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Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 February 2020 - 13 March 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

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:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
(R*,R*)-1,4-dimercaptobutane-2,3-diol
EC Number:
222-468-7
EC Name:
(R*,R*)-1,4-dimercaptobutane-2,3-diol
Cas Number:
3483-12-3
Molecular formula:
C4H10O2S2
IUPAC Name:
1,4-disulfanylbutane-2,3-diol
Specific details on test material used for the study:
Batch No.: 44606300 / 50774
Storage: refrigerator, under Nitrogen

Test animals

Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Species and strain: Han:WIST rats
- 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, 10 weeks old in first, second and third step
- Body weight range: at starting (first step): 198 - 200 g
- Body weight range at starting (second step): 202 - 207 g
- Body weight range at starting (third step): 199 - 204 g
- Acclimatization time: 19 days in first step, 20 days in second step and 21 days in third step
ENVIRONMENTAL CONDITIONS
- Animal health: Only healthy animals were used for the study.
- Room: 13/2
- Housing: Group caging (3 animals/cage)
- Cage type: Type III polypropylene/polycarbonate.
- Light: Artificial light, from 6 a.m. to 6 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 parameters 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.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
VEHICLE
- Name: Aqua purificata
- Batch number: 201910069
- Date of expiration: 21.04.2020
- Produced by: Magilab Kft.
- Formulation 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 and 30 mg/mL.
CLASS METHOD
- Justification of the doses.
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 female rats. All animals died in the first step at 2000 mg/kg bw dose level, so further three female rats were treated with the lower (300 mg/kg bw) dose. Only one animal died in second step, so further three female rats were treated with the same (300 mg/kg bw) dose. Only one animal died in third step, too. Test was finished, because the stopping criteria of Annex 2d of OECD Guideline No. 423 (presented in Appendix VII) was met.
Doses:
2000 mg/kg body weight (first step)
300 mg/kg body weight (second and third step)
No. of animals per sex per dose:
3 females per dose (first step)
3 females per dose (second step)
3 females per dose (third step)
Control animals:
no
Details on study design:
- PROCEDURE: 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. The groups were observed for up to min. 24 hours before the next step was treated.
- DURATION OF THE EXPERIMENTAL PERIOD: 19 days in first step, 20 days in second step and 21 days in third step of acclimatization, treatment’s day, 14 days post-treatment observation period, necropsy on the treatment day, Day1 and Day 15.
- FREQUENCY OF OBSERVATIONS FOR 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.
- FREQUENCY OF OBSERVATIONS FOR GENERAL STATE, EXTERNAL APPEARANCE, BEHAVIOR AND CLINICAL SYMPTOMS: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 once 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 WEIGHT: The body weights were recorded on day 0 (just before the treatment), on day 1, 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 were observed, and any abnormality was recorded with details of its location, colour, shape and size.
Statistics:
No statistical analysis was performed.

Results and discussion

Effect levels
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
>= 300 - < 2 000 mg/kg bw
Based on:
test mat.
Mortality:
All rats dosed at 2000 mg/kg bw 1,4-Dithiothreitol died on the treatment day 30 minutes after the treatment.
Altogether two animals treated with 300 mg/kg bw single oral dose of the test item died during the study. One rat (No.: 7288) of group 2 died on the treatment day 30 minutes after the treatment and one rat (No.: 7284) of group 3 died on Day 1, as well.
The deaths seemed to be consequences of systemic toxic effect of the test item.
Clinical signs:
In group 1 treated with 2000 mg/kg bw dose clinical sign of reaction comprised of tonic convulsion (3 cases of 3 observations) and clonic convulsion (3/3). These symptoms (score 4) were observed in all animals prior to deaths.
In group 2 treated with 300 mg/kg bw dose clinical sign of reaction comprised of decreased activity (10 cases of 39 observ.), tremor (4/39), incoordination (8/39), bedding chewing (7/39), closed eyes (10/39), piloerection (10/39), increased respiration rate (4/39), tonic convulsion (1/39) and clonic convulsion (1/39). Decreased activity (score -2, -3), tremor (score +2), incoordination (score +2), bedding chewing (score +2), closed eyes (score +1, +2, +3), piloerection (score +1, +2) and increased respiration rate (score +2) were detected in two animals (No.: 7286, 7287). Tonic convulsion (score +4) and clonic convulsion (score +4) were recorded in animal No.: 7288.
These symptoms were observed on the treatment day between 30 min and 4 h after the treatment.
In group 3 treated with 300 mg/kg bw dose clinical sign of reaction comprised of decreased activity (15 cases of 43 observ.), vocalisation (4/43), irritability (5/43), tremor (9/43), incoordination (9/43), bedding chewing (7/43), closed eyes (15/43), piloerection (15/43), increased respiration rate (8/43), tonic convulsion (6/43), clonic convulsion (6/43), abnormal gait (7/43) and lateral position (1/43). Decreased activity (score -2, -3), vocalisation (score +2), irritability (score +2), tremor (score +1, +2, +4), tonic convulsion (score +1, +2), clonic convulsion (score +1, +2), abnormal gait (score +2), incoordination (score +2), closed eyes (score +1, +2, +3), piloerection (score +1, +2) and increased respiration rate (score +1, +2) were detected in all animals. Bedding chewing (score +2) was recorded in two animals (No.: 7283, 7289). Lateral position (score +4) was found in animal No.: 7284.
These symptoms were observed on the treatment day between 30 min and 4 h after the treatment.
Body weight:
The mean body weight and body weight gain data of group 1 (2000 mg/kg bw) could not be evaluated, because of mortalities. The mean body weight of surviving animals treated with 300 mg/kg bw dose (group 2 and 3) corresponded to their species and age throughout the study.
Gross pathology:
Three rats (No.: 7280, 7281, 7282) treated with 2000 mg/kg bw dose and one animal (No.: 7288) treated with 300 mg/kg bw dose of the test item spontaneously died and were necropsied on the treatment day and one animal (No.: 7284) of the 300 mg/kg bw dose spontaneously died and was necropsied on Day 1, as well.
Four animals of the 300 mg/kg bw dose (No.: 7286, 7287, 7283, 7289) survived until the scheduled necropsy on Day 15.
An external necropsy finding as blood around the nose and internal necropsy findings as mucous membrane erosion and growing thin in the stomach and haemorrhages in the stomach lining were observed in animal No.: 7284 of group 3. Stomach was full of gas and the organs were partially autolysed in same animal.
Severe hydrometra was recorded in two animals (No.: 7286, 7287) of group 2.

Any other information on results incl. tables

EVALUATION: All of three animals treated with 2000 mg/kg bw dose of the test item died during the study.

Two out of six animals treated with 300 mg/kg bw dose of the test item died during the study.

The observed clinical signs were related to the systemic toxic effect of the test item.

There was no notable change in body weights of survivor animals.

The pathological alterations observed in 300 mg/kg bw dose were related to the effect of the test item, except autolysis. Autolysis is normal process after death.

Hydrometra was a physiological finding and connected to the oestrus cycle of the animals.

Applicant's summary and conclusion

Interpretation of results:
Category 4 based on GHS criteria
Conclusions:
In conclusion, the LD50 of the test item 1,4-Dithiothreitol (CAS No. 3483-12-3) is 500 mg/kg bodyweight by oral route in the rat, because there were three dead animals in first step at 2000 mg/kg bw dose. The GHS category is 4. The CLP category is 4.
Executive summary:

General Information:

All criteria for the validity of the performed experiments have been met.

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 female rats. All animals died in the first step at 2000 mg/kg bw dose level, so further three female rats were treated with the lower (300 mg/kg bw) dose. Only one animal died in second step, so further three female rats were treated with the same (300 mg/kg bw) dose. Only one animal died in third step, too. Test was finished, because the stopping criteria of Annex 2d of OECD Guideline No. 423 (presented in Appendix VII) was met.

Animals were weighed, observed for lethality and toxic symptoms for 14 days after the treatment. Gross pathological examination was carried out in animals that died on the treatment day and on Day 1, as well as 15th day after the treatment in survivor animals.

Lethality, Clinical Symptoms and Body weight:

In 2000 mg/kg bw 1,4-Dithiothreitol dose group, three rats died on the treatment day 30 minutes after the treatment.

In 300 mg/kg bw dose group, two out of six rats died on the treatment day 30 minutes after the treatment and on Day 1, as well. Four rats survived until the end of the 14-day observation period in the same dose group.

In the first step, CNS symptoms (tonic convulsion, clonic convulsion) were observed in animals on the treatment day prior to death.

In the second step, CNS symptoms (decreased activity, tremor, bedding chewing, closed eyes, tonic convulsion, clonic convulsion), a disturbance of coordination (incoordination) and disturbances of autonomic functions (piloerection, increased respiration rate) were observed in animals on the treatment day between 30 minutes and 4 hours after the treatment.

In the third step, CNS symptoms (decreased activity, vocalisation, irritability, tremor, bedding chewing, closed eyes, tonic convulsion, clonic convulsion), disturbances of coordination (incoordination, abnormal gait, lateral position) and disturbances of autonomic functions (piloerection, increased respiration rate) were observed in animals on the treatment day between 30 minutes and 4 hours after the treatment.

The body weight development was undisturbed in all survivor animals.

Gross Pathology:

Three animals treated with 2000 mg/kg bw dose and two animals treated with 300 mg/kg bw dose died spontaneously during the study. Four animals of the 300 mg/kg dose were sacrificed as scheduled during the study.

All organs of all animals in 2000 mg/kg bw dose proved to be free of treatment related gross pathological changes.

Autopsy revealed treatment related an external change (blood around the nose) and internal alterations in the stomach (full of gas, mucous membrane erosion, growing thin, heamorrhages) in one died animal of 300 mg/kg bw dose group.

All organs of all survivor animals proved to be free of treatment related gross pathological changes.

Evaluation:

The method used is not intended to allow the calculation of a precise LD50 value.

The test item was ranked into classes of Globally Harmonized Classification System (GHS) described in the OECD Guideline No. 423 as below:

 Dose (mg/kg bw)  Mortality (dead/treated)  LD50 (mg/kg bw)  GHS category
 2000  3/3  between 300 and 2000    4
 300  2/6

The test item was ranked into classes of the current EU Regulation on classification, labeling and packaging (CLP) (EC) No 1272/2008 CLP category as below:

 Dose (mg/kg bw)  Mortality (dead/treated)  LD50 (mg/kg bw)  CLP category
 2000  3/3     between 300 and 2000     4
 300  2/6

In conclusion, the LD50 of the test item 1,4-Dithiothreitol (CAS No. 3483-12-3) is 500 mg/kg bodyweight by oral route in the rat, because there were three dead animals in first step at 2000 mg/kg bw dose. The GHS category is 4. The CLP category is 4.