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Oral exposure (key studies):

BASF (1986) reported a 3-month subchronic drinking water study. Male and female Wistar rats were offered 5, 12, 30 or 75 ppm (= about 0.4, 1, 2.6 or 6 mg/kg/d) continuously via the drinking water. Neither mortality nor clinical signs were observed throughout the study. Food intake and mean body weights were unchanged over the study period. Water consumption was unchanged in the three lowest dose groups; in the highest dose group the water consumption was slightly, but only temporarily reduced in both sexes. The ophthalmological examinations which were carried out before and at the end of the study in the control and the highest dose groups showed no adverse effects. The clinicochemical and hematological investigations revealed reduced total protein and globulin concentrations in the plasma of both sexes in the highest dose group. In addition, the female animals in the highest dose group showed significantly lowered albumin concentrations at the end of the study. Other significant changes were regarded as not being related to the test substance. The mean kidney weights in males in the two highest dose groups were statistically significantly increased. However, no changes in the kidneys were observed on gross-pathological and histopathological examinations (no evidence for toxic fatty degeneration or degenerative parenchymal damage). All other observed gross and microscopic organ changes (mainly focal calcification in the kidneys) were assessed to be not substance-related. Based on the results of this study, the "no effect level" for male rats was in the range between 1 and 2.6 mg/kg/d (12 - 30 ppm) and for female rats in the range between 2.6 and 6 mg/kg/d (30 - 75 ppm).

A 90-day gavage study with Wistar rats was done by BASF (1986). Male and female animals were administered concentrations of 40, 60 or 100 mg/kg/d. None of the rats died during the study or had to be sacrificed other than when planned. Almost throughout the study, the food consumption by the male animals in 100 mg/kg bw test group was reduced, to varying extents compared with the control group but overall only slightly. In contrast, there was no noticeable difference between the mean daily food consumption of the male and female rats in the 40 and 60 mg/kg bw test group or the female animals in the 100 mg/kg bw test group and that of the controls. In contrast to the two other pilot studies (see below), in which the animals received the test substance continuously with drinking water and in which some animals exhibited a great reduction in the water consumption, water consumption was increased in a more or less dose-related manner. The figures for both the male and the female rats were clearly above those for the control animals. This effect was most pronounced in the female animals in the 100 mg/kg bw group, which consumed up to 60% more water during the study than did the control animals. For neither sex was there any marked difference between the control animals and the treated animals in the body weight gain. Merely the body weight of the animals of both sexes in the 100 mg/kg bw test group was marginally (about 5%) lower at the end of the study. No noteworthy clinical signs were found for any of the animals. The gamma-glutamyltransferase activities in liver homogenate at the end of the 3 months of administration were significantly raised in the female animals in all test groups. In contrast, there was only a tendency toward such an effect in the male animals. The increases in the liver enzyme activities found in both sexes must be related to the test substance, especially since earlier studies carried out with the test substance produced corresponding findings. The significant increase in the platelet count for both sexes in the mid and high dose test groups (60 and 100 mg/kg bw) is certainly related to the test substance, since analogous changes were also detected in earlier investigations on N-vinylpyrrolidone. However, the pathogenesis of an isolated finding of an increased platelet count is difficult to determine because of the lack of concomitant manifestations. Statistically significantly raised alpha2-globulin levels were found in the male animals in the 60 and 100 mg/kg groups at the end of the 3 months of administration. Although the alpha2-globulin concentration increased as a function of the dose, it is doubtful whether the cause of these changes is attributable to the administration of the test substance. On the one hand, a significant increase in the globulin levels was found for only one sex and, on the other hand, it has been shown in several previous studies that female animals react more sensitively to the test substance than do male rats. The dysproteinemia which was particularly observed in female animals in earlier investigations was mainly caused by a reduction in the alpha-globulin fraction and not - as in the present case - by a rise in the alpha2-globulin levels. Thus, it is improbable that this is an effect of the test substance. On the contrary, the increase in the alpha2-globulin levels appears to be an incidental finding. Likewise, no gross organ changes were detected in all animals which received the test substance by gavage for 3 months. However, the female animals in the 40 mg/kg bw test group and above were found to have a dose-dependent increase in the relative liver weight, which proved to be highly statistically significant. There was a dose-dependent increase in the relative liver weights for the male animals in the 60 mg/kg test group and above. This was considered to be related to the test substance. However, no evident changes explaining the increase in weight were detectable under the light microscope. On the other hand, foci of parenchymal alteration were found in the livers of 4 female animals and 1 male, and the morphology of these resembled that of the foci of cellular alteration found in earlier studies on this test substance. In one case the cells in one focus were basophilic (i.e. a basophilic focus). However, the foci were never numerous (never more than 3 foci in 3 liver samples examined) and were always rather small (about 10-25 cells in the histological section). No glycogen-positive foci were found with the PAS reaction of serial sections of the sections stained with hematoxylin and eosin. Nor was there any clear result of the test for gamma-GT on the histological section for the female animals (male animals were not investigated since female rats are more sensitive). Although there was a positive reaction in the periphery of the lobule, this was restricted to the bile capillaries and was found equally in the controls and at the highest concentration (100 mg/kg bw). A weakly positive reaction of hepatocytes adjacent to the biliary pole derived from "leakage" of the reaction from the bile capillaries into the cytoplasm of the hepatocytes. No unambiguously gamma-GT-positive foci of hepatocytes or positive individual cells were found in any of the sections examined. Despite the absence of glycogen storage and a negative gamma-GT reaction, the foci detected with the H.E. stain are regarded, on the basis of morphological criteria, as foci of cellular alteration equivalent to those detected in earlier investigations on the test substance. The LOAEL was found to be 40 mg/kg bw/day for both sexes.

Oral exposure (supporting studies):

BASF (1986) reported a 28-day subacute drinking water study. Male and female Wistar rats were offered continuously concentrations of 200, 400, 700, 1600 and 6400 ppm (= about 12, 22, 31, 15 -25, 35-44 mg/kg b.w./d). It was necessary to sacrifice the animals in the 1600 and 6400 ppm groups after 8 days because of their poor general state of health. One female rat of the 700 ppm group was sacrificed after 18 days for the same reason. All the animals treated with the test substance showed a dose-dependent reduction in the food consumption even in the first week of the study. Compared with the control group, the male and female rats in the 1600 and 6400 ppm groups ate only between 1/3 and 1/4 of the food offered. The dose-dependent loss of appetite persisted throughout the 21 days (200 and 700 ppm groups) or 28 days (400 ppm group) of the study. The reduction in the water consumption by the rats treated with NVP was even more pronounced than that in the food consumption. There was a marked relation to the dose in that the treated male animals drank between 91% (200 ppm) and 5.7% (6400 ppm), and the female rats consumed between 77% (200 ppm) and 8% (64000 ppm), of the amount of water in comparison to untreated control animals. Because of the dose-dependent diminution in food and water consumption all the animals treated with the test substance in this pilot study showed effects on the body weight, again dose-related. The male rats in the 200, 400 and 700 ppm groups and the female animals in the 200 ppm group still gained weight, although markedly less than control animals, during the study. In contrast, the animals in the other test groups (male rats: 1600 and 6400 ppm groups; female rats 400, 700, 1600 and 6400 ppm groups) lost weight during the study so that the weights of the animals determined before sacrifice were below their initial weights. Most prominent was the loss of weight in the animals on the 6400 ppm groups which as early as 1 week post start of administration had already lost 29% (male animals) and 27% (female animals) when compared to their respective initial weights at the start of the administration. Because of the - in some cases drastic - diminution in food and water consumption and the associated reduction in body weight, the male and female animals which received 1600 and 6400 ppm, and the female rats which received 700 ppm of the test substance, exhibited exsiccosis, a poor general state of health and shaggy, ruffled fur. In addition, one female animal of the 700 ppm group was found to have assumed a lateral position after 14 days of the study. No noteworthy clinical signs were found for any of the other animals. The animals which were offered 6400 or 1600 ppm test substance in drinking water were sacrificed after 8 days of the study. The gastrointestinal tracts of these animals were virtually empty. Nothing else was found in either sex in the 1600 ppm test group; and this was the only finding for all the female and for 2 male rats in the 6400 ppm test group. The other 3 male animals in this group also had gastritis, which was erosive-ulcerative in nature in two cases. It is impossible to say with certainty whether this was due to a toxic effect or was the consequence of a lack of palatability (e.g. endogenous exsiccosis). Two female animals in the 700 ppm test group had slight yellow-brown discoloration of the liver parenchyma. This was probably minor fatty infiltration. One 700 ppm female animal died in a cachectic and exsiccotic state after 18 days of the study, but there were no detectable gross organ changes which might have been responsible for the death of the animal. On the basis of the present results, this does not seem to be a connection between the administration of the test substance and the effects observed. A LOAEL was found to be about 12 mg/kg b.w. for both genders.

Another drinking water study was reported (BASF, 1986). Male and female Wistar rats were offered the test substance continuously for 21 days via the drinking water in concentrations of 50, 100 or 200 ppm (= about 5.5, 11 or 21 mg/kg/d). The following results were obtained: None of the rats died during the study or had to be sacrificed other than when planned. Marked diminution in the food consumption in female rats only in the 200 ppm test group was found. Although there was no clear relation to the dose, it appeared that the food consumption by the female animals in the 50 and 100 ppm groups was slightly diminished (about 8% less than the control animals). Whereas all male animals in the treatment groups consumed drinking water almost exactly as much water as the control animals, the consumption of water by the female rats at these doses was dose-dependently diminished. Thus, the water consumption after 21 days was about 16% (50 ppm), 20% (100 ppm) and 31% (200 ppm) below that of the control group. The body weights of all male animals in the treatment groups, which initially were almost the same as that of the control group, increased comparabely to the body weights of the control animals. In contrast, the female animals were found at the end of the study to have gained marginally less weight at 50 and 100 ppm and slightly less at 200 ppm. The mean body weights were about 5% (50 and 100 ppm) and 9% (200 ppm) lower than for the female control animals. No noteworthy clinical signs were found for any of the animals. As already shown in an earlier inhalation study on rats, repeated administration of N-vinylpyrrolidone leads to dysproteinemia, principally in the female animals. The primary cause of this is a reduction in the alpha-1-globulin fraction. Similar shifts in the protein composition were also observed in this study after administration of the test substance in the drinking water. Again the females reacted more sensitively than the males to the test substance. Thus, after 3 weeks of administration of the test substance, the female rats in the 200 ppm test group had significantly reduced figures for total protein and globulins. There was also a tendency toward this in the 100 ppm group. Again, the electrophoresis of the serum proteins showed that the decrease in the total protein and globulin concentration is attributable to a reduction in the alpha-1-globulin content. There was also an evident tendency for the alpha-1-globulin to be reduced in the male animals in the 100 ppm. This dysproteinemia is assumed to be caused by changes in the liver parenchyma, which may correspond to the cellular alteration in the liver found in the inhalation study. However, in the assessment of the hepatotoxic action of N-vinylpyrrolidone, account has to be taken of the clinicochemical findings which indicates that the activities of the other plasma enzymes, such as glutamic-pyruvic and glutamic-oxalacetic transaminases, alkaline phosphatase, gamma-glutamyltransferase, lactic dehydrogenase and plasma cholinesterase, were not changed in a way indicating liver dysfunction. This suggests that the test substance had no effect on the integrity of the hepatocytes or on their synthetic efficiency. On the contrary, in the present case there appears to be a specific action on proteins of the alpha1-globulin fraction. Dose-dependent increases in the creatinine concentrations were found at the end of the administration in the male animals in the 100 and 200 ppm groups, whereas no such changes were seen in the females. Furthermore, there were significant reductions in glucose for the male animals in the 100 ppm test group, and these were also clearly evident in the highest dose group - in spite of the absence of statistical significance. There was also a tendency for the glucose level to decrease in the female animals in the 100 and 200 ppm. The clear differences from the control group point to these changes being related to the test substance. However, this cannot be stated with complete certainty since no such changes had been observed in earlier studies. At necropsy, slight fatty infiltration of the liver parenchyma was also found in one female animal in both the 50 and 100 ppm test groups and in 2 female rats in the 200 ppm test group. These findings are not regarded as related to the test substance but are interpreted as being physiological and attributable to the feed which was available ad libitum. None of the other animals which were examined showed any gross organ changes. The NOAEL was about 5.5 mg/kg bw/day for male and female animals.

Inhalation exposure (key studies):

BASF (1988) reported a 3-month inhalation study. Male and female Sprague-Dawley rats were offered vapour concentrations of 0.023, 0.068, 0.204 or 0.545 mg/l (= 5, 15, 45 or 120 ppm) for 6 hours per day for 5 days per week (whole-body exposure). The effect on brain and hearth organ weights found is probably caused by the reduction in body weight, since, apart from water, these two organs are able to mobilize no (brain) or only small amounts of (heart) reserves when the animals are in a catabolic state. Accordingly, nothing relevant was found in these organs under the light microscope. There was a slight reduction in the absolute kidney weight in the male animals in test group 2. On the other hand, the relative weight was statistically slightly increased only in the female rats in all groups which received administration of the test substance. However, since there was no evident concentration-effect relation, only one sex was affected in each case, and there were no detectable gross-pathological or histopathological changes which might explain the increase in weight, the changes in kidney weight appear, from the viewpoint of pathology, to have no special biological relevance. On the basis of the results of this investigation, that 3 months of inhalation of N-vinyl-2-pyrrolidone in concentrations of 5, 15, 45 and 120 ppm by male and female Sprague-Dawley rats results in inflammatory changes, of variable severity, in the nasal cavity and atrophy of the olfactory epithelium and hyperplasia of the respiratory epithelium with the numbers and the intensity of the changes depending on the concentration. Apart from manifestations of toxicity, which caused the death of almost all the animals in the 120 ppm test group, at and above a concentration of 15 ppm there were liver changes related to the test substance. From the viewpoint of pathology, the no adverse effect level in this study was at a concentration of test substance below 5 ppm. When the results of the body weight gain and food consumption are compared, it is seen that a significant effect, or absence thereof, on the body weight gain was paralleled by a significant effect, or absence thereof, on the food consumption of all test groups, with the exception of a temporary effect only on the female animals in test group 1 (15 ppm). In the latter case, the body weight gain was significantly reduced, although the level of significance varied between 5 and 1%, but this was not paralleled by a similar change in food consumption. This finding may be explained by 15 ppm being the borderline concentration for an action on female rats, and although the animals still consume comparable amounts of feed they gain less weight because of the liver changes, which were detected in the histopathology and clinical chemistry. The NOAEC and the LOAEC were found to be 0.023 mg/l for both genders.

In another 3-month inhalation study (BASF, 1988), a vapour concentration of 0.0052 mg/l (= 1.14 ppm) was used. Male and female Sprague-Dawley rats were exposed whole-body for 6 hours per day and 5 days per week. The following findings were obtained: The behavior of the animals in both groups did not differ from normal at any time during the study. No animal died in either test group. There were no significant differences in the body weight or change in body weight between the test group and the control group. In the 3-month inhalation study on the rat with 5 ppm N-vinyl-2-pyrrolidone in the inhaled air (see study above), the female animals were found to have reduced levels of total protein, albumin and globulin (alpha1 fraction). In addition, these animals showed transient falls in the plasma concentrations of creatinine, urea and potassium. In the present supplementary study with 1 ppm N-vinyl-2-pyrrolidone in the inhaled air, no such changes were found for the female animals. In contrast, the levels of urea and alpha-1-globulin in the peripheral blood at the blood sampling 46 days after the start of administration were low for the male rats. It is doubtful whether these transient changes were caused by the test substance since statistically significant changes were detectable in the preceeding inhalation study (see study above) in the male animals only down to a concentration of 15 ppm. A concentration of 5 ppm had no effect on this sex. However, because of the relatively large concentration differences (factors of 3 and 5), a clear dependence on concentration of findings related to the test substance would be expected in the test groups. No relation between the effect and the concentration in the range between 15 ppm and 1 ppm was found. The other clinical, clinicochemical and histopathological examinations showed no specific changes. Neither the gross pathology nor the histopathology showed any changes in the nasal mucosa or in the liver which might be attributed to the test substance inhaled. At 1 ppm there were no findings comparable to the inflammatory and degenerative changes in the nasal mucosa (levels I and II) which were found even at 5 ppm in a preceeding study. The NOAEC was found to be 0.0052 mg/l for male and female animals.

BASF (1989) reported a 6-month inhalation (vapour) study using male and female Fischer-344 rats. The animals were offered a concentration of about 0.045 mg/l (= 10 ppm) for 6 hours per day and 5 days per week (whole-body exposure). The following findings were obtained: A significantly retardation in the body weight of the male animals in the test group (= 10 ppm) was found from day 56 onwards (with the exception of day 175). With the exception of day 91 (p < 0.05) the significance level was always p < 0.02. The effect of a reduced body weight was found in the female animals in the test group throughout the test-period. The significance level was p < 0.05 on day 7, p< 0.002 on days 42, 70, 77 and 175 and p < 0.02 on all other days. A significantly retarded body weight change was found in the male animals of the test group from day 21 onwards. This effect lasted throughout the test period. The significance level was p < 0.05 on days 21 and 175, p < 0.002 on day 77 and p < 0.02 on all other days. With the exception of days 7, 28 and 182 the significant reduction in the body weight change in the female animals of the test group was found throughout the test period. The significance level was p < 0.05 on days 49 and 147, p < 0.002 on day 21 and p < 0.02 on all other days. No signs related to the test substance occurred at any time during the test period. No deaths occurred at any time during the test period. The results of the hematological examinations reveal changes in the red blood picture (reduction in the hematocrit and decrease in the mean cell volume). In addition, a rise in the platelet count is found in the 10 ppm group. The decrease in the alanine aminotransferase activity is probably connected with the reduced body weight. The reduced total protein and albumin levels in the female animals and the decrease in the globulin concentration in the male animals were probably also due to administration of the test substance and, pathogenetically, are associated with hepatic dysfunction. The increased gamma-glutamyltransferase activities in both sexes and the rise in the glutathione content in female animals are also to be regarded as being connected with the liver dysfunction. The inhalation of the test substance did not result in any significant changes in the liver and lung weights of either the male or female animals in comparison to a control group. The changes found on histopathological examination of the liver are regarded as being spontaneous findings. The LOAEC was 0.045 mg/l for both sexes in this strain of rats.

In another 6-month inhalation (vapour) study (BASF, 1989), male and female C57BL mice were offered the same concentration (= 0.045 mg/l) for 6 hours per day and 5 days per week. The following results were found: No findings related to the test substance regarding clinical signs. No animal died during the study (two mice died after the sampling of blood before their subsequent sacrifice). Body weight and weight gain were reduced from the start of exposure onwards. Reduction in body weight of 15 % (female animals) and 17.5 % (male animals) at the end of the test. A significantly retarded body weight gain in the male animals in the test group was found on days 7 (p < 0.002), 21 (p < 0.02) and from day 35 onwards (day 35 p < 0.05, day 42 p < 0.02 and from day 49 to the end of study p < 0.002). In the female animals of the test group 1 this effect was found from day 7 up to the end of the study, with p always being < 0.002 with the exception of day 14 (p < 0.02). No substance-related changes were found concerning hematology. Clincal chemistry/hepatic dysfunction: reduced total protein and globulin levels, increased content of reduced glutathione in liver homogenate. Organ weights: The absolute lung weights of the male and female animals in the test group were slightly higher than those in the control group. No changes related to the test substance detectable on gross-pathological examination. The NOAEC found was 0.045 mg/l for male and female animals.

In a two-year cancer study (BASF, 1992), the inhalation of NVP vapour caused chronic toxicity in all dose groups, in a dose dependent manner. However, exposure to NVP for up to 2 years did not have a significant effect on survival. The most relevant non-neoplastic effects were hepatotoxicity and local toxic effects in the respiratory tract (nasal mucosa and larynx) consistent with irritation. Other exposure related effects included decreases in body weight, both sexes, minor changes in various red blood cell parameters consistent with slight anemia in females at the two highest doses, changes in plasma protein concentrations and serum enzyme levels consistent with liver dysfunction for males and females. A No Adverse Effect level (NOAEL) was not identified, the Low Adverse Effect Level (LOAEL) was 5 ppm based on effects in the liver and respiratory tract.

Inhalation exposure (supporting studies):

BASF (1988) reported a 90-day inhalation study using female Sprague-Dawley rats (whole body exposure). The animals were offered a concentration of 0.204 mg/l (= 45 ppm). The following findings were obtained: No substance-related clinical signs were reported. No death occurred during the period of exposure. Because of age-related changes animals were sacrificed in a moribund state in the observation period. A few animals died. The treatment with the test substance showed no effect on the lethality of the animals. A significant retardation in the body weight gain (reduction of body weight 2 - 7%) found in approximately the first third of the exposure period. A rise in the GT activity found in liver homogenate both during exposure (after 7 weeks) and in the recovery period. In contrast, a rise in GT activity in plasma was found only at the end of the recovery period. The content of reduced glutathione in liver homogenate was increased at the end of exposure and in the recovery period. Gross pathology/histopathology: After 7 weeks of inhalation centrolobular hepatocyte enlargement together with foci of atypical hepatocytes were found in the liver. The same findings of cellular alteration in the liver were found after 3 months, and the high glycogen content of the proliferating cells was also noteworthy. At the interim sacrifice after 9 months recovery period (12 months of the study), the findings in the liver were the same (cellular alteration), and this was also found in one control animal. There was no detectable progression or regression. At the sacrifice at the end of the study (after 24 months) neoplastic alterations were found in the livers of 4 of 6 surviving animals in the test group (hepatocellular carcinoma in 2 cases, neoplastic nodes in 2 cases). The animals exposed to NVP were found to have hypertrophied hepatocytes, and control animals and animals treated with the test substance were found to have foci and areas of cellular alteration. The glycogen content in these foci and areas of cellular alteration showed a tendency to increase from the first interim sacrifice after 7 weeks. All the other changes in liver tissue which were found on gross-pathological or histopathological examination are regarded as spontaneous or incidental findings or are attributable to the age of the animals. The other tumors in the liver tissue are spontaneous (hemangiosarcoma) or not related to the test substance (leukosis in one control animal). The LOAEC was found to be 0.204 mg/l for female animals.

In another 90-day vapour inhalation study (BASF, 1988) male and female hamsters were used (whole-body exposure). The animals were offered a concentration of 0.203 mg/l (= 45 ppm) for 6 hours per working day (total: 62 exposures). The following findings were assessed as being substance-related: Evidence of an irritant effect on the respiratory tract at the start of exposure; significant retardation in body weight gain in both sexes; marginal increases in the levels of glutathione (liver homogenate) and in gamma-GT activities (liver homogenate) were found only for the male hamsters. Whereas a parallel inhalation study on female rats showed marked increases in gamma-glutamyl-transferase activities and reduced glutathione concentrations in the liver during the 3-month exposure period, a marginal increase in these two parameters was found merely in the male hamsters in this study. The rise in the gamma-glutamyltransferase activity and concentration of reduced glutathione was so small that it is doubtful whether it had any connection with the administration of the test substance. It is obvious that there is a pronounced difference between the toxicity of the test material for hamsters and for rats. Since there appeared to be no morphological difference between the hamsters exposed to the test substance and the control animals, it was unnecessary to continue the hamster study to a final total of 18 months. The LOAEC found in this study was 0.203 mg/l for male and female hamsters.

A 7-week vapour inhalation study was done using male and female Fischer-344 rats (BASF, 1989). In this study, the animals were offered (whole body) concentrations of 0.023, 0.068 or 0.204 mg/l (= 5, 15, or 45 ppm) for 6 hours per day and 5 days per week. The following findings were obtained: Clinical signs and mortality: No death occurred throughout the test period. (Satellite group 1: Only the animals at 45 ppm showed findings related to the test substance, such as poor general state, apathy, atered breathing behavior and slight anemia. Satellite group 2: The initially pronounced symptoms lessened throughout the exposure to such an extent that the animals showed no reactions related to the test substance from day 13 of the test onwards. Main group: As in satellite group 2, changes were found only in the first few weeks of the test. No changes were observed after about 2 weeks.) The body weight was reduced only at the highest concentration (satellite group 2). Main group: Only the animals in the 45 ppm group showed a reduction in body weight related to the test substance up to the end of the test (reduction in the body weight gain). Hematology and clinical chemistry: Satellite group 1: Clinicochemical and hematological changes were found after only one week of exposure, and their cause was assumed to be hepatic dysfunction. The findings with 45ppm were: reduced total protein, globulin and albumin levels (male animals only); increase in the gamma-GT (male and female animals) and glutathione contents (male animals only) in liver homogenate; increased GPT (male and female animals), GOT (male and female animals) and AP activities (female animals); increased triglyceride, cholesterol (male animals) and bilirubin (male animals only) levels. The increased hemoglobin, erythrocyte and hematocrit values are possibly due to a reduction in the plasma volume as a result of an increased fluid loss. With 15 ppm, some of these changes were no longer detectable and some were less severe. No changes were found with 5 ppm. Satellite group 2: The clinicochemical changes after 3 weeks of exposure resembled those found after 1 week whereas the hematological findings were less severe (only in the female animals exposed to 45 ppm). Increased platelet counts were detectable in the male animals (45 ppm). The pathological findings revealed changes in the liver and nasal mucosa similar to those in satellite group 1. Main group: The results of the clinicochemical investigations generally revealed aleviation of the symptoms with increasing duration of exposure. In addition, the creatinine concentrations were lowered (correlating with the reduced body weight). After the longer period of exposure, the results of the hematological investigations revealed changes in the red blood picture in the form of lowered hemoglobin, erythrocyte and hematocrit levels (45 ppm and 15 ppm). The reduction in the MCV - the rise in the platelet count (male and female rats exposed to 45 ppm and 15 ppm), the increased occurrence of polychromatic erythrocytes (male rats 45 ppm) and the rise in the MCHC figures (female rats 15 ppm and 45 ppm) are also regarded as being connected with these anemic processes. Gross pathology: Satellite group 1: Changes in the liver (pale livers, increased absolute liver weight, central necrobiosis, centrilobular fatty infiltration, liver cells with nuclear hyperchromatism, polymorphism/mitosis) and in the nasal mucosa (atrophy of the olfactory epithelium) were particularly found with 45 ppm. Some of these changes no longer occurred with 15 ppm or were less severe and had disappeared completely with 5 ppm. Main group: On gross-pathological and histopathological examination, the same changes in the liver and nasal mucosa as occurred after a shorter exposure period were found. Furthermore, all the animals in the 45 ppm group had enlarged central liver cells and, usually mixed cell, foci and areas of cellular alteration. The LOAEC was found to be 0.204 mg/l.

Another 7-week vapour inhalation study is available (BASF, 1989). Male and female C57BL mice were offered concentrations of 0.022, 0.068 or 0.2 mg/l (= 4.9, 15 or 44 ppm) for 6 hours per day and 5 days per week (whole-body exposure). The following findings were obtained (SG = satellite group): Clinical signs and mortality: In the SG1 (1 week exposure): No signs in the low and mid dose group. In the high dose group, poor general state, and altered breathing behavior during the exposure were observed. Similar observations were reported in the SG2 (3 weeks) exposure and main test group (7 weeks exposure). No mortalities were observed in the SG1. Two animals of the high-dose group died in SG2. Two animals of the high-dose group were killed in extremis in the main test group. Reduced body weights were observed at 45 ppm in SG2 and main test group. Hematology: SG1: increased neutrophilic polymorphonuclears was observed in males of all dose groups. Additionally, decrease in the mean cell volume (male animals) in 45 ppm males. SG2: Increased platelet count (females 15 ppm and males + females 45 ppm) and decreased mean cell volume (males + females 45 ppm) were observed. Main test group: Reduced hemoglobin, erythrocyte and hematocrit levels (males 15 and 45 ppm), decreased mean cell volume (males 45 ppm), increased platelet count (males + females 15 and 45 ppm) and increased lymphocyte count (males 45 ppm) were observed. Clinical chemistry: SG1: increased glutathione content (males at 15 ppm; males and females at 45 ppm) was observed. SG2: Reduced total protein, globulin (males 15 ppm and males + females 45 ppm) and albumin (males + females, 15 and 45 ppm) levels, increased glutathione content (females 15 ppm and males + females 45 ppm) were observed. Main test group: Reduced total protein and albumin levels (males 15 ppm, males + females 45 ppm), reduced activity of alkaline phosphatase (females 45 ppm), increased glutathione content (males + females 45 ppm) were observed. Organ weights: Absolute lung weight was increased at 45 ppm in SG1. The absolute lung weights and the absolute liver weights (females only) were increased at 45 ppm in SG2. The absolute lung and liver weights were increased at 45 ppm in the main test group. Gross pathology: Pale centers in the liver lobes (male + females 45 ppm), reddish brown adrenals (one female + two males 15 ppm, and 4 females + all males 45 ppm). Histopathology (non-neoplastic): SG1: The pathological investigation showed centrilobular paleness, hypertrophied hepatocytes, central necrobiosis and single-cell necrosis in the livers at 45 ppm; epithelial proliferation with single-cell necrosis in the trachea at 45 ppm; perivascular and peribronchial infiltrates, dissarrangement of bronchial epithelium in the lungs at 45 ppm; catarrhal-purulent rhinitis in the nasal cavity and atrophy of the olfactory epithelium of the nasal mucosa, both at all concentration levels. SG2: The pathological evaluation revealed hypertrophied hepatocytes, polymorphic, enlarged and pale nuclei and increased glycogen content in the livers at 45 ppm; diffuse epithelial proliferation in the trachea of males at 45 ppm; dissarrangement of bronchial epithelium of the lungs at all concentrations; catarrhal-purulent rhinitis (15 and 45 ppm) in the nasal cavity; atrophy of the olfactory epithelium (all concentration levels), areas of hyperplasia in the respiratory epithelium (15 and 45 ppm) and hyperplasia of submucous glandular cells (all concentration levels) of the nasal mucosa. Main group: Pathology showed hypertrophied hepatocytes in the liver (45 ppm); diffuse epithelial proliferation in the trachea (45 ppm); dissarrangement of bronchial epithelium in the lungs (all concentration levels); catarrhal-purulent rhinitis in the nasal cavity; atrophy of the olfactory epithelium (all concentration levels), hyperplasia of submucous glandular cells (all concentration levels) and hyperplasia of the respiratory epithelium in the nasal mucosa. The NOAEC was found to be 0.0023 mg/l for male and female animals.

Two 10-day inhalation studies were reported (BASF; 1989). In the first study female mice were used. The animals were offered vapour concentrations of 0.023, 0.069, 0.204 mg/l (= 5, 15, 45 ppm) for 6 hours per day and 5 days per week. Hepatotoxic effects were demonstrated after only 2 exposures to 45 ppm. A pronounced reduction in body weight (45 ppm) was detectable in particular after the first 2 days of a 5-day exposure. There were indications that thereafter a plateau is reached. It was possible to detect only very slight induction of detoxifying mechanisms by pretreatment with 5 ppm or 15 ppm under the chosen test conditions: only some of the clinico-chemical parameters in the pretreated test groups were lower or unchanged. No rise in the glutathione level in the liver homogenate was found until after more than 5 exposures. The NOAEC was 0.069 mg/l. In the second 10-day inhalation study using male rats, the same concentrations and study conditions were used as in the first study. Hepatotoxic effects were detected after only 2 exposures to concentrations as low as 15 ppm test material. A marked reduction in the body weight (45 ppm) was detectable particularly after the first 2 days of 5-day exposure. Thereafter there are indications that a plateau was reached. Under the test conditions chosen it was not possible to demonstrate induction of detoxifying mechanisms by pretreatment with 5 ppm or 15 ppm of the test material. The NOAEC was found to be 0.023 mg/l.

To investigate the effects of N-Vinyl-2-pyrrolidone on cell proliferation in the liver and histological changes in the nasal cavity of rats, eight male Wistar rats per test group were whole-body exposed to vapor on 6 hours per day, 5 days a week for up to 28 days (BASF SE 2011, 2012). The target concentrations from N-Vinyl-2- pyrrolidone were 0.5, 1, 5 and 10 ppm. Control animals were exposed to conditioned air. To determine the time course of liver cell proliferation during the 4 week exposure period, satellite animals were sacrificed on study day 7, whereas the main group animals were sacrificed on study day 28. For each examination time point, a concurrent control group was conduced. BrdU minipumps were implanted 7 days before necropsy. On exposure days clinical examination was performed before, during and after exposure. Body weight of the animals was determined once prior to preflow, on start of exposure, once a week during exposure period and before necropsy. Animals were necropsied and the liver was investigated for cell proliferation and apoptotic cell death by BrdU staining and TUNEL labeling.The histological examination of the nasal cavity of main group animals (28 days of exposure) was performed after hematoxylin and eosin stain (H&E) by light microscopy. 

To establish a No Observed Adverse Effect Concentration (NOAEC) two additional groups were exposed to 0.2 and 0.5 ppm test substance to a later time point. For the later two groups, a concurrent control group was exposed to conditioned air.

The desired atmospheric concentrations were maintained throughout the study. There were no clinical signs of toxicity observed throughout the study. At the highestconcentration of 10 ppm, the body weight change was significantly reduced from study day 7 onward (not significant on study day 21). This effect indicates the slight general toxicity of the test substance and was considered to be an exposure–related adverse effect.

Concerning the determination of cell proliferation (S-Phase response), 7-day exposure with the test article led to an increased cell proliferation already at aconcentration of 1 ppm and, with a concentration-dependent enhancement, atconcentrations of 5 ppm and 10 ppm. No increased cell proliferation was noted when exposed with a concentration of 0.2 ppm.

Concerning the determination of apoptosis (TUNEL stain), 7-day exposure with the test article led to an increased apoptotic cell death at concentrations of 1 and 5 ppm. 28-day exposure with the test article led to an increased apoptotic cell death at concentrations of 1, 5 and 10 ppm. This increase is interpreted as a secondary, counter balancing induction of apoptosis due to the primary induction of cell proliferation. Exposure with a low concentration of 0.2 ppm of the test substance over a period of 28 days resulted in no increased apoptotic cell death.Treatment-related findings were noted in level I of the nasal cavity in animals at concentrations of 0.5 to 10 ppm.In level II, III and IV of the nasal cavity, findings were noted in all animals only at the highest concentration (10ppm). In level II, degeneration of the respiratory epithelium, degeneration of the olfactory epithelium, hyperplasia of the respiratory, olfactory and transitional epithelium and squamous metaplasia of the respiratory epithelium was found. These findings were graded minimal to slight. In level III and IV, degeneration of the olfactory epithelium was seen in most animals and hyperplasia of the basal cells of the olfactory epithelium was also seen in all animals of this group (10 ppm), mostly graded minimal to slight. There were no substance-related findings observed at 0.2 ppm (NOAEC).

Justification for classification or non-classification

N-vinylpyrrolidone (NVP) is currently classified under the EU DSD criteria (EU Directive 67/548/EEC) as R48/20 - Harmful: danger of serious damage to health by prolonged exposure through inhalation. Classification under the EU CLP criteria (Regulation (EC) 1272/2008) is curently STOT RE: Cat. 2; STOT SE: Cat. 3. These classifications are supported by multiple key studies (BASF, 1986 and BASF, 1992).