Carcinogenic activity of benzo[a]pyrene in a 2 year oral study in Wistar rats.

Because of the relatively high human oral exposure to polycyclic aromatic hydrocarbons (PAHs) compared to the inhalation exposure, the known carcinogenicity of this type of compounds and the limited data from oral studies available with polycyclic aromatic hydrocarbons, an oral carcinogenicity study was performed using benzo[a]pyrene (B[a]P) as a PAH representative. Wistar rats, 52 animals per sex and group were exposed daily (5 days a week) to 0, 3, 10 or 30 mg B[a]P/kg bw/day by gavage for 104 weeks and were subject to gross- and histopathology. The main tumours observed were hepatocellular carcinomas and forestomach tumours. Other tumours induced in this study were tumours of the auditory canal, skin and appendages, oral cavity, small intestine, kidney, and soft tissue sarcomas. For hepatocellular carcinomas and forestomach tumours, the BMDL10 were 3 and 1 mg/kg bw/day, respectively. The incidence of altered hepatic foci was increased in the 3mg/kg bw/day group. The increase in liver tumours is considered the most relevant effect for human risk assessment in terms of pathogenesis and sensitivity, and is proposed as the basis for human cancer risk assessment for oral PAH exposure.

Age-related differences in the toxicity of ochratoxin A in female rats.

Ochratoxin A (OTA) is a mycotoxin found in food and feedstuffs of plant and animal origin. OTA exposure is related to nephropathy in humans. Age-related differences, especially in nephro- and immunotoxicity of OTA, were investigated in young adult (aged 12 weeks) and old (aged 27-30 months) female SPF Wag rats, treated by gavage with 0, 0.07, 0.34 or 1.68 mg OTA/kg body weight for 4 weeks. In both age groups, survival was significantly decreased in the highest dose group. Clinical condition, body weight, clinical chemistry parameters (ALAT, ASAT, creatinin and urea) and target organs (as identified by weight and pathology - kidney, liver, adrenals, forestomach and brain) were affected by age and dose, but often more severely in old than in young rats. OTA induced primarily nephropathy. Old rats were more sensitive to induction of tubular karyomegaly and vacuolation/necrosis. In young rats, OTA induced a dose-related thickening of the basement membrane and reduction in splenic T-cell fraction. Decreased IgG levels were seen at 0.34 mg/kg OTA (young and old rats) and 1.68 mg/kg OTA (young rats). Vacuolation of the white brain matter (cerebellar medulla and ventral parts of the brain stem) was significantly increased in young rats at 0.34 and 1.68 mg/kg OTA and in old rats at 0.07 and 0.34 mg/kg OTA. It was concluded that: (1) the profiles of OTA toxicity for both age groups are similar, with the kidney and possibly the brain being primary target organs; (2) based on clinical and pathological data old rats are more sensitive to OTA than young rats; and (3) the immune system is probably not the primary target of OTA toxicity.

A new rat model of otitis media caused by Streptococcus pneumoniae: conditions and application in immunization protocols.

Streptococcus pneumoniae (pneumococcus [Pn]) can be cultured from up to 50% of acute otitis media (AOM) effusions, and these bacteria are the most common cause of AOM-related complications. With the recent advent of antibiotic-resistant Pn strains, treatment of Pn infections may meet with serious difficulties. Prevention through vaccination, notably for the four most common occurring Pn serotypes in humans (i.e., Pn 6B, Pn 14, Pn 19F, and Pn 23F), is a helpful alternative. Testing of vaccine efficacy should occur in an appropriate animal AOM model, which is presented here. The four involved Pn serotypes are not pathogenic to the rat, which was chosen as the experimental animal for practical reasons. To induce a natural infection (i.e., ascending through the eustachian tube), the mucociliary clearance of the eustachian tube was impaired by infusing histamine into the tympanic cavity on 2 consecutive days before intranasal inoculation of the bacteria. With this simple protocol, high and reproducible infection rates, as determined with bacterial cultures, of Pn-induced AOM (approximately 70%) with the two major Pn serotypes 14 and 19F (Pn 14 and Pn 19F) were obtained, whereas lower infection rates (25 to 50%) with Pn 6B and Pn 23F were obtained. In this model, intranasal priming with pneumococci, as well as subcutaneous vaccination with Pn 14 tetanus toxoid-conjugated polysaccharide, induced a protective effect against the induction of otitis media with these bacteria. This shows that immunity to Pn 14 AOM can be induced by both mucosal and systemic presentations of antigen. In conclusion, we have developed an animal model for Pn-induced AOM, which is suitable for the evaluation of the protecting effect of immunization.

Comparison of tobramycin nephrotoxicity in young adult and aged female rats.

A two week toxicity study was performed in rats to study the possible age-dependent toxicity of tobramycin, an aminoglycoside antibiotic with well known ototoxic and nephrotoxic properties in animals and man. Young adult female Wag/Rij rats aged 12 weeks (n = 10) and old female rats aged 23 to 26 months (n = 14) were treated subcutaneously with 0, 10, 40 or 160 mg tobramycin sulphate/kg/day. Clinical chemistry and urinalysis revealed significant changes in renal function in young adult rats mainly at 160 mg/kg, whereas in old rats significant changes were seen at 10, 40 and 160 mg/kg. Excretion of N-acetyl-beta-glucosaminidase, indicative for tubular dysfunction, was statistically significantly increased only in old animals at 160 mg/kg. Histopathology: At 40 mg/kg, tubular necrosis was increased in old animals and hyaline droplet formation in both age groups. At 160 mg/kg these lesions were increased in both age groups. For tubulonephrosis, interstitial nephritis and tubular regeneration, age-related differences were predominantly reflected in severity, for example, at 40 mg/kg, tubular regeneration in young animals was "moderate" in 7/10 and "marked" in 2/10, while in old animals the scores were 3/14 and 11/14, respectively. Secondary treatment-related lesions (in heart and adrenals) were also more increased in old animals. Chemistry and histopathology revealed the increased sensitivity to the toxic effects of tobramycin in old rats, which is important for the discussion of the most appropriate dosing regimen for aminoglycoside in humans. The once-daily dosing regimen for tobramycin should not be recommended for elderly, because high peak concentrations should be avoided to minimise nephrotoxicity.

The role of urocanic acid in UVB-induced suppression of immunity to Trichinella spiralis infection in the rat.

The naturally occurring trans-isomer of urocanic acid (trans-UCA), found in the stratum corneum, absorbs ultraviolet light (UV) and isomerizes to the cis-form. Cis-UCA has been shown to impair some cellular immune responses, and has been proposed as an initiator of the suppression that follows UV irradiation. UVB exposure leads to an increase in cis-UCA in the skin of rats from about 10% to 40% of the total UCA. Previously it has been demonstrated that UVB lowers immune responses to Trichinella spiralis after oral infection of rats with the parasitic worm. In the present study we investigated the role of cis-UCA in the control of this parasitic infection. Rats were infected orally with T. spiralis and injected with different doses of cis- or trans-UCA subcutaneously. Mitogenic responses and the mixed lymphocyte reaction were not affected by either isomer. In contrast, the number of T. spiralis larvae in muscle tissue of infected rats was increased significantly in the cis-UCA-treated animals compared with the trans-UCA-treated animals. In addition, delayed-type hypersensitivity (DTH) to T. antigen in infected rats was significantly impaired by cis-UCA but not by trans-UCA. If rats were injected with a monoclonal antibody with specificity for cis-UCA 2 hr prior to UVB exposure, the UVB-induced suppression in DTH to T. spiralis and the increase in larvae counts were significantly inhibited compared with rats that were similarly injected with a control antibody. Thus cis-UCA can inhibit the specific resistance to parasitic infections and acts as an important mediator of UVB-induced suppression of immunity to T. spiralis in the rat.

Methods for exposure of laboratory animals to ultraviolet radiation.

Two different sources of ultraviolet B (UVB) radiation, an electronically controlled UVB exposure unit, containing FS40 tubes, and a hand-held Kromayer lamp, were evaluated for actual irradiance in W/m2 and spectra (physical dosimetry and biological dosimetry (skin effects in rodents)). The technical studies of the FS40 sources demonstrated that the flux intensity of the lamps could be changed electronically, without affecting the spectrum. Thus it was possible to standardize UVB exposure electronically. The biologically effective doses of these sources were analysed in RIV-Tox Wistar rats and BALB/c mice. After low doses of UVB radiation, histopathological changes such as acanthosis, hyperkeratosis and dermal inflammation were observed in the skin without the presence of major side effects such as erythema and oedema. After higher doses of UVB radiation erythema and oedema were clearly visible. Quantitative studies showed that the minimal erythema dose, as a biological parameter, correlated well to the emission in J/m2. In addition, biological parameters such as acanthosis and inflammation in the skin correlated well to the actual exposure in J/m2 and were sensitive biomarkers for UVB-induced skin toxicity. Thus, in addition to minimal erythemal doses, acanthosis and inflammation may also be applied as biologically relevant doses for studies of the biological effects of UVB radiation.

Protection of UV-induced suppression of skin contact hypersensitivity: a common feature of flavonoids after oral administration?

In this study we investigated the effect of the dietary ingredients fruit and vegetable, green tea phenol extract (GTP) and the specific flavonoid components quercetin and chrysin on the UV-induced suppression of the contact hypersensitivity (CHS) response to picryl chloride (PCl). The SKH-1 mice were fed with test diet from 2 or 4 weeks before and during the UV irradiation (daily, 95 mJ/cm2) and tested for the CHS ear-swelling response 10 weeks after the onset of the irradiation. For the CHS, mice were immunized with PCl by epicutaneous application on nonirradiated sites. Four days after sensitization all mice were challenged on both sides of each ear by topical application of one drop PCl. In addition, from mice fed with the fruit and vegetable mixture the number of Langerhans cells (LC) were scored in the skin and from mice fed with quercetin, quercetin levels in plasma were measured at week 11 after the start of UV irradiation. It was found that fruit and vegetable (19% in the diet), GTP (0.1% and 0.01% in the drinking water), quercetin (1% in the diet) and chrysin (1% and 0.1% in the diet), prevented statistically significantly the UV-induced suppression of CHS to PCl. In the skin of mice fed with fruit and vegetables combined with UV irradiation the number of LC were comparable to the control mice, whereas the number of LC were significantly diminished in mice treated with UV only. This protective effect on the presence of LC in the epidermis after UV irradiation, which was also observed in a previous study with quercetin, may play a role in the prevention of UV-induced immunosuppression by the flavonoids tested. In conclusion, we found protection of flavonoids against UV-induced effects on CHS, which may be a common feature of most flavonoids.

Spontaneous liver tumors and benzo[a]pyrene-induced lymphomas in XPA-deficient mice.

Defects in the xeroderma pigmentosum complementation group A-correcting (XPA) gene, which encodes a component of the nucleotide excision repair (NER) pathway, are associated with the cancer-prone human disease xeroderma pigmentosum. We previously generated mice lacking the XPA gene, which develop normally but are highly sensitive to ultraviolet-B and 7,12-dimethylbenz[a] anthracene-induced skin tumors. Here we report that XPA-deficient mice spontaneously developed hepatocellular adenomas at a low frequency as they aged. Furthermore, oral treatment of XPA-deficient mice with the carcinogen benzo[a]pyrene (B[a]P) resulted in the induction of mainly lymphomas. These tumors appeared earlier and with a higher incidence than in B[a]P-treated wild-type and heterozygous mice. Our results show for the first time that XPA-deficient mice also displayed an increased sensitivity to developing tumors other than tumors of the skin.

Use of E mu-PIM-1 transgenic mice short-term in vivo carcinogenicity testing: lymphoma induction by benzo[a]pyrene, but not by TPA.

E mu-pim-1 transgenic mice are predisposed to develop lymphomas. Due to their low spontaneous tumour incidence and their increased sensitivity towards the lymphomagen ethylnitrosourea these mice may present an interesting model for short-term carcinogenicity testing. Here, we report on the further exploration of this transgenic mouse model with two additional carcinogens known to have, among others, the lymphohaematopoietic system as target, i.e. benzo[a]pyrene (B[a]P) and 12-O-tetradecanoylphorbol-13-acetate (TPA). B[a]P, given three times a week (by gavage) for 13 weeks at 4.3, 13 or 39 mg/kg body weight, resulted in a dose-related increase in lymphomas up to a 90% incidence in E(mu)-pim-1 mice during the observation period of 40 weeks. B[a]P also induced tumours of the forestomach within this observation period, though at a lower incidence and apparently equally effective in wildtype and transgenic mice. TPA, on the other hand, was unable to induce lymphomas (or tumours in any other organ) in either transgenic or wildtype animals within the observation period of 44 weeks, when applied dermally at the maximum tolerated dose of 3 microg/mouse, twice a week for 35 weeks. Molecular analysis showed that B[a]P-induced lymphomas in transgenic mice were of T-cell origin, 80% of which had elevated levels of c-myc expression. None of the lymphomas had increased N-myc expression and mutation analysis of the ras-gene family revealed a K-ras mutation in only one out of eight tumours investigated. Also, none of the lymphomas showed aberrant expression of p53 as determined by immunohistochemistry. It is concluded that the E mu-pim-1 mouse model will not be very suitable for short-term carcinogenicity testing in general: only genotoxic chemicals that have the lymphohaematopoietic system as target for carcinogenesis in wild-type mice, appear to be efficiently identified.

Quercetin prevents UV-induced local immunosuppression, but does not affect UV-induced tumor growth in SKH-1 hairless mice.

Ultraviolet is thought to induce skin tumors by its dual activity as a mutagenic agent and a suppressor of cell-mediated immunity. In the present study the effects of quercetin, a flavonoid-containing compound, on carcinogenesis and immunosuppression were studied in SKH hairless mice exposed to suberythemal doses of UV for up to 17 weeks. It was found that quercetin did not affect the onset or growth of non-melanoma skin tumors resulting from UV exposure. In contrast, it prevented the suppression in contact hypersensitivity (CHS) to picryl chloride induced by UV. The mechanism of this prevention might be explained by the observation that the decreased number of epidermal Langerhans' cells is partly prevented by the quercetin. Quercetin did not alter the effects of UV in increasing numbers of spleen and lymph node cells, only partly in decreasing the CD8-positive cells in spleen cell populations and decreasing the lymphoproliferative response of spleen cells to the mitogens concanavalin A and phytohemagglutinin. Thus oral quercetin did not prevent UV-induced carcinogenesis although it restored the skin-associated CHS response probably by protecting the antigen-presenting cells in the skin.

The effect of oral quercetin on UVB-induced tumor growth and local immunosuppression in SKH-1.

The effects of quercetin (4%) on UVB-induced carcinogenesis and immunosuppression were studied in hairless SKH-1 mice exposed daily to suberythemal UVB for 12/13 and 16/17 weeks. Macroscopic and microscopic examinations showed that quercetin did not affect the onset and growth of UVB-induced non-melanoma skin tumors. Quercetin prevented the UV-induced suppression of the contact hypersensitivity (CHS) and the reduction of the percentage of CD8-positive cells in spleen and lymph nodes. Other immunological parameters were not affected. Thus, the results indicate that oral intake of a high dose of quercetin does not prevent UVB-induced carcinogenesis, although it restores the skin-associated CHS response.

Risk assessment on the carcinogenic potential of hybridoma cell DNA: implications for residual contaminating cellular DNA in biological products.

The aim of this study was to evaluate the possible carcinogenic potential of residual DNA derived from immortalized and possibly tumorigenic cell lines due to activated oncogenic sequences (oncogenes). These cell lines have been used for the production of biologicals, i.e. monoclonal antibodies, lymphokines and vaccines. The authors used hybridoma DNA as a first model. For this reason experiments in two species were performed, namely in 3-4 week-old female Balb/c mice and newborn Riv:TOX rats. Doses of 250 micrograms DNA, derived from Balb/c hybridoma cells, were injected subcutaneously (s.c.) in 200 mice. These mice also received a s.c. injection of the solvent only (TE buffer) at another site of the back skin (negative control for local tumour development). An additional group of 50 mice was treated intraperitoneally (i.p.) with the solvent only to serve as a negative control group for possible systemic tumorigenic effects. Doses of 5 micrograms plasmid pPy1 DNA, containing the entire Polyoma virus genome, served as positive control and were injected s.c. and i.p. in 20 and 50 mice, respectively. Doses of 50 micrograms hybridoma DNA or 5 micrograms pPy1 DNA were injected s.c. in rats too, using nine animals per group. During the experiment, animals were observed weekly, especially for the occurrence of subcutaneous tumours at the injection sites. The mouse study was terminated after more than 2 years, the rat study after 1 year. Gross necropsy was performed on all animals and histopathological examination of grossly suspected neoplastic lesions was performed. In the mouse experiment, tumour development at the s.c. injection site of the DNA was observed in one out of 20 animals in the pPy1-treated positive control group (neurofibrosarcoma) and one out of 200 animals in the hybridoma DNA-treated group (haemangioma-like lesion). Tumour development at or near the s.c. injection site of the solvent only was observed in two out of 200 animals. In the rat study none out of nine hybridoma DNA-treated rats developed tumours at the injection site, while three out of nine rats of the positive control group, injected with the pPy1 DNA, showed local tumour development (benign and malignant soft tissue tumours.) It is concluded that, at the high dose and numbers of animals tested, parenteral administration of hybridoma DNA does not induce local tumour development. Furthermore, no indications were found for systemic carcinogenic potential of the hybridoma DNA used. Based on a worst case approach of our data, the oncogenic risk of 100 pg residual DNA was estimated to be 2 x 10(-9), a value intermediate of the estimations of the WHO (1987) and the Dutch Health Council (1988) 5 x 10(-11) and 2 x 10(-7), respectively. Therefore, it is unlikely that the risk of 100 pg of DNA derived from other immortalized cell lines will exceed the level of generally accepted cancer risk of 10(-6).

Effects of ultraviolet-B exposure on the resistance to Listeria monocytogenes in the rat.

A rat infection model using the bacterial pathogen Listeria monocytogenes was employed to analyze the immunosuppressive activity of UVB radiation. Rats were exposed to suberythemal doses of UVB radiation for 5 or 7 consecutive days, using Kromayer or FS40 lamps respectively. Subsequently, the rats were infected subcutaneously or intravenously with Listeria. Exposure to UVB resulted in an increased number of bacteria in the spleen 4 days after infection. Listeria-specific lymphocyte proliferation assays as well as delayed-type hypersensitivity reactions demonstrated that T cell-mediated immunity to Listeria was impaired by UVB as measured 4 and 8 days after infection. In addition, UVB exposure decreased phagocytotic activity of peripheral blood macrophages. This study demonstrated that suberythemal doses of UVB radiation caused a delay in the clearance of Listeria bacteria from the spleen of the rats and that this was probably caused by impaired nonspecific phagocytosis of Listeria by macrophages in addition to an impaired activity of Listeria-specific T cells.

Ototoxicity of tobramycin in young adult and old rats.

To study the possible age-dependent ototoxic effects of tobramycin a subacute toxicity study was performed. To young adult (3 months) and old (27-30 months) female Wag-Rij rats 0, 10, 40, and 160 mg tobramycin sulfate/kg was administered subcutaneously in two doses a day. After 14 days all animals were autopsied. The cochlea was fixed by perfusion through the opened oval window and prepared for scanning electron microscopy. The rat cochlea consists of two turns. In all animals of the young control group the row of inner hair cells (IHC) as well as the three rows of outer hair cells (OHC) of the organ of Corti were fully intact. IHC as well as OHC are provided with three rows of stereocilia, from inside to outside with increasing length. The stereocilia of OHC are arranged in a characteristic W-configuration. In the young low dose group effects were focally seen in only 1 of 10 rats, consisting of shortened, disoriented or partly disappeared stereocilia of IHC and OHC in the basal turn. In the young mid dose group 4 of 10 animals had focally shortened, disoriented, and less stereocilia in the first row of OHC in the basal turn, and once of IHC and OHC in the apical turn. Seven of 8 animals of the young high dose group showed effects consisting of focal loss or shortening of stereocilia of IHC and OHC of the apical turn (3x), of OHC in the basal turn (3x), and once of stereocilia of IHC only. In the old control group the stereocilia of IHC were fully intact. However, many OHC, independent of row or turn, had no stereocilia at all. The percentages of OHC without stereocilia in the three rows of the apical and basal turns were 44-10-50 and 40-20-50, respectively. In the old low, mid, and high dose group the percentages of OHC without stereocilia were nearly identical to those of the old control group. In the old low and high dose group a reduced number of stereocilia per IHC occurred in half of the number of animals, while in the mid dose group the IHC were fully intact. In young adult animals the number of mildly affected cochleae increased as well as the extent of the lesion increased with increasing dose of tobramycin. The lesions of IHC and OHC, consisting of a decrease in number or shortening of stereocilia, were restricted mainly to (the last part of) the basal turn. As old control rats showed already a large number of OHC without stereocilia, the possible ototoxic effects of tobramycin were not detected against this "background" of stereociliary loss, consistent with aging. The damage in old rats was certainly not greater than in young adult rats. In conclusion, old rats were not more sensitive to the possible ototoxic effects of tobramycin than young adult rats.

Increased susceptibility to ultraviolet-B and carcinogens of mice lacking the DNA excision repair gene XPA.

Xeroderma pigmentosum patients with a defect in the nucleotide-excision repair gene XPA are characterized by, for example, a > 1,000-fold higher risk of developing sunlight-induced skin cancer. Nucleotide-excision repair (NER) is involved in the removal of a wide spectrum of DNA lesions. The XPA protein functions in a pre-incision step, the recognition of DNA damage. To permit the functional analysis of the XPA gene in vivo, we have generated XPA-deficient mice by gene targeting in embryonic stem cells. The XPA-/-mice appear normal, at least until the age of 13 months. XPA-/-mice are highly susceptible to ultraviolet (UV)-B-induced skin and eye tumours and to 7,12-dimethylbenz[a]anthracene (DMBA)-induced skin tumours. We conclude that the XPA-deficient mice strongly mimic the phenotype of humans with xeroderma pigmentosum.

Investigation of a screening battery for immunotoxicity of pharmaceuticals within a 28-day oral toxicity study using azathioprine and cyclosporin A as model compounds.

The authors have investigated a panel of parameters for immunotoxicity that may be incorporated in routine screening for toxicity of pharmaceuticals. This panel comprises serum immunoglobulin concentrations, cellularity of bone marrow, weights and histopathology of thymus, spleen, and lymph nodes, histopathology of Peyers' patches, and FACScan analysis of lymphocyte subpopulations in the spleen, in addition to parameters of toxicity to other systems. To study the value of these assays for pharmaceuticals, the authors used the immunosuppressants azathioprine (AZP) and cyclosporin A (CsA) as model compounds with known immunotoxic activity. In two separate experiments, rats were treated by daily gastric intubation with 0, 5, 12.5, and 25 mg AZP/kg body wt or 0, 1.25, 5, and 20 mg CsA/kg body wt. In the AZP study, the histopathology of the thymus and the spleen were valuable parameters of immunotoxicity, since these organs showed microscopic alterations at relatively low dose levels. In the CsA experiment, both the histopathology of the thymus and the data provided by FACScan analysis were sensitive indicators of immunotoxicity detecting effects at the lowest dose level employed. The data indicate that the lymphoid system is the most sensitive target of toxicity after AZP or CsA administration. The authors conclude that their test battery yielded immunotoxicity profiles of AZP and CsA in rats that were consistent with published findings in the literature, indicating the usefulness of the test battery employed.

Evaluation of the OECD 421 reproductive toxicity screening test protocol using butyl benzyl phthalate.

The OECD421 reproductive toxicity screening test protocol was evaluated using the reproductive and developmental toxicant butyl benzyl phthalate (BBP). Female rats were orally exposed from 14 days premating to 6 days postpartum. Male rats were exposed for 29 days. At 1000 mg/kg bw/day effects were found on body weight gain and food consumption, on spermatogenesis, time to conception, pregnancy rate, postimplantation survival, and litter size and weight. Food consumption and pup weight were slightly affected at 500 mg/kg also. Effects occurred at expected dosages on the basis of literature data. These findings support the conclusion that the OECD421 test scores BBP correctly as a reproductive toxicant, both in a qualitative and in a quantitative sense.

Evaluation of the OECD 421 reproductive toxicity screening test protocol using 1-(butylcarbamoyl)-2-benzimidazolecarbamate (benomyl).

The OECD 421 reproductive toxicity screening test protocol was evaluated using the fungicide benomyl as a test compound at 10, 30, or 90 mg/kg per day. Male rats showed dose-dependent testicular degeneration after 28 days exposure, as expected on the basis of literature data. Dams in the high dose group, exposed from 14 days premating to postnatal day 6, had pups with decreased weights at postnatal days 1 and 6. Prenatal deaths were increased, but no malformations were found. In contrast, in a developmental toxicity test with exposure between gestation days 6 and 15, 90 mg/kg induced a high level of postimplantation loss, with ophthalmic malformations in the surviving offspring, in agreement with literature data. It is suggested that premating exposure in the OECD 421 protocol may have induced tolerance to the compound, e.g., by modulation of biotransformation in the dam. These findings indicate that the teratogenic potential of a compound need not necessarily be revealed using this screening test. The OECD 421 protocol appeared sufficiently sensitive to reveal the reproductive hazard of benomyl on the basis of prenatal deaths and testicular and pup weight effects. However, the absence of congenital anomalies in the offspring after benomyl treatment according to the OECD 421 protocol underscores the notion that lack of biological activity in the test should not be regarded as evidence of lack of activity of a compound on reproductive parameters.