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Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 May - 05 July, 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:
17 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:
Phenol, 2,4-dinitro-, sulfurized, thiosulfonated
EC Number:
215-445-8
EC Name:
Phenol, 2,4-dinitro-, sulfurized, thiosulfonated
Cas Number:
1326-83-6
Molecular formula:
not applicable
IUPAC Name:
phenol, 2,4-dinitro-, sulfurized, thiosulfonated
Test material form:
solid: particulate/powder
Details on test material:
Test item: Solubilised Sulphur Black 1
Appearance: black powder
CAS No: 1326-83-6
Specific details on test material used for the study:
Expiration date: 30.11.2021

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI rats (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 176 - 180 g; second step 200 - 202 g
- Fasting period before study: the day before administration
- Housing: Group caging (3 animals/cage) in Type II cages.
- Diet: ssniff® SM R/M-Z+H complete diet for rats and mice; ssniff Spezialdiäten GmbH, 59494 Soest, Germany, ad libitum
- Water: tap water from municipal supply, as for human consumption from bottle, ad libitum.
- Acclimation period: 6 days in the first step and 7 days in the 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.


Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Aqua purificata
Details on oral exposure:
VEHICLE
- Concentration in vehicle: 200 mg/mL
- Amount of vehicle: treatment volume of 10 mL/kg bw
- Batch no.: 1703-5503

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/kg bw
No. of animals per sex per dose:
6 females (3 per 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. 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 mean of the body weight and body weight gain were calculated by Excel spreadsheet software.
The method used is not intended to allow statistical evaluation and the calculation of a precise LD50 value.

Results and discussion

Effect levels
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
>= 2 000 - <= 5 000 mg/kg bw
Based on:
act. ingr.
Remarks on result:
other: GHS category: 5 or unclassified
Mortality:
No death occurred at 2000 mg/kg bw single oral dose. All female rats in step 1 and step 2 survived until the end of the 14-day observation period.
Clinical signs:
In group 1 treated with 2000 mg/kg bw dose clinical sign of reaction comprised black faeces (3 cases of 57 observations) and black colored diarrhoea (8/57). Black faeces (score +1) were observed in all animals on Day 1. Black colored diarrhoea (score +1, +2, +4) was recorded in two animals on the treatment day between 1 and 4 hours after the treatment.

In group 2 treated with 2000 mg/kg bw dose clinical sign of reaction comprised black faeces (3 cases of 57 observations) and black colored diarrhoea (4/57). Black faeces (score +1) were observed in all animals on Day 1. Black colored diarrhoea (score +1, +2, +4) was recorded in animal No.: 6185 on the treatment day between 1 and 4 hours after the treatment.

Body weight:
A body weight loss was observed in one female treated with 2000 mg/kg bw between Day 7 and Day 15. This body weight loss was below 10 % (approx. 9.4 %). The animal gained body weight again until the end of study, thus it can be assessed as an individual variation without toxicological meaning. The body weight development was not affected in all other animals.
Gross pathology:
All animals treated with 2000 mg/kg bw survived until the scheduled necropsy on Day 15.
At necropsy pale liver was observed in one animal. This alteration was not considered to be related to the test item, but was regarded as an individual variation. Slight hydrometra was found in one animal, which is a physiological finding and related to the estrous cycle of the animal. No pathological changes were found related to the treatment with the test item during the macroscopic examination of animals treated with 2000 mg/kg bw.

Any other information on results incl. tables

Table 2: Summary of Mortality, Post-treatment observation period (14 days) 

Groups

Treatment

Lethality

Test Item

Dose
(mg/kg bw)

Females

1

Solubilised Sulphur Black 1
Step 1

2000

0/3

2

Solubilised Sulphur Black 1
Step 2

2000

0/3

Table 3: Summary of Clinical Symptoms

Groups

Treatment

Symptoms

Incidence

Test Item

Dose
(mg/kg bw)

1

Solubilised Sulphur Black 1
Step 1

2000

Black faeces

3/57

Diarrhoea (black)

8/57

Normal

46/57

2

Solubilised Sulphur Black 1
Step 2

2000

Black faeces

3/57

Diarrhoea (black)

4/57

Normal

50/57

 

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 4: Summary of Body Weights (g)

 

FEMALES

 

Day 0

Day 7

Day 15

 

 

 

 

 

Group 1: Solubilised Sulphur Black 1 

                2000 mg/kg bw, Step 1

 

 

 

 

 

 

 

 

Group size:

 

3

3

3

Mean: (g)

 

177.7

205.0

222.0

SD:

 

2.08

7.94

2.00

 

 

 

 

 

FEMALES

 

Day 0

Day 7

Day 15

 

 

 

 

 

Group 2:  Solubilised Sulphur Black 1                  

                 2000 mg/kg bw, Step 2

 

 

 

 

 

 

 

Group size:

 

3

3

3

Mean: (g)

 

201.0

227.7

227.7

SD:

 

1.00

4.73

14.47

 

 

 

 

 

 

Table 5: Summary of Body Weight Gains (g)

 

FEMALES

 

Day 0-7

Day 7-15

Day 0-15

 

 

 

 

 

Group 1: Solubilised Sulphur Black 1               

                2000 mg/kg bw, Step 1

Group size:

 

3

3

3

Mean: (g)

 

27.3

17.0

44.3

SD:

 

6.35

6.56

2.52

 

 

 

 

 

FEMALES

 

Day 0-7

Day 7-15

Day 0-15

 

 

 

 

 

Group 2:  Solubilised Sulphur Black 1                                          

                 2000 mg/kg bw, Step 2

 

 

 

Group size:

 

3

3

3

Mean: (g)

 

26.7

0.0

26.7

SD:

 

4.62

19.16

14.57

 

 

 

 

 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
The method used is not intended to allow statistical evaluation and the calculation of a precise LD50 value.
Conclusions:
In an acute oral toxicity assay (acute toxicity class method, ATC) according to OECD guideline 423, an LD50 of above 2000 mg/kg bw was determined.
Executive summary:

The acute oral toxicity of the test item was determined in an in vivo toxicity test according to OECD guideline 423. The 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 young (8 weeks) female Wistar rats. No animal died in the first step at 2000 mg/kg bw, so treatment with 2000 mg/kg bw was repeated on further three female rats. No animal died in the second step, too. Since the stopping criteria of Annex 2d of OECD Guideline No. 423 were met, the test was finished.

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 mortality was noted at a single oral dose of 2000 mg/kg bw. In the first step and second step, disturbance of the autonomic functions (diarrhoea) was observed in animals on the treatment day between 1 and 4 hours after the treatment. Black coloured faces/diarrhea observed was related to the physical property of the test item.

A body weight loss was observed in one female of group 2 in the second week. This body weight loss was not strong (ca. 9.7 %) and the animal gained body weight until the end of the study, thus it can be assessed as an individual variation without toxicological meaning. The mean body weight of both groups corresponded to their species and age throughout the study. 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 LD50value. Therefore, the test item was ranked into classes of the Globally Harmonized Classification System (GHS) described in the OECD Guideline No. 423. According to the classification system of the GHS, a LD50 of above 2000 mg/kg bw was determined.

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