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Diss Factsheets

Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 March 2020 - 07 May 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Test conducted in accordance with OECD Test Guideline and in accordance with GLP. All validity criteria were met.

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:
2001
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)
Version / remarks:
EC No 440/2008, part B
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.1100 (Acute Oral Toxicity)
Version / remarks:
2002
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: JMAFF Guidelines
Version / remarks:
2000, including the most recent revisions.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test type:
acute toxic class method
Limit test:
no

Test material

Constituent 1
Test material form:
liquid
Details on test material:
Storage conditions: At room temperature protected from light
Physical description: Light orange liquid

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl: WI(Han). Condition: Outbred, SPF-Quality
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Young adult animals (approximately 8-10 weeks old) were selected
- Weight at study initiation: 143 to 178 g
- Fasting period before study: Animals were deprived of food overnight (for a maximum of 20 hours) prior to dosing and until 3-4 hours after administration of the test item. Water was available.
- Housing: On arrival and following assignment to the study, animals were group housed (up to 3 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon MIV type; height 18 cm.) containing sterilized wooden fibers as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. The room in which the animals were kept was documented in the study records.
Animals were separated during designated procedures/activities. Each cage was clearly labeled.
- Historical data: Not provided
- Diet: ad libitum
Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures. The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
- Water: ad libitum
Municipal tap-water was freely available to each animal via water bottles.
Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility. It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.
- Acclimation period: The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days before the commencement of dosing.
- Microbiological status when known: Not reported
- Method of randomisation in assigning animals to test and control groups: Not reported - animals were assigned to the study at the discretion of the coordinating biotechnician, with all animals within ± 20 % of the sex mean body weights. Animals in poor health or at extremes of body weight range were not assigned to the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Daily mean temperature during the study period was 20 to 21°C
- Humidity (%): Daily mean relative humidity of 43 to 52 %
- Air changes (per hr): => 10, with 100 % fresh air (no recirculation)
- Photoperiod (hrs dark / hrs light): 12 h light / 12 h dark

IN-LIFE DATES: From: 27 March 2020 To: 07 May 2020

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
VEHICLE
N/A

MAXIMUM DOSE VOLUME APPLIED: 2000 mg/kg body weight (bw)

DOSAGE PREPARATION (if unusual): The test item was dosed as received.
A single dose of test item was administered to the appropriate animals by oral gavage on Day 1, using a syringe with a plastic gavage cannula attached.
The dose volume for each animal was based on the body weight measurement prior to dosing. Dose volume (mL/kg body weight) was calculated as follows: Dose level (g/kg) / spec.gravity.
The dosing formulations were stirred continuously during dose administration protected from light.

CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose:
The dose levels were based on the OECD test guidelines and were selected from the series 5 (lowest dose level), 50, 300 and 2000 (highest dose level) mg/kg bw. The starting dose level should be the one that is likely to produce mortality in at least some of the animals and was selected based on available toxicity data of the test item.
Doses:
300 and 2000 mg/kg bw
No. of animals per sex per dose:
3 females at 300 mg/kg bw
6 females (in 2 groups) at 2000 mg/mg bw
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Post-dose observations were performed at periodic intervals on the day of dosing (at least three times) and once daily thereafter. Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.
Animals were weighed individually on Day 1 (pre-dose), 8 and 15. A fasted weight was recorded on the day of dosing.
- Necropsy of survivors performed: yes
- Clinical signs including body weight: yes
- Other examinations performed: Maroscopic abnormalities.

Post-dose observations:
All the animals were examined for reaction to dosing. The onset, intensity and duration of these signs was recorded (if appropriate). Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored.
Statistics:
No statistical analysis was performed (The method used is not intended to allow the calculation of a precise LD50 value).
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.
The oral LD50 value of the test item was ranked within the following ranges: 0-5, 5-50, 50- 300 or 300-2000 mg/kg b.w. or as exceeding 2000 mg/kg bw. The LD50 cut-off value was established based on OECD guideline 423.

Results and discussion

Preliminary study:
N/A
Effect levels
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
None
Clinical signs:
At 300 mg/kg, hunched posture and erected fur were noted for all animals on Day 1.
At 2000 mg/kg, hunched posture, erected fur, closed eyes, uncoordinated movements, decreased activity, shallow breathing and breathing with abnormal sounds were observed for the animals between Days 1 and 3. One animal showed a prostrate posture and was cold to the touch on Day 1.
Body weight:
The body weight gain shown by the animals over the study period was considered to be similar to that expected for normal untreated animals of the same age and strain.
Gross pathology:
No abnormalities were found at macroscopic post-mortem examination of the animals.
Other findings:
The oral LD50 value of the test item in Wistar Han rats was established to exceed 2000 mg/kg body weight.

Any other information on results incl. tables

Table 1. Individual mortality

Group

Dose Level (mg/kg bw)

Sex

Animal

Cage

Removal

Removal

Pathology

Day

Week

Date

Reason

1

300

Female

1

1

15

3

21Apr20

Terminal euthanasia

2

1

15

3

21Apr20

Terminal euthanasia

3

1

15

3

21Apr20

Terminal euthanasia

2

2000

Female

4

2

15

3

29Apr20

Terminal euthanasia

5

2

15

3

29Apr20

Terminal euthanasia

6

2

15

3

29Apr20

Terminal euthanasia

3

2000

Female

7

3

15

3

07May20

Terminal euthanasia

8

3

15

3

07May20

Terminal euthanasia

9

3

15

3

07May20

Terminal euthanasia

 

Table 2. Individual clinical observations

Group 1 Sex: Female 300 mg/kg

 

Observation Type: All Types

Day(s) Relative to Start Date

1
1 h

1
2 h

1
3 h

2

3

4

5

6

7

8

9

10

11

12

13

14

15

1

Hunched posture

X

X

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Fur, erected

X

X

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

2

Hunched posture

-

X

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Fur, erected

X

X

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

3

Hunched posture

-

X

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Fur, erected

X

X

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Group 2 Sex: Female 2000 mg/kg

 

Observation Type: All Types

Day(s) Relative to Start Date

1
1 h

1
2 h

1
3 h

2

3

4

5

6

7

8

9

10

11

12

13

14

15

4

Hunched posture

-

X

X

X

X

-

-

-

-

-

-

-

-

-

-

-

-

Fur, erected

X

X

X

X

-

-

-

-

-

-

-

-

-

-

-

-

-

Eye closed, left, completely

-

X

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Eye closed, right, completely

-

X

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Uncoordinated, slight

-

X

X

X

-

-

-

-

-

-

-

-

-

-

-

-

-

Activity decreased

-

X

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

5

Breathing, abnormal sounds

-

X

-

X

X

-

-

-

-

-

-

-

-

-

-

-

-

Breathing, shallow

-

-

-

X

-

-

-

-

-

-

-

-

-

-

-

-

-

Hunched posture

-

X

X

X

X

-

-

-

-

-

-

-

-

-

-

-

-

Fur, erected

-

X

X

X

-

-

-

-

-

-

-

-

-

-

-

-

-

Uncoordinated, slight

-

X

X

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Group 3 Sex: Female 2000 mg/kg

 

Observation Type: All Types

Day(s) Relative to Start Date

1
1 h

1
2 h

1
3 h

2

3

4

5

6

7

8

9

10

11

12

13

14

15

7

Hunched posture

-

X

X

X

X

-

-

-

-

-

-

-

-

-

-

-

-

Fur, erected

X

X

X

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Uncoordinated, moderate

-

X

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Uncoordinated, slight

-

-

X

-

-

-

-

-

-

-

-

-

-

-

-

-

-

8

Hunched posture

-

X

X

X

X

-

-

-

-

-

-

-

-

-

-

-

-

Fur, erected

X

X

X

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Uncoordinated, moderate

-

X

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Uncoordinated, slight

-

-

X

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Activity decreased

-

X

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

9

Hunched posture

-

X

X

X

X

-

-

-

-

-

-

-

-

-

-

-

-

Fur, erected

X

X

X

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Eye closed, left, completely

-

X

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Eye closed, right, completely

-

X

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Uncoordinated, moderate

-

X

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Uncoordinated, slight

-

-

X

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Activity decreased

-

X

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The oral LD50 value of the test item in Wistar Han rats was established to exceed 2000 mg/kg body weight.
Executive summary:

A study was performed in accordance with OECD 423 (2001), in order to determine the potential for acute oral toxicity of the test item.

The test item was administered in a single dose to female rats at two defined doses. Initially, the test item was administered by oral gavage to three female Wistar Han rats at 300 mg/kg bw. In a stepwise procedure two additional groups of three females were dosed at 2000 mg/kg bw. All animals were subjected to daily observations and weekly determination of body weight. Macroscopic examination was performed after terminal sacrifice (Day 15).

No mortality occurred in any of the animals. At 300 mg/kg, hunched posture and erected fur were noted for all animals on Day 1. At 2000 mg/kg, hunched posture, erected fur, closed eyes, uncoordinated movements, decreased activity, shallow breathing and breathing with abnormal sounds were observed for the animals between Days 1 and 3. One animal showed a prostrate posture and was cold to the touch on Day 1. The body weight gain shown by the animals over the study period was considered to be normal. No abnormalities were found at macroscopic post-mortem examination of the animals.

The oral LD50 value of the test item in Wistar Han rats was established to exceed 2000 mg/kg body weight. Based on the conditions of this test, the test item would not be classified for acute oral toxicity, in accordance with CLP Regulation No 1272/2008 on Classification, Labelling and Packaging of Substances and Mixtures.

 

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