Leuco polysulfided 4-[(2,4-dinitrophenyl)amino]phenol

Endpoint summary

Description of key information

In an in vivo acute toxicity assay (acute toxicity class method, ATC) according to OECD guideline 423, an LD50 of above 2000 mg dyestuff/kg bw, corresponnding to 2160 mg test item/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:

05 September - 29 September, 2016

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:

2001 December 17

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Council Regulation (EC) No 440/2008

Version / remarks:

2008 May 30

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Regulation (EC) No. 1272/2008

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

yes

Specific details on test material used for the study:

Expiration date: 29.04.2020
Correction factor: 1.08

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI, SPF

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: TOXI COOP ZRT. Cserkesz u. 90., 1103 Budapest, Hungary
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Young adult rat, 8 weeks old in first and second step
- Weight at study initiation: first step 185 - 189 g; second step 186 - 192 g
- Fasting period before study: the day before administration
- Housing: Group caging (3 animals/cage) in Type II cages
- Diet: ssniff Spezialdiäten GmbH, 59494 Soest, Germany, ad libitum
- Water: tap water from municipal supply, as for human consumption from bottle ad libitum.
- Acclimation period: 5 days in first step and 6 days in second step
- Animal health: Only healthy animals were used for the study. Health status was certified by the study director.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes: above 10 air exchanges/hour by central air-condition system.
- Photoperiod (hrs dark / hrs light): 12/12, from 6.00 a.m. to 6.00 p.m.

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Aqua purificata Ph.Hg. VIII, Parma Produkt Kft.

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 200 mg/mL
- Amount of vehicle: treatment volume of 10 mL/kg bw
- Batch no.: 1606-5508

CLASS METHOD
- Rationale for the selection of the starting dose: Starting dose was selected on the basis of the available information about the test item.

Doses:

2000 mg dyestuff/kg bw (corresponding to 2160 mg test item/kg bw)

No. of animals per sex per dose:

3 animals/group

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Animals were observed individually after dosing at least once during the first 30 minutes, then 1 h, 2 h, 3 h, 4 h after the treatment and twice each day for 14 days thereafter. The body weights were recorded on day 0 (just before the treatment), on day 7 and on day 15.
- Necropsy of survivors performed: yes. At the end of the observation period all survivor rats were sacrificed under isofluran anaesthesia (Batch No B49G15A. Piramal Healthcare UK Ltd). After examination of the external appearance, the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed, and any abnormality was recorded with details of its location, colour, shape and size.
- Other examinations performed: Individual observations were performed on the skin and fur, eyes and mucous membranes and also respiratory, circulatory, autonomic and central nervous system, somatomotor activity and behaviour pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

Statistics:

The method used is not intended to allow statistical evaluation and the calculation of a precise LD50 value.

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

act. ingr.

Mortality:

No death occurred at 2000 mg/kg bw after a single oral dose. All female rats in step 1 and step 2 survived until the end of the 14-day observation period.

Clinical signs:

other: In group 1 and 2 treated with 2000 mg/kg bw, no treatment related symptoms were observed throughout the 14-day post-treatment period.

Gross pathology:

All animals treated with 2000 mg/kg bw survived until the scheduled necropsy on Day 15. Moderate hydrometra was found in one animal of group 1. Hydrometra is physiological finding and connected to the cycle of the animal. No pathological changes were found related to the effect of the test item during the macroscopic examination of animals.

Table 2: GHS classification according to the results

Dose (mg/kg bw)	Mortality (dead/treated)	LD50 (mg/kg bw)	GHS category
2000	0/6	between 5000 and 2000	5

 

Table 3: Summary of Lethality. Post-treatment observation period (14 days) 

Groups	Treatment		Lethality
	Test Item	Dose (mg/kg bw)	Females
1	Leuco Sulfur Blue 11 Step 1	2000	0/3
2	Leuco Sulfur Blue 11 Step 2	2000	0/3

Table 4: Summary of Clinical Symptoms

FEMALES

Groups	Treatment		Symptoms	Incidence
	Test Item	Dose mg/kg bw
1	Leuco Sulfur Blue 11 Step 1	2000	Normal	57/57
2	Leuco Sulfur Blue 11 Step 2	2000	Normal	57/57

Remark:   Incidence = Number of symptoms/Summarized number of observations inside the group

                         Summarized number of observations inside the group =

(number of observations of first animal) + (number of observations of second animal) +(number of observations of third animal

Table 5: Summary of Body Weights (g)

 

FEMALES		Day 0	Day 7	Day 15
Group 1: Leuco Sulfur Blue 11
2000 mg/kg bw, Step 1
Group size:		3	3	3
Mean: (g)		186.7	231.7	249.7
SD:		2.08	7.09	13.32
FEMALES		Day 0	Day 7	Day 15
Group 2: Leuco Sulfur Blue 11
2000 mg/kg bw, Step 2
Group size:		3	3	3
Mean: (g)		188.3	233.0	252.7
SD:		3.21	14.80	17.95

Table 6: Summary of Body Weight Gains (g)

 

 

FEMALES		Day 0-7	Day 7-15	Day 0-15
Group 1:  Leuco Sulfur Blue 11
2000 mg/kg bw, Step 1
Group size:		3	3	3
Mean: (g)		45.0	18.0	63.0
SD:		7.55	6.24	13.53
FEMALES		Day 0-7	Day 7-15	Day 0-15
Group 2: Leuco Sulfur Blue 11
2000 mg/kg bw, Step 2
Group size:		3	3	3
Mean: (g)		44.7	19.7	64.3
SD:		11.72	3.51	14.98

Table 7: Summarized Gross Pathology Findings

Observations (Females)                     Leuco Sulfur Blue 11 (2000mg/kg bw)

Hydrometria                                                   1/6            

Normal                                                            5/6

Interpretation of results:

GHS criteria not met

Conclusions:

In an acute oral toxicity study (acute toxicity class method, ATC) according to OECD guideline 423, a LD50 of above 2000 mg dyestuff/kg bw, corresponding to 2160 mg test item/kg bw, was determined.

Executive summary:

In an acute oral toxicity study according to OECD 423, the LD50 of the test item after single oral gavage was determined. The acute toxic class method (ATC) was carried out involving a stepwise procedure starting at 2000 mg dyestuff/kg bw, corresponding to 2160 mg test item/kg bw, in three female Wistar rats. Animals were weighed, observed for lethality and toxic symptoms for 14 days after the treatment. Gross pathological examination was carried out on the 15th day after the treatment. No animal died in the first step at 2000 mg/kg bw dose level, so treatment with 2000 mg/kg bw was repeated on further three female rats. No animal died in the second step, too, so the test was finished, according to the stopping criteria of Annex 2d of OECD Guideline 423.

No lethality was noted at a single oral dose of 2000 mg dyestuff/kg bw. In the first and second step, no clinical symptoms were observed on the day of the treatment and during the 14-day observation period, the general state and behaviour of experimental animals were normal. The body weight development was undisturbed in all animals. Gross pathologically investigations of all organs of the treated animals showed no treatment related changes.

The test item was ranked into classes of Globally Harmonized Classification System (GHS) as described in the OECD Guideline No. 423. However, the method used is not intended to allow the calculation of a precise LD50 value. Therefore a LD50 of above 2000 mg dyestuff/kg bw, corresponnding to 2160 mg test item/kg bw, was determined.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

discriminating dose

Value:

2 000 mg/kg bw

Quality of whole database:

Guideline and GLP conform study.

Additional information

In an acute oral toxicity study according to OECD 423, the LD50 of the test item after single oral gavage was determined. The acute toxic class method (ATC) was carried out involving a stepwise procedure starting at 2000 mg dyestuff/kg bw, corresponding to 2160 mg test item/kg bw

in three femaleWistarrats. Animals were weighed, observed for lethality and toxic symptoms for 14 days after the treatment. Gross pathological examination was carried out on the 15th day after the treatment. No animal died in the first step at 2000 mg dyestuff/kg bw, so treatment with that dose level was repeated on further three female rats. No animal died in the second step, too, so the test was finished, according to the stopping criteria of Annex 2d of OECD Guideline 423.

No lethality was noted at a single oral dose of 2000 mg dyestuff/kg bw. In the first and second step, no clinical symptoms were observed on the day of the treatment and during the 14-day observation period, the general state and behaviour of experimental animals were normal. The body weight development was undisturbed in all animals. Gross pathologically investigations of all organs of the treated animals showed no treatment related changes.

The test item was ranked into classes of Globally Harmonized Classification System (GHS) as described in the OECD Guideline No. 423. However, the method used is not intended to allow the calculation of a precise LD50 value. Therefore a LD50 of above 2000 mg dyestuff/kg bw, corresponding to 2160 mg test item/kg bw was determined.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008 (CLP). As a result the test item is not considered to be classified for acute oral toxicity under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) No 2019/52.