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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:
Between 05 January 2012 and 07 February 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 420 (Acute Oral Toxicity - Fixed Dose Method)
Deviations:
yes
Remarks:
The bodyweight at death of one animal treated at a dose level of 2000 mg/kg was not recorded.
Qualifier:
according to
Guideline:
EU Method B.1 bis (Acute Oral Toxicity - Fixed Dose Procedure)
Deviations:
yes
Remarks:
The bodyweight at death of one animal treated at a dose level of 2000 mg/kg was not recorded.
GLP compliance:
yes (incl. certificate)
Test type:
fixed dose procedure
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Description : amber coloured viscous liquid
Purity : not supplied
Date received : 01 December 2011
Expiry date : 01 December 2012
Storage conditions: room temperature in the dark

The integrity of supplied data relating to the identity, purity and stability of the test item is the responsibility of the Sponsor.

A Certificate of Analysis supplied by the Sponsor is given in Appendix 3 (attachment 1)

Test animals

Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals and environmental conditions:
Female Wistar (RccHan™:WIST) strain rats were supplied by Harlan Laboratories UK Ltd., Oxon, UK. On receipt the animals were randomly allocated to cages. The females were nulliparous and non-pregnant. After an acclimatisation period of at least five days the animals were selected at random and given a number unique within the study by indelible ink marking on the tail and a number written on a cage card. At the start of the study the animals were eight to twelve weeks of age. The bodyweight variation did not exceed ±20% of the bodyweight of the initially dosed animal.
The animals were housed in groups of up to four in suspended solid floor polypropylene cages furnished with woodflakes. With the exception of an overnight fast immediately before dosing and for approximately three to four hours after dosing, free access to mains drinking water and food (2014C Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study. The diet, drinking water and bedding were routinely analysed and were considered not to contain any contaminants that would reasonably be expected to affect the purpose or integrity of the study.
The temperature and relative humidity were set to achieve limits of 19 to 25°C and 30 to 70% respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
All animals were dosed once only by gavage, using a metal cannula attached to a graduated syringe. The volume administered to each animal was calculated according to the fasted bodyweight at the time of dosing. Treatment of animals was sequential. Sufficient time was allowed between each dose level to confirm the survival of the previously dosed animals.
Doses:
Using available information on the toxicity of the test item, 2000 mg/kg was chosen as the starting dose.

A single animal was treated as follows:
Dose Level (mg/kg) Specific Gravity Dose Volume (ml/kg) Number of Rats (female)
2000 0.948 2.11 1

In the absence of mortality in the initial animal at a dose level of 2000 mg/kg, an additional group of animals was treated as follows:
Dose Level (mg/kg) Specific Gravity Dose Volume (ml/kg) Number of Rats (female)
2000 0.948 2.11 4

Due to mortality and signs of systemic toxicity at a dose level of 2000 mg/kg, an additional animal was treated as follows:
Dose Level (mg/kg) Concentration (mg/ml) Dose Volume (ml/kg) Number of Rats (female)
300 30 10 1

In the absence of mortality at a dose level of 300 mg/kg, an additional group of animals was treated as follows:
Dose Level (mg/kg) Concentration (mg/ml) Dose Volume (ml/kg) Number of Rats (female)
300 30 10 4
No. of animals per sex per dose:
5 female at 2000 mg/kg
5 females at 300 mg/kg
Control animals:
no
Details on study design:
Experimental Preparation
For the purpose of the 2000 mg/kg dose level, the test item was used as supplied. The specific gravity was determined and used to calculate the appropriate dose volume for the required dose level. For the purpose of the 300 mg/kg dose level, the test item was freshly prepared, as required, as a solution in arachis oil BP. Arachis oil BP was used because the test item did not dissolve/suspend in distilled water.
The test item was formulated within two hours of being applied to the test system. It is assumed that the formulation was stable for this duration.
No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

All animals were dosed once only by gavage, using a metal cannula attached to a graduated syringe. The volume administered to each animal was calculated according to the fasted bodyweight at the time of dosing. Treatment of animals was sequential. Sufficient time was allowed between each dose level to confirm the survival of the previously dosed animals.
Clinical observations were made ½, 1, 2, and 4 hours after dosing and then daily for up to fourteen days. Morbidity and mortality checks were made twice daily.
Individual bodyweights were recorded on Day 0 (the day of dosing) and on Days 7 and 14 or at death. Due to a technician error, the bodyweight at death of one animal treated at a dose level of 2000 mg/kg was not recorded. This deviation from the Study Plan was considered not to affect the purpose or integrity of the study.
At the end of the observation period the surviving animals were killed by cervical dislocation. All animals were subjected to gross necropsy. This consisted of an external examination and opening of the abdominal and thoracic cavities. The appearance of any macroscopic abnormalities was recorded. No tissues were retained.

Results and discussion

Preliminary study:
None
Effect levels
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Remarks on result:
other: 95% confidence limits not given in study report.
Mortality:
Dose Level - 2000 mg/kg
Four animals were found dead one or two days after dosing.

Dose Level - 300 mg/kg
There were no deaths.
Clinical signs:
Dose Level - 2000 mg/kg
Signs of systemic toxicity noted were increased salivation, hunched posture, pilo erection, ataxia, gasping or noisy respiration, lethargy, hypothermia, ptosis, diarrhoea and dehydration. Thickening of the skin on the top of the head and left ear was noted in one animal, seven to eleven days after dosing, with redness of the area where the thickening of the skin appeared and/or fur loss noted eleven to fourteen days after dosing.

Dose Level - 300 mg/kg
No signs of systemic toxicity were noted during the observation period.
Body weight:
Dose Level - 2000 mg/kg
Individual bodyweights and bodyweight changes are given in Table 2.
The surviving animal showed expected gains in bodyweight over the observation period.

Dose Level - 300 mg/kg
Individual bodyweights and bodyweight changes are given in Table 5.
All animals showed expected gains in bodyweight over the observation period.
Gross pathology:
Dose Level - 2000 mg/kg
Individual necropsy findings are given in Table 3.
Abnormalities noted at necropsy of animals that died during the study were abnormally red lungs, dark liver, dark kidneys, gaseous stomach, haemorrhage and epithelial sloughing of the gastric mucosa, haemorrhagic and reddened non glandular epithelium of the stomach, gaseous and fluid filled small intestine and off white substance adhered to the lining of the gastric mucosa and non glandular epithelium of the stomach. Ulcerated non glandular epithelium of the stomach was noted at necropsy of the animal that was killed at the end of the study.

Dose Level - 300 mg/kg
Individual necropsy findings are given in Table 6.
No abnormalities were noted at necropsy.
Other findings:
None

Any other information on results incl. tables

Data evaluations included the relationship, if any, between the animals' exposure to the test item and the incidence and severity of all abnormalities including behavioural and clinical observations, gross lesions, bodyweight changes, mortality and any other toxicological effects. If possible the signs of evident toxicity were also identified. Evident toxicity is defined as the toxic effects which are of a severity such that administration at the next highest level could result in mortality.

Using the mortality data obtained, an estimate of the acute oral median lethal dose (LD50) of the test item was made.

Table 1              Individual Clinical Observations and Mortality Data - 2000mg/kg

Dose Level mg/kg

Animal Number and Sex

Effects Noted After Dosing
(Hours)

Effects Noted During Period After Dosing
(Days)

½

1

2

4

1

2

3

4

5

6

7

8

9

10

11

12

13

14

2000

1-0

Female

0

0

0

0

0

0

0

0

0

0

0*

0*

0*

0*

Fl*

Fl·

Fl·

Fl·

2-0

Female

S

H

H

H

HPA

X

 

 

 

 

 

 

 

 

 

 

 

 

2-1

Female

0

0

0

0

X

 

 

 

 

 

 

 

 

 

 

 

 

 

2-2

Female

RnS

RnH

RnRg
SH

HAPL

X

 

 

 

 

 

 

 

 

 

 

 

 

 

2-3

Female

RnS

RnH

H

H

HoPPtDL
AHDhX

 

 

 

 

 

 

 

 

 

 

 

 

 


0=     No signs of systemic toxicity

S =      Increased salivation

H =      Hunched posture

P =      Pilo-erection

A =      Ataxia

Rg =    Gasping respiration

Rn =    Noisy respiration

L =      Lethargy

Ho =    Hypothermia

Pt =     Ptosis

D =      Diarrhoea

Dh =    Dehydration

* =       Thickening of the skin on the top of the head and left ear

·=       Redness of the area where the thickening of the skin was observed

Fl =     Fur loss

X =      Animal dead

Table 2              Individual Bodyweights and Bodyweight Changes - 2000mg/kg

Dose Level

mg/kg

Animal Number and Sex

Bodyweight (g) at Day

Bodyweight (g)
at Death

Bodyweight Gain (g) During Week

0

7

14

1

2

2000

1-0 Female

159

177

199

 

18

22

2-0 Female

165

-

-

139

-

-

2-1 Female

178

-

-

163

-

-

2-2 Female

189

-

-

178

-

-

2-3 Female

185

-

-

Ä

-

-

Table 3              Individual Necropsy Findings - 2000mg/kg

Dose Level
mg/kg

Animal Number
and Sex

Time of Death

Macroscopic Observations

2000

1-0 Female

Killed Day 14

Non-glandular epithelium of the stomach: ulcerated

2-0 Female

Found dead Day 2

Liver: dark

Kidneys: dark

Stomach: gaseous

Gastric mucosa: haemorrhagic

                         epithelial sloughing

                         off white substance adhered to stomach lining

Non-glandular epithelium of the stomach:   haemorrhagic

                                                              reddened

2-1 Female

Found dead Day 1

Lungs: abnormally red

Liver: dark

Kidneys: dark

Stomach: gaseous

Gastric mucosa: epithelial sloughing

                         off white substance adhered to stomach lining

Non-glandular epithelium of the stomach:   reddened

Small intestine: gaseous

                       fluid filled (clear yellow)

2000

2-2 Female

Found dead Day 1

Lungs: abnormally red

Liver: dark

Kidneys: dark

Stomach: gaseous

Gastric mucosa: epithelial sloughing

Non-glandular epithelium of the stomach: reddened

                                                            off white substance adhered to stomach lining

Small intestine: gaseous

                       fluid filled (opaque, off white)

2-3 Female

Found dead Day 1

Liver: dark

Kidneys: dark

Stomach: gaseous

Gastric mucosa: haemorrhagic

                         epithelial sloughing

Non-glandular epithelium of the stomach: reddened

Table 4              Individual Clinical Observations and Mortality Data - 300mg/kg

Dose Level mg/kg

Animal Number and Sex

Effects Noted After Dosing
(Hours)

Effects Noted During Period After Dosing
(Days)

½

1

2

4

1

2

3

4

5

6

7

8

9

10

11

12

13

14

300

3-0

Female

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

4-0

Female

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

4-1

Female

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

4-2

Female

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

4-3

Female

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0


0=     No signs of systemic toxicity

Table 5              Individual Bodyweights and Bodyweight Changes - 300mg/kg

Dose Level

mg/kg

Animal Number
and Sex

Bodyweight (g) at Day

Bodyweight Gain (g) During Week

0

7

14

1

2

300

3-0 Female

186

208

221

22

13

4-0 Female

151

180

196

29

16

4-1 Female

159

183

198

24

15

4-2 Female

184

210

221

26

11

4-3 Female

170

189

203

19

14

Table 6              Individual Necropsy Findings - 300mg/kg

Dose Level
mg/kg

Animal Number
and Sex

Time of Death

Macroscopic Observations

300

3-0 Female

Killed Day 14

No abnormalities detected

4-0 Female

Killed Day 14

No abnormalities detected

4-1 Female

Killed Day 14

No abnormalities detected

4-2 Female

Killed Day 14

No abnormalities detected

4-3 Female

Killed Day 14

No abnormalities detected

Applicant's summary and conclusion

Interpretation of results:
Category 4 based on GHS criteria
Remarks:
Migrated information
Conclusions:
The acute oral median lethal dose (LD50) of the test item in the female Wistar strain rat was estimated to be in the range of 300 - 2000 mg/kg bodyweight (Globally Harmonised Classification System - Category 4).
Executive summary:

Introduction.

The study was performed to assess the acute oral toxicity of the test item in the Wistar strain rat. The method was designed to be compatible with the following:

 OECD Guidelines for Testing of Chemicals No 420 “Acute Oral Toxicity - Fixed Dose Method” (2001)

 Method B1bisAcute Toxicity (Oral) of CommissionRegulation (EC) No. 440/2008

Method.

Following a sighting test at dose levels of2000 mg/kg and300 mg/kg, a further group of four fasted females was given a single oral dose of test item, as asolutioninarachis oil BP, at a dose level of300 mg/kg bodyweight. Clinical signs and bodyweight development were monitored during the study. All animals were subjected to gross necropsy.

Mortality.

Four animals treated at a dose level of 2000 mg/kg were found dead one or two days after dosing. There were no deaths noted at a dose level of 300 mg/kg.

Clinical Observations.

Signs of systemic toxicity noted in animals treated at a dose level of 2000 mg/kg were increased salivation, hunched posture, pilo-erection, ataxia, gasping or noisy respiration, lethargy, hypothermia, ptosis, diarrhoea and dehydration. Thickening of the skin on the top of the head and left ear, fur loss and redness were noted in one animal treated at a dose level of 2000 mg/kg. There were no signs of systemic toxicity noted at a dose level of 300 mg/kg.

Bodyweight.

Surviving animals showed expected gains in bodyweight.

Necropsy.

Abnormalities noted at necropsy of animals that died during the study were abnormally red lungs, dark liver, dark kidneys, gaseous stomach, haemorrhage and epithelial sloughing of the gastric mucosa, haemorrhagic and reddened non-glandular epithelium of the stomach, gaseous and fluid filled small intestine and off white substance adhered to the lining of the gastric mucosa and non‑glandular epithelium of the stomach. Ulcerated non-glandular epithelium of the stomach was noted at necropsy of one animal treated at a dose level of 2000 mg/kg that was killed at the end of the study. No abnormalities were noted at necropsy of animals treated at a dose level of 300 mg/kg.

Conclusion. 

The acute oral median lethal dose (LD50) of the test item in the female Wistar strain rat was estimated to be in the range of 300 ‑ 2000 mg/kg bodyweight (Globally Harmonised Classification System-Category 4).