Registration Dossier

Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 15, 2016 - December 6, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test type:
acute toxic class method
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Ethyl ethyl(phenyl)carbamate
EC Number:
213-796-1
EC Name:
Ethyl ethyl(phenyl)carbamate
Cas Number:
1013-75-8
Molecular formula:
C11H15NO2
IUPAC Name:
ethyl ethyl(phenyl)carbamate
Test material form:
liquid
Details on test material:
Colorless
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions:yes


Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
SPF Caw
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Elevage JANVIER LABS (F-53941 Le Genest St Isle)
- Females (if applicable) nulliparous and non-pregnant:yes
- Age at study initiation: 8-9 week old
- Weight at study initiation: average 213.0 g
- Housing:Housed by group of three in solid-bottomed clear polycarbonate cages with a stainless steel mesh lid.ach cage contains sawdust bedding which was changed at least 2 times a week. Each cage was installed in conventional air conditioned animal husbandry.
- Diet (ad libitum): ad libitum, ENVIGO - 2016. It consists of : macronutrients, minerals, amino acids, vitamins, fatty acids and cholesterol.
- Water (ad libitum):tap-water from public distribution system
Food was removed on D-1 and then redistributed 4 hours after the test item administration. Microbiological and chemical analyses of the water were carried out once every six months by Bureau Veritas – Eurofins (FRANCE).
- Acclimation period:5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25ºC
- Humidity (%): 30-70%
- Air changes (per hr): 10x per hour
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark

OTHERS:
Group treated (2000 mg/kg):
Rf0712 to Rf0714 (Step 1) - 3 female rats
Rf0724 to Rf0726 (Step 2) - 3 female rats

IN-LIFE DATES: From: To: November 15, 2016 - December 6, 2016

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
MAXIMUM DOSE VOLUME APPLIED: 1.95 mL/kg
DOSAGE PREPARATION: In the first and second step of the study, the test item was administrated by gavage under a volume of 1.95 mL/kg bw (corresponding to 2g/kg according the calculated density) using a suitable graduated syringe fifted with an oesophageal metal canula.

CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose: It is most likely to produce mortality in some of the dosed animals.
Doses:
2000 mg/kg
No. of animals per sex per dose:
6
Control animals:
yes
Remarks:
Current control study
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing:
First day : T0+30 minutes, T0+1hour, T0+3 hours, T0+4 hours, then the observations were done once a day for 14 days.
- Necropsy of survivors performed: yes (Rf0712 & Rf0714,Rf0725 & Rf0726)
- Other examinations performed:
Macroscopic obseravtiones: yes
Spontaneous activity, Preyer’s reflex (noise), Respiratory rate, Convulsions, Tremors, Body temperature, Muscle tone, Palpebral opening, Pupil appearance, Salivation, Lachrymation, Righting reflex, Back hair appearance

On D14, the animals were anaesthetised with sodium pentobarbital and administration continued to fatal levels.Only those organs likely to be modified in cases of acute toxicity were examined. No organ was removed and preserved in view to microscopic examinations.

Results and discussion

Effect levelsopen allclose all
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Key result
Sex:
female
Dose descriptor:
LD50 cut-off
Effect level:
ca. 2 500 mg/kg bw
Based on:
test mat.
Mortality:
Yes, 2 mortality were observed in animals treated at the dose of 2000 mg/kg body weight, one at 48 hours post dose during the 2nd step and one at day 3 during the 1st step. These mortality were preceded by an absence or decrease in spontaneous activity (2/2), Preyer’s reflex (2/2), muscle tones (2/2), righting reflex (2/2), associated with bradypnea (1/2), dyspnea (2/2), polypnea (1/2), hypothermia (2/2), eyes partly (2/2) and totaly (1/2) closed, myosis (1/2), mydriasis (2/2), an increase of salivation (1/2), lachrymation (1/2) and piloerection (1/2). Rigor mortis were noted before the necropsy.
Clinical signs:
In the surviving animals (4/6), a decrease or an absence in spontaneous activity (3/4), Preyer’s reflex (4/4), muscle tones (3/4) and righting reflex (3/4), associated with bradypnea (1/4), dyspnea (2/4), an hypothermia (3/4), eyes partly (1/4) and totally (2/4), an increase of lachrymation (2/4) and
piloerection (2/4) were noted during 48 hours. Two animals recovered a normal activity at 48 hours post dose.
Body weight:
An absence of body weight gain was noted on day 2 versus day 0. Then, the body weight evolution remained normal.
Gross pathology:
The macroscopic examination of this animal at the end of the study revealed thickening white forestomach (1/4).

Any other information on results incl. tables

D0: 15 November 2016 (step 1) & 22 November 2016 (step 2)

Table No.1: Observations

OBSERVATIONS

FEMALES

FEMALES

T0 + 30 minutes

Rf

Rf

Rf

Rf

Rf

Rf

0712

0713

0714

0724

0725

0726

Spontaneous activity

N

N

N

N

N

N

Preyer's reflex (noise)

N

N

N

N

N

N

Respiratory rate

N

N

N

N

N

N

Convulsions

N

N

N

N

N

N

Body temperature

N

N

N

N

N

N

Muscle tone

N

N

N

N

N

N

Palpebral opnening

N

N

N

N

N

N

Pupil appearance

N

N

N

N

N

N

Salivation

N

N

N

N

N

N

Lachrymation

N

N

N

N

N

N

Righting reflex

N

N

N

N

N

N

Back hair appearance

N

N

N

N

N

N

Tremors

N

N

N

N

N

N

MORTALITY

0

0

0

0

0

0

Table No.2: Observations

OBSERVATIONS

FEMALES

FEMALES

T0 + 1 HOUR

Rf

Rf

Rf

Rf

Rf

Rf

0712

0713

0714

0724

0725

0726

Spontaneous activity

D

N

N

D

N

N

Preyer's reflex (noise)

D

N

N

N

N

N

Respiratory rate

N

N

N

N

N

N

Convulsions

N

N

N

N

N

N

Body temperature

N

N

N

N

N

N

Muscle tone

D

N

N

N

N

N

Palpebral opnening

N

N

N

N

N

N

Pupil appearance

N

N

N

N

N

N

Salivation

N

N

N

N

N

N

Lachrymation

N

N

N

N

N

N

Righting reflex

D

N

N

N

N

N

Back hair appearance

N

N

N

N

N

N

Tremors

N

N

N

N

N

N

MORTALITY

0

0

0

0

0

0

Table No.3: Observations

OBSERVATIONS

FEMALES

FEMALES

T0 + 3 HOURS

Rf

Rf

Rf

Rf

Rf

Rf

0712

0713

0714

0724

0725

0726

Spontaneous activity

N

N

N

D

N

N

Preyer's reflex (noise)

N

N

N

N

N

N

Respiratory rate

N

N

N

N

N

N

Convulsions

N

N

N

N

N

N

Body temperature

N

N

N

N

N

N

Muscle tone

N

N

N

N

N

N

Palpebral opnening

N

N

N

N

N

N

Pupil appearance

N

N

N

N

N

N

Salivation

N

N

N

N

N

N

Lachrymation

N

N

N

N

N

N

Righting reflex

N

N

N

N

N

N

Back hair appearance

N

N

N

N

N

N

Tremors

N

N

N

N

N

N

MORTALITY

0

0

0

0

0

0

Table No.4: Observations

OBSERVATIONS

FEMALES

FEMALES

T0 + 4 HOURS

Rf

Rf

Rf

Rf

Rf

Rf

0712

0713

0714

0724

0725

0726

Spontaneous activity

N

0

D

D

N

N

Preyer's reflex (noise)

N

0

D

N

N

N

Respiratory rate

N

B

B

N

N

N

Convulsions

N

N

N

N

N

N

Body temperature

N

D

D

N

N

N

Muscle tone

N

0

0

N

N

N

Palpebral opnening

N

Pc

N

N

N

N

Pupil appearance

N

Ms

Ms

N

N

N

Salivation

N

N

N

N

N

N

Lachrymation

N

N

N

N

N

N

Righting reflex

N

0

0

N

N

N

Back hair appearance

N

N

N

N

N

N

Tremors

N

N

N

N

N

N

MORTALITY

0

0

0

0

0

0

Table No.5: Observations

OBSERVATIONS

FEMALES

FEMALES

D1

Rf

Rf

Rf

Rf

Rf

Rf

0712

0713

0714

0724

0725

0726

Spontaneous activity

0

0

0

D

D

N

Preyer's reflex (noise)

0

0

0

0

0

N

Respiratory rate

D

D

D

P

N

N

Convulsions

N

N

N

N

N

N

Body temperature

D

D

D

D

D

N

Muscle tone

0

0

0

0

D

N

Palpebral opnening

Cc

Cc

Cc

Pc

Pc

N

Pupil appearance

N

N

N

Md

N

N

Salivation

A

A

A

N

N

N

Lachrymation

A

A

A

N

N

N

Righting reflex

0

0

0

0

D

N

Back hair appearance

Pi

Pi

Pi

N

N

N

Tremors

N

N

N

N

N

N

MORTALITY

0

0

0

0

0

0

Table No.6: Observations

OBSERVATIONS

FEMALES

FEMALES

D2

Rf

Rf

Rf

Rf

Rf

Rf

0712

0713

0714

0724

0725

0726

Spontaneous activity

D

0

D

0

N

N

Preyer's reflex (noise)

N

0

N

0

N

N

Respiratory rate

N

B

N

D

N

N

Convulsions

N

N

N

N

N

N

Body temperature

N

D

N

D

N

N

Muscle tone

D

0

D

0

N

N

Palpebral opnening

N

N

N

Pc

N

N

Pupil appearance

N

Md

N

Md

N

N

Salivation

N

N

N

N

N

N

Lachrymation

N

N

N

N

N

N

Righting reflex

D

0

D

0

N

N

Back hair appearance

N

N

N

N

N

N

Tremors

N

N

N

N

N

N

MORTALITY

0

0

0

0

0

0

Rf0724: found dead after observations

Table No.7: Observations

OBSERVATIONS

FEMALES

FEMALES

D3-D14

Rf

Rf

Rf

Rf

Rf

Rf

0712

0713

0714

0724

0725

0726

Spontaneous activity

N

 

N

 

N

N

Preyer's reflex (noise)

N

 

N

 

N

N

Respiratory rate

N

 

N

 

N

N

Convulsions

N

 

N

 

N

N

Body temperature

N

 

N

 

N

N

Muscle tone

N

 

N

 

N

N

Palpebral opnening

N

 

N

 

N

N

Pupil appearance

N

 

N

 

N

N

Salivation

N

 

N

 

N

N

Lachrymation

N

 

N

 

N

N

Righting reflex

N

 

N

 

N

N

Back hair appearance

N

 

N

 

N

N

Tremors

N

 

N

 

N

N

MORTALITY

0

1

0

 

0

0

Rf0713: found dead on day 3

Table No.8: Body weight and weight gain in grams

 

D0          D2          D2-D0

D7     

      

241

DEAD

228

DEAD

251

248

242.0

 

10.2

D7-D0 

36   

DEAD

35

DEAD

32

28

32.8

 

3.6

D14

259

DEAD

261

DEAD

284

261

266.3

 

11.9

D14-D0

54

DEAD

68

DEAD

65

41

57.0

 

12.2

Rf 0712

Rf 0713

Rf 0714

205         199          -6

209         201          -8

193         196           3

232         220          -12

219         219           0

220         224           4

213.0     209.8       -3.2

 

13.6        12.4          6.5

Rf 0724

Rf 0725

Rf 0726

MEAN

Standart deviations

Table No.9

Sex / No. Animal: 1 female Rf0713

Necropsy: Necropsy date: 18 November 2016

Found dead=X

General appearance before autopsy: Rigor mortis, found dead on day 3

 

OBSERVED

ORGANS

Observations

GESOPHAGUS

X

N.t.R.

STOMACH

X

Thinning of the corpus

Cell lysis

Cell lysis

Cell lysis

Cell lysis

Cell lysis

Cell lysis

DUODENUM

-

JEJUNUM

-

ILEON

-

CAECUM

-

COLON

-

RECTUM

-

SPLEEN

-

LIVER

X

N.t.R.

THYMUS

X

N.t.R.

TRACHEA

X

N.t.R.

LUNGS

X

N.t.R.

HEART

X

N.t.R.

KIDNEYS

X

N.t.R.

URINARY BLADDER

X

N.t.R.

OVARIES

X

N.t.R.

UTERUS

X

N.t.R.

TREATMENT AREA

 -

   -

ADRENALS

X

N.t.R.

PANCREAS

X

N.t.R.

Body weight: 169 g

Table No.10

Sex / No. Animal: 1 female Rf0724

Necropsy date: 24 November 2016

Found dead =X

General appearance before autopsy: rigor mortis, found dead on day 2

 

OBSERVED

ORGANS

Observations

GESOPHAGUS

X

N.t.R.

STOMACH

X

Thinning of the corpus

Cell lysis

Cell lysis

Cell lysis

Cell lysis

Cell lysis

Cell lysis

DUODENUM

-

JEJUNUM

-

ILEON

-

CAECUM

-

COLON

-

RECTUM

-

SPLEEN

-

LIVER

X

N.t.R.

THYMUS

X

N.t.R.

TRACHEA

X

N.t.R.

LUNGS

X

N.t.R.

HEART

X

N.t.R.

KIDNEYS

X

N.t.R.

URINARY BLADDER

X

N.t.R.

OVARIES

X

N.t.R.

UTERUS

X

N.t.R.

TREATMENT AREA

 -

   -

ADRENALS

X

N.t.R.

PANCREAS

X

N.t.R.

Body weight:220g

Table No.11

Sex / No. Animal: 2 females Rf0712 & Rf0714

Necropsy date: 29 November 2016

Found dead =0

Euthanasia = x

At term = x

General appearance before autopsy: Normal

 

OBSERVED

ORGANS

Observations

GESOPHAGUS

X

N.t.R.

STOMACH

X

N.t.R.

DUODENUM

X

N.t.R.

JEJUNUM

X

N.t.R.

ILEON

X

N.t.R.

CAECUM

X

N.t.R.

COLON

X

N.t.R.

RECTUM

X

N.t.R.

SPLEEN

X

N.t.R.

LIVER

X

N.t.R.

THYMUS

X

N.t.R.

TRACHEA

X

N.t.R.

LUNGS

X

N.t.R.

HEART

X

N.t.R.

KIDNEYS

X

N.t.R.

URINARY BLADDER

X

N.t.R.

OVARIES

X

N.t.R.

UTERUS

X

N.t.R.

TREATMENT AREA

 -

   -

ADRENALS

X

N.t.R.

PANCREAS

X

N.t.R.

Particulars:NONE

Table No.12

Sex / No. Animal: 2 females Rf0725 & Rf0726

Necropsy date: 06 December 2016

Found dead =0

Euthanasia = x

At term = x

General appearance before autopsy: Normal

 

OBSERVED

ORGANS

Observations

GESOPHAGUS

X

N.t.R.

STOMACH

X

Rf0726:White thickening of the wall’s

forestomach (1/4), Rf0725: N.t.R.

DUODENUM

X

N.t.R.

JEJUNUM

X

N.t.R.

ILEON

X

N.t.R.

CAECUM

X

N.t.R.

COLON

X

N.t.R.

RECTUM

X

N.t.R.

SPLEEN

X

N.t.R.

LIVER

X

N.t.R.

THYMUS

X

N.t.R.

TRACHEA

X

N.t.R.

LUNGS

X

N.t.R.

HEART

X

N.t.R.

KIDNEYS

X

N.t.R.

URINARY BLADDER

X

N.t.R.

OVARIES

X

N.t.R.

UTERUS

X

N.t.R.

TREATMENT AREA

 -

   -

ADRENALS

X

N.t.R.

PANCREAS

X

N.t.R.

Particulars:NONE

Current control study:

Test item :destilled water

Study date: 20.09.2016-4.10.2016

3 animals, received distillated water, administered by gavage under volume of 10mL/kg bw using suitable syringe graduated fitted with an oesophageal metal canula.

Results: Clinical examinatiuon-nothing to report

             Body weight - normal during test

             Necropsy-No tretament related the changes were observed.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The LD50 of the test item is higher than 2000 mg/kg body weight by oral route in the rat.The LD50 cut-off of the test item may be considered to be 2500 mg/kg body weight by oral route in the rat.
Executive summary:

In accordance with GLP study and OECD TG 423, the test item was administered ,as supplied, to a group of 6 females rat Sprague Dawley to determine Acute toxicity. Animals were weighted on day D0 before administration then on day 2, D7 and D14. The dose used in this study was 2000mg/kg bw (limit test). Test item was administrated by gavage using the suitable graduated syringe fitted with and oesophageal metal canula. Two group of 3 females were tested ( 3 in STEP 1 and 3 in STEP 2). Clinic examinations were carried out during the study to identified any behavioural and toxic effects. During the examination two mortality were observed from each group (one at 48 hours post dose during the STEP 2 and one at day 3 during the STEP 1). The clinical signs, decrease or absence in spontaneous activity (3/4), Preyer’s reflex (3/4), muscle tones and righting reflex (3/4) associated with bradypnea (1/4), dyspnea (2/4), an hypothermia (3/4), eyes partly (1/4) and totally (2/4), an increase of lachrymation and piloerection (2/4) were noted during 48 h in survival animals. An absence of body weight gain were observed on day 2 and then body weight remained normal. On day 14, the animals were euthanised with sodium pentobarbital. The macroscopic examination of one animal at the end of the study revealed thickening white forestomach. In conclusion, the LD50 of the test item is higher than 2000 mg/ kg bw by oral route in the rat. The LD50 cut-off of the test item may be considered to be 2500 mg/kg bw by oral route in the rat.