Abstract

Background: 2,3-Benzofluorene (2,3-BF) is a member of the polycyclic aromatic hydrocarbon class of compounds to which humans are widely exposed. Toxicological information on this class of chemicals is sparse. A short-term, in vivo transcriptomic study was used to assess the biological potency of 2,3-BF.

Methods: A short-term in vivo biological potency study on 2,3-BF in adult male and female Sprague Dawley (Hsd:Sprague Dawley® SD®) rats was conducted. 2,3-BF was formulated in corn oil and administered once daily for 5 consecutive days by gavage (study days 0–4). 2,3-BF was administered at 10 doses (0, 0.15, 0.5, 1.4, 4, 12, 37, 111, 333, and 1,000 mg/kg body weight [mg/kg]). Blood was collected from animals dedicated for internal dose assessment in the 4 and 37 mg/kg groups. On study day 5, the day after the final dose was administered, animals were euthanized, standard toxicological measures were assessed, and the liver and kidney were assayed in gene expression studies using the TempO-Seq assay. Modeling was conducted to identify the benchmark doses (BMDs) associated with apical toxicological endpoints and transcriptional changes in the liver and kidney. A benchmark response of one standard deviation was used to model all endpoints.

Results: Several clinical pathology and organ weight measurements showed dose-related changes from which BMD values were calculated. In male rats, the effects included significantly decreased reticulocyte count, increased thyroid stimulating hormone concentration, and decreased total thyroxine concentration. The BMDs and benchmark dose lower confidence limits (BMDLs) were 11.837 (6.978), 44.526 (19.298), and 61.426 (24.276) mg/kg, respectively. In female rats, the effects included significantly increased thyroid stimulating hormone concentration, increased absolute liver weight, and increased cholesterol concentration. The BMDs (BMDLs) were 1.078 (0.267), 24.928 (7.768), and 288.242 (226.543), respectively. Average 2,3-BF plasma concentrations at 2 hours postdose were lower in female rats than in male rats. At 24 hours postdose, the concentration decreased and fell below the limit of detection of the analytical method in female rats and close to it in male rats. Half-lives in males, estimated using the two time points, were 25.3 and 4.4 hours for the 4 and 37 mg/kg groups, respectively.

In the liver of male and female rats, no Gene Ontology biological process or individual genes had BMD median values below the lower limit of extrapolation (<0.050 mg/kg). The most sensitive gene sets in male rats for which a reliable estimate of the BMD could be made were regulation of ossification and kidney development with median BMDs of 5.660 and 5.765 mg/kg and median BMDLs of 2.158 and 1.618 mg/kg, respectively. The most sensitive gene sets in female rats for which a reliable estimate of the BMD could be made were DNA conformation change and chromosome organization, both with a median BMD of 1.874 mg/kg and a median BMDL of 0.497 mg/kg. The most sensitive upregulated genes in male rats with reliable BMD estimates included G0s2, Trib3, Akr7a3, Nqo1, and Ephx1 with BMDs (BMDLs) of 3.451 (1.074), 16.895 (6.465), 19.850 (12.797), 24.112 (16.415), and 24.878 (15.487) mg/kg, respectively. The most sensitive downregulated genes in male rats with reliable BMD estimates were Zfp354a, Tsku, Egr1, C7, and Loc100911558/Spink1l with BMDs (BMDLs) of 3.267 (1.720), 6.126 (1.466), 8.263 (1.517), 13.843 (5.329), and 26.630 (12.428) mg/kg, respectively. The most sensitive upregulated genes in female rats with reliable BMD estimates included Kif22, Anln, Anlnl1, Asns, and Nr1d2 with BMDs (BMDLs) of 0.962 (0.282), 3.325 (1.105), 3.325 (1.105), 6.056 (2.744), and 9.460 (2.213) mg/kg, respectively. The most sensitive downregulated genes in female rats with reliable BMD estimates were Car3, Aass, A2m, Loc100911545/A2m, and Sez6 with BMDs (BMDLs) of 3.449 (2.131), 5.811 (2.605), 8.459 (1.870), 8.459 (1.870), and 10.639 (3.960) mg/kg, respectively.

In the kidney of male and female rats, no Gene Ontology biological process had BMD median values below the lower limit of extrapolation (<0.050 mg/kg). The most sensitive gene sets in male rats for which a reliable estimate of the BMD could be made were brain development and cellular process involved in reproduction in multicellular organism, with median BMDs of 6.167 and 12.334 mg/kg, and median BMDLs of 1.732 and 3.464 mg/kg, respectively. The most sensitive gene sets in female rats for which a reliable estimate of the BMD could be made were regulation of fibroblast proliferation and negative regulation of fibroblast proliferation with median BMDs of 14.574 and 14.763 mg/kg and median BMDLs of 7.161 and 9.430 mg/kg, respectively. In male rats, one individual kidney gene, Nefh, had a median BMD value <0.050 mg/kg and was downregulated. The next most sensitive downregulated genes with reliable BMD estimates included Top2a, Ect2, Hmgcs2, Nfil3, Mt1, and Cyp2c11 with BMDs (BMDLs) of 12.334 (3.464), 31.232 (16.473), 106.351 (59.870), 121.321 (82.552), 168.588 (103.554), and 216.743 (139.563) mg/kg, respectively. The most sensitive upregulated genes with reliable BMD estimates included Nqo1, Cyp1a1, and Rassf1 with BMDs (BMDLs) of 40.551 (21.583), 49.045 (39.236), and 903.848 (608.514) mg/kg, respectively. In female rats, no individual genes had median BMD values <0.050 mg/kg. The most sensitive upregulated genes in female rats with reliable BMD estimates included Cyp1a1, Gstp1, Cyp26b1, and Nqo1 with BMDs (BMDLs) of 12.886 (9.131), 14.763 (9.430), 18.187 (10.018), and 25.681 (7.739) mg/kg, respectively. The most sensitive downregulated genes in female rats with reliable BMD estimates included Vwf, Abcb1b, Npas2, Arntl, C4a, and Loc103689965/C4a with BMDs (BMDLs) of 19.676 (4.116), 20.794 (8.254), 21.088 (11.331), 22.913 (11.412), 24.209 (8.100), and 24.209 (8.100) mg/kg, respectively.

Summary: Taken together, in male rats, the most sensitive gene set BMD (BMDL) median, individual gene BMD (BMDL), and apical endpoint BMD (BMDL) values that could be reliably determined occurred at 5.660 (2.158), 3.267 (1.720), and 11.837 (6.978) mg/kg, respectively. The BMD (BMDL) could not be determined for one individual gene and was estimated to be <0.050 mg/kg. In female rats, the most sensitive gene set BMD (BMDL) median, individual gene BMD (BMDL), and apical endpoint BMD (BMDL) values that could be reliably determined occurred at 1.874 (0.497), 0.962 (0.282), and 1.078 (0.267) mg/kg, respectively.

Citation: Auerbach SS, Aillon KL, Ballin JD, Collins BJ, Cora MC, Duncan NS, Fostel JM, Kerns SP, Liu YF, Luh J, Machesky NJ, Pickett SJ, Prince LM, Roberts GK, Shipkowski KA, Skowronek AJ, Sparrow BR, Toy H, Waidyanatha S, Watson ATD. 2023. NIEHS report on the in vivo repeat dose biological potency study of 2,3-benzofluorene (CASRN 243-17-4) in Sprague Dawley (Hsd:Sprague Dawley® SD®) rats (gavage studies). Research Triangle Park, NC: National Institute of Environmental Health Sciences. NIEHS Report 09. [https://doi.org/10.22427/NIEHS-09 Auerbach SS, Aillon KL, Ballin JD, Collins BJ, Cora MC, Duncan NS, Fostel JM, Kerns SP, Liu YF, Luh J, Machesky NJ, Pickett SJ, Prince LM, Roberts GK, Shipkowski KA, Skowronek AJ, Sparrow BR, Toy H, Waidyanatha S, Watson ATD. 2023. NIEHS report on the in vivo repeat dose biological potency study of 2,3-benzofluorene (CASRN 243-17-4) in Sprague Dawley (Hsd:Sprague Dawley® SD®) rats (gavage studies). Research Triangle Park, NC: National Institute of Environmental Health Sciences. NIEHS Report 09.]