Carbonic acid disodium salt, reaction products with aniline, 4-nitrobenzenamine, p-phenylenediamine, sodium sulfide, sulfur and p-toluidine

Endpoint summary

Description of key information

The LD50 of the test item was found to be > 2000 mg dye/kg bw in rats (corrected dose).

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:

Sebtember 05 - October 04, 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:

17th December, 2001

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

yes

Species:

rat

Strain:

other: Crl:WI rats

Sex:

female

Details on test animals or test system and environmental conditions:

Source: TOXI COOP ZRT. Cserkesz u. 90. ,1103 Budapest, Hungary
Hygienic level at arrival: SPF
Hygienic level during the study: Good conventional
Justification of strain: The Wistar rats as a rodent is one of the standard species of acute toxicity studies
Number of animals: 3 animals/group
Sex: Female, nulliparous and non pregnant animals
Age of animals: Young adult rat, 8 weeks old in first and second step

Body weight range
at starting (first step): 171 - 174 g
Body weight range
at starting (second step): 173 - 176 g
Acclimatization time: 5 days in first step and 6 days in second step

Husbandry
Animal health: Only healthy animals were used for the study. Health status was certified by the study director.
Room: 13/2
Housing: Group caging (3 animals/cage)
Cage type: Type II polypropylene/polycarbonate.
Bedding: Laboratory bedding.
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 22 ± 3 °C
Relative humidity: 30 - 70 %
Ventilation: above 10 air exchanges/hour by central air-condition system.

The temperature and relative humidity were recorded daily during the study.

Food and Water Supply
Animals received ssniff® SM R/M-Z+H complete diet for rats and mice.
The diet and drinking water are periodically analysed and are considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

Identification
The individual identification was performed by numbers on the tail written by a permanent marker. The numbers were given on the basis of Toxi-Coop Zrt.'s master file, for each animal allocated to the study.
The boxes were identified by cards, holding information about study number, test item, sex, strain, dose group, date of arriving of animals, room number, cage number and individual animal numbers.

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

All doses were formulated in the vehicle. Concentration of formulations was adjusted to maintain a treatment volume of 10 mL/kg bw. The test item was applied in a concentration of 200 mg/mL. The correction factor was taken into consideration in the course of the making of solution. Formulations were prepared just before the administration and were stirred continuously during the treatment.
Vehicle
Name: Aqua purificata Ph.Hg. VIII.
Batch number:1606-5508
Date of expiration:08.12.2016
Produced by:Parma Produkt Kft.

Doses:

2000 mg/kg bw (act. ingredient)

No. of animals per sex per dose:

3 animals/group

Control animals:

no

Details on study design:

5 days in first step and 6 days in second step of acclimatization, treatment’s day, 14 days post-treatment observation period, necropsy on Day 15.

Mortality
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.

General state, external appearance, behavior and clinical symptoms

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.
Body weight
The body weights were recorded on day 0 (just before the treatment), on day 7 and on day 15 with a precision of 1 g.

Necropsy
At the end of the observation period all survivor rats were sacrificed under isofluran anaesthesia. 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.

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. 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 treated with 2000 mg/kg bw dose no treatment related symptoms were observed throughout the 14-day post-treatment period. In group 2 treated with 2000 mg/kg bw dose no treatment related symptoms were observed throughout the 14-day post-treatmen

Gross pathology:

All animals treated with 2000 mg/kg bw dose survived until the scheduled necropsy on Day 15.
Moderate hydrometra was found in two animals (No.: 2933, 2942) of group 1. Hydrometra is physiological finding and connected to the cycle of the animals. No pathological changes were found related to the effect of the test item during the macroscopic examination of animals treated with 2000 mg/kg bw dose.

Summary of Lethality

Post-treatment observation period (14 days)

 

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

Summary of Clinical Symptoms

FEMALES

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

Summary of Body Weights (g)

FEMALES		Day 0	Day 7	Day 15
Group 1:
2000 mg/kg bw, Step 1
Group size:		3	3	3
Mean: (g)		172.7	206.0	218.0
SD:		1.53	1.73	5.57
FEMALES		Day 0	Day 7	Day 15
Group 2:
2000 mg/kg bw, Step 2
Group size:		3	3	3
Mean: (g)		174.3	214.3	230.7
SD:		1.53	2.08	5.77

Summary of Body Weight Gains (g)

FEMALES		Day 0-7	Day 7-15	Day 0-15
Group 1:
2000 mg/kg bw, Step 1
Group size:		3	3	3
Mean: (g)		33.3	12.0	45.3
SD:		0.58	6.08	6.43
FEMALES		Day 0-7	Day 7-15	Day 0-15
Group 2:
2000 mg/kg bw, Step 2
Group size:		3	3	3
Mean: (g)		40.0	16.3	56.3
SD:		3.61	6.66	5.69

Interpretation of results:

GHS criteria not met

Conclusions:

The LD50 value of the test item was found to be >2000 mg/kg bw (corrected concentration).

Executive summary:

To assess the acute oral toxicity of the test item in rats, the acute toxic class method was carried out involving a stepwise procedure with the use of 2000 mg/kg bw (corrected concentration on the basis of the dye content, corresponding to 2210 mg/kg bw tst item) as the starting dose in three female rats. 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, the stopping criteria of Annex 2d of OECD Guideline No. 423 was met. Animals were weighed, observed for lethality and toxic symptoms for 14 days after the treatment. Gross pathological examination was carried out on day 15 after the treatment. No mortality and no clinical symptoms were observed on the day of the treatment and during the 14-day observation period, the general state and behaviour of the experimental animals were normal. The body weight development was uneffected in all animals. All organs of the animals treated with 2000 mg/kg bw proved to be free of treatment related gross pathological changes. The LD50 was found to be >2000 mg/kg bw.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

discriminating dose

Value:

2 000 mg/kg bw

Acute toxicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Acute toxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Oral:

To assess the acute oral toxicity of the test item in rats, the acute toxic class method was carried out involving a stepwise procedure with the use of 2000 mg dye/kg bw (corrected dose) as the starting dose in three female rats. 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, the stopping criteria of Annex 2d of OECD Guideline No. 423 was met. Animals were weighed, observed for lethality and toxic symptoms for 14 days after the treatment. Gross pathological examination was carried out on day 15 after the treatment. No mortality and no clinical symptoms were observed on the day of the treatment and during the 14-day observation period, the general state and behaviour of the experimental animals were normal. The body weight development was uneffected in all animals. All organs of the animals treated with 2000 mg/kg bw proved to be free of treatment related gross pathological changes. The LD50 was found to be >2000 mg/kg bw.

Inhalation:

The study does not need to be conducted as exposure of humans via inhalation is not the appropriate route of exposure. Furthermore, physicochemical and toxicological properties do not suggest a potential for a significant rate of respiratory absorption.

Dermal:

No study was conducted as the physiochemical and toxicological properties suggest no potential for a significant rate of absorption through the skin.

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 (EC) No 1272/2008. Based on available data on acute toxicity, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.