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Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 March 2017 to 12 April 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 No. 423. Acute Oral Toxicity – Acute Toxic Class Method. Adopted: 17 December 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:
6,6'-di-tert-butyl-2,2'-thiodi-p-cresol
EC Number:
202-009-7
EC Name:
6,6'-di-tert-butyl-2,2'-thiodi-p-cresol
Cas Number:
90-66-4
Molecular formula:
C22H30O2S
IUPAC Name:
6,6'-di-tert-butyl-2,2'-thiodi-p-cresol
Test material form:
solid: particulate/powder
Details on test material:
Name: 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol
Other name: LOWINOX® TBP-6
CAS number: 90-66-4
Batch/Lot Number: C034J0059 / C036K0111
Description: White powder
Purity*: 99.8%
Expiry date: 12 May 2017
Storage condition: Controlled room temperature (15-25 ºC, below 70 RH%)
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.
* No correction for purity of the test item was applied.
Specific details on test material used for the study:
No further details specified in the study report.

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI
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, Germany
Hygienic level at arrival: SPF
Hygienic level during the study: Standard housing conditions
Number of animals: 6 animals, 3 animals/group
Sex: Female, nulliparous and non-pregnant
Age of animals at dosing: Young healthy adult rats, 8 weeks old
Body weight at treatment: 185 – 202 g
Acclimatization 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: 522/4
Housing: 3 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.
Copies of the Certificates of Analysis are retained in the archive at CiToxLAB Hungary Ltd.
Lighting period: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 20.4 – 24.4 °C
Relative humidity: 30 – 64 %
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 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: 484 14771, expiry date: 30 June 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). The quality control results are retained in the archives at CiToxLAB Hungary Ltd.

.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:
methylcellulose
Details on oral exposure:
Vehicle: 1% methyl cellulose

Components of the vehicle:
Name: Distilled water
Manufacturer: Hungaro-Gal Kft.
Batch number: 802 0117
Expiry date: 23 July 2017

Name: Methyl cellulose
Manufacturer: Shin-Etsu Chemical Co., Ltd.
Batch number: 5115851
Expiry date: 27 November 2018

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.

Justification of the dose:
The initial dose level was selected by the Study Director to be that which is most likely to produce mortality in some of the dosed animals. In the lack of any preliminary toxicological information, 2000 mg/kg bw was selected to be the starting dose.

Initially, three female animals were treated with 2000 mg/kg bw of the test item. No mortality was observed, therefore further 3 animals were treated at the dose level of 2000 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:
The initial dose level was selected by the Study Director to be that which is most likely to produce mortality in some of the dosed animals. In the lack of any preliminary toxicological information, 2000 mg/kg bw was selected to be the starting dose.
No. of animals per sex per dose:
Initially, three female animals were treated with 2000 mg/kg bw of the test item. No mortality was observed, therefore further 3 animals were treated at the dose level of 2000 mg/kg bw. As no mortality was observed in this second dose group, further testing was not required.
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 levels
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
6,6’-di-tert-butyl-2,2’-thiodi-p-cresol did not cause mortality at a dose level of 2000 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 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol 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 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

7879

Symptom Free

+

+

+

+

+

+

+

+

+

+

+

+

+

20/20

7880

Symptom Free

+

+

+

+

+

+

+

+

+

+

+

+

+

20/20

7881

Symptom Free

+

+

+

+

+

+

+

+

+

+

+

+

+

20/20

2

7882

Symptom Free

+

+

+

+

+

+

+

+

+

+

+

+

+

20/20

7883

Symptom Free

+

+

+

+

+

+

+

+

+

+

+

+

+

20/20

7884

Symptom Free

+

+

+

+

+

+

+

+

+

+

+

+

+

20/20

Remarks:         + = present

                    h = hour (s)            ‘ = minute

                    Frequency of observation = 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

7879

219

197

226

239

-22

29

13

20

7880

212

200

241

257

-12

41

16

45

7881

212

202

221

229

-10

19

8

17

2

7882

196

185

204

218

-11

19

14

22

7883

196

190

209

229

-6

19

20

33

7884

206

200

224

237

-6

24

13

31

Mean:

206.8

195.7

220.8

234.8

-11.5

25.25

14.0

28.0

Standard deviation:

9.3

6.7

13.2

13.2

5.9

8.7

3.9

10.4

 

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

7879

11 April 2017

Day 14

No external observation recorded

No internal observations recorded

Not applicable

 

7880

11 April 2017

Day 14

No external observation recorded

No internal observations recorded

Not applicable

 

7881

11 April 2017

Day 14

No external observation recorded

No internal observations recorded

Not applicable

2

7882

12 April 2017

Day 14

No external observation recorded

No internal observations recorded

Not applicable

 

7883

12 April 2017

Day 14

No external observation recorded

No internal observations recorded

Not applicable

 

7884

12 April 2017

Day 14

No external observation 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 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol was found to be above 2000 mg/kg bw in female Crl:WI rats.
According the GHS criteria, 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol can be ranked as "Category 5 or Unclassified" for acute oral exposure.
Executive summary:

The single-dose oral toxicity of 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol 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.

 

Two groups of three female Crl:WI rats were treated with the test item at a dose level of 2000 mg/kg bw (Group 1 and Group 2).

 

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 1% methyl cellulose at a concentration of 200 mg/mL at a dose volume of 10 mL/kg bw.

 

Initially, three females (Group 1) were treated at a dose level of 2000 mg/kg bw. As no mortality was observed, a confirmatory 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.

 

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.

 

RESULTS

Mortality

6,6’-di-tert-butyl-2,2’-thiodi-p-cresol did not cause mortality at a dose level of 2000 mg/kg bw.

 

Clinical Observations

There were no systemic clinical signs noted in any animal throughout the study.

 

Body Weight and Body Weight Gain

There were no treatment related body weight changes. Body weight gains of 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol treated animals during the study showed no indication of a test item-related effect.

 

Macroscopic Findings

There was no evidence of the macroscopic observations 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 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol was found to be above 2000 mg/kg bw in female Crl:WI rats.

 

According the GHS criteria, 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol can be ranked as "Category 5 or Unclassified" for acute oral exposure.