Biochemical processes mediating neurotoxicity induced by synthetic food dyes: A review of current evidence.

The extensive use of synthetic food dyes in the food industry, primarily due to their durability and cost-effectiveness compared to natural colorants, has raised significant health concerns. Of particular concern are the potential neurotoxic effects of six commonly used synthetic food dyes: Tartrazine (E102/FD&C Yellow No. 5), Erythrosine (E127/FD&C Red No. 3), Brilliant Blue FCF (E133/FD&C Blue No. 1), Allura Red AC (E129/FD&C Red No. 40), Sunset Yellow FCF (E110/FD&C Yellow No. 6), and Indigo Carmine (E132/FD&C Blue No. 2). This review delves into the metabolic pathways and neurotoxicity mechanisms of each dye, highlighting their effects on oxidative stress, neurotransmitter imbalances, mitochondrial dysfunction, and inflammatory responses. The evidence suggests that these dyes can significantly impact brain function and overall neurological health. This review underscores the importance of continued research in this field, as it is crucial to fully comprehend the neurotoxic processes of synthetic food dyes and to inform regulatory decisions that are crucial for safeguarding public health.

Combine colorants of tartrazine and erythrosine induce kidney injury: involvement of TNF-α gene, caspase-9 and KIM-1 gene expression and kidney functions indices.

Twenty-five male Wistar rats (140-170 g) were partitioned into 5 groups (n = 5). 2.5 mg/kg, 5 mg/kg, 10 mg/kg and 20 mg/kg of combine Tartrazine and Erythrosine (T+E; 50:50) were administered for 23 days. Serum urea and creatinine, gene expression and profiling of pro-inflammatory cytokine (Tumor Necrosis Factor- α gene), Caspase-9 and Kidney injury molecule-1 (KIM-1) and histomorphological examination of the kidney were investigated. The fold change of relative gene expression of TNF-α gene showed significantly (p < 0.05) up-regulation in all the treated rats except for the 10 mg/kg T+E treated rats when compared to control rats. Casp-9 and KIM-1 genes were significantly (p < 0.05) up-regulated in low dose treatment (2.5 mg/kg T+E and 5 mg/kg T+E) and down-regulated in high dose treatment (10 mg/kg T+E and 20 mg/kg T+E). However, there was significant (p < 0.05) increase in serum urea concentration in the rats treated with 5 mg/kg T+E and 20 mg/kg T+E while the rats treated with 10 mg/kg T+E indicated a significant (p < 0.05) decrease. Conversely, serum creatinine concentration indicated significant (p < 0.05) increase in10mg/kg T+E and 20 mg/kg T+E treated rats versus the control. From the histomorphological examination of the kidney, there was hypertrophy of the glomeruli in relation to the size of Bowman's capsule in the 10 mg/kg T+E and 20 mg/kg T+E treated rats. Kidney function was impaired as evident in up-regulation of TNF-α gene, KIM-1 gene, and serum urea and creatinine concentration with down-regulation of Casp-9 gene. The combined treatment also tampers with the architecture of the kidney.

Cytotoxicity of dental disclosing solution on gingival epithelial cells in vitro.

OBJECTIVE: Coloring dental biofilm and plaque with a dental disclosing solution is visually effective in dental treatment and oral hygiene education. Despite continuous reports of the risk of the product ingredients, dental disclosing solution are widely used in dentistry. However, the cytotoxic mechanism of dental disclosing solution is not known. Here we elucidated the tissue dyeing range and investigated the cytotoxic mechanism of dental disclosing solution. MATERIALS AND METHODS: Gingival epithelial cells and mouse head and neck tissue were stained with dental disclosing solution. Changes in the cell cycle distribution by the dental disclosing solution treatment were analyzed. A deoxynucleotidyl transferase dUTP nick and labeling (TUNEL) assay was performed to examine the apoptotic features of the gingival epithelial cells. RESULTS: Dental disclosing solution stained the chromosome strongly, as well as both the hard and soft tissue of the mouse head and neck. The results of flow cytometric analysis and TUNEL analyses revealed that the cytotoxicity associated with dental disclosing solution was related to the induction of apoptosis. However, the staining of porcine skin by dental disclosing solution was not easily removed, even with a wide range of pH solutions. CONCLUSIONS: These results suggest that dental disclosing solution had strong cytotoxicity and safer alternatives are needed.

Erythrosine B and quinoline yellow dyes regulate DNA repair gene expression in human HepG2 cells.

Erythrosine B (ErB) is a cherry pink food colorant and is widely used in foods, drugs, and cosmetics. Quinoline yellow (QY) is a chinophthalon derivative used in cosmetic compositions for application to the skin, lips, and/or body surface. Previously, ErB and QY synthetic dyes were found to induce DNA damage in HepG2 cells. The aim of this study was to investigate the molecular basis underlying the genotoxicity attributed to ErB and QY using the RT2 Profiler polymerase chain reaction array and by analyzing the expression profile of 84 genes involved in cell cycle arrest, apoptosis, and DNA repair in HepG2 cells. ErB (70 mg/L) significantly decreased the expression of two genes ( FEN1 and REV1) related to DNA base repair. One gene ( LIG1) was downregulated and 20 genes related to ATR/ATM signaling ( ATR, RBBP8, RAD1, CHEK1, CHEK2, TOPB1), nucleotide excision repair ( ERCC1, XPA), base excision repair ( FEN1, MBD4), mismatch repair ( MLH1, MSH3, TP73), double strand break repair ( BLM), other DNA repair genes ( BRIP1, FANCA, GADD45A, REV1), and apoptosis ( BAX, PPP1R15A) were significantly increased after treatment with QY (20 mg/L). In conclusion, our data suggest that the genotoxic mechanism of ErB and QY dyes involves the modulation of genes related to the DNA repair system and cell cycle.

Correlation of Vascular Endothelial Growth Factor Production with Photochemical Reaction-induced Retinal Edema.

BACKGROUND: Retinal edema is the major complication of retinal vein occlusion and diabetic retinopathy; it can damage visual function by influencing macular region. This study was to establish a rat retinal edema model and explore the related VEGF expression and observe the responses to anti-VEGF drugs in this model. METHODS: A rat retinal edema model was established by inducing photochemical reaction using a 532 nm laser after the intravenous injection of Erythrosin B. Immediately after the laser treatment, models were given intravitreal injections of Ranibizumab or Conbercept to inhibit VEGF expression, and the changes of retinal thickness were measured. Retinal edema was observed using fundus photography (FP), optical coherence tomography (OCT), and fluoresce in fundus angiography (FFA) at 0, 1, 2, 4, 7 and 14 days after intervention. The retinal VEGF expression was measured using enzyme-linked immunosorbent assay (ELISA) and western blotting at each time point. The rat retinal edema model was also used to verify the function of anti-VEGF polypeptide ZY1. RESULTS: Both retinal edema and vascular leakage were clearly observed at 1, 2 and 4 days after photochemical induction and the retinal thickness increased notably over the same period. The retinal VEGF expression peaked at day 1 and retina became thickening simultaneously. After the interventions, the VEGF expression of the Ranibizumab and Conbercept groups decreased at each time point compared to the edema group (26.90 ± 3.57 vs. 40.29 ± 6.68, F = 31.269 on day 1 and 22.36 ± 1.12 vs. 29.92 ± 0.93 F = 163.789 on day 2, both P < 0.01); the mean RT (278 ± 4 vs. 288 ± 3, F = 134.190 on day 1 and 274 ± 7 vs. 284 ± 6, F = 64.367 on day 2, both P < 0.05) and vascular leakage in these groups also decreased. The same results were observed in the ZY1 group, particularly at day 2 (P < 0.05). CONCLUSIONS: This retinal edema model induced by a photochemical reaction is reliable and repeatable. Induced edema increases expression of VEGF. This model can be used to test new drugs.

Application of a non-hazardous vital dye for cell counting with automated cell counters.

Recent advances in automated cell counters enable us to count cells more easily with consistency. However, the wide use of the traditional vital dye trypan blue (TB) raises environmental and health concerns due to its potential teratogenic effects. To avoid this chemical hazard, it is of importance to introduce an alternative non-hazardous vital dye that is compatible with automated cell counters. Erythrosin B (EB) is a vital dye that is impermeable to biological membranes and is used as a food additive. Similarly to TB, EB stains only nonviable cells with disintegrated membranes. However, EB is less popular than TB and is seldom used with automated cell counters. We found that cell counting accuracy with EB was comparable to that with TB. EB was found to be an effective dye for accurate counting of cells with different viabilities across three different automated cell counters. In contrast to TB, EB was less toxic to cultured HL-60 cells during the cell counting process. These results indicate that replacing TB with EB for use with automated cell counters will significantly reduce the hazardous risk while producing comparable results.

Genotoxic and mutagenic effects of erythrosine B, a xanthene food dye, on HepG2 cells.

Erythrosine (ErB) is a xanthene and an US Food and Drug Administration approved dye used in foods, drugs and cosmetics. Although its utilization is permitted, ErB is described as inhibitor of enzymes and protein-protein interactions and is toxic to pituitary and spermatogenesis processes. However, the genotoxicity and mutagenicity of ErB is inconclusive in the literature. This study aimed to analyze the genotoxicity of this dye using the alkaline comet assay and is the first investigation to evaluate ErB mutagenicity using the cytokinesis block micronucleus cytome (CBMN-Cyt) assay in HepG2 cells. These cells were chosen because they produce phase I and phase II enzymes that can mimic in vivo metabolism. The cells were treated with seven concentrations (0.1-70.0 µg mL(-1)) of ErB, and the results showed genotoxicity at the two highest concentrations and mutagenicity at six concentrations. Furthermore, as micronuclei result from clastogenic and aneugenic processes, while comet assay is often considered more sensitive and detects DNA single strain breaks, we suggest that an aneugenic is responsible for the observed damage. Although ErB is approved for use in the food, cosmetic and pharmaceutical industries, it must be used carefully because it damages the DNA structure.

Evaluation of potential genotoxicity of five food dyes using the somatic mutation and recombination test.

In this study, different concentrations of five food dyes (amaranth, patent blue, carminic acid, indigotine and erythrosine) have been evaluated for genotoxicity in the Somatic Mutation and Recombination Test (SMART) of Drosophila melanogaster. Standard cross was used in the experiment. Larvae including two linked recessive wing hair mutations were chronically fed at different concentrations of the test compounds in standard Drosophila Instant Medium. Feeding ended with pupation of the surviving larvae. Wings of the emerging adult flies were scored for the presence of spots of mutant cells which can result from either somatic mutation or somatic recombination. For the evaluation of genotoxic effects, the frequencies of spots per wing in the treated series were compared to the control group, which was distilled water. The present study shows that carminic acid and indigotine demonstrated negative results while erythrosine demonstrated inconclusive results. In addition 25 mg mL(-1) concentration of patent blue and 12.5, 25 and 50 mg mL(-1) concentrations of amaranth demonstrated positive results in the SMART.

Cytogenetic evaluation and DNA interaction studies of the food colorants amaranth, erythrosine and tartrazine.

Food coloring agents, amaranth, erythrosine and tartrazine have been tested at 0.02-8mM in human peripheral blood cells in vitro, in order to investigate their genotoxic, cytotoxic and cytostatic potential. Amaranth at the highest concentration (8mM) demonstrates high genotoxicity, cytostaticity and cytotoxicity. The frequency of SCEs/cell was increased 1.7 times over the control level. Additionally, erythrosine at 8, 4 and 2mM shows a high cytotoxicity and cytostaticity. Finally, tartrazine seems to be toxic at 8 and 4mM. No signs of genotoxicity were observed. Reversely, tartrazine showed cytotoxicity at 1 and 2mM. Furthermore, spectroscopic titration studies for the interaction of these food additives with DNA showed that these dyes bind to calf thymus DNA and distinct isosbestic points are observed clearly suggesting binding of the dyes to DNA. Additionally DNA electrophoretic mobility experiments showed that these colorants are obviously capable for strong binding to linear dsDNA causing its degradation. PCR amplification of all DNA fragments (which previously were pre-treated with three different concentrations of the colorants, extracted from agarose gel after separation and then purified), seems to be attenuated with a manner dye concentration-dependent reflecting in a delayed electrophoretic mobility due to the possible binding of some molecules of the dyes. Evaluation of the data and curves were obtained after quantitative and qualitative analysis of the lanes of the gel by an analyzer computer program. Our results indicate that these food colorants had a toxic potential to human lymphocytes in vitro and it seems that they bind directly to DNA.

Involvement of high plasma corticosterone status and activation of brain regional serotonin metabolism in long-term erythrosine-induced rearing motor hyper activity in young adult male rats.

Long-term consumption of artificial food color(s) can induce behavioral hyperactivity in human and experimental animals, but no neurobiochemical mechanism is defined. This study investigates the role of brain regional serotonin metabolism including its turnover, MAO-A activity, and plasma corticosterone status in relation to behavioral disturbances due to an artificial food color, erythrosine. Long-term (15 or 30 consecutive days) erythrosine administration with higher dosage (10 or 100 mg/kg/day, p.o.) produced optimal hyperactive state in exploratory behavior (rearing motor activity) after 2 h of last erythrosine administration, in young adult male albino rats. Erythrosine-induced stimulation in brain regional (medulla-pons, hypothalamus, hippocampus, and corpus striatum) serotonin metabolism (measuring steady state levels of 5-HT and 5-HIAA, MAO-A activity), including its turnover (pargyline-induced 5-HT accumulation and 5-HIAA declination rate), as well as plasma corticosterone were also observed depending on dosage(s) and duration(s) of erythrosine administration under similar experimental conditions. The lower dosage of erythrosine (1 mg/kg/day, p.o.) under similar conditions did not affect either of the above. These findings suggests (a) the induction as well as optimal effect of long-term erythrosine (artificial food color) on behavioral hyperactivity in parallel with increase in 5-HT level in brain regions, (b) the activation of brain regional serotonin biosynthesis in accordance with plasma corticosterone status under such behavioral hyperactivity, and (c) a possible inhibitory influence of the enhanced glucocorticoids-serotonin interaction on erythrosine-induced rearing motor hyperactivity in young adult mammals.

Toxicity of xanthene food dyes by inhibition of human drug-metabolizing enzymes in a noncompetitive manner.

The synthetic food dyes studied were rose bengal (RB), phroxine (PL), amaranth, erythrosine B (ET), allura red, new coccine, acid red (AR), tartrazine, sunset yellow FCF, brilliant blue FCF, and indigo carmine. First, data confirmed that these dyes were not substrates for CYP2A6, UGT1A6, and UGT2B7. ET inhibited UGT1A6 (glucuronidation of p-nitrophenol) and UGT2B7 (glucuronidation of androsterone). We showed the inhibitory effect of xanthene dye on human UGT1A6 activity. Basic ET, PL, and RB in those food dyes strongly inhibited UGT1A6 activity, with IC(50) values = 0.05, 0.04, and 0.015 mM, respectively. Meanwhile, AR of an acidic xanthene food dye showed no inhibition. Next, we studied the inhibition of CYP3A4 of a major phase I drug-metabolizing enzyme and P-glycoprotein of a major transporter by synthetic food dyes. Human CYP3A4 and P-glycoprotein were also inhibited by basic xanthene food dyes. The IC(50) values of these dyes to inhibit CYP3A4 and P-glycoprotein were the same as the inhibition level of UGT1A6 by three halogenated xanthene food dyes (ET, PL, and RB) described above, except AR, like the results with UGT1A6 and UGT2B7. We also confirmed the noninhibition of CYP3A4 and P-gp by other synthetic food dyes. Part of this inhibition depended upon the reaction of (1)O(2) originating on xanthene dyes by light irradiation, because inhibition was prevented by (1)O(2) quenchers. We studied the influence of superoxide dismutase and catalase on this inhibition by dyes and we found prevention of inhibition by superoxide dismutase but not catalase. This result suggests that superoxide anions, originating on dyes by light irradiation, must attack drug-metabolizing enzymes. It is possible that red cosmetics containing phloxine, erythrosine, or rose bengal react with proteins on skin under lighting and may lead to rough skin.

Short-term erythrosine B-induced inhibition of the brain regional serotonergic activity suppresses motor activity (exploratory behavior) of young adult mammals.

Previous studies showed that repeated ingestion of erythrosine B (artificial food color) developed behavioral hyperactivity, but nothing is known about its single administration effect as well as the neurochemical (s) involvement. The present study provides evidence that a single higher dosage (10, 100 or 200 mg/kg, p.o.) of erythrosine administration to young adult male rats reduced motor activity (MA) maximally at 2 h and brain regional (medulla-pons, hippocampus and hypothalamus) serotonergic activity (measuring steady-state levels of 5-HT and 5-HIAA, pargyline-induced 5-HT accumulation and 5-HIAA declination rate and 5-HT receptor binding) under similar experimental condition. The degree of erythrosine-induced inhibition of both MA and brain regional serotonergic activity was dosage dependent. Lower dosage (1 mg/kg, p.o.) did not affect either of the above. Erythrosine (100 or 200 mg/kg, p.o.)-induced MA suppression was also observed in the presence of specific MAO-A inhibitor, clorgyline (5 mg/kg, i.p.) or MAO-B inhibitor, deprenyl (5 mg/kg, i.p.); but their co-application (5 mg/kg, i.p., each) effectively prevented the erythrosine-induced motor suppression. Altogether these results suggest that a single higher dosage of erythrosine (10-200 mg/kg, p.o.) may reduce MA by reducing serotonergic activity with modulation of central dopaminergic activity depending on the brain regions.

Ability of 13 chemical agents used in dental practice to induce sister-chromatid exchanges in Syrian hamster embryo cells.

To evaluate the genotoxic potential of 13 chemical agents used in dental practice, the abilities of these agents to induce sister-chromatid exchanges (SCEs) were examined using Syrian hamster embryo (SHE) cells. Statistically significant increases in the frequencies of SCEs were observed in SHE cells treated with all seven of the chemical agents used as endodontic medicaments: p-chlorophenol, m-cresol, formaldehyde, guaiacol, hydrogen peroxide, p-phenolsulfonic acid, and sodium hypochlorite (P < 0.01; Student t test). Assessment of two chemical agents that are applied to the oral mucosa as antiseptics showed that SCEs were induced by iodine (P < 0.01), but not by chlorhexidine. Of three chemical agents that are used as dyes for disclosing dental plaque, erythrosine B had no effect on SCE induction, while acid fuchsin and basic fuchsin increased the SCE frequencies in SHE cells (P < 0.01). Glutaraldehyde, which is used as a disinfectant for dental instruments and impressions, also induced SCEs (P < 0.01). Because SCE assays are used as a sensitive indicator for evaluating genetic toxicity of chemicals, the chemical agents that had a positive response in the present study are potentially genotoxic to mammalian cells.

Response characteristics of cutaneous mechanoreceptors in neuropathic rats.

The activity of single myelinated afferents was recorded from dorsal roots L4-5 in normal Sprague-Dawley rats and animals that developed mechanical hypersensitivity following ischemic injury to the sciatic nerve. The mechanical response properties and conduction velocity of afferents conducting through the injury site (about 50% of units) were similar to controls. However, the majority of afferents not conducting through the injury site exhibited ongoing activity. The results suggest that mechanical allodynia may be at least partly due to the central integration of activity arising from these two populations of afferents in neuropathic rats.

Reproductive and neurobehavioural toxicity study of erythrosine administered to mice in the diet.

Erythrosine was given in the diet to provide levels of 0 (control), 0.005, 0.015 and 0.045% from 5 weeks of age of the F(0) generation to 9 weeks of age of the F(1) generation in mice, and selected reproductive and neurobehavioural parameters were measured. There were no adverse effects of erythrosine on either litter size, litter weight or sex ratio at birth. The average body weight of the offspring was significantly increased in the middle-dose group in both sexes during the lactation period. In behavioural developmental parameters, any variables showed no significant adverse effects in either sex in the lactation period. In movement activity of exploratory behaviour, several parameters were significantly changed in the high-dose group, and those effects were dose related in adult females in the F(0) and F(1) generations and in male offspring in the F(1) generation. The dose level of erythrosine in the present study produced few adverse effects in reproductive and neurobehavioural parameters in mice.

Cold exposure enhances tactile allodynia transiently in mononeuropathic rats.

A laser and erythrosin-B-induced sciatic nerve injury decreases thresholds of a mechanically induced paw withdrawal reflex and enhances cold-induced withdrawal behavior of the affected limb. Exposure of the affected paw to a normally innocuous cold stimulus results in a transient decrease in the threshold of the mechanically evoked paw withdrawal reflex in neuropathic but not in intact rats. The present data suggest that in an experimental neuropathic state a normally innocuous cold stimulus may further sensitize spinally mediated withdrawal reflexes to stimuli of another stimulus modality, in this case, to innocuous tactile stimuli. Therefore, testing mechanical allodynia in neuropathic rats immediately after testing cold allodynia may produce artifactual results.

Correlation of mechanistic data and histopathology in the evaluation of selected toxic endpoints of the endocrine system.

The objective of this review is to correlate endocrinologic data from mechanistic studies with quantitative histopathology in selected examples of toxic endpoints of the endocrine system in laboratory animals. Mechanistic data can aid in the interpretation of animal toxicology findings and help clarify their significance in risk assessment. Endocrine organs of rodents frequently undergo proliferative changes with advancing age and following chronic exposure to large doses of xenobiotic chemicals, and the sensitivity of rodent endocrine tissues appears to be increasing. Many xenobiotic chemicals in large doses disrupt thyroid function in rodents either by a direct effect on the thyroid influencing synthesis of thyroid hormones or by adversely influencing their peripheral metabolism. A number of chemicals disrupt thyroid function by inhibiting the important enzyme, thyroperoxidase (TPO). A contemporary example of a chemical acting as TPO-inhibitor is sulfamethazine. In short-term mechanistic studies in rats there was a log-dose response relationship in circulating levels of thyroid and pituitary hormones plus a similar non-linear dose-response in morphologic changes in thyroid follicular cells. Endocrinologic data from mechanistic studies and histopathologic/ultrastructural findings will also be presented for the effects of the food color, FDC Red No. 3 (Erythrosine), on the thyroid gland in rats and parathyroid hormone-related protein (a major causative factor in cancer-associated hypercalcemia) on parathyroid chief cells in mice.

Short-term toxicity study of erythrosine on wistar rats hepatic mitochrondrial respiration

El colorante alimentario xanteno eritrosina presentó, en investigación anterior realizada in vitro, un fuerte efecto inhibitorio en mitocondrias aisladas de hígado y de riñones de ratas. Por ese motivo, fue seleccionado para una investigación del mismo efecto después de su administración por vía oral en ratas wistar. La eritrosina fue administrada en el agua de bebida, durante 90 días, a ratas machos y hembras recién destetadas, en las dosis de 0,100,500 y 1000 mg del colorante/kg depeso corporal por día. Al final del experimento, la función respiratoria de las mitocondrias aisladas del hígado de los animales que consumieron eritrosina, no fue distinta (p>0,05) del grupo control. Durante el período de estudio no hubo diferencia significativa (p>0,05) en la ganacia de peso de los animales. La observación al microscopio de los sistemas digestivo, respiratorio, urinario y linfoide no mostró anomalías. Preparaciones histológicas indicaron dilatación del cecum y una moddrada adherencia del colorante de la mucosa intestinal

A study on the reproductive toxicity of erythrosine in male mice.

Worldwide usage of different colouring agents in the food industry prompted us to study their toxicity. The potential adverse effects of erythrosine (ER, FD & C Red No. 3) on the spermatogenesis process were investigated in adult mice. Testicular lactic dehydrogenase isoenzyme activity (LDH-X), a pachytene spermatocyte marker of testicular toxicity, was significantly decreased to 71.8% and 68.6% of the control value after daily p.o. administration of ER (21 days) in doses of 68 and 136 mg kg-1 respectively. At the same time, the normal average epididymal sperm count as well as the percentage of motile sperms were significantly inhibited by about 50% and 57% respectively. Moreover, ER was shown to disrupt the normal morphology of the sperm head. Thus, after 5 daily p.o administrations of ER in doses of 680 and 1360 mg kg-1 (equivalent to 10 and 20% of its LD50) it increased the incidence of sperms with abnormal head by about 57% and 65% respectively. The induced increase in sperm abnormalities could enhance the spermatogenic dysfunction and germ cell mutagenicity. These findings indicate that ER in the used doses has a potential toxic effect on spermatogenesis in mice and in turn, it may affect its testicular function and reproductive performance.

An assessment of the in vivo clastogenicity of erythrosine.

In an investigation of the in vivo clastogenic potential of the food colouring erythrosine (ER), male B6C3F1 mice were treated by ip injection at doses of 50, 100 and 200 mg/kg, repeated 24 hr apart. Signs of toxicity were observed at the highest dose of ER administered. The three cytogenetic endpoints analysed were sister chromatid exchanges (SCEs) in peripheral blood lymphocytes (PBLs), micronuclei in bone marrow polychromatic erythrocytes (PCEs), and micronuclei in peripheral blood reticulocytes (PBRs). SCE frequencies in PBLs were 4.13, 4.58, 4.33 and 4.60 SCE/cell at 0, 50, 100 and 200 mg ER/kg, respectively. At the same doses, the frequencies of micronucleated PCEs were 3.5, 3.2, 2.0 and 2.5/1000 PCEs. Micronuclei in PBRs ranged from 1.2 to 3.6 and from 1.4 to 3.0/1000 PBRs in control and treated mice, respectively. These results indicate that ER is inactive as a clastogen in mouse blood and marrow cells. This result supports the hypothesis of a non-genotoxic mechanism for ER carcinogenicity.