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Description of key information

In an acute oral toxicity study in rats the acute oral LD50 was determined to be > 2000 mg dye/kg bw (corresponding to > 2300 mg product/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:
07 December 2016 - 16 March 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Version / remarks:
2001
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test type:
acute toxic class method
Limit test:
yes
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI rats
Sex:
female
Details on test animals 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): 218 - 224 g
Body weight range at starting (second step): 216 - 218 g
Acclimatization time: 6 days in the first step and 7 days in the 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 produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany and tap water from municipal supply, as for human consumption from bottle ad libitum.
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.
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 (1.15) was taken into consideration in the course of the preparation 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: 1608-5511
Date of expiration: 11.02.2017
Produced by: Parma Produkt Kft.
Doses:
2000 mg/kg bw
No. of animals per sex per dose:
3 animals/group
Control animals:
no
Details on study design:
A single oral administration - followed by a fourteen-day observation period - was performed by gavage. The day before treatment the animals were fasted. The food but not water was withheld overnight. Animals were weighed before the application and the food was given back 3 hours after the treatment.

Duration of the experimental period
6 days in the first step and 7 days in the second step of acclimatization, treatment’s day, 14 days post-treatment observation period and 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 surviving 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 lethality was noted at a single oral dose of 2000 mg/kg bw.
Clinical signs:
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.
Body weight:
The body weight development was undisturbed in all animals.
Gross pathology:
All organs of the animals treated with 2000 mg/kg bw proved to be free of treatment related gross pathological changes.
Other findings:
No death occurred after the single 2000 mg/kg bw oral dose of the test item. There were no toxic clinical signs or any treatment related effects of the test item found in body weights and body weight gains during the study. Autopsy revealed no treatment related pathological changes.

Table 1: Summary of Clinical Symptoms

Groups

Treatment

Symptoms

Incidence

Test Item

Dose
mg/kg bw

1

Blue Sema
Step 1

2000

Normal

57/57

2

Blue Sema
Step 2

2000

Normal

57/57

Table 2: Summary of Body Weights (g)

 

Day 0

Day 7

Day 15

 

 

 

 

 

Group 1:

2000 mg/kg bw, Step 1

 

 

 

 

 

 

 

 

Group size:

 

3

3

3

Mean: (g)

 

221.0

248.7

261.7

SD:

 

3.00

6.66

6.35

 

 

 

 

 

 

Day 0

Day 7

Day 15

 

 

 

 

 

Group 2:            

2000 mg/kg bw, Step 2

 

 

 

 

 

 

 

Group size:

 

3

3

3

Mean: (g)

 

216.7

237.0

248.7

SD:

 

1.15

10.00

9.07

 

 

 

 

 

Interpretation of results:
GHS criteria not met
Conclusions:
The oral LD50 was determined to be > 2000 mg dye/kg bw (corresponding to > 2300 mg product/kg bw).
Executive summary:

An acute toxic class method was carried out involving a stepwise procedure with the use of 2000 mg/kg bw as the starting dose in three female rats. The dose refers to the dye content (87 %, correction factor of 1.15) and corresponds to 2300 mg product/kg bw. 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 the 15th day after the treatment. No lethality was noted at a single oral dose of 2000 mg/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. All organs of the animals treated with 2000 mg/kg bw proved to be free of treatment related gross pathologicakem changes. The method used is not intended to allow the calculation of a precise LD50 value. The oral LD50 was determined 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

Acute oral toxicity:

An acute toxic class method was carried out involving a stepwise procedure with the use of 2000 mg/kg bw as the starting dose in three female rats. The dose refers to the dye content (87 %, correction factor of 1.15) and corresponds to 2300 mg product/kg bw. 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 the 15th day after the treatment. No lethality was noted at a single oral dose of 2000 mg/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. All organs of the animals treated with 2000 mg/kg bw proved to be free of treatment related gross pathologicakem changes. The method used is not intended to allow the calculation of a precise LD50 value. The oral LD50 was determined to be > 2000 mg/kg bw.

Acute inhalation toxicity:

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.

Acute dermal toxicity:

According to REACH Annex VIII point 8.5.3 the study does not need to be conducted because the substance does not meet the criteria for classification as acute toxicity or STOT SE by the oral route and no systemic effects have been observed in in vivo studies with dermal exposure (e.g. skin irritation, skin sensitisation).

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 oral toxicity, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the twelfth time in Regulation (EU) No 2019/521.