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Toxicological information

Acute Toxicity: oral

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Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 May 2017 to 24 May 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:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Version / remarks:
OECD Guidelines for Testing of Chemicals No. 423. Acute Oral Toxicity – Acute Toxic Class Method. Adopted: 17 December 2001
Deviations:
yes
Remarks:
See "Any other information" for details
Qualifier:
according to
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:
yes
Remarks:
See "Any other information" for details
Qualifier:
according to
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:
yes
Remarks:
See "Any other information" for details
GLP compliance:
yes (incl. certificate)
Test type:
acute toxic class method
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals 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, Germany
Hygienic level at arrival: SPF
Hygienic level during the study: Standard housing conditions
Justification of strain: The Wistar rat as a rodent is one of the standard strains of acute toxicity studies.
Number of animals: 3 animals
Sex: Female, nulliparous and non-pregnant
Age of animals at dosing: Young healthy adult rats, 8 weeks old
Body weight at treatment: 183 – 206 g
Acclimatisation period: 5-6 days

Husbandry
Animal health: Only healthy animals were used for the test. The health status was certified by the staff Veterinarian.
Number of animal room: 522/5
Housing: 1-2 animals / cage
Cage type: Type II. polypropylene/polycarbonate
Bedding and nesting: “Lignocel 3/4-S Hygienic Animal Bedding” and “Arbocel crinklets natural” nesting 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 a.m. to 6.00 p.m.
Temperature: 20.2 – 24.5 °C
Relative humidity: 32 – 65 %
Ventilation: 15-20 air exchanges/hour
Enrichment: Rodents were housed with deep wood sawdust bedding to allow digging and other normal rodent activities.
The temperature and relative humidity were recorded twice daily during the acclimatisation period and throughout 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 number: 285 17890, expiry date: 31 August 2017), ad libitum, and tap water from the municipal supply, as for human consumption from a 500 mL bottle, 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:
corn oil
Details on oral exposure:
Formulation
The test item was freshly formulated at a concentration of 500 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 the start of the treatment.

Justification of the dose:
The starting dose level was selected at the request of the Sponsor. The starting dose level was 5000 mg/kg bw of LUPEROX® 520M50 which is equivalent to 2465 mg/kg bw of 2,2-di-(tert-amylperoxy)butane.

Initially, one female animal was treated with 5000 mg/kg bw of the test item. No mortality was observed, therefore further 2 animals were treated at the dose level of 5000 mg/kg bw. As no mortality was observed in this second dose group, further testing was not required according to the test guidelines (OECD 423, Commission Regulation (EC) No 440/2008 of 30 May 2008, B.1.tris).
Doses:
5000 mg/kg bw
No. of animals per sex per dose:
Initially, one female animal was treated. No mortality was observed, therefore further 2 animals were treated.
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 returned 3 hours after the treatment.

OBSERVATIONS
Clinical Observations
Clinical observations were performed on all animals at 30 minutes, 1, 2, 3, 4 and 6 hours after dosing and daily for 14 days thereafter. 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) and weekly thereafter.

NECROPSY
Macroscopic examination was performed on all animals. The animals were sacrificed by exsanguination under pentobarbital anaesthesia (Release, 300 mg / kg pentobarbital sodium; Lot number: 106075, Expiry date: 31 July 2018, Produced by: Wirtschaftsgenossenschaft deutscher Tierärzte eG, Germany). After examination of the external appearance, the cranial, thoracic and the abdominal cavities were opened and the organs and the tissues were observed. Macroscopic abnormalities were recorded.
Statistics:
Not required

Results and discussion

Effect levelsopen allclose all
Sex:
female
Dose descriptor:
LD0
Effect level:
> 5 000 mg/kg bw
Based on:
test mat.
Sex:
female
Dose descriptor:
LD0
Effect level:
> 2 465 mg/kg bw
Based on:
act. ingr.
Remarks:
as 2,2-di-(tert-amylperoxy)butane
Mortality:
LUPEROX® 520M50 did not cause mortality at a dose level of 5000 mg/kg bw.
Clinical signs:
There were no systemic clinical signs noted in any animal throughout the study.
Body weight:
There were no treatment related body weight changes. Body weight gains of LUPEROX® 520M50 treated animals during the study showed no indication of a test item-related effect.
Gross pathology:
There was no evidence of the macroscopic observations at a dose level of 5000 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

8786

Symptom Free

+

+

+

+

+

+

+

+

+

+

+

+

+

20/20

2

8787

Symptom Free

+

+

+

+

+

+

+

+

+

+

+

+

+

20/20

8788

Symptom Free

+

+

+

+

+

+

+

+

+

+

+

+

+

20/20

Remarks:        + = present

                    h = hour (s)          ‘ = minute

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

 

BODY WEIGHT DATA

DOSE LEVEL: 5000 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

8786

226

206

232

241

-20

26

9

15

2

8787

197

183

213

222

-14

30

9

25

8788

210

194

212

231

-16

18

19

21

Mean:

211.0

194.3

219.0

231.3

-16.7

24.7

12.3

20.3

Standard deviation:

14.5

11.5

11.3

9.5

3.1

6.1

5.8

5.0

 

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

8786

23 May 2017

Day 14

No external observations recorded

No internal observations recorded

Not applicable

2

8787

24 May 2017

Day 14

No external observations recorded

No internal observations recorded

Not applicable

8788

24 May 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 LD0 value of the test item LUPEROX® 520M50 was found to be equal to or above 5000 mg/kg bw in female Crl:WI rats.
Executive summary:

The single-dose oral toxicity of LUPEROX® 520M50 (49.3% of 2,2-di-(tert-amylperoxy)butane in isododecane) was performed according to the acute toxic class method (OECD 423) in Crl:WI rats. Three female Crl:WI rats were treated with LUPEROX® 520M50 at a dose level of 5000 mg/kg bw. 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 corn oil at a concentration of 500 mg/mL at a dose volume of 10 mL/kg bw.  Initially, one female was treated at a dose level of 5000 mg/kg bw. As no mortality was observed, a confirmatory group of two females 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. 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 14 (before necropsy). All animals were subjected to a necropsy and a macroscopic examination.  LUPEROX® 520M50 did not cause mortality at a dose level of 5000 mg/kg bw. There were no systemic clinical signs noted in any animal throughout the study. Body weight gains of LUPEROX® 520M50 treated animals during the study showed no indication of a test item-related effect. There was no evidence of the macroscopic observations at a dose level of 5000 mg/kg bw.  Under the conditions of this study, the acute oral LD0 values of LUPEROX® 520M50 and 2,2-di-(tert-amylperoxy)butane were found to be higher than 5000 and 2465 mg/kg bw in female Crl:WI rats, respectively.