Table 4.

Summary of Studies Evaluating Styrene/SO in in vitro Gene Mutation Assays

Assay	Methods	Results	Comments	Reference
Styrene
HPRT‐V79 cells	Methods poorly described. One hour treatment. 8.5 and 17 mM styrene. Three expression periods (72, 90, and 114 hr). 8‐azaguanine selection.	Styrene, even at 17 mM showed little cytotoxicity and little increase in MF.	Not OECD TG476 compliant. Unclear whether sufficient cells were used. Expression period too short. Does not appear that cells were subcultured during expression. Very low number of viable cells reported in the cultures (Table 2 of the publication). Uninterpretable.	Loprieno et al. (1976)
HPRT‐V79 cells	Both liver perfusion system and S9 experiments. 240 and 480 μM styrene used in perfusion study. 250, 480, and 960 μM styrene used with and without S9. Concentrations of styrene and SO evaluated in the perfusion mixture. Unclear how many cells treated, but only 4 × 105 cells carried during expression. 6‐thioguanine selection.	The exposed cultures (both methods) showed little cytotoxicity. While there was some increase in the MF with exposure in the liver perfusion, there was substantial variability. The with and without S9 showed little to no increase in MF and there was substantial variability. The styrene was metabolized within two hour. During this time, the SO was measured at only 2%–4% of the styrene concentration at the beginning of the experiments.	Not OECD TG476 compliant. Uninterpretable.	Beije and Jenssen (1982)
Styrene Oxide
Mouse Lymphoma‐TK TK+/− 3.7.2C cells	Agar version. Three hour treatment with and without S9. Without S9: nine doses (0–103.6 μg/mL). With S9: nine doses (0–10.5 μg/mL). Two‐day expression. TFT selection. Automatic counter set to count only large colonies.	Without S9: MF from 40 to 590 × 106 at RTGs above 10%. With S9: MF from 58 to 94 × 106 at RTGs above 10%.	Not OECD TG490 compliant. Did not count the small colony mutants; however, the result was positive without S9 (showed a dose response and exceeded the GEF) based solely on the large colony MF. With S9, result was uninterpretable.	Amacher and Turner (1982)
HPRT‐V79 cells	Methods poorly described. One hour treatment. 4.25, 8.5, 17, and 25 mM SO. Three expression periods (72, 90, and 114 hr). 8‐azaguanine selection.	SO was cytotoxic at 17 and 25 mM and there was an increase in MF.	Not OECD TG476 compliant. Unclear whether sufficient cells were used. Expression period too short. Does not appear that cells were subcultured during expression. Very low number of viable cells reported in the cultures (Table 2). Uninterpretable.	Loprieno et al. (1976)
HPRT‐V79 cells	Methods indicated as that of Loprieno et al. (1976). The focus of the study was to examine expression time. 8.5 and 17 mM with one hour exposure. Different expression times (0, 66, 90, 114, 162, and 210 hr). EMS as a positive control, but no negative control.	The observed MF increases and then decreases with increasing expression time in some experiments, but not in others. There are some cultures with apparently “high” MFs, but no negative control and insufficient number of concentrations to assess.	Not OECD TG476 compliant. No negative control. Cloning efficiencies for some cultures that appear to be positive are too low (8–25%). Three of the five experiments used concentrations (17 mM) that are substantially above the recommended maximum (10 mM). Uninterpretable.	Bonatti et al. (1978)
HPRT‐V79 cells	Cytotoxicity and mutagenicity assessed in different treated cultures. Unclear how many cells treated, but does not appear to be sufficient. Four hour treatment. Eight‐day expression. Six TG selection.	Data presented graphically, not possible to evaluate response.	Not OECD TG476 compliant. Data only presented graphically and could not be evaluated. Uninterpretable.	Sugiura et al. (1979)
HPRT‐V79 cells	One hour treatment. Different expression times up to 210 hr. Six TG selection.	Data presented graphically, and the SO data presented appear to be from another publication.	Not OECD TG476 compliant. SO data not clearly presented. Uninterpretable.	Turchi et al. (1981)
HPRT‐V79 cells	Both liver perfusion system and S9 experiments. 240 μM SO used in perfusion study. 104 and 208 μM SO used without S9. Unclear how many cells treated, but only 4 × 105 cells carried during expression.	In the liver perfusion study, there was little cytotoxicity and no increase in MF. The SO was totally metabolized within 30 min. Without activation, the SO induced cytotoxicity, but little increase in MF.	Not OECD TG476 compliant. Uninterpretable.	Beije and Jenssen (1982)
HPRT‐V79 cells	Insufficient detail for the methods. This was a 40 compound study. Unclear how many cells were used but appears not to be sufficient. Six‐day expression, six TG selection.	Data presented graphically and impossible to interpret.	Not OECD TG476 compliant. Uninterpretable.	Nishi et al. (1984a)
HPRT‐V79 cells	Insufficient detail for the methods. Focus was the association between the induction of SCEs and mutation. Multiple chemicals tested—basically the same set as in Nishi et al. (1984a). Three hour treatment. Six‐day expression, six TG selection. Unclear how many cells were used but appears to be insufficient.	Data presented graphically comparing the number of induced SCEs/cell vs. the number of induced mutants per 106 viable cells. Although in the text, the authors indicate that SO was a weak inducer of mutation, it is impossible, in the graph, to see any real increase in the number of mutants, particularly when compared with the mutant frequencies that are clearly induced by other known mutagens such as methyl nitrosourea, ethyl nitrosourea, ethyl methanesulfonate, ICR 191 and ICR 170.	Not OECD TG476 compliant. Uninterpretable.	Nishi et al. (1984b)
Human T‐lymphocytes	Exposed to SO for 24 hr or 6 days and 8 days for expression. Nine different experiments with nine different donors.	While the authors indicate that increases in the hprt mutant frequency were observed, a close look at the data indicates that most of the cell cultures had very poor (less than 20%) survival after the end of treatment. Furthermore, the number of cell doublings during the eight‐day expression time was very low, ranging from 1.2 to 3.7 (primarily in the untreated cultures).	Not OECD TG476 compliant. Appears that the number of cells used was inadequate. Very poor cell survival/growth. Uninterpretable.	Bastlová et al. (1995)
Two human B‐cell lymphoblastoid cell lines differing in glutathione‐S‐transferase gene status	The methods in one paper (Shield and Sanderson, 2004) were reasonably well‐detailed and it was possible to determine that sufficient cells were exposed and an appropriate expression time and selective agent were used. In the second paper (Shield and Sanderson, 2001), an insufficient number of cells were treated.	In both studies, based on the graphical presentation of the data, the cell lines deficient in GSTM1 showed a greater degree of cytotoxicity (assessed by % population growth over eight days) and higher mutant frequency than the cell lines expressing GSTM1 activity.	Not OECD TG476 compliant. The recommended cytotoxicity method for assessing the appropriateness of concentration selection was not used in either study making it impossible to determine if appropriate concentrations (per TG476) were used. Uninterpretable.	Shield and Sanderson (2001, 2004)