Subchronic heavy metal and alcohol treatment in rats: changes in the somatosensory evoked cortical activity.

Young adult male Wistar rats were treated, by gavage, with 80 or 320 mg/kg Pb2+ (lead acetate), 0.4 or 1.6 mg/kg Hg2+ (mercuric chloride) or both by combining the lower doses. For combination with alcohol, ethanol was added to the rats' drinking water in 5 v/v %. After 12 weeks of treatment, electrophysiological recording was made from the somatosensory cortex in urethane anaesthesia. Evoked potentials obtained by stimulation of the whiskers were recorded. Both metals, and alcohol alone, increased significantly the latency of the evoked response. Alcohol seemed to abolish the effect of Pb, but not of Hg. Fatigue, calculated form the response amplitude, was increased by Pb and Hg treatment and this effect of Hg was reduced by ethanol. Evoked activity and its dynamic characteristics were sensitive to the complex neurotoxic effect induced in the rats and can provide a basis for developing functional markers.

A study on immunotoxicological effects of subacute amitraz exposure in rats.

The effects of amitraz, a formamidine pesticide, were investigated in four-week old outbred male Wistar rats on certain classic toxicological and haematological parameters as well as on specific immune functions. The animals were treated, per os by gavage for 28 days, in a five-day treatment two days break system, with 26.5, 21.1, 10.6 and 5.29 mg/kg/day amitraz. On the 29th day, organ weights of the thymus, heart, lung, spleen, liver, kidneys, adrenals, testicles and popliteal lymph node; WBC and RBC counts, Ht, MCV, haemoglobin; and cell content of the femoral bone marrow were determined. In two separate groups, the effects of amitraz on the PFC content of the spleen, and on the maximum level and time course of DTH reaction, were investigated. Amitraz in 26.5 mg/kg dose increased relative adrenal weight, and decreased relative liver weight, MCV value, PFC content of the spleen, and the maximum level of DTH reaction. The 21.1 mg/kg dose decreased only MCV value, while 10.6 mg/kg elevated the liver-to-brain weight ratio. Based of these findings, a NOEL dose of 5.29 mg/kg was determined for amitraz in this experimental system; while the LOEL doses were 10.6 mg/kg for the general toxicological, 21.1 mg/kg for the haematological and 26.5 mg/kg for the immune function parameters. The results show that the exposure sensitivity of these immune functions to amitraz is lower than that of some other toxicological and haematological parameters.

Neurotoxic and general effects of combined subchronic exposure of rats to insecticides and heavy metals.

Three different insecticides: dimethoate, cypermethrin and amitraz were given, alone or combined with the heavy metals Pb, Hg and Cd, to male Wistar rats per os for 12 weeks from their 4th week of life. After the treatment period, the left hemisphere of the rats was exposed in urethane anaesthesia, and spontaneous and evoked cortical activity was recorded from the primary sensory areas. The effects of dimethoate on the spontaneous activity, and of dimethoate and amitraz on the evoked responses, were increased by the metal combination treatment, whereby the metals alone had no effect on the spontaneous and mild effect on the evoked activity. Finally, the animals were dissected, organ weights measured, and relative organ weights calculated. The weight gain of all treated groups was significantly retarded compared to the control. Several organ weights were also significantly reduced, mainly in groups receiving insecticide plus metal treatment. The toxic interactions observed in this work indicate that combined human exposure to environmental pesticide residues and heavy metals may have unexpectedly severe effects.

Immunotoxicological investigation of subacute combined exposure with low doses of Pb, Hg and Cd in rats.

Detectable interactions between NOEL (No Observed Effect Level) doses of Pb, Hg and Cd in general toxicological, hematological, and immune function parameters were investigated. The metals (Pb-acetate, 20 mg/kg; HgCl2, 0.40 mg/kg; CdCl2, 1.61 mg/kg) were combined. First, the rats received the combination Pb + Hg + Cd for 4 weeks per os. Significant difference vs. control was found only in the weight of lung and popliteal lymph node (PLN). The Pb + Hg and Pb + Cd combinations significantly decreased the PLN to 100 g body weight and PLN to brain weight ratio, and Pb+Hg also decreased the relative adrenal weight. After 12 weeks treatment with the same doses, effects on the thymus, kidney, and adrenal weights in the Pb + Hg, and thymus weight in the Pb + Cd, combination were seen. Pb + Cd also affected the white and red blood cell count and hematocrit. Combined with Hg or Cd, NOEL dose Pb showed toxicity, indicating that exposure limits may be inefficient in combined exposure situations.

Comparative investigation of behavioral, neurotoxicological, and immunotoxicological indices in detection of subacute combined exposure with methyl parathion and propoxur in rats.

The effects of 6 weeks of oral exposure to propoxur (PR; at doses of 0.851 and 8.51 mg/kg body wt.), methylparathion (MP; at doses of 0.218 and 0.872 mg/kg body wt.), and their combinations were investigated in male Wistar rats. Measurement endpoints of the investigation were certain general toxicological parameters (body weight gain, organ weights), plaque-forming cell (PFC) count from the spleen, open field (OF) behavior, auditory startle response (ASR), prepulse inhibition (PPI), rotarod performance, somatosensory and auditory cortical evoked potentials, and peripheral nerve conduction velocity. The treated rats did not show any sign of acute intoxication during the 6 weeks of exposure. The higher dose of PR, but not of MP, significantly decreased the relative liver weight. Both agents produced a significant dose-dependent increase of OF activity, with larger expression after 2 weeks than after 6 weeks. The number of ASR responses and the ASR amplitude increased. The amplitude after PPI was increased by MP but only minimally altered by PR and the combinations. There was a small, but with high-dose PR significant, increase in the latency of the somatosensory evoked potentials. Neither of the two substances alone had any effect on the PFC response. The effect of the combination of high-dose PR and low-dose MP was significantly different from that of high-dose PR alone on the liver weight, on the ASR amplitude, and on the PFC/10(6) cell and PFC/spleen counts. With high-dose MP and low-dose PR, no such interaction was observed. According to the results, the noneffective dose of MP can influence the toxicity of the effective dose of PR in a combined exposure situation.

Immuno- and neurotoxicological investigation of combined subacute exposure with the carbamate pesticide propoxur and cadmium in rats.

In the present study, the effects of subchronic per os exposures to cadmium chloride (CdCl(2)), and a carbamate insecticide, propoxur (Pr), were investigated in male Wistar rats on general toxicological (body weight gain, relative organ weights) haematological (RBC, WBC, Ht, MCV, cell content of the femoral bone marrow) immune function (plaque forming cell (PFC) assay, delayed type hypersensitivity (DTH) reaction) and neurotoxicological (spontaneous and stimulus-evoked cortical activity, nerve conduction velocity) parameters. The animals were treated for 4, 8 and 12 weeks with 6.43 mg/kg CdCl(2), 8.51 mg/kg Pr, or with a combination of 6.43 mg/kg CdCl(2)+0.851 mg/kg Pr or 8.51 mg/kg Pr+1.61 mg/kg CdCl(2). Cadmium exposure affected the relative thymus, liver, and adrenal weight, RBC count, haematocrit and MCV, and there was an increase in nerve conduction velocity and a decrease in the cortical evoked potential latency. Pr induced a decrease in thymus weight, had some effect on the liver weight but none on the electrophysiological parameters. A significant interaction between Cd and Pr was detected by the following parameters: RBC, Ht, PFC, and nerve conduction velocity. The results indicate that combined exposures in humans may result in a shift in the apparent detection limits and/or in the LOEL of the single substances. The latter raises the necessity to reconsider exposure limits in situations where the risk of combined exposure is high.

A study on behavioral, neurotoxicological, and immunotoxicological effects of subchronic arsenic treatment in rats.

Male Wistar rats were treated for 4, 8, and 12 wk with 3.33, 6.66, 13.3, or 26.6 mg/kg of inorganic arsenic (NaAsO(2)) per os by gavage. Changes in behavioral and electrophysiological parameters (spontaneous open-field exploration; electrocorticogram mean frequency and power spectrum; latency and duration of somatosensory, visual, and auditory evoked potentials; conduction velocity; and relative and absolute refractory period of a peripheral nerve) were determined. Treated rats exhibited hypoactivity of horizontal ambulation in the open field and showed depressed rates of grooming. The electrophysiological data, recorded from anesthetized rats, did not show any significant dose- and time-dependent changes. Changes in humoral immune response, tested after 4 wk of treatment, were not marked. The weight of organs responsible for immune response (thymus, spleen, adrenals), was significantly reduced, as were delayed-type hypersensitivity (DTH) reaction and mean cell volume (MCV) of red blood cells a hematological parameter. Plaque-forming cell (PFC) assay proved to be insensitive in this short-time exposure. These results suggest that subchronic low-level exposure to arsenic can affect immune responses and/or spontaneous behavior of rats.

A study on geno- and immunotoxicological effects of subacute propoxur and pirimicarb exposure in rats.

A 28-day oral exposure with 8.51, 3.40, and 0.851 mg/kg propoxur (PR) and 4.67, 1.87, and 0.467 mg/kg pirimicarb (PI) was performed in male Wistar rats, and the occurrence of numerical and structural chromosome aberrations and the changes in certain immune function parameters (plaque-forming cell (PFC) assay, delayed-type hypersensitivity reaction) and in some basic toxicological (body weight gain and weights of brain, thymus, lung, heart, liver, spleen, kidneys, adrenals, and popliteal lymph node) and hematological (white blood cells, red blood cells, hematocrit (Ht), mean cell volume of red blood cells (MCV) cell content of the femoral bone marrow) parameters were investigated. The high dose of PR increased the relative liver weight and the cell content of femoral bone marrow, and all three doses increased Ht and MCV. The applied doses of PI decreased the relative adrenal weight in a dose-dependent manner, and its highest dose increased the relative liver weight. Among the immune function parameters tested, PFC content of the spleen was decreased by high-dose PR and elevated by high-dose PI, whereas the maximum and the time course of the delayed-type hypersensitivity reaction showed no changes in this dose range. In the genotoxicological investigations only the high PR dose increased the number of numerical, but not the structural, chromosome aberrations. In addition to the changes in relative adrenal weight following PI treatment, the PFC assay showed the highest sensitivity for detection of the 4-week exposure with these carbamates. On the basis of our results, the immunotoxicological approach seems to have the same (PR) or higher (PI) sensitivity in early detection of the repeated low-dose exposure by these carbamates compared to the genotoxicological approach.

Immunotoxicological investigations on rats treated subacutely with dimethoate, As3+ and Hg2+ in combination.

Effects of combined exposure with dimethoate (DM), HgCl2 (Hg), and NaAsO2 (As) were investigated following a 28 - day oral exposure in male Wistar rats. In preliminary experiments, the LOEL (Lowest Observed Effect Level) and NOEL (Non Observed Effect Level) doses of the substances were determined using the same experimental system [determination of body weight gain, organ weights of brain, thymus, heart, lung, kidneys, adrenals, spleen, testicles, popliteal lymph node, white blood cell (WBC) and red blood cell (RBC) count, haematocrit (Ht), mean cell volume (MCV) of RBCs, cell content of the femoral bone marrow, IgM-plaque forming cell (PFC) content of the spleen, delayed type hypersensitivity (DTH) reaction] and animal strain. In the combination studies, LOEL dose of DM (28.2 mg/kg) was combined with NOEL doses of the heavy metals ( HgCl2 = 0.40 mg/kg, NaAsO2 = 3.33 mg/kg), and vice versa (DM = 7.04 mg/kg, HgCl2 = 3.20 mg/kg, NaAsO2 = 13.3 mg/kg). In the DM-Hg combinations, significant alterations were found versus the corresponding high- dose internal control in the body weight gain, relative liver and kidney weights, and in the PFC response. When DM was combined with As, interactions were indicated by changes of relative liver weight, MCV value, and the PFC content of the spleen. These results support the theory that the interactions between pesticides and heavy metals may modify the toxic effects of the single substances, and may also change the functional detection limits of the exposure. The changes in the functional detection limits, if they occur, can lead to false-positive and false-negative results in human epidemiological studies.

Detection of the effects of repeated dose combined propoxur and heavy metal exposure by measurement of certain toxicological, haematological and immune function parameters in rats.

In the present study, an immunotoxicity test system, containing general toxicological (body weight gain, organ weights), haematological (WBC,RBC, Ht, mean cell volume of the RBCs, cell content of the femoral bone marrow), and immune function (PFC assay, DTH reaction) investigations, was used for detection the effects of a 4 weeks repeated low dose combined oral exposure of male Wistar rats with propoxur and the heavy metals arsenic or mercury. Two doses of the compounds were used: a higher one (the lowest dose which resulted in significant change of at least one parameter examined in previous dose-effect experiments), and a lower one (the highest dose which proved to be non-effective). The applied doses were: 8.51 and 0.851 mg kg(-1) of propoxur, 13.3 and 3.33 mg kg(-1) of NaAsO(2), and 3.20 and 0.40 mg kg(-1) of HgCl(2). In the combination treatment, the high dose of propoxur was combined with the low dose of arsenic or mercury, and the high doses of each heavy metals were combined with the low dose of propoxur. The main finding of this study was that some of the combinations significantly altered the relative weight of liver, adrenals and kidneys, related to both the untreated and the high dose internal control. Among the immune functions examined, only the PFC content of the spleen showed a trend of changes in certain combinations versus the corresponding high dose control. According to the present results, combined exposure with propoxur and the heavy metals examined can modify the detection limit of the single compounds and/or may alter their toxic effects.

Immunotoxicological investigation of subacute combined exposure by permethrin and the heavy metals arsenic(III) and mercury(II) in rats.

Effects of combined 28 days of oral exposure to the insecticide Permethrin (Pe), alone or in combination with arsenic-III (As) or Hg-II (Hg), were investigated on certain toxicological (body weight, organ weights), haematological (white blood cell (WBC) and red blood cell (RBC) counts, haematocrit (Ht), mean cell volume (MCV), cell content of the femoral bone marrow) and immune function (IgM-PFC, delayed-type hypersensitivity (DTH) reaction) parameters of male Wistar rats. Immunotoxic (H = high) and NOEL (L = low) doses of the three substances were determined in preliminary experiments under identical experimental conditions. In the present study, the immunotoxic dose of Pe (126 mg/kg) was combined with the NOEL dose of As (3.33 mg/kg) or Hg (0.40 mg/kg), and the NOEL dose of Pe (12.6 mg/kg) with the immunotoxic dose of As (13.3 mg/kg) or Hg (3.20 mg/kg). A separate group of animals, treated with the appropriate high dose component only, was used as internal control. Significant interactions were observed in the liver weight of the animals treated with Pe(H)-As(L) or As(H)-Pe(L), in the cell content of the femoral bone marrow in case of Pe(H)-As(L) and Pe(H)-Hg(L) combinations, as well as in the number of PFCs formed from 10(6) spleen cells in the Pe(H)-As(L) and in the maximum of DTH reaction in the Hg(H)-Pe(L) combination. The results show that combined exposures by the investigated substances modify the toxic (including immunotoxic) effects of the single compounds. These findings rise the probability that the interactions observed can also be present in human situations altering the health hazard of this three chemicals.

Simultaneous geno- and immunotoxicological investigations for early detection of organophosphate toxicity in rats.

Detectability of toxic effects by repeated doses of dimethoate (DM) and methylparathion (MPT) were investigated by geno-and immunotoxicological methods in male Wistar rats following a 28-day oral exposure. In the dose range of 28.2, 14.1, and 7.04, and 7.04 mg/kg/day DM, the two higher doses decreased the body weight gain. The top dose increased the weight of liver, kidneys, and testicles; the white blood cell count; and the cell content of the femoral bone marrow. From immune function parameters measured [IgM-plaque forming cells (PFC) assay, delayed-type hypersensitivity (DTH) reaction] only the maximum of the DTH reaction decreased at the top dose. Of the MPT doses (0.872, 0.436, and 0.218 mg/kg/day) the two higher ones increased the liver weight, and a dose-dependent increase was found in the MCV value. No evaluable changes in the examined immune function parameters were observed. Both substances increased the number of numerical but not the structural chromosome aberrations at lower dose levels (the two larger doses of DM, and all the three doses of MPT) than those ones which caused changes in the examined immune function parameters. According to these results, the genotoxicological approach seems to be more sensitive for detection of repeated-dose oral toxicity of the investigated two organophosphates than the immunotoxicological one.

Immunotoxicological effects of repeated combined exposure by cypermethrin and the heavy metals lead and cadmium in rats.

The immunotoxic effect of a 28 days oral exposure by 55.4, 22.2, and 11.1 mg/kg cypermethrin (CY) was investigated in 4 weeks old male Wistar rats. The applied test system involved the determination of general toxicological parameters (body weight gain, organ weight of thymus, heart, lung, liver, spleen, kidneys, adrenals and the popliteal lymph node), haematological parameters (white blood cell count, red blood cell count, haematocrit, mean cell volume of red blood cells, cellularity of the femoral bone marrow), as well as immune function assays (splenic plaque forming cell assay, delayed type hypersensitivity reaction). The highest dose resulted in a significant increase of the relative liver weight, and all three doses resulted in (although inconsistent) changes in the haematocrit and MCV values. The maximum of DTH reaction decreased at all three doses. On combination of the highest CY dose with non-effective doses of lead or cadmium the immunotoxic effects of the former were modified. When immunotoxic doses of Cd or Pb were combined with the lowest CY dose, further interactions were observed on the examined parameters. The alterations of the immunotoxic effects of CY by simultaneous exposure with Cd or Pb, as described here, can lead to unexpected health consequences and/or can lead to false positive or negative results in human epidemiological studies.

Comparison of detection sensitivity of immuno- and genotoxicological effects of subacute cypermethrin and permethrin exposure in rats.

Immuno- and genotoxicological effects of a 28-day oral treatment by equitoxic (1/10, 1/25, 1/50 LD50) doses of cypermethrin (55.4, 22.2, and 11.1 mg/kg) and permethrin (125.7, 50.3, and 12.6 mg/kg) were compared on male Wistar rats. Humoral and cell-mediated immunity were investigated by PFC assay and delayed type hypersensitivity (DTH) reaction (footpad swelling assay), and the genotoxic effects were studied by structural and numerical chromosome aberrations in bone marrow cells. The experimental system also involved certain general toxicological (body weight gain, organ weights) and haematological [white blood cell (WBC), red blood cell (RBC), haematocrit (Ht) and cell content of the femoral bone marrow] investigations. Among the immune function assays, only DTH reaction decreased at the two higher cypermethrin (CY) doses. These doses also increased the number of numerical chromosome aberrations of the bone marrow cells but did not change the number of structural aberrations. All CY doses decreased the mean cell volume (MCV) of RBCs and the Ht value. The two higher doses also reduced the WBC count in the peripheral blood. Permethrin (PE), in the applied dose range, had no effect on the examined immune function parameters, but all three doses increased the number of numerical chromosome aberrations. A dose-dependent increase in the liver weight, decreased MCV value, and elevated cell content of the femoral bone marrow were also observed. Under these experimental conditions, examination of chromosome aberrations proved to be less sensitive in detection of exposure by cypermethrin than applied immune function assays did. Permethrin, on the contrary, increased the number of numeric aberrations at all dose levels but had no effect on the immune function parameters examined.

Immunotoxicological examination of repeated dose combined exposure by dimethoate and two heavy metals in rats.

The immunotoxicity of 28 days combined oral exposure by dimethoate (DM) and two heavy metals (Pb or Cd) was investigated in male Wistar rats. Immunotoxic and no-effect doses of DM (28.2 and 7.04 mg/kg) were combined with immunotoxic and no-effect doses of CdCl2 (6.43 and 1.61 mg/kg) or lead acetate (80.0 and 20.0 mg/kg) in such a way that the high dose of each substance was given in combination with the no-effect dose of the other. To examine the interactions of these agents, general toxicological (body weight gain, organ weights), haematological (absolute and differential WBC, RBC, MCV, Ht. cell content of the femoral bone marrow), and immune function (splenic PFC number. DTH reaction) parameters were measured. Treatment with the combination of Pb or Cd and DM did not result in a reduction of humoral (PFC) and cellular (DTH) immune responses, whereas treatment with the substances alone did result in immune suppression. This protecting effect can probably be attributed to an effect on the kinetics of the compounds tested rather than on the immune system itself. Further interactions were found in both combinations, DM-Cd and DM-Pb, in the body weight gain and in the relative liver weight; the DM-Pb combination also affected the relative thymus weight and the MCV value. These findings show that the immunotoxic effects of the investigated materials, including their detectability and health consequences, can be modified in case of combined exposure.

Extension of the protocol of OECD guideline 407 (28-day repeated dose oral toxicity test in the rat) to detect potential immunotoxicity of chemicals.

To indicate the immunotoxic potential of chemicals the examinations prescribed by OECD Guideline 407 were extended by the following additional toxicological, haematological, histopathological, and immune function examinations: absolute and relative organ weight of spleen, thymus, popliteal lymph nodes, lung and brain; histopathology of thymus, mesenteric lymph nodes, popliteal lymph nodes, bone marrow (femur), Peyer's patches (ileum), lungs and colon; PFC assay (spleen), T cell proliferation and NK cell assay. Two well known immunosuppressants Azathioprine (AZA) and Cyclosporine A (CysA) were chosen as model compounds at a dose range which do not cause visible toxic signs on the animals during a 28 days treatment period. The results show that the applied experimental system is much more sensitive in detection of the immunotoxic potential of these two compounds in a low dose range than the examination required by OECD Guideline 407 are.

Immunotoxicity study of repeated small doses of dimethoate and methylparathion administered to rats over three generations.

Immunotoxic effects of chronic, equitoxic doses (1/50, 1/75, and 1/100 LD50) of two organophosphorus pesticides dimethoate (DM) and methylparathion (MPT) (14.1, 9.39, and 7.04 mg kg-1 DM; and 0.436, 0.291, and 0.218 mg kg-1 MPT) were investigated in a three generation study in outbred Wistar rats. Treatment of the first generation (G1) with these doses began in animals 4 weeks of age; the parental males were dosed until separation of females, and after mating the females were treated until separation of their G2 offspring (at the age of 4 weeks), and the G3 generation was produced in the same way from treated parental G2 animals. Selected 4 week old males from each generation were also treated with DM and MPT for 4 weeks (experimental groups) before determination of certain conventional toxicological (body weight gain, birth weight and number, organ weights), haematological (absolute and differential WBC, RBC, Ht, MCV, nucleated cell content of femoral bone marrow), and immune function parameters (IgM-PFC number of spleen, DTH reaction). Effects of both substances on immunological variables were detectable at the 1/75 LD50 dose level, but different parameters were affected in the three consecutive generations.

Immunotoxicological investigation of SCMF, a new pyrethroid pesticide in mice.

The toxicity of a new pyrethroid pesticide Supercypermethrin Forte (SCMF) was studied in male CFLP mice using classic toxicological (body weight, organ weights) and haematological (white blood cell count, haematocrit, nucleated cell content of femoral bone marrow) methods and immune function tests (PFC assay, DTH reaction). Four weeks of oral treatment in a 5 days per week system at doses of 1/10, 1/20, or 1/40 x LD50 did not cause evaluable changes in the measured parameters. When single calculated LD20, LD10, or LD5 doses of SCMF were administered on different days before termination to different groups of mice the two higher doses caused a time- and dose-dependent decrease in the splenic PCF number. Apart from some temporary toxic signs and an increase of haematocrit at the top dose the other examined parameters did not show evaluable changes. Under these experimental conditions toxic changes appeared only at the high dose range and, of those applied, the PFC assay proved to be the most sensitive method for detecting the toxicity of SCMF.

The diverse modification of N-butyl-N-(4-hydroxybutyl) nitrosamine induced carcinogenesis in urinary bladder by dibromodulcitol and dianhydrodulcitol.

The effects of intravesical therapy with adriamycin, 1.6-dibromo-1.6-dideoxydulcitol (DBD) or with 1.6-dianhydrodulcitol (DAD) on bladder carcinogenesis were investigated in rats. To induce premalignant lesions in the urinary bladder female Sprague-Dawley rats received 0.05% N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) in their drinking water for 4 weeks and then post-treated intravesically with one of the antitumor drugs and examined once a week for 3 months. These antitumor drugs in healthy rats (i.e. without the pre-administration of BBN) did not cause any significant morphological changes in the urinary bladder after intravesical application once a week for 3 months. In the applied dose BBN alone induced only premalignant lesions in the urinary bladder. However, neoplastic lesions occurred in the groups treated with BBN and adriamycin (9 papillomas and 3 carcinomas in 14 animals). Similarly intravesical application of DBD after BBN administration resulted 5 carcinomas among the 11 animals. On the contrary no urinary bladder tumor was found in the animals treated with BBN and DAD. As DAD is one of the conversion products of DBD it is conceivable that the difference between DBD and DAD action may be due to the formation of other solvolytic product from DBD than DAD.