Registration Dossier

Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was performed between 02 December 2008 and 30 December 2008
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:
2009

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 420 (Acute Oral Toxicity - Fixed Dose Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)).
Test type:
fixed dose procedure
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Limited, Bicester, Oxon, UK
- Age at study initiation: 8 to 12 weeks.
- Weight at study initiation: 162-210 g (the bodyweight variation did not exceed +/- 20% of the initial/mean bodyweight of any previously dosed animal(s)
- Fasting period before study: Overnight fast immediately before dosing and for approximately three to four hours after dosing.
- Housing: The animals were housed in groups of up to four in suspended solid-floor polypropylene cages furnished with woodflakes.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: At least five days.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): Set to achieve limits of 19 to 25°C
- Humidity (%): Set to achieve limits of 30 to 70%
- Air changes (per hr): At least fifteen changes per hour
- Photoperiod (hrs dark / hrs light): The lighting was controlled by a time switch to give twelve hours continous light (06:00 to 18:00) and twelve hours darkness.


IN-LIFE DATES: From: Day 0 To: Day 14

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
VEHICLE
- Concentration in vehicle:
Dose level 2000 mg/kg - Concentration 200 mg/ml
Dose level 300 mg/kg - Concentration 30 mg/ml
- Amount of vehicle (if gavage): The volume administered to each animal was calculated according to the fasted bodyweight at the time of dosing.
- Justification for choice of vehicle: For the purpose of the study the test material was freshly prepared, as required, as a suspension in arachis oil BP. Acrachis oil BP was used because the test material did not dissolve/suspend in distilled water.



MAXIMUM DOSE VOLUME APPLIED: 10 ml/kg


DOSAGE PREPARATION: All animals were dosed once only by gavage, using a metal cannula attached to a graduated syringe.



- Rationale for the selection of the starting dose: Using available information on the toxicity of the test material, 2000 mg/kg was choosen as the starting dose.
Doses:
2000 mg/kg and 300 mg/kg.
No. of animals per sex per dose:
2000 mg/kg - A total of five female animals were treated at a dose level of 2000 mg/kg.
300 mg/kg - A total of five female animals were treated at a dose level of 300 mg/kg.
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Clinical observations were made at 1/2, 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.
- Necropsy of survivors performed: yes
- Other examinations performed: 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.
Statistics:
Using the mortality data obtained, an estimate of the acute oral median lethal dose (LD50) of the test material was made.

Results and discussion

Preliminary study:
Using available information on the toxicity of the test material 2000mg/kg was chosen as the starting dose. In the absence of mortality two days after dosing, at a dose level of 2000mg/kg, an additionla group of animals was treated.
Effect levels
Sex:
female
Dose descriptor:
LD50
Effect level:
300 - 2 000 mg/kg bw
Remarks on result:
other: 95% CL not stated.
Mortality:
Dose Level - 2000 mg/kg
One animal was found dead three to four hours after dosing. Two animals were killed in extremis one or four 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 hunched posture, lethargy, ataxia, decreased respiratory rate, laboured respiration, tiptoe gait, dehydration, pilo-erection, loss of righting reflex, hypothermia and emaciation.
Surviving animals appeared normal six days after dosing.

Dose Level - 300 mg/kg
Signs of systemic noted during the day of dosing in one animal were hunched posture, lethargy and pilo-erection. No other signs of systemic toxicity were noted during the observation period.
Body weight:
Dose Level - 2000 mg/kg
The surviving animals showed expected gains in bodyweight over the observation period.

Dose Level - 300 mg/kg
All animals showed expected gains in bodyweight over the observation period.
Gross pathology:
Dose Level - 2000 mg/kg
Abnormalities noted at necropsy of animals that died or were killed in extremis during the study were abnormally red lungs, dark liver or patchy pallor of the liver, dark kidneys and gaseous stomach. No abnormalities were noted at necropsy of animals that were killed at the end of the study.

Dose level - 300 mg/kg
No abnormalities were noted at necropsy.
Other findings:
None.

Any other information on results incl. tables

The acute oral median lethal dose (LD50) of the test material in the female Wistar strain rate was estimated to be in the range of 300 - 2000 mg/kg bodyweight.

Table1              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

HLARd

HARd

H

0

0

H

HLAWt
DhRdPX*

 

 

 

 

 

 

 

 

 

 

2-0

Female

HLA

HLARd

HLARd

HLARdRl

RrHoDh
EmRdRl X*

 

 

 

 

 

 

 

 

 

 

 

 

 

2-1

Female

HA

HLARd

HLARd

X

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2-2

Female

0

HLARd

HLARd

HA

HA

H

H

H

H

0

0

0

0

0

0

0

0

0

2-3

Female

HA

HA

HA

HA

HA

H

H

H

H

0

0

0

0

0

0

0

0

0


Table2              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

200

-

-

146

-

-

2-0 Female

162

-

-

150

-

-

2-1 Female

167

-

-

162

-

-

2-2 Female

188

199

212

 

11

13

2-3 Female

184

197

217

 

13

20

-


Table3              Individual Necropsy Findings -2000mg/kg

Dose Level
mg/kg

Animal Number
and Sex

Time of Death

Macroscopic Observations

2000

1-0 Female

Killedin extremisDay 4

Liver: patchy pallor

Stomach: gaseous

2-0 Female

Killedin extremisDay 1

Liver: patchy pallor

2-1 Female

Found dead Day 0

Lungs: abnormally red

Liver: dark

Kidneys: dark

2-2 Female

Killed Day 14

No abnormalities detected

2-3 Female

Killed Day 14

No abnormalities detected


Table4              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

HL

HLP

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

 


Table5              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

169

193

217

24

24

4-0 Female

183

200

217

17

17

4-1 Female

165

203

209

38

6

4-2 Female

197

203

216

6

13

4-3 Female

210

235

249

25

14

 


Table6              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


0= No signs of systemic toxicity

H = Hunched posture

L = Lethargy

A = Ataxia

Rd = Decreased respiratory rate

Rl = Laboured respiration

Wt = Tiptoe gait

Dh = Dehydration

P = Pilo-erection

Rr = Loss of righting reflex

Ho = Hypothermia

Em = Emaciation

X = Animal dead

X* = Animal killedin extremis

-= Animal dead

0= No signs of systemic toxicity

H = Hunched posture

L = Lethargy

P = Pilo-erection

Applicant's summary and conclusion

Interpretation of results:
harmful
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The acute oral median lethal dose (LD50) of the test material in the female Wistar strain rate was estimated to be in the range of 300 - 2000 mg/kg bodyweight.
Executive summary:

Introduction.

The study was performed to assess the acute oral toxicity of the test material in the Wistar strain rat. The method was designed to meet the requirements of the following:

  • OECD Guidelines for Testing of Chemicals No 420 "Acute Oral Toxicity - Fixed Dose Method" (adopted 17 December 2001)
  • Method B1 bis Acute Toxicity (Oral) of Commision Directive 2004/73/EC

Method.

Following a sighting test at a dose level of 2000 mg/kg and 300 mg/kg, a further group of four fasted females was given a single oral dose of test material, as a suspension in arachis oil BP, at a dose level of 300 mg/kg bodyweight/ Clinical signs and bodyweight development were monitored during the study. All animals were subjected to gross necropsy.

Mortality.

One animal treated at a dose level of 2000 mg/kg was found dead and two animals treated at a dose level of 2000 mg/kg were killed in extremis during the study. 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 hunched posture, lethargy, ataxia, decreased respiratory rate, laboured respiration, tiptoe gait, dehydration, pilo-erection, loss of righting reflex, hypothermia and emaciation. Signs of systemic toxicity noted in one animal treated at a dose level of 300 mg/kg were hunched posture, lethargy and pilo-erection.

Bodyweight.

Surviving animals showed expected gains in bodyweight.

Necropsy.

Abnormalities noted at necropsy of animals that died or were killed in extremis during the study were abnormally red lungs, dark liver or patchy pallor of the liver, dark kidneys and gaseous stomach. No abnormalities were noted at necropsy of animals that were killed at the end of the study.

Conclusion.

The acute oral median lethal dose (LD50) of the test material in the female Wistar strain rat was estimated to be in the range of 300 - 2000 mg/kg bodyweight.