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 March 2018 and 25 April 2018.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

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)
Test type:
fixed dose procedure

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of Rel-(3aR,4S,7R,7aS)-1-(methoxymethylene)octahydro-1H-4,7-methanoindene and Rel-(3aR,4S,7R,7aS)-2-(methoxymethylene)octahydro-1H-4,7-methanoindene
EC Number:
951-275-7
Molecular formula:
C12H18O
IUPAC Name:
Reaction mass of Rel-(3aR,4S,7R,7aS)-1-(methoxymethylene)octahydro-1H-4,7-methanoindene and Rel-(3aR,4S,7R,7aS)-2-(methoxymethylene)octahydro-1H-4,7-methanoindene
Specific details on test material used for the study:
Identification: FRET 14-0383
Physical state / Appearance: Clear colourless to very pale yellow liquid
Storage Conditions: 4 °C in the dark

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 of any previously treated animals.

Animal Care and Husbandry
The animals were housed in groups of up to four in suspended solid floor polypropylene cages furnished with wood flakes. 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.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 2000 mg/kg dose level: no vehicle, 300 mg/kg dose level: arachis oil BP
Details on oral exposure:
At 300 mg/kg
- Concentration in vehicle: 30 mg/mL
- Justification for choice of vehicle: Arachis oil BP was used because the test item did not dissolve/suspend in distilled water.

MAXIMUM DOSE VOLUME APPLIED: 10 mL/kg

DOSAGE PREPARATION (if unusual): 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 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.

CLASS METHOD (if applicable)
- In the absence of data regarding the toxicity of the test item, 300 mg/kg was chosen as the starting dose.

At 2000 mg/kg
MAXIMUM DOSE VOLUME APPLIED: 1.99 mL/kg

DOSAGE PREPARATION (if unusual): 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.

CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose: In the absence of mortality at a dose level of 300 mg/kg, an additional animal was treated at 2000 mg/kg dose level.
Doses:
300 mg/kg and 2000 mg/kg
No. of animals per sex per dose:
300 mg/ kg: 1 female treated per dose
2000 mg/kg: 1 female treated per dose
In the absence of mortality at a dose level of 2000 mg/kg, an additional group of animals was treated: 4 females at 2000 mg/kg
Control animals:
no
Details on study design:
Study Design
In the absence of data regarding the toxicity of the test item, 300 mg/kg was chosen as the starting dose.
A single 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 animal was treated as follows:
Dose Level(mg/kg) Specific Gravity Dose Volume(mL/kg) Number of Rats(Female)
2000 1.007 1.99 1

In the absence of mortality 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 1.007 1.99 4

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 up to 14 days. Morbidity and mortality checks were made twice daily, early and late during normal working days, and once daily at weekends and public holidays.
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.

Results and discussion

Effect levels
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
Dose Level - 300 mg/kg
There were no deaths.

Dose Level - 2000 mg/kg
There were no deaths.
Clinical signs:
Dose Level - 300 mg/kg
Signs of systemic toxicity noted up to 2 hours after dosing were tiptoe gait and/or hunched posture.
The animal appeared normal 4 hours after dosing.

Dose Level - 2000 mg/kg
Hunched posture was noted in one animal during the day of dosing.
The remaining four animals appeared normal throughout the observation period.
Body weight:
Dose Level - 300 mg/kg
The animal showed an expected gain in body weight over the observation period.

Dose Level - 2000 mg/kg
All animals showed expected gains in body weight over the observation period.
Gross pathology:
Dose Level - 300 mg/kg
No abnormalities were noted at necropsy.

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

HWt

H

H

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0


0      =  No signs of systemic toxicity

H    =   Hunched posture

Wt  =   Tiptoe gait

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

159

176

207

17

31

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

H

H

H

H

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

H =  Hunched posture

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

184

195

206

11

11

3-0 Female

195

202

207

7

5

3-1 Female

180

184

197

4

13

3-2 Female

206

207

221

1

14

3-3 Female

206

220

231

14

11

 

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:
Criteria used for interpretation of results: EU
Conclusions:
The acute oral toxicity test for FRET 14-0383 showed an LD50 of >2000 mg/kg bw.
Executive summary:

In this study, 1 female rat was administered the substance at dose levels of 300 mg/kg bw and 5 female rats were administered the substance at dose levels of 2000 mg/kg bw. The rats showed no mortality, body weight, necropsy at both dose levels. At 300 mg/kg, signs of systemic toxicity noted up to 2 hours after dosing were tiptoe gait and/or hunched posture. The animal appeared normal 4 hours after dosing. At 2000 mg/kg bw, hunched posture was noted in one animal during the day of dosing. The remaining four animals appeared normal throughout the observation period.

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).