azo]benzenesulfonic acid, sodium salts

Endpoint summary

Description of key information

The acute toxicity of the substance has been determined in adequate studies in the rat following oral administration and in the rat and rabbit with dermal administrations. No studies are available for inhalation.

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:

1993

Reliability:

2 (reliable with restrictions)

Qualifier:

according to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

other: WHO/VBC 88.953

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

yes

Species:

rat

Strain:

other: TIf: RAI f (SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CIBA-GEIGY Limited Animal Production 4332 Stein / Switzerland
- Weight at study initiation: 177 to 206g
- Fasting period before study: overnight
- Housing: in Macrolon cages type 4, with standardized soft wood beeding.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%):55 ± 10%
- Air changes (per hr): 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hour /day light cycle.

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

Administration of the test article: One single oral dose, by gastric intubation (gavage)

VEHICLE
Distilled water
- Concentration in vehicle: 10 ml/kg body weight
- Amount of vehicle (if gavage): 2000 mg/kg
- Lot/batch no. (if required): 623
- Purity: 27%

MAXIMUM DOSE VOLUME APPLIED: 2000 mg/kg

DOSAGE PREPARATION (if unusual):

CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose:

Doses:

2000 mg/kg (males and females)
Volume applied: 10 ml/kg bw

No. of animals per sex per dose:

5 males, 5 females (total number of animals: 10)

Control animals:

yes

Details on study design:

Limit test

Observations and records
Mortality: daily; a.m. and p.m. on working days, a.m. on weekend days.
Signs and symptoms; daily for 14 days.
Body weight: immediately before administration and on days 7 and 14.
Necropsies: The animals were submitted to a gross necropsy at the end of the observation period.

Sex:

male/female

Dose descriptor:

LD50

Effect level:

2 000 mg/kg bw

Based on:

test mat.

Mortality:

No mortalities occurred in this study.

Clinical signs:

other: Piloerection, hunched posture, and dyspnea were seen, being common symptoms in acute tests . Additionally, diarrhea was observed in the females. The animals recovered within 5 days.

Gross pathology:

At necropsy, a spotted thymus was found in one female. No deviations from normal morphology were found in the remaining animals.

Dose mg/kg   Number of animals  Number of deaths  Males  2000  5  0  Females  2000  5  0

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Upon an acute oral administration and a 14 day post-treatment observation period, the following LD50 (with 95% confidence limits calculated , where possible) was determined for the tested material.

LD50 in male rats: greater than 2000 mg/kg body weight
LD50 in female rats: greater than 2000 mg/kg body weight
LD50 in rats of both sexes: greater than 2000 mg/kg body weight

Executive summary:

10 young adult rats (5 males and 5 females Tif: Rai f) were housed in Macrolon cages type 4 and acclimatized at least for 5 days before administration. 2000 mg/kg of the test article solved in water was administrated in one single oral dose by gastric intubation. During 14 days the mortality, signs and symptoms were observed daily. The body weight was measured immediately before administration and on days 7 and 14 of the study and necropsies at the end of the observation period.

In conclusion, after 14 day post-treatment observation period, the LD50 in rats of both sexes is greater than 2000 mg/kg body weight.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Quality of whole database:

Three studies available, one of them according to GLP standards (key study). The two supporting studies are not performed following GLP.

Acute toxicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Acute toxicity: via dermal route

Link to relevant study records

Reference

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

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:

Five male and five female Wistar (RccHanÔ:WIST) strain rats were supplied by Harlan Laboratories UK Ltd., Oxon, UK. On receipt the animals were randomly allocated to cages.
The females were nulliparous and non-pregnant. After an acclimatization period of at least five days the animals were selected at random and given a number unique within the study by indelible ink-marking on the tail and a number written on a cage card. At the start of the study the animals weighed at least 200 g, and were eight to twelve weeks of age. The weight variation did not exceed ±20% of the mean weight for each sex.
The animals were housed in suspended solid floor polypropylene cages furnished with woodflakes. The animals were housed individually during the 24-Hour exposure period and in groups of five, by sex, for the remainder of the study. Free access to mains drinking water and food (2014C Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study. The diet, drinking water and bedding were routinely analyzed and were considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
The temperature and relative humidity were set to achieve limits of 19 to 25 °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 (06:00-18:00) and twelve hours darkness.
The animals were 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.

Type of coverage:

semiocclusive

Vehicle:

water

Duration of exposure:

24-hour contact period

Doses:

2000 mg/kg

No. of animals per sex per dose:

five (5) male and five (5) female

Details on study design:

On the day before treatment the back and flanks of each animal were clipped free of hair.
Using available information on the toxicity of the test item, a group of five male and five female rats was treated with the test item at a dose level of 2000 mg/kg.
The appropriate amount of test item, moistened with distilled water, was applied as evenly as possible to an area of shorn skin (approximately 10% of the total body surface area). A piece of surgical gauze was placed over the treatment area and semi occluded with a piece of self adhesive bandage. The animals were caged individually for the 24-Hour exposure period.
Shortly after dosing the dressings were examined to ensure that they were securely in place.
After the 24-Hour contact period the bandage was carefully removed and the treated skin and surrounding hair wiped with cotton wool moistened with distilled water to remove any residual test item. The animals were returned to group housing for the remainder of the study period.
The animals were observed for deaths or overt signs of toxicity ½, 1, 2 and 4 hours after dosing and subsequently once daily for fourteen days.
After removal of the dressings and subsequently once daily for fourteen days, the test sites were examined for evidence of primary irritation and scored according to the scale 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.31.

Any other skin reactions, if present were also recorded.

Individual body weights were recorded prior to application of the test item on Day 0 and on Days 7 and 14.
At the end of the study the animals were killed by cervical dislocation. All animals were subjected to gross necropsy. This consisted of an external examination and opening of the abdominal and thoracic cavities. The appearance of any macroscopic abnormalities was recorded. No tissues were retained.

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

There were no deaths

Clinical signs:

other: No signs of systemic toxicity were noted during the observation period.

Gross pathology:

No abnormalities were noted at necropsy

Other findings:

Orange coloured staining, not preventing evaluation of skin responses, was noted at the test sites of all animals during the study.
Physical damage caused on bandage removal was noted at the test site of one male 1 to 10 days after dosing. Small superficial scattered scabs and glossy skin were also noted at this test site 11 and 12 days after dosing with glossy skin persisting 13 and 14 days after dosing.
There were no signs of dermal irritation noted at the test sites of the remaining nine animals.

Individual Dermal Reactions - Males

Dose level mg/kg	Animal number and sex	Observation	Effects noted after initation of exposure (days)
			1	2	3	4	5	6	7	8	9	10	11	12	13	14
2000	1 -0 male	Erythema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		Edema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		Other	STAPd	STAPd	STAPd	STAPd	STAPd	Pd	Pd	Pd	Pd	Pd	SsG	SsG	G	G
	1 -1 male	erythema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		edema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		other	STA	STA	STA	STA	STA	0	0	0	0	0	0	0	0	0
	1 -2 male	erythema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		edema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		other	STA	STA	STA	STA	STA	STA	STA	STA	0	0	0	0	0	0
	1 -3 male	erythema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		edema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		other	STA	STA	STA	STA	STA	0	0	0	0	0	0	0	0	0
	1 -4 male	erythema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		edema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		other	STA	STA	STA	STA	STA	STA	STA	STA	STA	0	0	0	0	0

Individual Dermal reactions - females                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  

0: no reactions; STA: Orange coloured staining; Pd: Physical damage caused on bandage removal; Ss: Small superficial scattered scabs; G: Glossy skin.

Individual Dermal reactions - females

Dose level mg/kg	animal number and sex	observation	Effects noted after initiation of exposure (days)
			1	2	3	4	5	6	7	8	9	10	11	12	13	14
	2 -0 female	erythema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		edema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		other	STA	STA	STA	STA	STA	0	0	0	0	0	0	0	0	0
	2 -1 female	erythema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		edema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		other	STA	STA	STA	STA	STA	STA	0	0	0	0	0	0	0	0
	2 -2 female	erythema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		edema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		other	STA	STA	STA	STA	STA	STA	STA	0	0	0	0	0	0	0
	2 -3 female	erythema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		edema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		other	STA	STA	STA	STA	STA	0	0	0	0	0	0	0	0	0
	2 -4 female	erythema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		edema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		other	STA	STA	STA	STA	STA	STA	STA	STA	0	0	0	0	0	0

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The acute dermal median lethal dose (LD50) of the test item in the Wistar strain rat was found to be greater than 2000 mg/kg body weight

Executive summary:

A group of ten animals (five males and five females) was given a single, 24 hour, semi-occluded dermal application of the test item to intact skin at a dose level of 2000 mg/kg bodyweight. Clinical signs and body weight development were monitored during the study. All animals were subjected to gross necropsy (No abnormalities were noted at necropsy).

The observation on mortality showed that there were no deaths. Clinical observations showed no signs of systemic toxicity. Observation on dermal irritation showed a physical damage caused on bandage removal, small superficial scattered scabs and glossy skin were confined to the test site of one male. There were no signs of dermal irritation noted at the test sites of the remaining nine animals. Observations in body weight gave as a result that one female showed expected gain in bodyweight during the first week but body weight loss during the second week. Two females showed bodyweight loss or no gain in body weight during the first week with expected gain in bodyweight during the second week. Remaining animals showed expected gains in body weight during the study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Quality of whole database:

Two studies available, one of them according to GLP standards (key study). The supporting study confirms the result found and used for assessment.

Additional information

Acute oral toxicity:

Two oral acute toxicity studies are available for CAS 1325-54-8 / EC 215-397-8. One of them is a limit test done according to OECD Guideline 401, Hartmann HR (1993), where rats were administered orally (gavage) 0 and 2000 mg/kg bw. No remarcable clinical signs were observed (male/female), no deaths occured and no remarkable macroscopic changes were observed in both male/female. With this study a LD50 > 2000 mg/kg bw was determined.

In an older test from Thomann P (1973) the rats were feeded with different doses of substance from 1000 to 10.000 mg/kg and a LD50 of 8700 mg/kg was obtained by calculation of probit analysis.

In addition one study performed with CAS 1195028-55-7 and following test method EU method B.1, Alvarez i Genoher N (2003), gave a result of LD50 > 2500 mg/kg bw. This substance has an identical structure to CAS 1325-54-8 in respect of the anionic components . It is considered that both substances have the same mode of action.

The study of Hartmann HR (1993) has been chosen as key study.

 

Acute inhalation toxicity:

There isn't any test available for acute inhalation toxicity. The exposure of humans via inhalation is unlikely taking into account the vapour pressure of the substance and/or the possibility of exposure to aerosols, particles or droplets of an inhalable size.

 

Acute dermal toxicity:

Two studies of dermal toxicity of CAS 1325-54-8 / EC 215-397-8 are available. One of the studies Morena M (1980), was done following the guidances of 16 CFR Part 1500 - HAZARDOUS SUBSTANCES AND ARTICLES; ADMINISTRATION AND ENFORCEMENT REGULATIONS. The substance was applied dissolved into the skin of rabbit and observations were done on toxicity and mortality. An LD50 >2000 mg/kg bw was determined.

The other study of Pooles A (2013) followed OECD Guideline 402 and EU Method B.2. In this study the substance was applied to the skin of Wistar rat at a limit dose of 2000 mg/kg bw. Erythema and edema were monitored and scored. No deaths occured during or/and after the observation period and no signs of systemic toxicity were observed. Also no abnormalities were found at necropsy. As a remark, and as consquence of the nature of the substance, an orange coloured staining was seen in the site of application, but this effect didn't prevent to evaluate the skin responses. With this study an LD50 > 2000 mg/kg bw has been determined.

The study of Pooles A (2013) has been chosen as key study.

 

Justification for selection of acute toxicity – oral endpoint
The study is a GLP compliant and has Klimisch score 2.

Justification for selection of acute toxicity – dermal endpoint
The study is a GLP compliant and has Klimisch score 1

Justification for classification or non-classification

Acute oral toxicity

Based on the results of acute oral toxicity testing, CAS 1325-54-8 / EC 215-397-8 is not classified as "Harmful if swallowed" according to the EU DSD classification criteria (EU Directive 67/548/EEC). According to the EU CLP classification criteria (EU Regulation 1272/2008),CAS 1325-54-8 / EC 215-397-8 is not classified as hazardous.

The criteria in CLP Regulation establishes the limit for classification as Acute Tox Oral Category 4 in 2000 mg/kg, and the value obtained is >2000 mg/kg bw.

 

Acute inhalation toxicity

There isn't any test available for acute inhalation toxicity. The exposure of humans via inhalation is unlikely taking into account the vapour pressure of the substance and/or the possibility of exposure to aerosols, particles or droplets of an inhalable size.

 

Acute dermal toxicity

Based on the results of acute dermal toxicity testing CAS 1325-54-8 / EC 215-397-8 is not classified as harmful for skin according to the EU DSD classification criteria (EU Directive 67/548/EEC). According to the EU CLP classification criteria (EU Regulation 1272/2008),CAS 1325-54-8 / EC 215-397-8 is not classified as hazardous for this hazard class.