2 Journal of Toxicology Table 1: Isoﬂavone constituents of SE5-OH. Daidzein Reproductive toxicity study and developmental Isoﬂavone ingredient∗ Reproductive toxicity study (Lot 060106P)∗∗ (mg/g) toxicity study (Lot 051206P)∗∗ (mg/g)∗ 0.323 ± 0.0015 0.275 ± 0.0025 Genistein 0.293 ± 0.0017 0.287 ± 0.0032 Glycitein 1.65 ± 0.015 2.04 ± 0.010 Dihydrodaidzein 0.362 ± 0.0021 0.337 ± 0.0036 Equol 5.16 ± 0.070 5.24 ± 0.025 ∗The amount of each isoﬂavone ingredient is the average of three separate measurements, mean ± standard deviation. ∗∗Both lots were used in the reproductive study, while only Lot 051206P was used in the developmental study. HO O O Daidzein OH Fermentation by Lactococcus garvieae HO Figure 1: Daidzein metabolism to equol. O Equol OH to produce equol [18–20]. Serum levels of equol in equol producers that consume daidzein have been noted at up to 76 ± 137 nmol/L. The content of equol in the soy-based product (SE5- OH) has been enriched as a result of fermentation of soy germ with the bacterium L. garvieae (Otsuka Pharmaceutical Co., Ltd., Chiyoda-ku, Tokyo, Japan). L. garvieae is not a normal member of the human gut ﬂora, but is typically found in ﬁsh, and within ﬁsh aquacultures, and is associated with decreased growth rates, an unmarketable appearance, and mortality, but is not known to produce toxins [21]. L. garvieae is a rare pathogen with a low virulence in humans with gastrointestinal disorders that consumed raw ﬁsh, also has been found in several diﬀerent cheeses, and may be an important fermentation microbe in the production of several cheeses, including artisanal Italian and traditional Egyptian Domiati cheeses [22–25]. The controlled process of soy fermentation with L. garvieae is capable of converting approximately 50% of the daidzein contained within the soy to equol. SE5-OH is autoclaved to kill the bacteria, and subsequently analyzed to ensure that SE5-OH does not contain any living L. garvieae; therefore, the live organism will not be ingested by the consumer. Previously, we have reported acute and subchronic toxic- ity studies of SE5-OH in the adult rat model, and determined the NOAEL to be 2000 mg/kg/day following administration for 90 days [26]. The objective of the studies described herein was to investigate the eﬀect of the speciﬁc equol- enriched soy-based SE5-OH product on rat development and reproduction over two generations. 2. Methods and Materials 2.1. Test Articles. The test article, referred to as SE5-OH, was prepared by a controlled fermentation of a daidzein- rich soy germ containing isoﬂavones with L. garvieae (Otsuka Pharmaceutical Co., Ltd., Chiyoda-ku, Tokyo, Japan). SE5- OH contains approximately 0.65% equol, 0.024% daidzein, 0.022% genistein, and 0.30% glycitein. The test article is a light brown powder that includes ﬁve speciﬁc isoﬂavones; the amounts in the two lots of test article used in these studies are presented in Table 1. The test article control was hydroxypropylmethylcellulose (HPMC), supplied by Shin- Etsu Chemical Co., Ltd (Chiyoda-ku, Tokyo, Japan). 2.2. Animals. Male and/or female Crl:CD(SD) rats, supplied by the Atsugi Breeding Center of Charles River Laboratories (CRL) Japan, Inc., (Kohoku-ku, Yokohama, Japan) were used for both the developmental and reproductive toxicity studies. All animals were clinically monitored at the time of delivery to the testing facility and during the acclimation period. Housing consisted of stainless steel or polycarbonate cages, with a twelve-hour light period (7:00 AM to 7:00 PM). Temperature was maintained at 20.1–23.4◦C with a relative humidity of 49.6–69.2% and 6–20 air changes/h. The bedding, which was used only in the reproductive toxicity study, was of the Beta-Chip brand supplied by Charles River Laboratories Japan, Inc. The rats were six weeks of age at the start of dosing in the reproductive toxicity study and twelve weeks of age at mating in the developmental toxicity study. Rats were acclimatized for ﬁve or six days before use. 2.3. Guidelines. The developmental toxicity and reproductive toxicity studies were carried out under the guidelines of Redbook 2000 and FDA’s Toxicological Principles for the Safety Assessment of Food Ingredients [27, 28]. Both studies were conducted within the Good Laboratory Practice (GLP) standards for the conduction of nonclinical studies on the safety of drugs (Ministry of Health and Welfare (MHW), Japan, Ordinance Number 21, 26 March 1997, [http://pdg-a258.koyosha.co.jp/jsqa2/en/glp/glp424e.html]). This study was approved by the Committee of Kashima