In vitro and in vivo evaluation of the genotoxicity of titanium dioxide, GST.

Titanium dioxide (TiO2) was used in various applications in a wide range of products including food, cosmetics and photocatalyst. General toxicity studies of titanium dioxide, GST (Green Sludge Titanium) have been investigated in several reports, whereas studies concerning mutagenicity and genotoxicity have not been elucidated. Herein, we investigated the potential mutagenicity and genotoxicity of GST by genetic toxicology testing. The bacterial reverse mutation test was conducted by the pre-incubation method in the presence and absence of metabolic activation system (S9 mixture). The chromosome aberration test was performed using cultured Chinese hamster lung cell line in the absence and presence of S9 mixture. The micronucleus test was performed by using specific pathogen-free male ICR mice. Genotoxicity tests were conducted following the test guidelines of the Organisation for Economic Cooperation and Development with application of Good Laboratory Practice. No statistically significant increases were found in the bacterial reverse mutation test, in vitro chromosome aberration test, and in vivo micronucleus test when tested for induction of genotoxicity in GST. These results suggest that GST did not induce mutagenicity and genotoxicity in both in vitro and in vivo system.

Toxicity of a 90-day repeated oral dose of a collagen peptide derived from skate (Raja kenojei) skin: a rat model study.

Collagen peptides are widely employed as therapeutic materials due to their numerous beneficial properties, including for the following uses: antiaging, antioxidant applications, antibacterial applications, wound healing, tissue engineering, medication delivery, and cosmetics. Although collagen peptides are useful in these applications, to our knowledge, few published studies have been undertaken on their repeated-dose toxicity. We evaluated the possible subchronic toxicity of a collagen peptide derived from skate (Raja kenojei) skin (CPSS) in Sprague-Dawley rats by administering repeated oral doses over 90 days. Rats of both sexes were assigned randomly to one of four experimental groups, respectively receiving 0, 500, 1000, or 2000 mg/kg/day of CPSS. At all doses tested, repeated oral CPSS administration had no treatment-related adverse effects in terms of clinical signs, body weight, food consumption, detailed clinical observation, sensory reactivity, functional assessment, urinalysis, ophthalmic examination, gross pathology, hematology, serum biochemistry, hormone analysis, organ weight, and histopathology. Even though there were some alterations in hematologic parameters, serum biochemistry parameters, organ weight, and histopathological findings, these did not follow a dose-response pattern and were within historical limits for control rats. The oral no-observed-adverse-effect level (NOAEL) of the CPSS was 2000 mg/kg/day for both male and female rats in the applied experimental circumstances, and no target organs were identified.

Reproductive and developmental toxicity screening test of new TiO2 GST in Sprague-Dawley rats.

TiO2 nanoparticles are widely used in paints, plastics, cosmetics, printing ink, rubber, food products, pharmaceuticals and other products (photocatalyst, etc.). However, there is little toxicological information during reproduction and developmental period. This study was performed to obtain safety data for new TiO2 powder, GST (Green Sludge Titanium) produced through sludge recycling of the sewage treatment plant for Reproduction/developmental toxicity screening test in Sprague-Dawley (SD) rats in according to the OECD test guideline (TG 421). Based on the results of the dose-range finding study (14-day repeated toxicity), GST was orally administered to rats at doses of 0, 500, 1000, and 2000 mg/kg B.W/day. Males were dosed for 35 days beginning 14 days before mating, and females for a maximum of 53 days beginning 14 days before mating to day 13 of lactation, including throughout the mating, gestation and lactation periods. In the reproductive and developmental examinations, there were no marked toxicities in terms of general clinical signs, body weight, food consumption, organ weights, macroscopic / microscopic findings, stages of spermatogenesis in the testis, reproductive finding (estrous cycle, copulation-fertility-gestation index), developmental finding (number of corpora lutea and implantations, pups parameters including live birth and viability index). The NOAEL for reproductive/developmental screening toxicity was concluded to be 2000 mg/kg/day under the present study conditions.

Repeated-dose 28-day dermal toxicity study of TiO2 catalyst (GST) in Sprague-Dawley rats.

TiO2 have been studied on inhalation and skin exposure due to the properties of the materials' use (cosmetics, paints and other products) and the additional safety information on other intake routes for the potential risk assessment is limited. The aim of this study was to obtain dose-range for subchronic study (repeated 90-day dermal toxicity) new TiO2 powder, GST produced through sludge recycling of the sewage treatment plant through repeated-dose toxicity in Sprague-Dawley (SD) rats. Three test groups for the GST were administered at 500, 1000, 2000 mg/kg B.W/day in addition to a control group (distilled water for injection). 5 male and 5 female rats were included in each group, and we examined the clinical signs, body weights, food consumption, necropsy (organ weights, macroscopic findings), hematological / biochemical parameters and histopathological findings (eye, skin). As a result of observations, there were no treatment-related effects including clinical signs, mortality, necropsy findings etc. Therefore, the present results suggest that the TiO2-related effects were not observed for dermal during 28-day and dose selection for repeated 90-day study was considered to be 500, 1000 and 2000 mg/kg B.W/day under the present study conditions.

Repeated-dose 90-day oral toxicity study of GST in Sprague-Dawley rats.

TiO2 have been studied on inhalation and skin exposure due to the properties of the materials' use (cosmetics, paints and other products) and the additional safety information on other intake routes for the potential risk assessment is limited. Therefore, the aim of this study was to obtain safety data for new TiO2 powder, GST produced through sludge recycling of the sewage treatment plant through repeated-dose toxicity in Sprague-Dawley (SD) rats in according to the OECD test guideline (TG 408). Based on the results of the dose-range finding study (28-day repeated toxicity), GST was orally administered to rats at doses of 0, 500, 1000, and 2000 mg/kg B.W/day for 90-day and reversibility of effects of 2000 mg/kg bw/day was assessed after 4 weeks. In clinical signs, compound-colored stool was observed in all animals of treatment group (low: day 14 or 15, middle: day 8, high: day 8) and continuously observed up to the end of administration or day 1 of recovery period (high dose group). Also, the test substance retention in gastro-intestinal tract was observed in all animals of treatment group in gross finding at necropsy and foreign materials in lumen of these organs (stomach, duodenum, ileum, cecum, colon, rectum) likely indicative for the presence of test material in histopathological examination. In addition, no test substance-related adverse effects were noted in the detailed clinical observations, sensory reactivity/ functional assessments, body weight, food consumption, urinary analysis, ophthalmological examination, hematological / biochemical parameters, organ weights, histopathological findings. Therefore, the present results show that the NOAEL (no observed adverse effect level) of new TiO2 powder, GST was considered to be 2000 mg/kg B.W/day in rats after repeated oral administration for 90-day under the present study conditions and no target organs were identified.

Evaluation of potential eye or skin irritation/corrosion in rabbit exposed to TiO2 photocatalyst (GST).

TiO2 NPs photocatalyst is widely used in a variety of applications and products in the environmental and energy fields, including self-cleaning surfaces, air and water purification systems, sterilization, hydrogen evolution, and photoelectrochemical conversion. The possible biological and safety effects of TiO2 dermal exposure and absorption have not been well studied and more investigations on the potential health hazards of the TiO2 are needed. This study aimed to investigate potential effect of local lesions (eye and skin irritation/corrosion) for new TiO2 material powder, GST produced through sludge recycling of the sewage treatment plant in according to the OECD test guideline (TG 404, 405) and imaging evaluation (micro-computed tomography analysis), histopathology examination. Also, P-25, commercial photocatalyst was used to compare with GST. For the eye or skin irritation/corrosion test, the test substances (GST, P-25) showed no irritation/corrosion for local lesions and the GHS category was identified as a "No hazard class". The imaging analysis indicated that GST did not penetrate or distribute in the local lesions (eye, skin) and the treatment-related effect was not observed in histopathology. Therefore, the present study revealed that new TiO2 powder, GST was considered to be no potential effects (irritation/corrosion), penetration or distribution in the local lesions (eye, skin).

In vitro acute inhalation toxicity for TiO2 (GST) using 3D human tissue model (EpiAirwayTM).

The present study was performed to screen in vitro potential acute inhalation toxicity using an EpiAirwayTM tissue model (human tracheal/bronchial tissue) for the nano-sized titanium dioxide, GST manufactured as a photocatalyst through of sludge recycling and to compare with P-25 a commercialized photocatalytic material. According to the protocol provided by in vitro tissue manufacturer, the GST was exposure to the tissue for 3 hours in 450, 500, 650, 850 mg/mL concentration after preliminary dose range finding study and then tissue viability (%, IC75) was calculated using the MTT assay. Besides, the histopathological observation was performed to compare to the MTT assay. As a result of study, IC75 could not be confirmed at 850 mg/mL in both GST and P-25 and the grade was confirmed to be IC75> 600 mg/mL in vitro model tissue category. Therefore, it was considered that the GHS category could be classified as 'No classification' in screening method for potential acute inhalation toxicity. Also, not the morphological effects of epithelial cells in tissue model were observed compared with the vehicle control and histological findings were similar to the results of MTT Viability assay. Based on these results, the potential acute inhalation toxicity for GST produced through sludge recycling using in vitro tissue model inhalation toxicity showed that it could be non-hazardous substance. However, further study (in vivo study, etc.) is thought to be needed to ascertain whether GST is a toxic effect or safe.

Acute toxicity assessment for TiO2 photocatalyst (GST) made from wastewater using TiCl4 in rat.

TiO2 was a photocatalyst that used to the most common product because of the high efficiency. TiO2 (P-25, commercial nanomaterial product) is the most typical photocatalyst product and TiO2 (GST) was a sludge recycling product. This study was reported to evaluate an acute toxicity of TiO2 (P-25 and GST) according to OECD test guideline 402 and 423 in Sprague-Dawley (SD) female rats via route of oral and dermal. There was investigated the lethal dose (LD50), and mortality, clinical signs, body weight changes and gross findings were continually monitored for 14 days following the single administration. After administration, TiO2 (P-25) was calculated that LD50 was considered to be a dose of over 2000 mg/kg body weight for both different route of exposure, and TiO2 (GST) was the same. Other items were no observed an adverse effect between P-25 and GST; no mortality and clinical signs, accidental body weight loss, no gross findings. On the basis of the above results, the toxicity of the GST was almost equal to that of the commercial product, P-25 and there was no toxicological evidence.

A 13-week subchronic toxicity study of an Eriobotrya japonica leaf extract in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Eriobotrya japonica leaf is widely used in traditional medicine, and exhibits various beneficial effects such as anti-inflammatory, antiviral, antioxidant, and antitumor activities. However, limited data are available on the potential adverse effects of E. japonica. AIM OF THE STUDY: This study investigated the potential subchronic toxicity of an E. japonica leaf extract (EJE) through a 13-week repeated oral dose experiment in Sprague-Dawley rats. MATERIALS AND METHODS: Forty male and 40 female rats were randomly assigned to four experimental groups: three treatment groups receiving 250, 500, and 1000 mg/kg/day of EJE and a vehicle control group receiving sterile distilled water for 13 weeks. RESULTS: Repeated oral administration of EJE for 13 weeks did not cause any treatment-related adverse effects with respect to clinical symptoms, body weight, food and water consumption, urinalysis, ophthalmology, necropsy findings, hematology, serum biochemistry, organ weight, and histopathological examination at any dose tested. Although some changes were observed in clinical symptoms, organ weight, hematology, and histopathology, these findings did not show a dose-response relationship and were within normal historical ranges for control rats. CONCLUSION: Under the present experimental conditions, the no-observed-adverse-effect level of EJE was > 1000 mg/kg/day in both sexes and no target organs were identified. The results suggest that the EJE is a safe traditional medicine for clinical applications at proper dose.

Acute high-level toluene exposure decreases hippocampal neurogenesis in rats.

Toluene is an organic solvent that is used in various industrial applications. Despite its usefulness, toluene has toxic effects on the brain and is a substance that is commonly abused. Toluene causes behavioral and functional abnormalities such as decreased memory capacity, cognitive impairment, and depression-like symptoms. However, the target sites and toxic mechanisms of inhaled toluene in the brain are poorly understood. In this study, we subjected Sprague-Dawley (SD) rats to acute high-level toluene exposure (7000 ppm) to investigate its neuronal toxicity, and in particular, its effect on neurogenesis in the hippocampus. In order to assay the effects of inhaled toluene on hippocampal neurogenesis, we measured the levels of neurogenesis markers Ki-67 and doublecortin (DCX) in the hippocampus 1, 2, 5, and 8 days after cessation of toluene exposure. In addition to assaying clinical signs, body weight, and bronchoalveolar lavage fluid, the liver, lungs, and kidneys were subjected to histopathological examination to investigate the toxic effects of high-level toluene exposure. Although abnormal neurological signs were observed after toluene exposure, these disappeared within 24 h and no toluene-related toxicological effects were observed in the liver, lungs, or kidneys. The animals exposed to toluene showed significantly decreased hippocampal neurogenesis, which persisted until the 8th and final day of measurement. Thus, acute high-level toluene exposure inhibited hippocampal neurogenesis and produced transient abnormal neurological signs, but did not produce toxicity in the other organs studied.

Toxicity of colloidal silica nanoparticles administered orally for 90 days in rats.

This study was undertaken to investigate the potential toxicity and establish the no observed adverse effect level (NOAEL) and target organ(s) of negatively charged colloidal silica particles of different sizes, ie, SiO2 (EN20(-)) (20 nm) or SiO2 (EN100(-)) 2(100 nm), administered by gavage in Sprague-Dawley rats. After verification of the physicochemical properties of the SiO2 particles to be tested, a preliminary dose range-finding study and 90-day repeated dose study were conducted according to the Organisation for Economic Cooperation and Development test guideline. Based on the results of the 14-day dose range-finding study, a high dose was determined to be 2,000 mg/kg, and middle and low doses were set at 1,000 and 500 mg/kg, respectively. In the 90-day toxicity study, there were no animal deaths in relation to administration of SiO2 particles of either size. In addition, no treatment-related clinical changes or histopathological findings were observed in any of the experimental groups. Moreover, no difference in toxic effects from chronic exposure to SiO2 (EN20(-))(20 nm) or SiO2 (EN100(-)) (100 nm) was observed. The results of this study indicate that the NOAEL for SiO2 (EN20(-)) and SiO2 (EN100(-)) would most likely be 2,000 mg/kg, and no target organ was identified in rats of either sex.

Evaluation of silica nanoparticle toxicity after topical exposure for 90 days.

Silica is a very common material that can be found in both crystalline and amorphous forms. Well-known toxicities of the lung can occur after exposure to the crystalline form of silica. However, the toxicities of the amorphous form of silica have not been thoroughly studied. The majority of in vivo studies of amorphous silica nanoparticles (NPs) were performed using an inhalation exposure method. Since silica NPs can be commonly administered through the skin, a study of dermal silica toxicity was necessary to determine any harmful effects from dermal exposures. The present study focused on the results of systemic toxicity after applying 20 nm colloidal silica NPs on rat skin for 90 days, in accordance with the Organization for Economic Cooperation and Development test guideline 411 with a good laboratory practice system. Unlike the inhalation route or gastrointestinal route, the contact of silica NPs through skin did not result in any toxicity or any change in internal organs up to a dose of 2,000 mg/kg in rats.

Amifostine ameliorates recognition memory defect in acute radiation syndrome caused by relatively low-dose of gamma radiation.

This study examined whether amifostine (WR-2721) could attenuate memory impairment and suppress hippocampal neurogenesis in adult mice with the relatively low-dose exposure of acute radiation syndrome (ARS). These were assessed using object recognition memory test, the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay, and immunohistochemical markers of neurogenesis [Ki-67 and doublecortin (DCX)]. Amifostine treatment (214 mg/kg, i.p.) prior to irradiation significantly attenuated the recognition memory defect in ARS, and markedly blocked the apoptotic death and decrease of Ki-67- and DCX-positive cells in ARS. Therefore, amifostine may attenuate recognition memory defect in a relatively low-dose exposure of ARS in adult mice, possibly by inhibiting a detrimental effect of irradiation on hippocampal neurogenesis.

Toluene inhibits hippocampal neurogenesis in adult mice.

Toluene, a representative industrial solvent and abused inhalant, decreases neuronal activity in vitro and causes mental depression and cognitive impairment in humans. However, the effects of toluene on brain function and the sites of its action are poorly understood. This study investigated the temporal changes of neurogenesis in the hippocampus of adult C57BL/6 mice after acute administration of toluene using two immunohistochemical markers for neurogenesis, Ki-67 and doublecortin (DCX). In addition, after toluene treatment, depression-like behaviors and learning and memory tasks were examined to assess hippocampal neurogenesis-related behavioral dysfunction. The number of Ki-67- and DCX-positive cells in the dentate gyrus of adult hippocampi declined acutely between 0 h and 24 h after toluene treatment (500 mg/kg, i.p.) and increased gradually from 2 to 8 days post-administration. The level of Ki-67 and DCX immunoreactivity decreased in a dose-dependent manner within the range of toluene administered (0-1000 mg/kg). In tail suspension and forced-swim tests performed at 1 and 4 days after toluene treatment (500 mg/kg), mice showed significant depression-like behaviors compared to the vehicle-treated controls. In the contextual fear conditioning and object recognition memory test, the mice trained at 1 and 4 days after toluene treatment showed significant memory defects compared to the vehicle-treated controls. This study suggests that acute exposure to toluene reduces the rate of adult hippocampal neurogenesis and can cause hippocampal dysfunction such as depression and cognitive impairment.

LC50 Determination of tert-Butyl Acetate using a Nose Only Inhalation Exposure in Rats.

tert-Butyl acetate (TBAc) is an organic solvent, which is commonly used in architectural coatings and industrial solvents. It has recently been exempted from the definition of a volatile organic compound (VOC) by the Air Resources Board (ARB) . Since the use of TBAc as a substitute for other VOCs has increased, thus its potential risk in humans has also increased. However, its inhalation toxicity data in the literature are very limited. Hence, inhalation exposure to TBAc was carried out to investigate its toxic effects in this study. Adult male rats were exposed to TBAc for 4 h for 1 day by using a nose-only inhalation exposure chamber (low dose, 2370 mg/m(3) (500 ppm) ; high dose, 9482 mg/m(3) (2000 ppm) ) . Shamtreated control rats were exposed to clean air in the inhalation chamber for the same period. The animals were killed at 2, 7, and 15 days after exposure. At each time point, body weight measurement, bronchoalveolar lavage fluid (BALF) analysis, histopathological examination, and biochemical assay were performed. No treatment-related abnormal effects were observed in any group according to time course. Based on those findings, the median lethal concentration (LC50) of TBAc was over 9482 mg/m(3) in this study. According to the MSDS, the 4 h LC50 for TBAc for rats is over 2230 mg/m(3). We suggested that this value is changed and these findings may be applied in the risk assessment of TBAc which could be beneficial in a sub-acute study.

Activation of Ca2+/calmodulin-dependent protein kinase II alpha in the spinal cords of rats with clip compression injury.

Ca(2+)/calmodulin-dependent protein kinase II alpha (CaMKIIalpha) is abundant in the central nervous system, where it plays important roles in regulating neuronal plasticity and survival. However, the role of CaMKIIalpha activation in traumatically injured spinal cords remains unclear. This study examined the effects of clip compression injury on levels of phosphorylated CaMKIIalpha (pCaMKIIalpha) and its cellular localization in rat spinal cords. Western blot analysis showed that the pCaMKIIalpha levels in both rostral (days 7, 14, and 21 post-injury) and caudal (days 4, 7, 14, and 21 post-injury) areas of the injury site were more than twice the levels in the non-injured controls. Immunohistochemical examination revealed constitutive localization of pCaMKIIalpha in the superficial lamina of the dorsal horn and neurons in normal spinal cord controls. After spinal cord injury, levels of the same components were markedly increased in both rostral and caudal regions approximately 3 mm from the center of the spinal cord lesions. However, pCaMKIIalpha was very rare in inflammatory cells in the injured spinal cords. In this animal model, CaMKIIalpha may play an important role in the spontaneous reversal of spinal cord dysfunction, thus restoring locomotor activity, possibly by functioning in the reconstruction of synaptic transmission and in protecting neurons from spinal cord injury.

Immunohistochemical analysis of cAMP response element-binding protein in mouse testis during postnatal development and spermatogenesis.

Basal activity and cellular localization of cAMP response element-binding protein (CREB) was examined in mouse testis during postnatal development and spermatogenesis. Testes of ICR mice sampled on postnatal day (PND) 3, 7, 14, 21, 28, 35, 42, and 49 were analyzed using Western blotting. Basal CREB activity was significantly higher in early phase (PND 3-7) developing testes than in intermediate- and late-phase developing (PND 14-42) and adult testes (PND 49). Furthermore, immunohistochemical analysis demonstrated the change of CREB phosphorylation in various testicular cell types during postnatal development. In particular, CREB phosphorylation in seminiferous tubules of the adult testis varied according to the spermatogenic cycle, while phosphorylation was evident in spermatogonia during all stages. Phosphorylation was moderate in pachytene spermatocytes of stages I-III and intense in round and elongate spermatids of spermiogenesis in stages XII-IX. These results suggest that CREB plays an important role in cell proliferation and differentiation in the early phase of postnatal development and spermatogenesis of mouse testis.

Photoprotective effect of red ginseng against ultraviolet radiation-induced chronic skin damage in the hairless mouse.

To evaluate the ability of red ginseng (RG) to protect the skin from photodamage, the gross and microscopic changes in the skin of hairless mice and RG-treated mice exposed chronically to UV were examined. The skin of the UV-irradiated mice showed characteristic signs of photoaging, such as deep wrinkles across the back, increased epidermal thickness, numerous cell infiltration, and many enlarged keratinizing cysts. The RG-treated mice showed a significantly decreased wrinkling score, minimal epidermal hyperplasia, slightly increased dermal cellularity and lack of proliferation of cysts. By week 22, 88.9% (i.p. with saline) or 60.0% (topical administration with cream base) of the UV-irradiated mice developed at least one tumor. RG delayed tumor onset significantly. RG was also effective in reducing the occurrence of UV radiation-induced skin tumors and reduced the number of tumors per mouse. After 22 weeks of treatment, 57.1% (i.p.) or 85.7% (topical administration) of the mice treated with RG were tumor-free. Tumor multiplicity was reduced by 89.3% (i.p.) or 92.2% (topical administration) in the RG treated groups. It is noted that skin that is chronically exposed to UV is subject to photoaging and photocarcinogenesis and the regular use of RG would prevent these photodamaging effects of UV.

The expression and localization of inhibin isotypes in mouse testis during postnatal development.

Inhibin, which is important for normal gonadal function, acts on the pituitary gonadotropins to suppress folliclestimulating hormone (FSH) secretion. The level and cellular localization of the inhibin isotypes, alpha, betaA and betaB, in the testis of mice were examined during postnatal development in order to determine if inhibin expression is related to testicular maturation. Mouse testes were sampled on postnatal days (PNDs) 1, 3, 6, 18, 48 and 120, and analyzed by Western blotting and immunofluorescence. Western blot analysis showed very low levels of inhibin alpha, betaA and betaB expression in the testes at days 1 to 6 after birth. The levels then increased gradually from PND 18 to 48-120, and there were significant peaks at PND 48. Inhibin alpha, betaA and betaB were detected in testicular cells during postnatal development using immunohistochemistry. The immunoreactivity of inhibin alpha was rarely observed in testicular cells during PND 1 to 6, or in the cytoplasmic process of Sertoli cells surrounding the germ cells and interstitial cells during PND 18 to 120. Inhibin betaA and betaB immunoreactivity was rarely observed in the testis from PND 1 to 6. On the other hand, it was observed in some spermatogonial cells, as well as in the interstitial space between PND 48 and PND 120. We conclude that the expression of inhibin isotypes increases progressively in the testis of mice with increasing postnatal age, suggesting that inhibin is associated with a negative feedback signal for FSH in testicular maturation.

Lack of adaptive response of gamma radiation for protection against neutron-induced teratogenesis.

BACKGROUND: Although there are some reports on neutron teratology, there is little information on the adaptive response of gamma radiation for protection against neutron-induced teratogenesis. This study examined whether or not a low dose of gamma radiation can induce an adaptive response in mouse fetuses exposed to a subsequent dose of neutrons in vivo. METHODS: Pregnant ICR mice were exposed to a priming dose of 0.3 Gy (0.9 Gy/min) of gamma rays on day 10.5 of gestation and challenged with 0.8 Gy (0.94 Gy/minute) of neutrons 24 h later. The mice were sacrificed on day 18.5 of gestation. The fetuses were examined for mortality, growth retardation, and other morphologic abnormalities. RESULTS: The tail length in the 0.3 Gy of gamma rays + 0.8 Gy of neutrons group was significantly shorter than in the 0.8 Gy of neutrons group. Although there was no significant difference compared with the 0.8 Gy of neutrons group, the number of live fetuses in the 0.3 Gy of gamma rays +0.8 Gy of neutrons group was lower. There was no evidence of primed exposure-related reductions in the malformed fetuses. Although there was no significant difference compared with the unprimed group, the number of malformed offspring in the primed group was higher. Furthermore, the incidence of kinked tail and adactyly was significantly higher in the primed mice than in the unprimed mice. CONCLUSIONS: Overall, this study shows that exposure to 0.3 Gy of gamma rays failed to induce an adaptive response of fetogenesis to a neutron challenge dose.