Registration Dossier

Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 September 2017 - 23 November 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Test item: Black DB
Appearance: black powder

Test animals

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 rat 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 the first and second step
Body weight range at starting (first step): 194 - 200 g
Body weight rangeat starting (second step): 188 - 192 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: 13/3
Housing: Group caging (3 animals/cage)
Cage type: Type III 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. Copies of the relevant Certificates of Analysis are maintained in Toxi-Coop Zrt.’s archive.


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.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Aqua purificata Ph.Hg. VIII
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. Formulations were prepared just before the administration and were stirred continuously during the treatment.
Doses:
2000 mg/kg bw
No. of animals per sex per dose:
5 females/dose (3 females/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.

Study design:
6 days in the first step and 7 days in the second step of acclimatization, day of treatment, 14 days post-treatment observation period and necropsy on Day 15.

Observations:
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. 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 weights:
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 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 the calculation of a precise LD50 value. The mean of the body weight and body weight gain were calculated by Excel spreadsheet software.

Results and discussion

Effect levels
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
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 the experimental animals were normal.
Body weight:
The body weight development was not affected 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.

Any other information on results incl. tables

Summary of Lethality

Post-treatment observation period (14 days)

Groups

Treatment

Lethality

Test Item

Dose
(mg/kg bw)

Females

1

Black DB
Step 1

2000

0/3

2

Black DB
Step 2

2000

0/3

  Summary of Clinical Symptoms

FEMALES

Groups

Treatment

Symptoms

Incidence

Test Item

Dose
mg/kg bw

1

Black DB
Step 1

2000

Normal

57/57

2

Black DB
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)

Summary of Body Weights (g) 

FEMALES

 

Day 0

Day 7

Day 15

 

 

 

 

 

Group 1: Black DB 

                2000 mg/kg bw, Step 1

 

 

 

 

 

 

 

 

Group size:

 

3

3

3

Mean: (g)

 

198.0

240.0

264.7

SD:

 

3.46

14.73

17.01

 

 

 

 

 

FEMALES

 

Day 0

Day 7

Day 15

 

 

 

 

 

Group 2:  Black DB                

                2000 mg/kg bw, Step 2

 

 

 

 

 

 

 

Group size:

 

3

3

3

Mean: (g)

 

190.3

222.3

238.0

SD:

 

2.08

3.51

12.53

 

 

 

 

 

 

Summary of Body Weight Gains (g) 

FEMALES

 

Day 0-7

Day 7-15

Day 0-15

 

 

 

 

 

Group 1: Black DB              

                2000 mg/kg bw, Step 1

Group size:

 

3

3

3

Mean: (g)

 

42.0

24.7

66.7

SD:

 

13.45

3.21

16.17

 

 

 

 

 

FEMALES

 

Day 0-7

Day 7-15

Day 0-15

 

 

 

 

 

Group 2: Black DB                                         

                2000 mg/kg bw, Step 2

 

 

 

Group size:

 

3

3

3

Mean: (g)

 

32.0

15.7

47.7

SD:

 

2.65

14.64

12.06

 

 

 

 

 

 


Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The acute oral LD50 value of the test item was found to be > 2000 mg/kg bw.
Executive summary:

The acute toxic class method according to OECD guideline 423 was carried out involving a stepwise procedure with the use of 2000 mg/kg bw 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 were 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 the experimental animals were normal. The body weight development was not affected 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 method used is not intended to allow the calculation of a precise LD50 value. The acute oral LD50 of the test item was determined to be > 2000 mg/kg bw.