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- Cp REPORT SANITIZED ACUTE ORAL TOXICITY WITH 1,1,3,3-TETRAMETHYLBUTYL PEROXYPIVALATE IN THE RAT (FIXED DOSE METHOD) /"-- NOTOX Project

(

. NOTOX **Substanc ~/**  - page

1 of 15

-1,1,3,3-TETRAMETHYLBUTYL PEROXYPIVALATE CONFIDENTIALITY STATEMENT
**~** NOTOX Projech_,

· -...____/ This report contains the unpublished results
of research sponsored

by Akzo Nobel Chemicals

B

. V .. Reproduction, issue or disclosure

to third parties

in any form is not permitted without prior written authorization
from the sponsor. - Page

2
--- **:~~,**

I **lT-~.-;;3,--3--T-E-T--RAMR-H¥-LB-'df-Y-t:.** -?ERtJ-)tYPIV A'cATE,___ __________ **-NIJTOX~PtOject** ------------

- **~--/** STATEMENT

OF GLP COMPLIANCE NOTDX B.V., 's-Hertogenbosch,

The Netherlands The study described

in this report has been correctly reported and was conducted in compliance with the most recent edition of: The OECD Principles

of Good Laboratory Practice which are essentially

in conformity with: The United States Food and Drug Administration. Title 21 Code of Federal Regulations Part 58. The United States Environmental Protection Agency (FIFRA). Title 40 Code of Federal Regulations Part 160. The United States Environmental Protection Agency (TSCA). Title 40 Code of Federal Regulations Part 792. study Director:
Dr.Ir. W.R. Pels Rijcken **~--~** ------ Management:

Drs. W.J.A.M. Frieling Section Head General and Industrial Toxicology - Page

3
-1,1,3,3-TETRAMETHYLBUTYL PEROXYPIVALATE

NOTOX **Pcojoo~** QUALITY ASSURANCE STATEMENT NOTOX B.V., 's-Hertogenbosch,

The Netherlands This report was audited

by the NOTOX Quality Assurance Unit to ensure that the methods and results accurately reflect the raw data. The dates of Quality Assurance inspections

and audits are given below. During the on-site inspections procedures applicable

to this type of study were inspected. DATES
OF QAU INSPECTIONS/ REPORTING DATES AUDITS on-site inspection(s) 19 December 1996 protocol inspection(s) 2 December 1996 report audit(s) 29 January 1997

19 December 1996 2 December 1996 29 January 1997 Quality Assurance Manager: C.J. Mitchell B.Sc. - Page 4 -SUMMARY Acute oral toxicity with 1,1,3,3-TETRAMETHYLBUTYL PEROXYPIVALATE

in the rat (Fixed dose method), The study was carried out in accordance with OECD Guideline
No. 420, "Acute Oral Toxicity

- Fixed dose method" and EEC Directive 92/69/EEC, Part 8.1-bis, "Acute Toxicity-Oral, Fixed dose method". Initially, 1,1,3,3-TETRAMETHYLBUTYL PEROXYPIVALATE

was administered

by oral gavage
to one female
at 2000 mg/kg body weight.

As no mortality occurred and no signs of toxicity were observed,
the main study was conducted with

a fixed dose of 2000 mg/kg body weight administered

to five rats of each sex. The animals were subjected

to daily observations.
Body weights were determined

on days 1, 2, 3, 4, 8 and 15. Macroscopic examination

was performed

at the end of the experimental period. No mortality occurred. Hunched posture and uncoordinated movements were observed

in one female, which had recovered from the symptoms

by day 4. The body weight gain shown by the animals over the study period was considered to be normal. No abnormalities
were found at macroscopic past mortem examination

of the animals. The minimum oral lethal dose of 1,1,3,3-TETRAMETHYLBUTYL PEROXYPIVALATE

in rats was established

as exceeding 2000 mg/kg body weight. Based
on these results and according

to the EEC criteria
for classification

and labelling requirements

for dangerous substances
and preparations (Guidelines

in Commission Directive 93121/EEC, 27th April 1993), 1,1,3,3-TETRAMETHYLBUTYL PEROXYPIVALATE
does not have to be classified
and has no obligatory labelling requirement.

- Page

5
-1,1,3,3-TETRAMETHYLBUTYL PEROXYPIVALATE PREFACE Sponsor Study Monitor Testing Facility Study
Oi rector study Plan TEST SUBSTANCE

AKZO Nobel Chemicals B.V. P.O. Box 247 3800 AE AMERSFOORT The Netherlands **-------=---~** NOTOX B.V. Hambakenwetering

3 5231 DO 's-Hertogenbosch The Netherlands Start Pilot Study

4 December 1996 Main Study

: 10 December 1996 End

: 24 December 1996 The sponsor

is responsible

for the completeness

and GLP Compliance

of all test substance data. Identification Chemical name CAS No. Description Batch Purity Test substance storage Stability under storage conditions Expiry date Density Preparation PURPOSE AND RATIONALE 1,1,3,3-tetramethylbutyl peroxypivalate Propaneperoxic acid, 2,2-dimethyl-,1,1,3,3- tetramethylbutyl
ester (9CI) 22288-41-4 **~olourless** liquid 

### (__



# '

. See-eer·Joi-rtcate

of Ana
1 ys is In freezer

in the dark Stable 01 June 1997 0.87 g/ml (Determined

at NOTOX) The test substance
was dosed undiluted

as delivered by the sponsor. The objective

of this study was to assess
at which maximum fixed dose level 1,1,3,3-TETRAMETHYLBUTYL PEROXYPIVALATE causes evident toxicity,
but no mortality, when administered

to rats as a single oral dose. This study should provide part of a rational basis for risk assessment

in man. The oral route was selected

as it is a possible route of human exposure during manufacture, handling

or use of 1,1,3,3-TETRAMETHYLBUTYL PEROXYPIVALATE. - Page

6
-GUIDELINES The study procedures described

in this report are based on the following guidelines: Directive 92/69/EEC, Annex

V of the EEC directive 67/548/EEC; Part
B, Methods for the Determination

of Toxicity,

81 bis: "Acute toxicity-oral, Fixed Dose Method". Official Journal
of the European Communities, L383, December 1992. DECO "Guidelines

for Testing
of Chemicals", Section

4, Health Effects, No.420, "Acute Oral Toxicity- Fixed Dose Method", Paris Cedex, adopted

17 July, 1992. ARCHIVING NOTOX B.V. will archive the following data for at least 10 years: raw data, protocol, report and test substance reference sample. All magnetic media produced

on this study will be maintained indefinitely. TEST SYSTEM Test System Age at start of Treatment Body weight
at start . of treatment Total number of animals Identification ANIMAL HUSBANDRY Conditions

Rat, Wistar strain Crl:(WI)

BR (outbred, SPF-Quality) Recognised

by international guidelines

as the recommended test system (e.g. DECO, EEC). Source

: Charles River, Sulzfeld, Germany. Approx.

6 weeks. Within +1- 20% of the sex mean. Pilot study:

1 female. Main study

: 5 males and
5 females. Earmark Air-conditioned

room with approximately

15 air changes per hour and the environment contra

11 ed wi. th optima

1 conditions considered

as being

a temperature of 21°C and

a relative humidity

of 50%. Fluctuations
from these optimal conditions were noted, but were considered
not to have affected study integrity. Lighting was 12 hours artificial fluorescent light and 12 hours dark per day. Accommodation Individual housing
of animals

in the Pilot study and group housing
of five animals per sex per cage in the Main study in labelled polycarbonate cages containing purified sawdust

as bedding material (Woody SPF, supplied

by B.M.I., Helmond, The Netherlands). Certificates

of analysis were examined and then retained

in the NOTOX archives. Acclimatisation period was at least

5 days before start of treatment under laboratory conditions. Diet Free access
to standard pelleted laboratory animal diet (from Carfil Quality BVBA, Oud-Turnhout, Belgium). Certificates

of analysis are examined and then retained

in the NOTOX archives.

- Page

7
-._,........-------1, 1,3,3-TETRAMETHYLBUTYLPEROXYPIVALATENOTOXPro}ec_tWaterFreeaccesstotap-water.Certificatesofanalysis(performedquarterly)areexaminedandthenretainedintheNOTOXarchives.DOSESELECTIONInitiallya"Pilotstudy",i.e.NOTOXProject192847wasconductedinwhichonefemaleratwasorallydosedwith1,1,3,3-TETRAMETHYLBUTYLPEROXYPIVALATEat2000mg/kg(2.3ml/kg)bodyweight.Nomortalityoccurredatthisdoselevel.Basedontheresults,themainstudywasselectedwithonedoselevelandfivemaleandfivefemaleanimalsdosedat2000mg/kgbodyweight.TREATMENTAhealthinspectionwasperformedpriortocommencementoftreatment,toensurethattheanimalswereinagoodstateofhealth.MethodFastingFrequencyDoselevel/volumeOBSERVATIONSPeriodMortality/ViabilityBodyweightsPilotstudyMainstudyClinicalsigns(Pilotandmainstudy)Necropsy(Mainstudy)INTERPRETATIONOralgavageusingstainlesssteelstomachtube.Foodwaswithheldovernightpriortodosinguntilapproximately3-4hoursafteradministrationofthetestsubstance.Once,onday1.Group1:2000mg/kg(2.3ml/kg)bodyweight.Dosevolumecalculatedasfollows:doselevel(g/kg):density(g/ml).Pilotstudy:8days.Mainstudy:15days.Twicedaily·(Pilotandmainstudy).Days1(pre-administration)and8.Days12,3,4,8and15.Atperiodicintervalsonthedayofdosing(day1)andoncedailythereafter.Thetimeofonset,degreeanddurationwererecorded.Allanimalssurvivingtotheendoftheobservationperiodweresacrificedbyoxygen/carbondioxideasphyxiation.Allanimalsassignedtothestudyweresubjectedtonecropsyanddescriptionsofallmacroscopicabnormalitiesrecorded.TheresultswereevaluatedaccordingtotheEECcriteriaforclassificationandlabellingrequirementsfordangeroussubstancesandpreparations(GuidelinesinCommissionDirective93/21/EEC,27thApril1993).-Page8-**-~-----:l,.l,b3-TETRAMETHYLB.uJJ'LP£R!lXY_?llALAI£**

__

_ RESULTS PRELIMINARY SIGHTING STUDY CPILOT STUOY)

. -- /.----

.. ,_"'

I **~~~---<<.OHlX-P-1'-o~~~---~)-**

-- -- '-.___j 2000 mg/kg:

No mortality occurred and no clinical signs were observed during this study period. MAIN STUDY Mortality No mortality occurred

in the animals during the study. Clinical Signs Hunched posture and uncoordinated movements were observed

in one female, which had recovered from the symptoms

by day 4. Body Weight The body weight gain shown by the animals over the study period was considered to be similar

to that expected

of normal untreated animals
of the same age and strain. Macroscopic Findings No abnormalities
were found at macroscopic post mortem examination

of the animals. CONCLUSION The minimum oral lethal dose of 1,1,3,3-TETRAMETHYLBUTYL PEROXYPIVALATE

in rats was established

as exceeding 2000 mg/kg body weight. Based on these results and according

to the EEC criteria for classification and labelling requirements

for dangerous substances
and preparations (Guidelines

in Commission Directive 93/21/EEC, 27th April 1993), 1,1,3,3- TETRAMETHYLBUTYL PEROXYPIVALATE
does not have to be classified
and has no obligatory labelling requirement.

- Page 9 -....--------...___NOTOX)1,1,3,3-TETRAMETHYLBUTYLPEROXYPIVALATEPILOTSTUDYMORTALITYFEMALES(NOTOXNUMBEROFDECEDENTSAFTERTREATMENTTestday1 1 1 234 5 6 7 89101112l31415Hoursaftertreatment024Group1(2000MG/KG)CLINICALSIGNSFEMALESGROUP1(2000MG/KG)TestdayTimeaftertreatment.Hours:ANlMALNUMBERSIGNS1NOCLINICALSIGNSNOTEDMAX.GRADEBODYWEIGHTS(GRAM)GROUPISEXGROUP1IFEMALES(2000MG/KG)ANIMAL1DAY11451 1 1 234561s9101112131415024DAYS184-Page10-**/~-~ ------'-,J.,3~3JB.RAMEJliYLBUT't'-L**  PEROX'if'Tl!AL!l.JE **NOWX-f'r-{l;jeG~** ---')--- -- **~~** CLINICAL SIGNS MALES

MAIN STUDY GROUP

1 (2000 MG/KG) Test daY Time after treatment. Hours: ANIMAL NUMBER SIGNS 1

NO CLINICAL SIGNS NOTED 2

NO CLINICAL SIGNS NOTED 3

NO CLINICAL SIGNS **NOT~D** 4

NO CLINICAL SIGNS NOTED 5

NO CliNICAL SIGNS NOTED FEMALES

MAX. GRADE 1 1 1 2

3 4 5
6 7
8
9 10 11 12 13 14 15 0
2 4 GROUP

1 (2000 MG/KG) Test day

. Time after treatment. Hours: ANIMAL NUMBER SIGNS 6

NO CLINICAL SIGNS NOTED 7

NO CLINICAL SIGNS NOTED 8

NO CLINICAL SIGNS NOTED 9

NO CLINICAL SIGNS NOTED 10 POSTURE

MAX. GRADE 1 1 1 2 3 4

s
6 7
8 9 10 11 12 13 14 15 0
2
4 HUNCHED POSTURE, ...........

(1)

-

-

l 1 1 1

- GAIT

I MOTILITY UNCOORDINATED MOVEMENTS.,,.

(3) - **~** sign not observed

I

• = observation
not performed

I + **~** animal dead - Page 11

-1,1,3,3-TETRAMETHYLBUTYLPEROXYPIVALATENOTOXProje{;__.---)\.,.....______BODYWEIGHTS(GRAM)GROUPSEXAN'!MALOAY1OAY2DAYJDAY4OAYBDAY15GROUP1IMALES1174!82200209250304(2000MGIKG)2167176195208240295316617819220124230041701882042132553185168179195206245315MEAN169181197207246306ST.OEV.3554610N;55555GROUP1 IFEMALES61%148!58.162183212(2000MG/KG)714415316417018320481481571711761972119138146!6417-1!S920110141 147!50160189217MEAN141150161168 188209ST.OEV.558766N555555-Page12-MACROSCOPIC FINDINGS MALES GROUP

1 (2000 MG/KG) ANIMAL

1 NO FINDINGS NOTED ANIMAL

2 NO FINDINGS NOTED ANIMAL

l' NO FINDINGS NOTED ANIMAL

4 NO FINDINGS NOTED **ANII~AL**

5 NO FINDINGS NOTED FEMALES GROUP

1 (2000 MG/KG) ANIMAL

6 NO FINDINGS NOTED ANIMAL

7 NO FINDINGS NOTED ANIMAL

8 NO FINDINGS NOTED ANIMAL

9 NO FINDINGS NOTED ANIMAL
10 NO FINDINGS NOTED
{SCHEDULED NECROPSY, 24-0EC-96,
DAY 15 AFTER TREATMENT) (SCHEDULED NECROPSY, 24-DEC-96,
DAY 15 AFTER TREATMENT) (SCHEDUlED NECROPSY, **24-0EC~96,**
DAY 15 AFTER TREATMENT) (SCHEDULED NECROPSY, 24-DEC-96,
DAY 15 AFIER TREATMENT) (SCHEDULED NECROPSY, 24-DEC-96,
DAY 15 AFTER TREATMENT) (SCHEDULED NECROPSY

1 24-DEC-96

1 DAY 15 AFTER TREATMENT) {SCHEOULEO NECROPSY, 24-DEC-96,
DAY 15 AFIER TREATMENT) (SCHEDULED NECROPSY, 24-DEC-96,
DAY 15 AFTER TREATMENT) (SCHEDULED NECROPSY, 24-DEC-96,
DAY 15 AFTER TREATMENT) (SCHEDULED NECROPSY, 24-DEC-96,
DAY 15 AFTER TREATMENT) - Page 13

-1,1,3,3-TETRAMETHYLBUTYL PEROXYPIVALATE

(----- NOTOX **Proje~~** APPENDIX TEST SUBSTANCE CERTIFICATE

OF ANALYSIS

- Page 14

-fJ 47<'- AKZONOBEL Certificate

of Analysis ReffCS-331 .. Product nama **i:'f;t~e** _nama **B~!chnumbar**

: 1,1 ,3,3-tetramethylbutyl peroxypiva!ate :-Ttig.QllilX 425 C75 . . . :·-.

':

: ·. . ..

;, **Ari~IYtfcal documanta~c>"n** 

· §

( **Fii~cad~**

-

-l Autfiorlzad **bv---~-----------­** Na(na Function **D~ta** Signature

- Page 15

-

Central Research