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Toxicological information

Acute Toxicity: oral

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Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 January 2014 to 06 February 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 425 (Acute Oral Toxicity: Up-and-Down Procedure)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test type:
up-and-down procedure
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- Appearance: Off-white solid
- Storage Conditions: room temperature in the dark, in a closed container under nitrogen

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Sprague-Dawley (Hsd:Sprague Dawley(TM) CD(TM))
- Age at study initiation: Eight to twelve weeks of age
- Weight at study initiation: 164 to 195 g. The body weight variation did not exceed ±20 % of the body weight of the initially dosed animal.
- Fasting period before study: Yes; animals were subjected to an overnight fast immediately before dosing and for approximately three to four hours after dosing.
- Housing: The animals were individually housed in suspended solid-floor polypropylene cages furnished with woodflakes. The animals were provided with environmental enrichment items.
- Diet: ad libitum
- Water: ad libitum access to mains drinking water
- Acclimation period: At least five days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 25 °C
- Humidity: 30 to 70 % (relative)
- Air changes: The rate of air exchange was at least fifteen changes per hour
- Photoperiod: Lighting was controlled by a time switch to give twelve hours of continuous light (06:00 to 18:00) and twelve hours of darkness.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: distilled water
Details on oral exposure:
VEHICLE
- Concentration in vehicle: The concentration of test material in the vehicle was 17.5, 55 and 2000 mg/mL for the 175, 500 and 2000 mg/kg dose levels, respectively.

MAXIMUM DOSE VOLUME APPLIED: The dose volume was 10 mL/kg
Doses:
175, 500 and 2000 mg/kg
No. of animals per sex per dose:
6 females were dosed overall. The first animal was dosed at 175 mg/kg. Further animals were then treated based on the short-term results of the previously treated animal. Overall, 2 animals were dosed at 175 mg/kg, 3 animals were dosed at 550 mg/kg and 1 animal was dosed at 2000 mg/kg.
Control animals:
no
Details on study design:
- Dosing regimen: Treatment of animals was sequential (Table 1). Sufficient time (at least 48 hours) was allowed between each individual animal to confirm the outcome of the previously dosed animals.
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: The animals were observed for deaths or overt signs of toxicity 0.5, 1, 2 and 4 hours after dosing and subsequently once daily for up to fourteen days. Individual body weights were recorded prior to dosing and seven and fourteen days after treatment or at death.
- Necropsy of survivors performed: Yes. At the end of the observation period the surviving animals were killed by cervical dislocation. All animals were subjected to gross pathological examination. This consisted of an external examination and opening of the abdominal and thoracic cavities for examination of major organs. The appearance of any macroscopic abnormalities was recorded. No tissues were retained.
Statistics:
The oral LD50 was calculated by the maximum likelihood method. Data evaluations also included the relationship, if any, between the exposure of the animal to the test material and the incidence and severity of all abnormalities including behavioural and clinical observations, gross lesions, body weight changes, mortality and any other toxicological effects.
Using the mortality data obtained, an estimate of the acute oral median lethal dose (LD50) of the test material was calculated by the statistical program.

Results and discussion

Effect levels
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
550 mg/kg bw
Based on:
test mat.
95% CL:
88.94 - 2 430
Mortality:
The mortality data are summarised in Table 2. Two animals dosed at 550 mg/kg and the single animal dose at 2000 mg/kg died.
Clinical signs:
- At the 2000 mg/kg dose level, signs of systemic toxicity noted were hunched posture, ataxia, ptosis, occasional body tremors and cyanosis.
- At the 550 mg/kg dose level, signs of systemic toxicity noted in first and third treated animals were hunched posture, ataxia and laboured and noisy respiration. Additional signs of systemic toxicity noted in the third treated animal were pilo-erection, occasional body tremors, pallor of the extremities, emaciation, fasciculations and red/brown staining around the snout. Lethargy, decreased respiratory rate and ptosis were also noted in the first treated animal.
No signs of systemic toxicity were noted in the second treated animal during the observation period.
- At the 175 mg/kg dose level, no signs of systemic toxicity were noted during the observation period.
Body weight:
Individual body weights and body weight changes are given in Table 3. Surviving animals showed expected gains in body weight over the observation period.
Gross pathology:
Individual necropsy findings are given in Table 4.
Abnormalities noted at necropsy of animals that died or were humanely killed during the study were dark liver, gaseous stomach, clear liquid present in the stomach and haemorrhagic gastric mucosa and non-glandular epithelium of the stomach. No abnormalities were noted at necropsy of animals that were killed at the end of the study.

Any other information on results incl. tables

Table 2: Summary of Mortality Data

Dose Level

(mg/kg)

No. of Animals that Survived

No. of Animals that Died

Total No. of Animals

175

2

0

2

550

1

2

3

2000

0

1

1

All doses

3

3

6

 

Table 3: Individual Body weights and Body Weight Changes

Dose level (mg/kg)

Animal No.

Body weight (g) at Day

Body Weight (g) at Death

Body weight Gain (g) During week

0

7

14

1

2

175

1 / 1-0

174

195

202

NA

21

7

550

2 / 2-0

176

-

-

178

-

-

175

3 / 3-0

164

189

209

NA

25

20

550

4 / 4-0

195

206

217

NA

11

11

2000

5 / 5-0

164

-

-

162

-

-

550

6 / 6-0

181

-

-

175

-

-

 

Table 4: Individual Necropsy Findings

Dose level (mg/kg)

Animal No.

Time of Death

Macroscopic Observations

175

1 / 1-0

Killed Day 14

No abnormalities detected

550

2 / 2-0

Found dead Day 0

Liver: dark

Stomach: clear liquid present

Gastric mucosa: haemorrhagic

Non-glandular epithelium of the stomach: haemorrhagic

175

3 / 3-0

Killed Day 14

No abnormalities detected

550

4 / 4-0

Killed Day 14

No abnormalities detected

2000

5 / 5-0

Found dead Day 0

Gastric mucosa: haemorrhagic, severe

Non-glandular epithelium of the stomach: haemorrhagic, severe

550

6 / 6-0

Humanely killed Day 0

Stomach: gaseous; clear liquid present

Gastric mucosa: haemorrhagic

Applicant's summary and conclusion

Interpretation of results:
other: Classified as Category 4 in accordance with EU criteria
Conclusions:
Under the conditions of this study, the acute oral LD50 of the test material was determined to be 550 mg/kg bodyweight (95 % confidence limits 88.94 to 2430 mg/kg bodyweight). In accordance with EU criteria, the test material requires classification for Acute Toxicity as Category 4.
Executive summary:

The study was performed to assess the acute oral toxicity of the test material in the Sprague-Dawley strain rat using the up and down procedure. It was conducted in accordance with the standardised guideline OECD 425 under GLP conditions.

A total of six female animals were dosed individually in sequence with sufficient time (at least 48 hours) between each animal, at dose levels ranging from 175 mg/kg body weight to 2000 mg/kg body weight.

The test material was administered orally by gavage as a solution in distilled water. Clinical signs and body weight development were monitored during the study. All animals were subjected to gross necropsy.

The animal treated at a dose level of 2000 mg/kg and one animal treated at a dose level of 550 mg/kg were found dead on the day of dosing. One other animal treated at a dose level of 550 mg/kg was killed in extremis four hours after dosing. There were no deaths at a dose level of 175 mg/kg.

Signs of systemic toxicity noted in the animal treated at a dose level of 2000 mg/kg were hunched posture, ataxia, ptosis, occasional body tremors and cyanosis. Signs of systemic toxicity noted at a dose level of 550 mg/kg were hunched posture, lethargy, ataxia, ptosis, pilo-erection, decreased respiratory rate, laboured and noisy respiration, occasional body tremors, pallor of the extremities, emaciation, fasciculations and red/brown staining around the snout. There were no signs of systemic toxicity noted in one animal treated at 550 mg/kg and in the animals treated at the dose level of 175 mg/kg. Surviving animals showed expected gains in body weight.

At necropsy, abnormalities noted in animals that died or were humanely killed during the study were dark liver, gaseous stomach, clear liquid present in the stomach and haemorrhagic gastric mucosa and non-glandular epithelium of the stomach. No abnormalities were noted at necropsy of animals that were killed at the end of the study.

Under the conditions of this study, the acute oral LD50 of the test material was determined to be 550 mg/kg bodyweight (95 % confidence limits 88.94 to 2430 mg/kg bodyweight). In accordance with EU criteria, the test material requires classification for Acute Toxicity as Category 4.