Registration Dossier

Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 27 October 2015 and 24 November 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 420 (Acute Oral Toxicity - Fixed Dose Method)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.1 bis (Acute Oral Toxicity - Fixed Dose Procedure)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese MAFF, 2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test type:
fixed dose procedure
Limit test:
yes

Test material

Constituent 1
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals or test system and environmental conditions:
Female Wistar (RccHan™:WIST) strain rats were supplied by Envigo RMS (UK) Limited, Oxon, UK. On receipt the animals were randomly allocated to cages. The females were nulliparous and non pregnant. After an acclimatization period of at least 5 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 8 to 12 weeks of age. The body weight variation did not exceed ±20% of the mean body weight at the start of treatment.
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 3 to 4 hours after dosing, free access to mains drinking water and food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study. The diet, drinking water and bedding were routinely analyzed 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. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 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.


Justification
Rats are the preferred species of choice as historically used for safety evaluation studies and are specified in the appropriate test guidelines.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
Test Item Formulation and Experimental Preparation
For the purpose of the study the test item was freshly prepared, as required, as a solution in distilled water.

The test item was formulated within 2 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.
Doses:
A single animal was treated at 300 mg/kg at a concentration of 30 mg/mL.
In the absence of toxicity at a dose level of 300 mg/kg, an additional animal was treated at 2000 mg/kg at a concentration of 200 mg/mL.
In the absence of toxicity at a dose level of 2000 mg/kg, an additional group of 4 animals were treated at 2000 mg/kg.
No. of animals per sex per dose:
A single animal was treated at 300 mg/kg.
Five animals were treated at 2000 mg/kg.
Control animals:
no
Details on study design:
Procedure
In the absence of data regarding the toxicity of the test item, 300 mg/kg was chosen as the starting dose. A single female animal was treated with 300 mg/kg at a concentration of 30 mg/mL (dose volume 10 mg/kg).

In the absence of toxicity at a dose level of 300 mg/kg, an additional animal was treated at 2000 mg/kg at a concentration of 200 mg/mL (dose volume 10 mg/kg). In the absence of toxicity at a dose level of 2000 mg/kg, an additional group of 4 female animals were treated at 2000 mg/kg at a concentration of 200 mg/mL (dose volume 10 mg/kg).

A total of five animals were therefore treated at a dose level of 2000 mg/kg in the study.

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 body weight at the time of dosing. Treatment of animals was sequential. Sufficient time was allowed between each dose group to confirm the survival of the previously dosed animals.

Clinical observations were made 30 minutes, 1, 2, and 4 hours after dosing and then daily for 14 days. Morbidity and mortality checks were made twice daily.

Individual body weights were recorded on Day 0 (the day of dosing) and on Days 7 and 14.

At the end of the observation period the 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.


Evaluation of Data
Evaluation of data included identification of the number of animals that died during the study (or that were killed for humane reasons), and determination of the nature, severity, onset and duration of the toxic effects. If possible, the signs of evident toxicity were described. Evident toxicity refers to the toxic effects of sufficient severity that administration of the next higher dose level could result in development of severe signs of toxicity and probable mortality. Effects on body weights and abnormalities noted at necropsy were also identified.

Using the mortality data obtained, an estimate of the acute oral median lethal dose (LD50) of the test item was made.
Statistics:
No data

Results and discussion

Preliminary study:
Dose Level - 300 mg/kg

Mortality
There was no mortality.

Clinical Observations
No signs of systemic toxicity were noted during the observation period. Orange colored stained urine was noted during the day of dosing.

Body Weight
The animal showed expected gains in body weight over the observation period.

Necropsy
No abnormalities were noted at necropsy.
Effect levels
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
Dose Level - 2000 mg/kg: There were no deaths.
Clinical signs:
Dose Level - 2000 mg/kg: No signs of systemic toxicity were noted during the observation period.
Body weight:
Dose Level - 2000 mg/kg: All animals showed expected gains in body weight over the observation period.
Gross pathology:
Dose Level - 2000 mg/kg: No abnormalities were noted at necropsy.

Any other information on results incl. tables

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

1-0

Female

0U

0U

0U

0U

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0 = No signs of systemic toxicity

U = Orange colored stained urine

Individual Body Weights and Body Weight Changes -300mg/kg

Dose Level

mg/kg

Animal Number
and Sex

Body Weight (g) at Day

Body Weight Gain (g)
During Week

0

7

14

1

2

300

1-0 Female

190

207

226

17

19


 Individual Necropsy Findings -300 mg/kg

Dose Level
mg/kg

Animal Number
and Sex

Time of Death

Macroscopic Observations

300

1-0 Female

Killed Day 14

No abnormalities detected

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

2-0

Female

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

3-0

Female

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

3-1

Female

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

3-2

Female

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

3-3

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

Individual Body Weights and Body Weight Changes -2000mg/kg

Dose Level

mg/kg

Animal Number
and Sex

Body Weight (g) at Day

Body Weight Gain (g) During Week

0

7

14

1

2

2000

2-0 Female

150

166

176

16

10

3-0 Female

164

181

202

17

21

3-1 Female

179

197

214

18

17

3-2 Female

178

199

219

21

20

3-3 Female

176

192

207

16

15

Individual Necropsy Findings -2000mg/kg

Dose Level
mg/kg

Animal Number
and Sex

Time of Death

Macroscopic Observations

2000

2-0 Female

Killed Day 14

No abnormalities detected

3-0 Female

Killed Day 14

No abnormalities detected

3-1 Female

Killed Day 14

No abnormalities detected

3-2 Female

Killed Day 14

No abnormalities detected

3-3 Female

Killed Day 14

No abnormalities detected

Applicant's summary and conclusion

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The acute oral median lethal dose (LD50) of the test item in the female Wistar strain rat was estimated to be greater than 2000 mg/kg body weight (Globally Harmonized Classification System - Unclassified).
Executive summary:

Introduction

The study was performed to assess the acute oral toxicity of the test item in the female Wistar strain rat.

 

Methods

Following a sighting test at dose levels of 300 mg/kg and 2000 mg/kg, a further group of four fasted females was given a single oral dose of test item, as a solution in distilled water, at a dose level of 2000 mg/kg body weight. Clinical signs and body weight development were monitored during the study. All animals were subjected to gross necropsy.

 

Results

Mortality. There were no deaths.

Clinical Observations. There were no signs of systemic toxicity. Orange colored stained urine was noted in the initial animal, treated at a dose level of 300 mg/kg body weight.

Body Weight. All animals showed expected gains in body weight.

Necropsy. No abnormalities were noted at necropsy.

 

Conclusion

The acute oral median lethal dose (LD50) of the test item in the female Wistar strain rat was estimated to be greater than 2000 mg/kg body weight (Globally Harmonized Classification System-Unclassified).