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Dunnick JK, Malarkey DE, Bristol DW, et al. NTP Genetically Modified Model Report on the Toxicology and Carcinogenesis Study of Benzene (CASRN 71-43-2) in Genetically Modified Haploinsufficient p16Ink4a/p19Arf Mice (Gavage Study): NTP GMM 08 [Internet]. Research Triangle Park (NC): National Toxicology Program; 2007 Oct.

Cover of NTP Genetically Modified Model Report on the Toxicology and Carcinogenesis Study of Benzene (CASRN 71-43-2) in Genetically Modified Haploinsufficient p16Ink4a/p19Arf Mice (Gavage Study)

NTP Genetically Modified Model Report on the Toxicology and Carcinogenesis Study of Benzene (CASRN 71-43-2) in Genetically Modified Haploinsufficient p16Ink4a/p19Arf Mice (Gavage Study): NTP GMM 08 [Internet].

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APPENDIX DCHEMICAL CHARACTERIZATION AND DOSE FORMULATION STUDIES

Procurement and Characterization of Benzene

Benzene was obtained from Sigma Aldrich Chemical Co. (Milwaukee, WI) in one lot (00358CS). Identity and purity analyses were conducted by the analytical chemistry laboratory, Research Triangle Institute (RTI) (Research Triangle Park, NC) and the study laboratory, Battelle Columbus Operations (Columbus, OH). Reports on analyses performed in support of the benzene study are on file at the National Institute of Environmental Health Sciences.

Lot 00358CS, a colorless liquid, was identified as benzene by RTI using infrared (IR) spectroscopy and proton nuclear magnetic resonance (NMR) spectroscopy and by the study laboratory using IR spectroscopy. Spectra were in agreement with the structure of benzene, literature spectra (Aldrich, 1981,1983,1985), and spectra from a frozen reference standard of the same lot. Representative IR and NMR spectra are presented in Figures D1 and D2.

Figure D1. Infrared Absorption Spectrum of Benzene.

Figure D1

Infrared Absorption Spectrum of Benzene.

Figure D2. Proton Nuclear Magnetic Resonance Spectrum of Benzene.

Figure D2

Proton Nuclear Magnetic Resonance Spectrum of Benzene.

The purity of lot 00358CS was determined by RTI using gas chromatography (GC) by system A (Table D1) and by the study laboratory using GC by system B. GC by system A indicated one major peak and one impurity peak with an area that was less than 0.1% of the total peak area. The overall purity of lot 00358CS was determined to be greater than 99%.

To ensure stability, the bulk chemical was stored at room temperature (25° C) in amber glass bottles with Teflon®-lined lids, protected from light. Stability was monitored twice during the 27-week study with GC by system B; no degradation of the bulk chemical was detected. GC by system B indicated one major peak with a purity of 99.3% relative to a frozen reference standard of the same lot.

Preparation and Analysis of Dose Formulations

The dose formulations were prepared by mixing benzene with corn oil (Spectrum Chemicals and Laboratory Products, Inc., Gardena, CA) to give the required concentrations (Table D2). Because animal room sample analyses indicated probable evaporative loss of benzene from the dose formulations during administration, dose formulations prepared on January 4, 2000, or later were prepared with 5% less corn oil than specified in the original SOP for dose formulation. The dose formulations were stored at room temperature (25° C) in amber glass bottles capped with Teflon®-lined lids for up to 35 days.

Stability studies of a 1.0 mg/mL dose formulation of benzene in corn oil were performed by RTI with GC by system C. Stability was confirmed for up to 35 days for dose formulations stored in the dark in sealed glass bottles at refrigerator temperature (5° C) and for up to 3 hours exposed to air and light under simulated dosing conditions.

Periodic analyses of the dose formulations of benzene were conducted by the study laboratory using GC by system B. During the 27-week study, the dose formulations were analyzed four times and postadministration animal room samples were analyzed once (Table D3). All 12 dose formulations analyzed and used in the study were within 10% of the target concentrations; all four animal room samples analyzed were within 15% of the target concentration. Periodic analyses of the corn oil vehicle by the study laboratory demonstrated peroxide concentrations below the acceptable limit of 3 mEq/kg.

Table D1Gas Chromatography Systems Used in the 27-Week Gavage Study of Benzenea

Detection SystemColumnCarrier GasOven Temperature Program
System A
Flame ionizationJ&W DB-5, 30 m × 0.53 mm, 5.0-μm film (J&W Scientific, Folsom, CA)Helium at 2.0 mL/minute

50° C for 5 minutes, then

5° C/minute to 225° C

System B
Flame ionizationSPB-5, 30 m × 0.53 mm, 3.0-μm film (Supelco, Inc., Bellefonte, PA)Helium at 4.0 mL/minute

45° C for 3 minutes, then

5° C/minute to 135° C, held for 2 minutes

System C
Flame ionizationJ&W DB-5, 30 m × 0.53 mm, 5.0-μm film (J&W Scientific)Nitrogen at 4.0 mL/minute

75° C for 3 minutes, then

5° C/minute to 135° C

a

The gas chromatograph for system C was manufactured by Hewlett-Packard (Palo Alto, CA).

Table D2Preparation and Storage of Dose Formulations in the 27-Week Gavage Study of Benzene

Preparation
Formulations were prepared by pipetting the appropriate amounts of benzene into a calibrated mixing bottle containing corn oil, bringing to volume with corn oil, capping, shaking vigorously, stirring on a stir plate for approximately 30 minutes, then shaking vigorously again. To compensate for evaporative loss of benzene from the dose formulations during administration, dose formulations prepared on January 4, 2000, or later were prepared with 5% less corn oil than specified in the original SOP for dose formulation. The dose formulations were prepared approximately every 4 weeks or as needed.
Chemical Lot Number
00358CS
Maximum Storage Time
35 days
Storage Conditions
Dose formulations were stored in amber glass bottles sealed with Teflon®-lined lids at room temperature (25° C).
Study Laboratory
Battelle Columbus Operations, Columbus, OH

Table D3Results of Analyses of Dose Formulations Administered to Haploinsufficient p16Ink4a/p19Arf Mice in the 27-Week Gavage Study of Benzene

Date PreparedDate AnalyzedTarget Concentration (mg/mL)Determined Concentrationa (mg/mL)Difference from Target (%)
October 7, 1999October 8–9, 19992.52.352−6
54.726−5
109.543−5
2018.47−8
November 11–12, 1999b2.52.224−11
54.510−10
108.541−15
2017.68−12
December 28, 1999cDecember 28–29, 19992.52.229−11
54.365−13
108.634−14
2016.09−20
January 4, 2000dJanuary 4–5, 20002.52.466−1
54.834−3
109.9670
2020.57+3
March 20, 2000March 22–23, 20002.52.443−2
55.040+1
109.602−4
2019.89−1
a

Results of duplicate analyses. Dosing volume=10 mL/kg; 2.5 mg/mL=25 mg/kg, 5 mg/mL=50 mg/kg, 10 mg/mL=100 mg/kg, 20 mg/mL=200 mg/kg

b

Animal room samples

c

Remixed; not used in study

d

Results of remix. To compensate for evaporative loss of benzene, dose formulations mixed on this date or later were prepared with 5% less corn oil than specified in the original SOP for dose formulation.

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This is a work of the US government and distributed under the terms of the Public Domain

Bookshelf ID: NBK576952
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