Registration Dossier

Diss Factsheets

Toxicological information

Acute Toxicity: oral

Currently viewing:

Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 June 2017 to 05 July 2017
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)
Version / remarks:
OECD GUIDELINES FOR TESTING OF CHEMICALS (423, adopted at 17th Dec. 2001)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)
Version / remarks:
Commission Regulation (EC) No 440/2008 of 30 May 2008, B.1.tris
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.1100 (Acute Oral Toxicity)
Version / remarks:
EPA Health Effects Test Guidelines (OPPTS 870.1100), United States, EPA 712-C-98-190 (1998)
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:
2-Propanone, reaction products with diphenylamine
EC Number:
270-192-0
EC Name:
2-Propanone, reaction products with diphenylamine
Cas Number:
68412-48-6
Molecular formula:
Variable.
IUPAC Name:
N-cyclohexylcyclohexanamine; propan-2-one
Test material form:
solid: particulate/powder
Details on test material:
Name: AMINOX®
Chemical name: 2-Propanone, reaction products with diphenylamine
Batch/Lot number: EL5D06K347
Appearance: Green to brown powder
CAS number: 68412-48-6
Purity: 100% (as a UVCB)
Retest date: 05 April 2018
Storage conditions: Room temperature
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.
Specific details on test material used for the study:
No further details specified in the study report.

Test animals

Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals or test system and environmental conditions:
Species and strain: CRL:(WI) Wistar rats
Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633 Sulzfeld
Hygienic level at arrival: SPF
Hygienic level during the study: Standard housing conditions
Number of animals: 6 animals
Sex: Female, nulliparous and non-pregnant
Age of animals at dosing: Young healthy adult rats, 9 weeks old
Body weight at treatment: 203 – 232 g
Acclimatization period: at least 12 days

Husbandry
Animal health: Only healthy animals were used for the test. The Veterinarian certified health status.
Number of animal room: 522/9
Housing: 3 animals / cage
Cage type: Type II polypropylene/polycarbonate
Bedding/Nesting: “Lignocel 3/4-S Hygienic Animal Bedding” and “Arbocel crinklets natural” nest building material produced by J. Rettenmaier & Söhne GmbH & Co.KG (D-73494 Rosenberg, Germany) were available to animals during the study.
Lighting period: 12 hours daily, from 6.00 am to 6.00 pm
Temperature: 22 ± 3 °C
Relative humidity: 30-70%
Ventilation: 15 – 20 air exchanges/hour
Enrichment: Animals were housed by group to allow social interaction and with deep wood sawdust bedding to allow digging and other normal rodent activities.
The temperature and relative humidity were recorded twice daily during the study.

Food and Water Supply
Animals received ssniff® SM R/M "Autoclavable complete diet for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany (Batch no.: 285 17890, expiry date: 30 August 2017), ad libitum, and tap water from the municipal supply, as for human consumption from 500 ml bottles, ad libitum. The food is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
Water quality control analysis is performed once every three months and microbiological assessment is performed monthly, by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A.u.36., Hungary).

Animal Identification
Animals were individually identified using numbers written on the tail with an indelible marker pen. The numbers were given on the basis of CiToxLAB Hungary Ltd.'s Master File, for each animal allocated to the treatment groups. The cages were identified by cards, with information about study code, sex, dose group, cage number and individual animal numbers.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
DMSO
Details on oral exposure:
Formulation
The test item was freshly formulated at a concentration of 200 mg/mL in the vehicle, in the Pharmacy of CiToxLAB Hungary Ltd. on the day of administration. The formulation container was stirred continuously up to finishing the treatment.

Vehicle Selection
The selection of the vehicle was made during trial formulations with the test item. The final choice of vehicle was approved by the Sponsor.
On the basis of the trial formulations with the test item, the vehicle used was DMSO.
Vehicle: Dimethyl sulfoxide (DMSO)
Batch number: STBG8411
Expiry date: 29 February 2020
Dose volume: 10 mL/kg bw

Justification of the dose:
A limit of 2000 mg/kg bw dose was selected by the Sponsor.
Initially, three females (assigned to Group 1) were treated at a dose level of 2000 mg/kg bw. The test item did not cause mortality in this group and a second group (Group 2) was treated at the same dose level. No mortality was observed in the confirmatory group, therefore no further testing was required according to OECD 423 and Commission Regulation (EC) No 440/2008 of 30 May 2008, B.1.tris.
Doses:
A limit of 2000 mg/kg bw dose was selected by the Sponsor.
No. of animals per sex per dose:
Initially, three females (assigned to Group 1) were treated at a dose level of 2000 mg/kg bw. The test item did not cause mortality in this group and a second group (Group 2) was treated at the same dose level.
Control animals:
no
Details on study design:
Procedure
A single oral gavage administration was followed by a 14-day observation period. On the night before treatment, the animals were fasted. The food but not water was withheld during an overnight period. Animals were weighed just before treatment. The test item was administered by oral gavage in the morning. The food was made available again at about 3 hours after the treatment.

OBSERVATIONS
Clinical Observations
Clinical observations were performed at 30 minutes, 1, 2, 3, 4 and 6 hours after dosing and daily for 14 days thereafter, as applicable. Individual observations were performed on the skin, fur, eyes, mucous membranes, respiratory, circulatory, autonomic and central nervous system, somatomotor activity and behaviour pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

Body Weight Measurement
The body weight was recorded on the day before treatment (Day -1), on the day of the treatment (Day 0), Day 7 and Day 14.

NECROPSY
All animals were subjected to a necropsy and a macroscopic examination. The animals were exsanguinated after verification of narcosis following an injection of pentobarbital sodium (Release; Lot No.: 106075, Expiry Date: July 2018, Produced by: Wirtschaftsgenossenschaft deutscher Tierärzte eG, Siemensstr. 14, 30827 Garbsen, Germany). After examination of the external appearance, the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed. All gross macroscopic changes were recorded for each animal.
Statistics:
Not specified.

Results and discussion

Effect levels
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
AMINOX® did not cause mortality at a dose level of 2000 mg/kg bw.
Clinical signs:
other: Clinical signs were observed in 3/6 animals treated at the dose level of 2000 mg/kg bw with AMINOX® which included hunched back. There was no other test item related effects on the animals.
Gross pathology:
There was no evidence of the macroscopic observations at necropsy at a dose level of 2000 mg/kg bw.
Other findings:
No further findings detailed in the study report.

Any other information on results incl. tables

CLINICAL OBSERVATIONS

DOSE LEVEL: 2000 mg/kg bw, Treatment on Day 0     SEX: FEMALE

Cage No.

Animal Number

Observations

Observation days

Frequency

0

1

2

3

4

5

6

7-14

30’

1h

2h

3h

4h

6h

1

9631

Symptom free

+

+

-

-

-

+

+

+

+

+

+

+

+

17/20

Hunched back

-

-

+

+

+

-

-

-

-

-

-

-

-

3/20

9632

Symptom free

+

+

-

-

-

+

+

+

+

+

+

+

+

17/20

Hunched back

-

-

+

+

+

-

-

-

-

-

-

-

-

3/20

9633

Symptom free

+

+

-

-

-

+

+

+

+

+

+

+

+

17/20

Hunched back

-

-

+

+

+

-

-

-

-

-

-

-

-

3/20

2

9634

Symptom free

+

+

+

+

+

+

+

+

+

+

+

+

+

20/20

9635

Symptom free

+

+

+

+

+

+

+

+

+

+

+

+

+

20/20

9636

Symptom free

+

+

+

+

+

+

+

+

+

+

+

+

+

20/20

Remarks:              + = present           - = absent

                          h = hour              ‘ = minute

                          Frequency of observations = number of occurrence of observation / total number of observations

 

BODY WEIGHT DATA

DOSE LEVEL: 2000 mg/kg bw, Treatment on Day 0     SEX: FEMALE

Cage No.

Animal Number

Body weight (g)

Days

Body Weight Gain (g)

-1

0

7

14

-1-0

0-7

7-14

-1-14

1

9631

243

227

235

250

-16

8

15

7

9632

233

217

233

231

-16

16

-2

-2

2633

239

227

237

243

-12

10

6

4

2

9634

241

232

240

261

-9

8

21

20

9635

212

203

227

239

-9

24

12

27

9636

220

207

230

247

-13

23

17

27

Mean:

231.3

218.8

233.7

245.2

-12.5

14.8

11.5

13.8

Standard deviation:

12.6

11.8

4.7

10.2

3.1

7.3

8.3

12.5

 

NECROPSY FINDINGS

DOSE LEVEL: 2000 mg/kg bw, Treatment on Day 0     SEX: FEMALE

Cage No.

Animal Number

Necropsy Date/ Necropsy Day

External Observations

Internal Observations

Organ/Tissue

1

9631

04 July 2017

Day 14

No external observations recorded

No internal observations recorded

Not applicable

9632

04 July 2017

Day 14

No external observations recorded

No internal observations recorded

Not applicable

9633

04 July 2017

Day 14

No external observations recorded

No internal observations recorded

Not applicable

2

9634

05 July 2017

Day 14

No external observations recorded

No internal observations recorded

Not applicable

9635

05 July 2017

Day 14

No external observations recorded

No internal observations recorded

Not applicable

9636

05 July 2017

Day 14

No external observations recorded

No internal observations recorded

Not applicable

 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of this study, the acute oral LD50 value of the test item AMINOX® was found to be above 2000 mg/kg bw in female CRL:(WI) rats.
Executive summary:

The single-dose oral toxicity of AMINOX® was performed according to the acute toxic class method (OECD 423 and Commission Regulation (EC) No 440/2008 of 30 May 2008, B.1.Tris) in CRL:(WI) rats.

 

Initially, three females (assigned to Group 1) were treated at a dose level of 2000 mg/kg bw. The test item did not cause mortality in this group and a second group (Group 2) was treated at the same dose level. No mortality was observed in the confirmatory group, therefore no further testing was required according to OECD 423 and Commission Regulation (EC) No 440/2008 of 30 May 2008, B.1.tris.

 

A single oral treatment was carried out by gavage for each animal after an overnight food withdrawal. Food was made available again 3 hours after the treatment. The test item was administered formulated in Dimethyl sulfoxide (DMSO) at a concentration of 200 mg/mL at a dosing volume of 10 mL/kg bw.

 

Clinical observations were performed at 30 minutes, 1, 2, 3, 4 and 6 hours after dosing and daily for 14 days thereafter. Body weight was measured on Days -1, 0 and 7 and Day 14.

All animals were subjected to a necropsy and a macroscopic examination.

 

Results

Mortality

AMINOX® did not cause mortality at a dose level of 2000 mg/kg bw.

 

Clinical Observations

Clinical signs were observed in 3/6 animals treated at the dose level of 2000 mg/kg bw with AMINOX® which included hunched back. There was no other test item related effects on the animals.

 

Body Weight and Body Weight Gain

One animal at dose level 2000 mg/ kg bw (No: 9632) showed a reduced body weight gain in the second week of the observation period. This change was considered incidental and not ascribed to treatment. There was no other treatment related effects on body weight or body weight gain during the observation period.

 

Macroscopic Findings

There was no evidence of the macroscopic observations at necropsy at a dose level of 2000 mg/kg bw.

 

Conclusion:

Under the conditions of this study, the acute oral LD50 value of the test item AMINOX® was found to be above 2000 mg/kg bw in female CRL:(WI) rats.