1,2-Ethandiylbis(oxy-2,1-ethandiyl)-dimethansulfonat

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3 May - 1 June 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Version / remarks:

17 December 2001

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.1 (Acute Toxicity (Oral))

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-[2-(2-hydroxyethoxy)ethoxy]ethanol,methanesulfonic acid

Cas Number:

80322-82-3

Molecular formula:

C8H18O8S2

IUPAC Name:

2-[2-(2-hydroxyethoxy)ethoxy]ethanol,methanesulfonic acid

impurity 1

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Oxidane

impurity 2

Reference substance name:

1,2-Ethandiylbis(oxy-2,1-ethandiyl)-monomethanesulfonat

IUPAC Name:

1,2-Ethandiylbis(oxy-2,1-ethandiyl)-monomethanesulfonat

Test material form:

liquid

Details on test material:

Appearance: Dark yellow liquid
Batch No.: 01110008
Storage: -5 – 40 °C, container tightly closed, protected from light

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, 8 weeks old in first and second step, 8-9 weeks old in third and fourth step and 9-10 weeks old in fifth and sixth step, as well
- Body weight range: at starting (first step): 171 - 173 g
- Body weight range at starting (second step): 170 - 171 g
- Body weight range at starting (third step): 172 - 174 g
- Body weight range at starting (fourth step): 174 - 181 g
- Body weight range at starting (fifth step): 178 - 183 g
- Body weight range at starting (sixth step): 193 - 198 g
- Acclimatization time: 5 days in the first step, 7 days in the second step, 11 days in the third step, 12 days in the fourth step, 18 days in the fifth step and 19 days in the sixth step

ENVIRONMENTAL CONDITIONS
- Animal health: Only healthy animals were used for the study. Health status was certified by the study director.
- Room: 13/4
- 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:

other: Helianthi annui oleum raffinatum

Details on oral exposure:

VEHICLE
- Name: Helianthi annui oleum raffinatum
- Batch number: 2111-4480
- Date of expiration: 28.02.2023
- Produced by: Parma Produkt 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 of 200, 30 and 5 mg/mL. Formulations were prepared just before the administration and stirred continuously during the treatment.

CLASS METHOD
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. As two animals survived on Day 1, three further female rats were treated with the same dose of 2000 mg/kg bw. However, delayed deaths were observed between Day 4 and Day 5. All animals of step 1 died, one on Day 1, one on Day 4 and one on Day 5, as well as three animals of step 2 died on Day 5. Therefore, the test had to be continued, dosing further three female rats with the next lower dose of 300 mg/kg bw. As all animals survived on Day 1, three further female rats were treated with the same dose of 300 mg/kg bw. However, delayed deaths were observed between Day 6 and Day 7. All animals of step 3 died, one on Day 6 and two on Day 7, as well as three animals of step 4 died, one on Day 5 and two on Day 6. Therefore, the test had to be continued, dosing further three female rats with the next lower dose of 50 mg/kg bw. As no animal died in this fifth step, three further female rats were treated with the same dose of 50 mg/kg bw. No animal died in this sixth step as well, thus, the test was finished, as the stopping criteria of Annex 2d of OECD Guideline No. 423 was met.

Doses:

50, 300, 2000 mg/kg bw

No. of animals per sex per dose:

3 females per dose for each 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
5 days in the first step, 7 days in the second step, 11 days in third step, 12 days in fourth step, 18 days in fifth step and 19 days in sixth step of acclimatisation, treatment’s day, 14 days post treatment observation period, necropsy on Day 1, on Day 4, on Day 5, on Day 6, on Day 7 and on 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.

MEASUREMENT OF BODY WEIGHT
The body weights were recorded on day 0 (just before the treatment), on day 1, on day 4, on day 5, on day 6, 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:

200 mg/kg bw

Based on:

test mat.

Mortality:

All of three animals of group 1 (step 1) treated with a test item dose of 2000 mg/kg bw died during the study. One animal died on Day 1, one animal died on Day 4 and one animal died on Day 5. All of three animals of group 2 (step 2) treated with the same dose as group 1 died on Day 5. All of three animals of group 3 (step 3) treated with a test item dose of 300 mg/kg bw died during the study. One animal died on Day 6, two animals died on Day 7. All of three animals of group 4 (step 4) treated with the same dose as group 3 died during the study. One animal died on Day 5 and two animals died on Day 6. The delayed deaths seemed to be the consequence of systemic toxic effects of the test item.
No death occurred after the single oral dose of 50 mg/kg bw.

Clinical signs:

convulsions

diarrhoea

lethargy (hypoactivity)

observations of tremors

Body weight:

other body weight observations

Remarks:

The mean body weight and body weight gain data of group 1 (2000 mg/kg bw), group 2 (2000 mg/kg bw), group 3 (300 mg/kg bw), as well as group 4 (300 mg/kg bw) could not be evaluated, because of mortalities. A body weight loss was observed in one female treated with a single dose of 50 mg/kg bw between Day 7 and Day 15. This body weight loss was very slight (0.5 %) and the body weight of this animal exceeded the original body weight by the end of the study. Thus, it can be evaluated as an individual variation without toxicological meaning. The body weight development was undisturbed in all other animals of 50 mg/kg bw dose.

Gross pathology:

All of six rats treated with a dose of 2000 mg/kg bw and 300 mg/kg bw of the test item spontaneously died and were necropsied on the day found dead. All animals dosed with 50 mg/kg bw survived until the scheduled necropsy on Day 15.

In group 1, external and internal necropsy findings were observed. Anus was contaminated with faeces (external finding) in two animals. The internal findings were as follows: punctiform haemorrhages in the stomach, empty stomach, empty small intestines, pale liver and autolysis.

In group 2, external and internal necropsy findings were observed. Anus was contaminated with faeces (external finding) in all animals and the forelegs were bloody in two animals. The internal findings observed in all animals were as follows: smaller than normal spleen, attenuated stomach wall, erosions in the stomach, punctiform haemorrhages in the stomach and intestines full of liquor. Besides, autolysis was found in two animals.

In group 3, external and internal necropsy findings were observed. External findings were as follows: anus contaminated with faeces or bloody faeces and bloody forelegs. The internal findings were as follows: punctiform haemorrhages or haemorrhages in the stomach, colon full of bloody faeces, smaller than normal spleen, attenuated stomach wall, perforated cardia, slightly autolysed organs.

In group 4, external and internal necropsy findings were observed. External findings were as follows: anus contaminated with faeces and bloody forelegs. The internal findings were as follows: smaller than normal spleen, punctiform haemorrhages or haemorrhages in the stomach, attenuated stomach wall.

No pathological changes were found during the macroscopic examination in animals of groups 5 and 6.

Any other information on results incl. tables

Evaluation

All of six animals dosed with 2000 and 300 mg/kg bw Triethylene glycol dimethanesulfonate died during the study. No death occurred after the single oral dose of 50 mg/kg bw.

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

There was not any test item related effect found in body weights and body weight gains of animals dosed with 50 mg/kg bw during the study.

The internal and external findings observed in animals treated with 2000 and 300 mg/kg bw could be related to the effect of the test item, except autolysis, which is a normal process after death.

 

Autopsy revealed no treatment related pathological changes in animals dosed with 50 mg/kg bw.

Applicant's summary and conclusion

Interpretation of results:

Category 3 based on GHS criteria

Conclusions:

In conclusion, the LD50 of the test item Triethylene glycol dimethanesulfonate is 200 mg/kg bw by oral route in the rat, because there were three dead animals in the first and second step dosed with 300 mg/kg bw and no deaths occurred in the animals treated with 50 mg/kg bw. The GHS category is 3. The CLP category is 3.

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 body weight (bw) of the test item Triethylene glycol dimethanesulfonate as the starting dose in three female rats. As two animals survived on Day 1, three further female rats were treated with the same dose of 2000 mg/kg bw. However, delayed deaths were observed between Day 4 and Day 5. All animals of step 1 died, one on Day 1, one on Day 4 and one on Day 5, as well as three animals of step 2 died on Day 5. Therefore, the test had to be continued, dosing further three female rats with the next lower dose of 300 mg/kg bw. As all animals survived on Day 1, three further female rats were treated with the same dose of 300 mg/kg bw. However, delayed deaths were observed between Day 6 and Day 7. All animals of step 3 died, one on Day 6 and two on Day 7, as well as three animals of step 4 died, one on Day 5 and two on Day 6. Therefore, the test had to be continued, dosing further three female rats with the next lower dose of 50 mg/kg bw. As no animal died in this fifth step on day 1, three further female rats were treated with the same dose of 50 mg/kg bw. No animal died in this sixth step after 14 days, thus, the test was finished, as 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 dosage. Gross pathological examinations were carried out in animals that died on the 1^st, 4^th, 5^th, 6^th and 7^th day, as well as 15th day after dosage in surviving animals.

 

Lethality, Clinical Symptoms and Body weight

In the 2000 mg/kg bw dose group, all of six rats died between Day 1 and Day 5.
In the 300 mg/kg bw dose group, all of six rats died between Day 5 and Day 7.
No lethality was noted after a single oral dose of 50 mg/kg bw.

 

Following a single dose of 2000 mg/kg bw in the first step, CNS (central nervous system)- and emotion symptoms (decreased activity, tremor, tonic convulsion, clonic convulsion), disturbances of coordination (prone position, incoordination), decreased righting reflex, a decreased muscular tension (grip- and limb tone) and disturbances of autonomic functions (diarrhoea, piloerection) were observed between 30 minutes and 4 hours after the dosage and between Day 1 and Day 4, as well.

Following a single dose of 2000 mg/kg bw in the second step, CNS- and emotion symptoms (decreased activity, tremor, closed eyes), a disturbance of coordination (incoordination), decreased righting reflex, a decreased muscular tension (grip- and limb tone), disturbances of autonomic functions (diarrhoea, piloerection) and bloody forelegs were observed between 30 minutes and 4 hours after the dosage and between Day 1 and Day 4, as well.

 

Following a single dose of 300 mg/kg bw in the third step, a CNS- and emotion symptom (decreased activity), disturbances of autonomic functions (diarrhoea, piloerection) blood around the nose and bloody forelegs were observed between Day 4 and Day 6.

 

Following a single dose of 300 mg/kg bw in the fourth step, a CNS- and emotion symptom (decreased activity), disturbances of autonomic functions (diarrhoea, piloerection), bloody forelegs and blood around the nose were observed between Day 3 and Day 5.

 

Following a single dose of 50 mg/kg bw in steps fifth and sixth, no systemic toxic 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.

 

A body weight loss was observed in one female of group 5 (50 mg/kg bw) in the second week. This body weight loss was very slight, and the body weight of this animal exceeded the original body weight by the end of the study, thus it can be evaluated as an individual variation without toxicological meaning.

 

Gross Pathology

Six animals treated with 2000 mg/kg bw and 300 mg/kg bw died spontaneously during the study. Six animals treated with 50 mg/kg bw were sacrificed as scheduled during the study.

Autopsy revealed treatment related external and internal alterations in animals dosed with 2000 mg/ kg bw.

External findings were as follows:

• Anus: contaminated with faeces


• Forelegs: bloody

Internal findings were as follows:

• Stomach: haemorrhages, empty, erosion, attenuated wall


• Small intestines: empty


• Intestines: full of liquor


• Liver: pale


• Spleen: smaller than normal

 

Autopsy revealed treatment related external and internal alterations in animals dosed with 300 mg/kg body weight.

External findings were as follows:

• Anus: contaminated with faeces or bloody faeces


• Forelegs: bloody

Internal findings were as follows:

• Stomach: haemorrhages, attenuated wall, perforated cardia


• Colon: full of bloody faeces


• Spleen: smaller than normal

 

These external and internal changes were related to the presumable effect of the test item.

 

All organs of animals dosed with 50 mg/kg bw proved to be free of treatment related gross pathological changes.

 

Conclusion

The method used is not intended to calculate a precise LD₅₀ value. The test item was ranked into classes of the Globally Harmonized Classification System (GHS) described in the OECD Guideline No. 423 and given below:

Dose (mg/kg bw)	Mortality (dead/treated)	LD50 (mg/kg bw)	CLP category
2000	6/6	between 50 and 300	3
300	6/6
50	0/6

 

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

Dose (mg/kg bw)	Mortality (dead/treated)	LD50 (mg/kg bw)	CLP category
2000	6/6	between 50 and 300	3
300	6/6
50	0/6

 

In conclusion, the LD₅₀ of the test item Triethylene glycol dimethanesulfonate is 200 mg/kg bw by oral route in the rat, because there were three dead animals in the first and second step dosed with 300 mg/kg bw and no deaths occurred in the animals treated with 50 mg/kg bw. The GHS category is 3. The CLP category is 3.