Registration Dossier

Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Feb - Mar 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Version / remarks:
December 17, 2001
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Landesanstalt für Umwelt Baden-Württemberg
Test type:
acute toxic class method
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: L2018-137
- Expiration date of the Batch: February 01, 2021
- Purity: 99.6 % (tolerance ± 1 %)
- Physical state / color: solid / yellowish

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was prepared for each test group shortly before administration by stirring with a high-speed homogenizer (Ultra-Turrax) and a magnetic stirrer. The homogeneity of the test item preparation during administration was ensured by stirring with a magnetic stirrer.

FORM AS APPLIED IN THE TEST (if different from that of starting material) : suspension

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI (Han) SPF
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Wiga GmbH, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approx. 9 - 10 weeks
- Weight at study initiation: animals of comparable weight (± 20 % of the mean weight)
- Fasting period before study: feed was withdrawn from the animals at least 16 hours before administration, but water was available ad libitum
- Housing: single housing
- Diet (e.g. ad libitum): R/M maintenance, low phytoestrogen; Ssniff, Spezialdiäten GmbH (Experimental Animal Diets Inc., 59494 Soest, Germany), ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: at least 5 days before administration

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ° C ± 3 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
Ph.Eur.
Details on oral exposure:
VEHICLE
- Concentration in vehicle: 6 and 40 g/100 mL
- Amount of vehicle (if gavage): 5 mL/kg bw
- Justification for choice of vehicle: good homogeneity of the test substance in corn oil Ph.Eur.

MAXIMUM DOSE VOLUME APPLIED: 2000 mg/kg bw

DOSAGE PREPARATION (if unusual): The test item was prepared for each test group shortly before administration by stirring with a high-speed homogenizer (Ultra-Turrax) and a magnetic stirrer.

CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose: By request of the sponsor a starting dose of 300 mg/kg bw was chosen in the first step with 3 female animals.
Doses:
300 and 2000 mg/kg bw
No. of animals per sex per dose:
3
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Individual body weights shortly before administration (day 0), weekly thereafter, on the last day of observation and on the day of death starting with study day 1. Clinical signs for each animal were recorded several times on the day of administration and at least once during each workday thereafter.
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight,organ weights, gross-pathology
Statistics:
Calculations were performed using Microsoft Excel 2010 and checked with a calculator.

Results and discussion

Effect levels
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
No mortality occurred in either the single 300 mg/kg bw test group or in the second 2000 mg/kg bw test group.
In the first 2000 mg/kg bw test group, one animal was found dead on study day 1.
Clinical signs:
No clinical signs were observed in the first 300 mg/kg bw and the second 2000 mg/kg bw test group during clinical examination.
In two animals of the first 2000 mg/kg bw test group reduced defecation was seen on day 1 only.
In the other animal of this test group, which died on day 1, dyspnea, staggering and impaired general state was observed from hour 4 until hour 5 after administration.
Body weight:
All animals gained weight in a normal range throughout the study period.
Gross pathology:
The following macroscopic pathologic findings were observed in the single animal that died in the first 2000 mg/kg bw test group: dark red spotted discoloration of the liver.
There were no macroscopic pathological findings in any animal sacrificed at the end of the observation period (2000 mg/kg bw: 5 females; 300 mg/kg bw: 3 females).

Any other information on results incl. tables

Table 2: Mortality

Dose [mg/kg bw]

2000

2000

300

Sex

Female

Female

Female

Administration

1

2

1

No. of animals

3

3

3

Mortality [animals]

1

No mortality

No mortality

Table 3: Maximum incidence of clinical signs

Dose [mg/kg bw]

2000

2000

300

Sex

Female

Female

Female

Administration

1

2

1

No. of animals

3

3

3

Animal No.

R 482

R 483

R 484

R 485

R 486

R 487

R 476

R 477

R 478

Abnormalities

 

 

 

-

-

-

-

-

-

Dyspnoea

-

h4 – h5

-

-

-

-

-

-

-

Impaired general state

-

h4 – h5

-

-

-

-

-

-

-

Staggering

-

h4 – h5

-

-

-

-

-

-

-

Reduced defecation

d1

-

d1

-

-

-

-

-

-

Mortality

-

d1

-

-

-

-

-

-

-

h hour

d day

Table 4: Individual body weights

Dose [mg/kg bw]

2000

2000

300

Administration

1

2

1

Animal No.

R 482

R 483

R 484

Mean

SD

R 485

R 486

R 487

Mean

SD

R 476

R 477

R 478

Mean

SD

Body weight at study day [g]:

0

1

7

14

 

183

-

200

203

 

183

174

-

-

 

184

-

196

203

 

183.3

-

198.0

203.0

 

0.58

-

2.83

0.00

 

182

-

201

207

 

185

-

198

210

 

183

-

197

212

 

183.3

-

198.7

209.7

 

1.53

-

2.08

2.52

 

178

-

199

211

 

173

-

199

207

 

174

-

201

208

 

175.0

-

199.7

208.7

 

2.65

-

1.15

2.08

SD Standard deviation

Table 5: Gross pathology

Dose [mg/kg bw]

2000

2000

300

Administration

1

2

1

No. of animals

3

3

3

Animal No.

R 482

R 483

R 484

R 485

R 486

R 487

R 476

R 477

R478

Macroscopic pathologic abnormalities

-

Liver: dark red, spotted discolored

-

-

-

-

-

-

-

Applicant's summary and conclusion

Interpretation of results:
Category 5 based on GHS criteria
Conclusions:
Under the conditions of this study the median lethal dose of the test substance after oral administration was found to be > 2000 mg/kg bw in rats.
Executive summary:

In an acute oral toxicity study performed according to the Acute Toxic Class Method, doses of 2000 and 300 mg/kg bw of the test item (preparations in corn oil Ph.Eur.) were administered by gavage to three test groups of three fasted Wistar rats each (2000 mg/kg bw in 6 females and 300 mg/kg bw in 3 females).

The following test substance-related clinical observations were recorded, clinical signs occurred within one day after administration:

2000 mg/kg (first test group):

- Mortality in one animal

- Reduced defecation in two animals

- Dyspnoea in one animal

- Staggering in one animal

- Impaired general state in one animal

- Macroscopic pathological findings in the animal that died:

- Dark red spotted discoloration of the liver

2000 mg/kg (second test group):

- No mortality occurred

- No clinical signs were observed

300 mg/kg (single test group):

- No mortality occurred

- No clinical signs were observed.

The body weights of the surviving animals increased within the normal range throughout the study period.

There were no macroscopic pathological findings in any animal sacrificed at the end of observation period (8 females).

The acute oral LD50 was calculated to be: LD50, oral, rat > 2000 mg/kg bw.