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Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 August 2017 - 09 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
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Version / remarks:
17th December 2001
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)
Version / remarks:
30 May 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test type:
acute toxic class method
Limit test:
yes

Test material

Constituent 1
Reference substance name:
Acetamide, N-(2,4-dinitrophenyl)-, reaction products with 1-methyl-2,4-dinitrobenzene and sodium sulfide (Na2(Sx)), leuco derivatives
EC Number:
288-896-1
EC Name:
Acetamide, N-(2,4-dinitrophenyl)-, reaction products with 1-methyl-2,4-dinitrobenzene and sodium sulfide (Na2(Sx)), leuco derivatives
Cas Number:
85940-08-5
Molecular formula:
not applicable
IUPAC Name:
Reaction product of N-(2,4-dinitrophenyl)acetamide with 1-methyl-2,4-dinitrobenzene and polysulfide, leuco derivatives
Test material form:
solid: particulate/powder
Details on test material:
Test item: Leuco Sulphur Brown 46
Appearance: Brown powder
CAS No: 85940-08-5

Test animals

Species:
rat
Strain:
Wistar
Remarks:
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 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): 203 - 214 g
Body weight range at starting (second step): 209 - 210 g
Acclimatization time: 5 days in the first step and 6 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 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.

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.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Aqua purificata Ph.Hg.
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 (refer to the substance purity degree). Correction of concentrations for active component content was made (correction factor: 1.33). Formulations were prepared just before the administration and were stirred continuously during the treatment.

Vehicle: Aqua purificata Ph.Hg; Batch number: 1707-5506; Date of expiration: 06.01.2018; 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
5 days in the first step and 6 days in the second step of acclimatization, day of treatment, 14 days post-treatment observation period and necropsy on Day 15.
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 were recorded on day 0 (just before the treatment), on day 7 and on day 15 with a precision of 1 g.

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:
No statistics was used in the study.

Results and discussion

Effect levels
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 the experimental animals were normal.
Body weight:
The body weight development was not affected in all animals.
Gross pathology:
At necropsy pale kidneys were observed in oen animal of group 1 and in two animals of group 2. This alteration was not considered to be related to test item toxicity, but was regarded as an individual variation. Most likely the observation is a congenital anomaly. Slighthydrometra was found in one animal of group 1. Hydrometra is a physiological finding and related to the estrous cycle of the animal. 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.
Other findings:
The method used is not intended to allow the calculation of a precise LD50 value.

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

Leuco Sulphur Brown 46
Step 1

2000

0/3

2

Leuco Sulphur Brown 46
Step 2

2000

0/3

Summary of Clinical Symptoms

FEMALES

Groups

Treatment

Symptoms

Incidence

Test Item

Dose
mg/kg bw

1

Leuco Sulphur Brown 46
Step 1

2000

Normal

57/57

2

Leuco Sulphur Brown 46
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: Leuco Sulphur Brown 46 

                2000 mg/kg bw, Step 1

 

 

 

 

 

 

 

 

Group size:

 

3

3

3

Mean: (g)

 

207.3

243.7

261.0

SD:

 

5.86

8.62

13.11

 

 

 

 

 

FEMALES

 

Day 0

Day 7

Day 15

 

 

 

 

 

Group 2:  Leuco Sulphur Brown 46                

                 2000 mg/kg bw, Step 2

 

 

 

 

 

 

 

Group size:

 

3

3

3

Mean: (g)

 

209.7

236.0

249.0

SD:

 

0.58

5.29

5.29

 

 

 

 

 

 

Summary of Body Weight Gains (g)

 

FEMALES

 

Day 0-7

Day 7-15

Day 0-15

 

 

 

 

 

Group 1: Leuco Sulphur Brown 46              

                2000 mg/kg bw, Step 1

Group size:

 

3

3

3

Mean: (g)

 

36.3

17.3

53.7

SD:

 

10.41

4.51

14.15

 

 

 

 

 

FEMALES

 

Day 0-7

Day 7-15

Day 0-15

 

 

 

 

 

Group 2: Leuco Sulphur Brown 46                                         

                2000 mg/kg bw, Step 2

 

 

 

Group size:

 

3

3

3

Mean: (g)

 

26.3

13.0

39.3

SD:

 

5.86

0.00

5.86

 

 

 

 

 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The acute oral LD50 was determined to be > 2000 mg dye/kg bw (corresponding to > 2660 mg product/kg bw).
Executive summary:

An acute oral toxicity study was performed according to OECD guideline 423. The acute toxic class method was carried out involving a stepwise procedure with the use of 2000 mg dye/kg bw (corresponding to 2660 mg product/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. 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 was determined to be > 2000 mg/kg bw.