Tobacco Documents Online

Key Messages • Compared to the 1 R4F the cytotoxicity of the particulate phase of smoke from the EHCSS-AMP is remarkably low. • AMP in the overwrap lowers the cytotoxicity of the particulate phase. S6£S6L580Z SAB Meeting 7 to 8 Nov. 2001 Slide 2 Confidential INBIFO/CRC

AMP in the EHCSS - Overview of Cytotoxicity Testing Ewald Roemer Presentation at the SAB Meeting, McLean, VA, Nov. 7-8, 2001 Confidential ti4£S61S88Z Slide 1 INBIFO/CRC

Methods, Test Substance Generation • Test Substances mainstream smoke fractions - total particulate matter (TPM) - water soluble fraction of the gas/vapor phase (GVP) - 3 batches/cigarette type and smoke fraction 30-port INBIFO smoking machine 0 0o ice bath H gas wash bottle with 36 ml PBS - dose range - EHCSS: 7 to 72 cig./I cell culture medium - 1 R4F: 2 to 20 cig./I cell culture medium 94£S6LS80Z collection site for GvP SAB Meeting 7 to 8 Nov. 2001 Slide 3 Confidential INBIFO/CRC

Summary • Compared to the 1 R4F, the particulate phase of smoke from the electrically heated cigarettes was 3/4 lower in cytotoxicity. The cytotoxicity of the water solubles of the gas/vapor phase was approximately the same. • The addition of AMP to the overwrap lowered the cytotoxicity of the particulate phase by 1/3. The cytotoxicity of the water solubles of the gas/vapor phase was not influenced. 64£S6LS80Z SAB Meeting 7 to 8 Nov. 2001 Slide 6 Confidential INBIFO/CRC

Methods, Assay Conditions, and Evaluation • Assay neutral red uptake assay (according to INVITTOX protocol 3a, 1999) mouse embryo BALB/c 3T3 cells, clone A31 exposure time: 24 h photometrical determination of neutral red taken up by viable cells ~ evaluation 6o~ - 1/EC from sigmoid logistic 50 40-~ function fitted to the concentration response curve 20 --~ statistics overall: one-way analysis of variance followed by a pairwise comparison according to Tukey between EHCSS cigarettes: t-test 0~ ~ : 10-May-2000 0 : 17-Mey-2000 13 : n-Mey-2000 r 0 T 30 EHCSS-CaCO3 TPM 60 90 120 9575-1 TPM CONC. (cig./I) SAB Meeting 7 to 8 Nov. 2001 L 4£S6LS80Z Slide 4 Confidential INBIFO/CRC

Results: Cytotoxicity (1/EC50) Equal TPM Basis 4-I EHCSS-CaC03 F EHCCSS-AMP-F 1R4F * Cigarette Basis 40 -~ 20 -~ *4 8 GJ 4 S smoke I a fracton TPM I I GVP 1 1PM *: statistically significant difference between 2 cigarettes; mean ± SE 1 I GVP I SAB Meeting 7 to 8 Nov. 2001 g 4£S61580Z Slide 5 Confidential INBIFO/CRC