Tomoko Kawamura

Validation of the (Q)SAR combination approach for mutagenicity prediction of flavor chemicals

Most exposure levels of flavor in food are considered to be extremely low. If at all, genotoxic properties should be taken into account in safety evaluations. We have recently established a (quantitative) structure-activity relationship, (Q)SAR, combination system, which is composed of three individual models of mutagenicity prediction for industrial chemicals. A decision on mutagenicity is defined as the combination of predictive results from the three models. To validate the utility of our (Q)SAR system for flavor evaluation, we assessed 367 flavor chemicals that had been evaluated mainly by JECFA and for which Ames test results were available. When two or more models gave a positive evaluation, the sensitivity was low (19.4%). In contrast, when one or more models gave a positive evaluation, the sensitivity increased to 47.2%. The contribution of this increased sensitivity was mainly due to the result of the prediction by Derek for Windows, which is a knowledge-based model. Structural analysis of false negatives indicated some common sub-structures. The approach of improving sub-structural alerts could effectively contribute to increasing the predictability of the mutagenicity of flavors, because many flavors possess categorically similar functional sub-structures or are composed of a series of derivatives.

Reproductive and developmental toxicity screening test of 3-cyanopyridine in rats

Crl:CD(SD)rats were given 3-cyanopyridine by gavage at 0, 5, 30 or 180 mg/kg/day. Males were dosed for 42 days beginning 14 days before mating, and females for 40-53 days beginning 14 days before mating to day 3 of lactation, including throughout the mating and gestation periods. General toxicity, mainly liver damage, was observed in males at ≥30 mg/kg/day and in females at ≥5 mg/kg/day. Sertoli cell vacuolation was observed at 180 mg/kg/day, and spermatocyte damages were observed at ≥30 mg/kg/day. Effects on estrous cycles, corpora lutea and implantations, and unsuccessfully mated females, despite additional mating, were observed at 180 mg/kg/day. Delayed initiation of delivery, dystocia, and deaths or moribundities of pregnant females were observed at 180 mg/kg/day, and only two pregnant rats delivered live pups at that dose. The NOAEL for reproductive/developmental toxicity was concluded to be 30 mg/kg/day.

Initial hazard assessment of benzyl salicylate: In vitro genotoxicity test and combined repeated-dose and reproductive/developmental toxicity screening test in rats

ABSTRACT Benzyl salicylate is used as a fragrance ingredient and an ultraviolet light absorber, but its toxicity is unknown. Therefore, toxicity tests and hazard classification were conducted for screening assessment under the Japanese Chemical Substances Control Law. Benzyl salicylate was found to be non‐genotoxic in vitro based on the chromosomal aberration test using Chinese hamster lung cells. However, the combined repeated‐dose and reproductive/developmental screening toxicity test, in which male and female rats were administered benzyl salicylate by gavage at 0, 30, 100, or 300mg/kg/day for 42 and 41–46 days, respectively, from 14 days before mating until postnatal Day 4, showed that repeated doses had major effects on the thymus, liver, epididymis, and femur at 100 and/or 300mg/kg/day. Furthermore, although benzyl salicylate had no effect on the estrus cycle, fertility, corpus lutea, or implantation rate, embryonic resorption, offspring mortality, and neural tube defects were observed at 300mg/kg/day, and the offspring had lower body weights at 30 and 100mg/kg/day, suggesting teratogenicity similar to other salicylates. Based on the developmental toxicity, this chemical was classified as hazard class 2, with a lowest observed adverse effect level (LOAEL) of 30mg/kg/day and a D‐value of 0.003mg/kg/day. HIGHLIGHTSBenzyl salicylate was non‐genotoxic in vitro.Marked embryotoxicity and neural tube defects were indicated at 300mg/kg/day.LOAEL was 30mg/kg/day based on lower body weights of offspring.D‐value of 0.003mg/kg/day with hazard class 2 was proposed for benzyl salicylate.

Initial hazard assessment of 4-benzylphenol, a structural analog of bisphenol F: Genotoxicity tests in vitro and a 28-day repeated-dose toxicity study in rats

ABSTRACT 4‐Benzylphenol (CAS No. 101–53–1), a structural analog of bisphenol F, has estrogenic activity in vitro and in vivo, as is the case with bisphenol F. 4‐Benzylphenol is used in plastics and during organic synthesis. Since its safety is largely unknown, we conducted toxicity tests as part of screening risk assessment in an existing chemical safety survey program. Based on results of the Ames test and the chromosomal aberration test using Chinese hamster lung cells (OECD TG 471 and 473), 4‐benzylphenol was determined to be non‐genotoxic in vitro. In a 28‐day repeated‐dose toxicity study, Crl:CD (SD) rats were administrated 4‐benzylphenol by gavage at 0, 30, 150, or 750mg/kg/day (OECD TG 407). Consequently, body weight was lower in males at 750mg/kg/day. In the liver, relative organ weights were increased in both sexes at 750mg/kg/day, and centrilobular hepatocellular hypertrophy was observed in males at 150 and 750mg/kg/day. In the forestomach, hyperkeratosis and hyperplasia of squamous cells were observed in males at 150 and 750mg/kg/day, and in females at 750mg/kg/day. Based on these results, we identified the NOAEL for 4‐benzylphenol as 30mg/kg/day, with a hazard assessment value (D‐value) of 0.05mg/kg/day corresponding to hazard class 3. HIGHLIGHTS4‐Benzylphenol was non‐genotoxic in vitro.The NOAEL for 28‐day repeated dose toxicity was 30mg/kg/day.The D‐value of 0.05mg/kg/day was derived for 4‐benzylphenol. Abbreviations: CHL: Chinese hamster lung fibroblastic cell; CSCL: The Japanese Chemical Substances Control Law; JECDB: Japan Existing Chemical Data Base; QSAR: quantitative structure‐activity relationship; LOAEL: lowest observed adverse effect level; MHLW: The Ministry of Health, Labour, and Welfare; NOAEL: no observed adverse effect level; OECD: Organization for Economic Cooperation and Development; UF: uncertainty factor.

In silico prediction of chromosome damage: comparison of three (Q)SAR models

Two major endpoints for genotoxicity tests are gene mutation and chromosome damage (CD), which includes clastogenicity and aneugenicity detected by chromosomal aberration (CA) test or micronucleus (MN) test. Many in silico prediction systems for bacterial mutagenicity (i.e. Ames test results) have been developed and marketed. They show good performance for prediction of Ames mutagenicity. On the other hand, it seems that in silico prediction of CD does not progress as much as Ames prediction. Reasons for this include different mechanisms and detection methods, many false positives and conflicting test results. However, some (quantitative) structure-activity relationship ((Q)SAR) models (e.g. Derek Nexus [Derek], ADMEWorks [AWorks] and CASE Ultra [MCase]) can predict CA test results. Therefore, performances of the three (Q)SAR models were compared using the expanded Carcinogenicity Genotoxicity eXperience (CGX) dataset for understanding current situations and future development. The constructed dataset contained 440 chemicals (325 carcinogens and 115 non-carcinogens). Sensitivity, specificity, accuracy or applicability of each model were 56.0, 86.9, 68.6 or 89.1% in Derek, 67.7, 61.5, 65.2 or 99.3% in AWorks, and 91.0, 64.9, 80.5 or 97.7% in MCase, respectively. The performances (sensitivity and accuracy) of MCase were higher than those of Derek or AWorks. Analysis of predictivity of (Q)SAR models of certain chemical classes revealed no remarkable differences among the models. The tendency of positive prediction by (Q)SAR models was observed in alkylating agents, aromatic amines or amides, aromatic nitro compounds, epoxides, halides and N-nitro or N-nitroso compounds. In an additional investigation, high sensitivity but low specificity was noted in in vivo MN prediction by MCase. Refinement of test data to be used for in silico system (e.g. consideration of cytotoxicity or re-evaluation of conflicting test results) will be needed to improve performance of CD prediction.