Subchronic Oral Study Research Articles

The short-term toxicity and metabolome of dicyclopentadiene

Dicyclopentadiene (DCPD) was investigated in a 14-day oral rat toxicity study based on the OECD 407 guideline in combination with plasma metabolomics. Wistar rats received the compound daily via gavage at dose levels of 0, 50 and 150 mg/kg bw. The high dose induced transient clinical signs of toxicity and, in males only, reduced body weight gain. High dose liver changes were characterized by altered clinical chemistry parameters in both sexes and pathological changes in females. In high dose males an accumulation of alpha-2 u-globulin in the kidney was noted. Comparing the DCPD metabolome with previously established specific metabolome patterns in the MetaMap® Tox data base suggested that the high dose would result in liver enzyme induction leading to increased breakdown of thyroid hormones for males and females. An indication for liver toxicity in males was also noted. Metabolomics also suggested an effect on the functionality of the adrenals in high dose males, which together with published data, is suggestive of a stress related effect in this organ. The results of the present 14-day combined toxicity and metabolome investigations were qualitatively in line with literature data from subchronic oral studies in rats with DCPD. Importantly no other types of organ toxicity, or hormone dysregulation beyond the ones associated with liver enzyme induction and stress were indicated, again in line with results of published 90-day studies. It is therefore suggested that short term “smart” studies, combining classical toxicity with ‘omics technologies, could be a 2 R (refine and reduce) new approach method allowing for the reduction of in vivo toxicity testing.

Toxicological evaluation of Propyl-propane-thiosulfinate (PTS) by 90-days oral subchronic study in rats

Toxicological evaluation of Propyl-propane-thiosulfinate (PTS) by 90-days oral subchronic study in rats

Sex-specific accumulation of silver nanoparticles in rat kidneys is not ovarian hormone regulated but elimination limited

Preferential Ag deposition in female kidneys has been reported in subacute and subchronic oral gavage and inhalation studies of various sizes of silver nanoparticles (AgNPs). In these in vivo animal studies, female kidneys were shown to accumulate 3–4 times more silver than male kidneys. The current 28-day oral gavage study of AgNPs administered to ovariectomized female rats investigated the effect of sex-dependent hormones. Ovariectomized rats were allowed to recover for 14 days, then AgNPs (60 nm) were administered by gavage for 28 days based on 500 mg/kg body weight. After this subacute oral exposure, the rats were sacrificed and the Ag content in the kidneys was measured using an atomic absorption spectrophotometer. No statistically significant difference was noted in the Ag content in the kidneys among the AgNP administered, AgNP administered sham, and AgNP administered ovariectomized rats, although the female kidneys showed a statistically significant higher accumulation of Ag than the male kidneys. Reanalysis of the clearance of Ag accumulated in the kidneys showed 2 different phases; fast (T1/210.4 days) and slow (T1/2 61.9 days) for males, while the clearance of females showed (T1/2 20.8 days) and slow (T1/2 154 days). The higher accumulation of Ag in female kidneys is due to slower clearance of Ag in female kidneys than those of males. Therefore, the current results indicate that the preferential silver accumulation in female kidneys is not regulated by ovarian hormones, but by elimination rate differences between males and females.

Acute and Subchronic Oral and Dermal Toxicological Studies of Salvia Verbenaca Extracts in Mice and Rats

ABSTRACTThe acute and subchronic toxicological effects of Salvia verbenaca extracts in hexane, ethyl acetate, n-butanol, were investigated. The extracts were not toxic at 2000 mg Kg−1. A normal body weight evolution was observed during the 14 treatment days where there was no difference between the control and the treated groups. No abnormalities were seen in the haematological and biochemical parameters with the exception of an increase of the platelets. S. verbenaca may be recommended to boost platelets formation when compromised. Biochemical analysis revealed normal physiological levels of alanine transaminase (ALAT) and aspartate transaminase (ASAT). Histopathological examination showed a normal architecture with no morphological changes in the major vital examined organs.

Subchronic toxicity of lyophilized apoaequorin protein powder in Sprague-Dawley rats

Apoaequorin is a bioluminescent calcium-binding apoprotein endogenous to the Aequorea species of jellyfish and is commercially available in a dietary supplement in support of brain and cognitive health. Results from a previous 90-day subchronic oral gavage study established the no-observed-adverse-effect-level (NOAEL) of lyophilized apoaequorin protein powder (LAPP) at 666.7 mg/kg/day. The current 90-day oral gavage study in Sprague-Dawley rats administered dose levels of 1000, 2000, and 4000 mg/kg/day of test substance as received. These doses are expressed as milligram of supplement with the amounts of apoaequorin based on the analysis of the percentage of active ingredient. The corresponding amounts of apoaequorin protein are 603, 1206, and 2412 mg/kg/day. These dose levels target approximately 4221, 8442, and 16,844 times more than the expected human oral intake. There were no mortalities, clinical observations, ophthalmological, clinical pathology, or histopathological changes attributable to LAPP administration. Changes in mean body weight and feed efficiency, without other correlating clinical or pathological or other toxicologically relevant findings, were considered to be of little toxicological significance. Therefore, the NOAEL for LAPP administered orally up to 90 days was 4000 mg/kg/day (2412 mg/kg/day based on 603 mg/g or 60.3% active ingredient, apoaequorin protein), the highest dose tested in male and female rats.

Acute and 13-week subchronic toxicological evaluations of turanose in mice

BACKGROUND/OBJECTIVESTuranose, α-D-glucosyl-(1→3)-α-D-fructose, is a sucrose isomer which naturally exists in honey. To evaluate toxicity of turanose, acute and subchronic oral toxicity studies were conducted with ICR mice.MATERIALS AND METHODSFor the acute oral toxicity study, turanose was administered as a single oral dose [10 g/kg body weight (b.w.)]. In the subchronic toxicity study, ICR mice were administered 0, 1.75, 3.5, and 7 g/kg b.w. doses of turanose daily for 13 weeks.RESULTSNo signs of acute toxicity, including abnormal behavior, adverse effect, or mortality, were observed over the 14-day study period. In addition, no changes in body weight or food consumption were observed and the median lethal dose (LD50) for oral intake of turanose was determined to be greater than 10 g/kg b.w. General clinical behavior, changes in body weight and food consumption, absolute and relative organ weights, and mortality were not affected in any of the treatment group for 13 weeks. These doses also did not affect the macroscopic pathology, histology, hematology, and blood biochemical analysis of the mice examined.CONCLUSIONNo toxicity was observed in the acute and 13-week subchronic oral toxicology studies that were conducted with ICR mice. Furthermore, the no-observed-adverse-effect level is greater than 7 g/kg/day for both male and female ICR mice.

A Study of 2-Mercaptobenzothiazole (2-MBT) and HBM values for 2-MBT in the urine of adults and children: Opinion of the "Human Biomonitoring" Commission of the German Federal Environment Agency

2-Mercaptobenzothiazole (2-MBT, CAS No. 149-30-4) is mainly used as a vulcanization accelerator in the production of rubber. Other applications are as a fungicide in paints and varnishs and for the external treatment of animals. Because of its manifold application in consumer products, for example in soothers, exposure of the general population to 2-MBT can't be excluded. For the purpose of a toxicological evaluation of a possible body burden, the German HBM-commission derived HBM-I values for 2-MBT in the urine of children and adults. The no observed adverse effect level of 94 mg/kg bw/d from a subchronic oral study with mice was used as the point of departure. After considering a total assessment factor of 350, a tolerable daily intake of 0.3 mg/kg bw/d was deduced for humans. Considering further the percentage of 2-MBT and its glucuronide excreted in urine together with the body weight proportional urine volume leads to an HBM-I value for 2-MBT of 4.5 mg/l urine for children and 7 mg/l urine for adults.

Genotoxicity and subchronic toxicity of Sophorae radix in rats: Hepatotoxic and genotoxic potential

Although Sophorae radix (SR) has been traditionally used as a treatment for various clinical symptoms, a comprehensive investigation of its safety has not yet been carried out. Therefore, we present an evaluation of the toxicity of the SR extract that was performed according to the Organization for Economic Cooperation and Development test guidelines for subchronic toxicity and genotoxicity. In an oral subchronic study for 13weeks, the repeated treatment of rats with 429 or 1500mg/kg of the SR extract induced a dose-related change in body weight. In particular, the SR extract was observed to exert a significant increase in liver weight along with an increase in serum alkaline phosphatase and alanine transaminase. A small but statistically significant reductions in red blood cell, hemoglobin, and hematocrit levels in the SR extract-treated rats suggest the possibility that anemia, accompanied by liver injury, was at least partially induced. These findings indicate the no-observed-adverse-effect-level for the SR extract was considered to be 10mg/kg/d. And, the data obtained from the chromosome aberration assay showed that SR extract might be considered to be a weak clastogen although no significant micronucleus induction was observed in vivo. Despite the benefits that SR extract can exhibit, this study indicates that SR extract may possess hepatotoxic and genotoxic potential.

Scientific Opinion on the safety assessment of the substance, 5‐norbornene‐2, 3‐dicarboxylic anhydride, CAS No 826‐62‐0, for use in food contact materials

This scientific opinion of the EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids deals with the risk assessment of 5-norbornene-2, 3-dicarboxylic anhydride CAS 826-62-0, FCM substance No 978, for a co-monomer in polyester based coatings for metal cans. Based on the residual content of the substance in the final coating, the calculated worst case migration of the two isomers of the substance and their hydrolysis products was 27 µg/kg food. From the same experiment the overall migration was 24 mg/kg food. LMWF was found to make up 8.8 mg/kg food, out of this the LMWF which contains the substance was 1.54 mg/kg food. Based on negative results from two in vivo genotoxicity tests, the Panel considered that the substance and the LMWF oligomers containing the substance moieties are non-genotoxic. The systemic toxicity of the substance-containing LMWF oligomers was assessed in a subchronic oral rat study. The comparison between the NOAEL, derived from this study and a daily intake gives a large margin of safety. The CEF Panel concluded that the substance 5-norbornene-2, 3-dicarboxylic anhydride is not of a safety concern for the consumer if it is used as a co-monomer in polyester based can coatings at levels of up to 18.3 weight % of the final dry coating, in contact with all food excluding beverages, at sterilization conditions and long term contact. Migration of the substance and its hydrolysis products should in total not exceed 50 µg/kg food.

Relevance of non-guideline studies for risk assessment: The coverage model based on most frequent targets in repeated dose toxicity studies

A common challenge for human risk assessment is the quality of the available animal studies. Non-guideline studies are often limited for different aspects of study design and documentation. Within this publication the relevance of a limited scope of examination is discussed. Preliminary analyses of the RepDose database have shown that liver, body weight, kidney and clinical symptoms are frequently affected in oral repeated dose toxicity in rats and mice (Bitsch et al., 2006), while many other targets are seldom affected.As most of these targets are investigated frequently also in non-guideline studies, it is likely that they provide a reliable NOEL, although the full spectrum of endpoints has not been covered. Based on RepDose data we investigate the relevance of individual targets for determining the LOEL and the consequences for risk assessment. The resulting coverage model for subchronic oral rat studies includes up to six targets and an additional assessment factor for LOEL extrapolation. It can be applied to assess the reliability of non-guideline studies with respect to the scope of examination. Furthermore the application of the coverage model to other databases will increase and/or specify the chemical domain and reveal respective targets as well as effects.

Sub-Chronic Oral Exposure to Iridium (III) Chloride Hydrate in Female Wistar Rats: Distribution and Excretion of the Metal

Iridium tissue distribution and excretion in female Wistar rats following oral exposure to iridium (III) chloride hydrate in drinking water (from 1 to 1000 ng/ml) in a sub-chronic oral study were determined. Samples of urine, feces, blood and organs (kidneys, liver, lung, spleen and brain) were collected at the end of exposure. The most prominent fractions of iridium were retained in kidney and spleen; smaller amounts were found in lungs, liver and brain. Iridium brain levels were lower than those observed in other tissues but this finding can support the hypothesis of iridium capability to cross the blood brain barrier. The iridium kidney levels rose significantly with the administered dose. At the highest dose, important amounts of the metal were found in serum, urine and feces. Iridium was predominantly excreted via feces with a significant linear correlation with the ingested dose, which is likely due to low intestinal absorption of the metal. However, at the higher doses iridium was also eliminated through urine. These findings may be useful to help in the understanding of the adverse health effects, particularly on the immune system, of iridium dispersed in the environment as well as in identifying appropriate biological indices of iridium exposure.

8:2 fluorotelomer alcohol: A one-day nose-only inhalation toxicokinetic study in the Sprague-Dawley rat with application to risk assessment

8:2 fluorotelomer alcohol (8:2 FTOH) inhalation exposure was investigated to (1) compare plasma metabolites to oral data, (2) conduct a route-to-route extrapolation (oral to inhalation), (3) develop a human equivalent air concentration (HEC) from a 90-day oral sub-chronic study in rats using BMD analysis, and (4) calculate a margin of exposure (MOE) between the HEC and measured air concentrations. Male and female rats were exposed nose-only for 6h at 3 or 30mg/m3. Blood was collected at 1, 3 and 6h during exposure and 6 and 18h post exposure. Alcohol, perfluorocarboxylic acid and polyfluorinated acid metabolites were determined in plasma by LC–MS/MS. 8:2 FTOH was <LOQ (32nM) at the low exposure and quantifiable (37–69nM) at the high exposure. The quantifiable metabolites in plasma were dose proportional and comprised mainly of 8:2 FTCA, 7:3 Acid, and PFOA. By kinetic modeling, the yields of the terminal products 7:3 Acid (1.6–2.1 and 0.9mol%) and PFOA (1.0–1.2 and 0.3mol%) of the inhaled dose were low for male and female rats, respectively. The kinetic yield of PFOA after oral dosing was similar (1.1–1.7-fold) for male rats and greater (8–9-fold) for female rats relative to inhalation exposure, an observation confirmed by non-compartmental analysis. A BMDL10% (3.7mg/kg/day) was derived for mild hepatic necrosis observed in male rats following a 90-day oral dose study with 8:2 FTOH. The corresponding HECs were 1.8 and 3.7mg/m3, which gave MOE values ranging from 1.8×104 to 6.1×106 based on reported ambient air concentrations of 0.3–209ng/m3. These findings demonstrate rapid 8:2 FTOH uptake and clearance by the inhalation route and a consistent metabolite profile between inhalation and oral exposures in rats. No toxicity is expected in humans from typical ambient 8:2 FTOH air exposures.

Toxicology and risk assessment of acrolein in food

Acrolein is an α,β-unsaturated aldehyde formed by thermal treatment of animal and vegetable fats, carbohydrates and amino acids. In addition it is generated endogenously. As an electrophile, acrolein forms adducts with gluthathione and other cellular components and is therefore cytotoxic. Mutagenicity was shown in some in vitro tests. Acrolein forms different DNA adducts in vivo, but mutagenic and cancerogenous effects have not been demonstrated for oral exposure. In subchronic oral studies, local lesions were detected in the stomach of rats. Systemic effects have not been reported from basic studies. A WHO working group established a tolerable oral acrolein intake of 7.5 μg/kg body weight/day. Acrolein exposure via food cannot be assessed due to analytical difficulties and the lack of reliable content measurements. Human biomonitoring of an acrolein urinary metabolite allows rough estimates of acrolein exposure in the range of a few μg/kg body weight/day. High exposure could be ten times higher after the consumption of certain foods. Although the estimation of the dietary acrolein exposure is associated with uncertainties, it is concluded that a health risk seems to be unlikely.

Derivation of a reference dose and drinking water equivalent level for 1,2,3-trichloropropane

In some US potable water supplies, 1,2,3-trichloropropane (TCP) has been present at ranges of non-detect to less than 100ppb, resulting from past uses. In subchronic oral studies, TCP produced toxicity in kidneys, liver, and other tissues. TCP administered by corn oil gavage in chronic studies produced tumors at multiple sites in rats and mice; however, interpretation of these studies was impeded by substantial premature mortality. Drinking water equivalent levels (DWELs) were estimated for a lifetime of consumption by applying biologically-based safety/risk assessment approaches, including Monte Carlo techniques, and with consideration of kinetics and modes of action, to possibly replace default assumptions. Internationally recognized Frameworks for human relevance of animal data were employed to interpret the findings. Calculated were a reference dose (=39μg/kgd) for non-cancer and Cancer Values (CV) (=10–14μg/kgd) based on non-linear dose–response relationships for mutagenicity as a precursor of cancer. Lifetime Average Daily Intakes (LADI) are 3130 and 790–1120μg/person-d for non-cancer and cancer, respectively. DWELs, estimated by applying a relative source contribution (RSC) of 50% to the LADIs, are 780 and 200–280μg/L for non-cancer and cancer, respectively. These DWELs may inform establishment of formal/informal guidelines and standards to protect public health.

Acute, subacute, and subchronic exposure to 2A-DNT (2-amino-4,6-dinitrotoluene) in the northern bobwhite (Colinus virginianus)

2-Amino-4,6-dinitrotoluene (2A-DNT) is a metabolite of the explosive 2,4,6-trinitrotoluene (TNT) which is present in the soil at numerous U.S. Army installations as the result of TNT manufacture or training activities. Although many avian species are known to inhabit areas where 2A-DNT has been found in the environment, no published studies of the effects of 2A-DNT exposure in birds are available. In this study, we conducted an evaluation of the oral toxicity of 2A-DNT in a representative ground foraging species of management concern, the northern bobwhite (Colinus virginianus). Subacute (14 days) and subchronic (60 days) oral gavage exposure studies were conducted following determination of the median acute lethal dose (LD50 = 1167 mg/kg). In the subacute study, survival occurred at 50 mg/kg/day. This helped to determine dose groups for the subchronic study: 0, 0.5, 3, 14, and 30 mg 2A-DNT/kg body weight-d in corn oil. The lowest observed adverse effects level (LOAEL) was determined to be 14 mg/kg/day based on mortality, and the no observed adverse effects level (NOAEL) was determined to be 3 mg/kg/day based on lack of effects at this exposure level.

Kinetics of 8-2 fluorotelomer alcohol and its metabolites, and liver glutathione status following daily oral dosing for 45 days in male and female rats

Fluorotelomer alcohols (FTOHs) are raw materials used in the manufacture of polymeric and surfactant products. Based on previous findings from single oral dosing in rats with radiolabeled 8-2 FTOH, glutathione (GSH) depletion and/or the presence of perfluorinated/polyfluorinated acids and aldehyde metabolites was hypothesized to account for the hepatocellular lesions observed in male rats from a 90-day subchronic oral dosing study. Further, the reported nephropathy in female rats from the subchronic experiment was hypothesized to have been initiated by a thiol metabolite produced by degradation of GSH conjugates. In the current investigation, the kinetics of 8-2 FTOH and its metabolites along with liver GSH status were evaluated in the rat following daily oral dosing with 8-2 FTOH for 45 days at 5 and 125 mg/kg/day. Liver GSH stores 1–2 h after dosing were unaffected, suggesting that GSH depletion is not likely a relevant mode of action in the liver. The tissue metabolite data indicate that the liver toxicity mode of action is likely associated with elevated levels of perfluoroalkyl acids found in males, since other polyfluorinated metabolites and 8-2 FTOH were present in livers from female rats at comparable or higher levels. Detection of the N-acetyl cysteine conjugate of the unsaturated parent telomer alcohol in urine from female rats and not male rats provides some evidence to support the mechanistic basis for the observed kidney effects. Further, the increasing levels of perfluorooctanoic acid (PFOA) in plasma from female rats over the 45-day dosing phase, while unexpected, may reflect an increased net absorption of 8-2 FTOH, slow elimination of intermediates in the metabolic pathway between 8-2 FTOH and PFOA, or altered kidney clearance. The results of this study have enhanced our understanding of 8-2 FTOH kinetics and metabolism and potential modes of action in the rat, which will guide the design of future studies for FTOHs and our need to define the mechanistic basis for the observed effects.

Safety profile of a food dextrin: Acute oral, 90-day rat feeding and mutagenicity studies

Nutriose ® is a glucose polysaccharide produced by the chromatographic separation of a dextrin fraction derived from maize, wheat or other edible starches. Animal safety studies conducted on Nutriose ®FB are reported. They include an acute oral and a 90-day study in rats and short-term in bacteria (Ames test) and a mutation assay at the TK locus in L5178Y mouse lymphoma cells. An acute oral study in Sprague–Dawley rats established the LD 50 as greater than 2000 mg/kg. In a 90-day, oral subchronic study, Sprague–Dawley rats were administered Nutriose ®FB in their diet at doses of 0, 1.25%, 2.5% or 5% for 13 weeks. Neither mortality nor significant behavioral changes occurred during the study. The consumption of Nutriose ®FB did not have any effect on body weight or on feed or water consumption. Blood coagulation and hematology and blood and urine biochemistry did not reveal any toxic effect of the compound. No treatment-related histopathological differences were observed between control and test groups. Adverse clinical observations, including opthalmological observations, were marginal and not considered treatment-related. There was no effect of Nutriose ®FB on relative or absolute organ weight of rats of either sex, except for the increase in caecum content and caecum mucosa. The increase in caecum weight is considered a physiological adaptation seen after the ingestion of indigestible carbohydrates and is not considered a toxicological effect. The No-Observed-Adverse-Effect-Levels (NOAELs) were established by the highest tested doses: 4.4 g/kg bw/day in males and 6.5 g/kg bw/day in females. Mutation assays in bacteria (Ames tests) and in mammalian cells (tk locus in mouse lymphoma cells) were negative with Nutriose ®FB.

Final Amended Report on the Safety Assessment of Methylparaben, Ethylparaben, Propylparaben, Isopropylparaben, Butylparaben, Isobutylparaben, and Benzylparaben as used in Cosmetic Products

Final Amended Report on the Safety Assessment of Methylparaben, Ethylparaben, Propylparaben, Isopropylparaben, Butylparaben, Isobutylparaben, and Benzylparaben as used in Cosmetic Products

Himematsutake (Iwade Strain 101) extract (ABM-FD): Genetic toxicology and a 3-month dietary toxicity study in rats

Agaricus blazei Murrill, an edible mushroom, is used as a functional food due to medicinal effects of (1→6)-β-D-glucan protein complex which has been shown to have anti tumour activity in mice. A 13week oral subchronic study in rats performed at 500, 1000 or 2000mg/kg/day caused, at the highest dose, reduced erythrocyte numbers and high mean cell volume in males, high creatinine and urea concentrations in both sexes and low spleen weights in females, but no histopathological change. The findings suggested low level chronic toxicity at 2000mg/kg/day and a no observed adverse effect level (NOAEL) of 1000mg/kg/day. Genotoxicity tests on the aqueous extract were negative in the bacterial reverse mutation test, either with or without S9 mix, up to 5000μg/plate and in a rat bone marrow micronucleus test up to 2g/kg bodyweight. The extract was positive at acceptable levels of toxicity in an L5178Y mouse lymphoma assay following 24h exposure in the absence of S9 and this was associated with an increase in the number of small colonies, suggesting possible clastogenic activity or aneuploidy, rather than point mutation. The aqueous extract of A. blazei is therefore of low subchronic toxicity and did not cause any direct effect upon the DNA molecule and the weak positive in the L5178 mouse lymphoma test was likely due to large deletions or the loss of the whole chromosomes rather than to direct damage to the DNA.

Final Amended Report on the Safety Assessment of Methylparaben, Ethylparaben, Propylparaben, Isopropylparaben, Butylparaben, Isobutylparaben, and Benzylparaben as used in Cosmetic Products

Parabens is the name given to a group of p-hydroxybenzoic acid (PHBA) esters used in over 22,000 cosmetics as preservatives at concentrations up to 0.8% (mixtures of parabens) or up to 0.4% (single paraben). The group includes Methylparaben, Ethylparaben, Propylparaben, Isopropylparaben, Butylparaben, Isobutylparaben, and Benzylparaben. Industry estimates of the daily use of cosmetic products that may contain parabens were 17.76 g for adults and 378 mg for infants. Parabens in cosmetic formulations applied to skin penetrate the stratum corneum in inverse relation to the ester chain length. Carboxylesterases hydrolyze parabens in the skin. Parabens do not accumulate in the body. Serum concentrations of parabens, even after intravenous administration, quickly decline and remain low. Acute toxicity studies in animals indicate that parabens are not significantly toxic by various routes of administration. Subchronic and chronic oral studies indicate that parabens are practically nontoxic. Numerous genotoxicity studies, including Ames testing, dominant lethal assay, hostmediated assay, and cytogenic assays, indicate that the Parabens are generally nonmutagenic, although Ethylparaben and Methylparaben did increase chromosomal aberrations in a Chinese Hamster ovary cell assay. Ethylparaben, Propylparaben, and Butylparaben in the diet produced cell proliferation in the forestomach of rats, with the activity directly related to chain length of the alkyl chain, but Isobutylparaben and Butylparaben were noncarcinogenic in a mouse chronic feeding study. Methylparaben was noncarcinogenic when injected subcutaneously in mice or rats, or when administered intravaginally in rats, and was not cocarcinogenic when injected subcutaneously in mice. Propylparaben was noncarcinogenic in a study of transplacental carcinogenesis. Methylparaben was nonteratogenic in rabbits, rats, mice, and hamsters, and Ethylparaben was nonteratogenic in rats. Parabens, even at levels that produce maternal toxicity, do not produce fetal anomalies in animal studies. Parabens have been extensively studied to evaluate male reproductive toxicity. In one in vitro study, sperm were not viabile at concentrations as low as 6 mg/ml Methylparaben, 8 mg/ml Ethylparaben, 3 mg/ml Propylparaben, or 1 mg/ml Butylparaben, but an in vivo study of 0.1% or 1.0% Methylparaben or Ethylparaben in the diet of mice reported no spermatotoxic effects. Propylparaben did affect sperm counts at all levels from 0.01% to 1.0%. Epididymis and seminal vesicle weight decreases were reported in rats given a 1 % oral Butylparaben dose; and decreased sperm number and motile activity in F 1 offspring of rats maternally exposed to 100 mg/kg day -1 were reported. Decreased sperm numbers and activity were reported in F 1 offspring of female rats given Butylparaben (in DMSO) by subcutaneous injection at 100 or 200 mg/kg day -1 , but there were no abnormalities in the reproductive organs. Methylparaben was studied using rats at levels in the diet up to an estimated mean dose of 1141.1 mg/kg day -1 with no adverse testicular effects. Butylparaben was studied using rats at levels in the diet up to an estimated mean dose of 1087.6 mg/kg day -1 in a repeat of the study noted above, but using a larger number of animals and a staging analysis of testicular effects-no adverse reproductive effects were found. .