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Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study with acceptable restricctions
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report Date:
1992

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
other: OECD Guideline 451 (Carcinogenicity Studies)
Qualifier:
equivalent or similar to
Guideline:
other: EPA OTS 798.3300 (Carcinogenicity)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): N-vinylpyrrolidone-2 (NVP)
- Substance type:
- Physical state: liquid
- Analytical purity: 99.9%
- Impurities (identity and concentrations): water - 0.02 -0.09%, kerobit stabilizer 3ppm
- Purity test date:
- Lot/batch No.:
- Expiration date of the lot/batch:
- Stability under test conditions: stabile
- Storage condition of test material:
- Other:

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Wiga, Sulzfeld, FRG
- Age at study initiation: 6-8 weeks
- Fasting period before study:
- Housing: Wire mesh cage, type D III of Becker Co.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 -24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12 hours ark / 12 hours light

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
air
Details on inhalation exposure:
For the 5ppm exposure group, the test substance was supplied to a vaporizer using a continuous infusion pump and heated by means of a circulating thermostat and evaporated. In exposure groups 10 ppm and 20 ppm, the test substance was atomized by means of a two component atomizer and a stream of compressed air measured with a rotameter into an ultrafine aerosol within the vaporizer and evaporated. The vapors were taken up in a stream of fresh air measured by means of a rotameter, passed through the vaporizer and subsequently conducted over heated pipes. After evaporation, a further stream of fresh air (diluting air) was introduced.

Total supply air 80 - 90 m3/hour. Humidity was maintained between 30 and 70% and temperature in the chamber was regulated between 21 and 27 C.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The nominal concentrations of NVP in the inhalation chambers were calculated from the amounts of test material and air per unit time. The actual concentrations in the inhalation chambers were determined with total hydrocarbon analyzers (Gas chromatography with Flame Ionization detector). The value measured in the middle of the chamber was defined as the chamber concentration.
Duration of treatment / exposure:
6 hours
Frequency of treatment:
5 days per week, except holidays
Doses / concentrations
Remarks:
Doses / Concentrations:
0ppm, 5 ppm, 10 ppm, or 20 ppm
Basis:
nominal conc.
No. of animals per sex per dose:
Main Group (2 years exposure period) 60 animals/sex/group for exposed animals, 70 animals/sex for control group.
Satellite Group 1 (3 months exposure) 20 animals/sex/group for exposed animals, 10 animals/sex for control group.
Satellite Group 2 (12 months exposure) 10 animals/sex/group.
Satellite Group 3 (18 months exposure, 6 months post exposure) 10 animals/sex/group.
Control animals:
yes, sham-exposed

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Each workday before and then after exposure.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Palpations usually every 4 weeks during the first 6 months, then every 2 weeks, and finally the day before sacrifice.

BODY WEIGHT: Yes
- Time schedule for examinations: The day before the first exposure, then once per week in the first 13 weeks, and then every 4 weeks.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, food consumption was monitored every week for the first 13 weeks, then monthly thereafter in satellite group 3 only.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to initiation of study and just prior to sacrifice for all satellite groups
- Dose groups that were examined: All dose groups of all of the satellite groups.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Prior to sacrifice
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: All animals of all satellite groups and amin test group/
- Parameters checked . White blood cells, red blood celss, hemoglobin, hematocrit, mean cell volume, mean hemoglobin content per erythrocyte (MCH), mean corpuscular henoglobin concentration (MCHC), platelets, differential blood count, reticulocytes.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Prior to sacrifice
- Animals fasted: No
- How many animals: All animals
- Parameters examined. Prothrombin time (clotting), serum enzymes (alanine-aminotransferase, aspartate-aminotransferase, alkaline phosphatase), Liver homogenate - gamma-glutamyl transferase, glutathione-SH, Blood chemistry

URINALYSIS: Yes
- Time schedule for collection of urine: after 3, 6 and 12 months in satelite group 3 only.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: Nitrite, pH value, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Blood, Volume, Specific Gravity, Sediment.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals were sacrificed by exsanguination from the abdominal aorta and vena cava under Narcoren anesthesia. Animals were necropsied and assessed by gross pathology. Liver, kidneys, adrenals, lungs, brain, and testes from all animals sacrificed were weighed,

HISTOPATHOLOGY: Yes
The following tissues from the main study were examined: Brain, Pituitary, Thyroid and Parathyroid glands, Thymus, Lung, Nasal cavity Levels I, II, III, Larynx, trachea, Heart, Salivary glands, Liver, Spleen, Kidneys, Adrenals, Tongue, Esophagus, Stomach, Small intestine (duodenum, jejunum, ileum), Large Intestine (cecum, colon, rectum), Uterus, Urinary bladder, Lymph nodes (mandibular, mediastinal, mesenteric), Pancreas, Testes, Ovaries, Prostate/seminalvesicle/epididymides, Feamle maamary gland, Skin, Sciatic nerve, Spinal cord (cervical, mid-thorasic and lumbar), Sternum with marrow, Bone marrow (femur), Eyes, Aorta, All gross lesions, Target organs.
Statistics:
Means and standard deviations were calculated for single data. Statistical evaluation was performed by an analysis of covariance, Anova and Dunnett's test for body weight. For clinical chemistry and hematology (with groups of 8 or more animals) the means of the dosed groups were compared to the means of the control group using the analysis of variance (Anova and Dunnetts test) to test if results from individual test groups were statistically significantly different from controls. For groups of less than 8 animals a statistical one-way analysis of variance was done via the Kruskal-Wallis-h-test. If the resulting p-value was equal or less than 0.05 a pairwise comparison of each dose group with the control group was carried out. This comparison was done using Mann-Whitney-U-Test for the hypothesis of equal medians. The assessment to determine whether certain characteristics of Urinalysis differed between treated and control groups was carried out using the chi-squared test in appropriate two-by-two contingency tables. Statistical analysis was carried out using the chi-squared analysis by the Peto method (life table analyses) for the hypothesis of no treatment related effects on lethality. The test was performed one-sided.
Statistical analysis for tumor incidence data was carried out using the "RoeLee System" of the firm P.N. Lee Statistics and Computing LTD, which is included as a statistical package in the acopat patholgy system.

Results and discussion

Results of examinations

Details on results:
Body weight

Main group

In male rats a statistically significant decrease body weight was found in the high dose group as early as one week after the start of exposure and this persisted up to week 36. At the mid-dose a statistically significant decreased body weight was found as early as one week after the start of exposure and this persisted (except on test day 41) up to week 17. At the lowest dose a statistically significant decrease bodyweight was first observed 2 weeks after the start of exposure and the deficit remained significant (except on days 27 and 41) up to week 20. After 3 months of exposure body weight decreases were approximately11, 6.5, and 5.9% compared with the control group for the high mid and low dose, respectively.

In female rats a statistically significant decrease body weight was found in the high dose group as early as one week after the start of exposure and this persisted ( except on day 41) up to week 17. At the mid-dose a statistically significant decreased body weight was found as early as 2 weeks after the start of exposure and this persisted (except on test day 41, 48, 76) up to week 17. At the lowest dose a statistically significant decrease bodyweight was first observed 3 weeks after the start of exposure. Thereafter the deficit was found to be significant only on some weighing dates, indicating that the effect was borderline.(except on days 27 and 41) up to week 20. After 3 months of exposure body weight decreases were approximately 5, 5.6, and 3.4% compared with the control group for the high mid and low dose, respectively

No treatment related effects were noted on food consumption. No substance induced effects were noted on lethality for any of the dose periods.

Hematology

3 month exposure period
At 20 ppm and 10 ppm a dose0dependant increase in platelet count was found in both sexes. No substance-induced effects were seen on the red blood cells.

12 month exposure period
Changes in the white blood cell counts were only reported in female rats of the high dose group. They consisted of increases in leukocytes, lymphocytes, monocytes and neutrophilic polymorphnuclear granulocytes and were probably indicative of persistent inflammation. In red blood cells, there was a significant decrease in hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and an increase in mean corpuscular hemoglobin concentration (MCHC) and in reticulocytes were found. Increased anisocytosis and microcytosis in red blood cells were observed. These changes in red blood cell parameters are indicative of a slight anemic process.

24 month exposure period
Morphologically no substance-induced variations were detected in the white blood cells of the differential blood count. In red blood cells increased incidence of anisocytosis and microcytosis and macrocytosis were seen in female rats of the high and mid-dose groups.

Clinical Chemistry

3 month exposure period
Changes in plasma-protein concentrations (total protein, albumin, globulins) were the main clinical findings. At least one decreased parameter was found at all concentrations in male and female rats, indicating liver dysfunction. In liver homogenate increased levels of reduced glutathione (GSH) was found at 20 ppm (males and females) and at 10 ppm (males) an increased activity of Zr-glutamyltransferase (IF-GT) was also measured at 20 ppm (females).

12-month exposure period
Changes in plasma-protein concentrations (total protein, albumin, globulins) were the main clinical findings. The effects were more pronounced in female rats and were observed in the mid and high dose group. In addition, an increased cholesterol level (20 ppm females) and a slight reduction in alanine aminotransferase activity (10 and 20 ppm females) was probably caused by liver dysfunction. Levels of reduced glutathione (GSH) concentrations and Zr-glutamyltransferase activities in liver homogenate of both sexes at 20 ppm were increased and were likely related to liver dysfunction

18 month exposure period and 6 month recovery
No changes were reported on reduced glutathione (GSH) concentration whereas S’-glutamyltransferase activities were increased in the liver homogenate, especially in male rats at 10 and 20 ppm.

Urinalysis
No substance induced changes were noted in any exposure group.

Organ weights
3-month exposure period
The mean relative liver weight was statistically significantly and dose-dependently increased at 10 and 20 ppm in males and in females, the mean absolute and relative liver weights were statistically significantly increased at 20 ppm.

12-month exposure period
In males, the mean absolute liver weight was statistically significantly increased in all treatment groups, but without a clear dose-response relationship, and the mean liver weight was statistically significantly increased at 20 ppm. In females, the mean absolute liver weight was statistically and dose-dependently increased at 10 and 20 ppm, and the mean relative liver weight was statistically significantly increased at 20 ppm.

18-month exposure period and 6-months post exposure
There were no statistically significantly changes in mean liver weight.

24- month exposure period
The mean absolute and relative liver weights were statistically significantly increased in the high dose group for both sexes.

Macroscopic findings

3-month exposure period
Dark colored foci were observed in the liver of 2 males of the high dose group. This finding was reported in one female of group 1.

12-month exposure period
Colored foci were observed in the liver of one control male, one male in the 10 ppm exposure group, and 2 males and one female in the 20 ppm exposure group.

18-month exposure period and 6 month post exposure
Masses were observed in the liver in 2 males exposed to 5 ppm, and in one male and 2 females exposed to 20 ppm. Colored foci were seen in the livers of females more often in treated groups than in control group, but there was no dose-response relationship.

24-month exposure period
Masses were observed in the liver of one control male, 3 males in the 5 ppm group, 4 males in the 10 ppm group and 15 males in the 20 ppm group. In females, 2 control animals, 4 rats in the 5ppm group, 5 rats in the 10 ppm group and 25 rats in the 20 ppm group had masses in the liver. Colored foci were noted in the livers of 23 control males, 22 males in the 5 ppm group, 34 males in the 10 ppm group, and 45 males in the 20 ppm group. In females, 20 control animals, 32 animals in the 5 ppm group,40 animals in the10 ppm group and 45 animals in the 20 ppm group had such foci.

Masses were also reported in the nasal cavity of one male of the 10 ppm group and 2 males and 2 females of the 20 ppm group.

Histopathology (non-neoplastic)

3-month exposure period

Liver – Clear cell areas were noted in the livers of 6 control males, 3 5 ppm exposed males, 10 males in the 10 ppm group, and all of the males in the 20 ppm group. In females the clear cell areas were noted in 3 rats in the 10 ppm group and 17 rats in the 20 ppm group. The clear cell areas consisted of slightly enlarged hepatocytes, the cytoplasm of which was somewhat marginated with perinuclear region being left relatively clear of stainable material. The nuclei of some of the cells within these areas showed early degenerative changes. The appearance of these areas suggest that they are degenerative rather than proliferative.

Nasal cavity – Minimal to slight mucopurulent inflammation was seen in 2 male rats exposed to 5 ppm , 5 males exposed to 10 ppm and 5 males exposed to 20 ppm. While in females, inflammation was noted in one control animal, 5 females in the 5 ppm group, 4 rats exposed to 10 ppm and 3 rats exposed to 20 ppm.

Focal atrophy of the olfactory epithelium was observed in 4, 6, and 16 male animals and 1, 15, and 19 female animals in the 5, 10, and 20 ppm dose groups, respectively.

Minimal to slight focal hyperplasia was observed in males and females in a dose related fashion.

12-month exposure group

Liver- Clear cell areas were present in the livers on 9 control males and all exposed males. In females, the number of animals with clear cell areas and the number of clear cell areas per animal was increased in a dose related fashion.

Spongiosis hepatic was observed in 2 control males, 6 males in the 5 ppm group, 4 males in the 10 ppm group, and all of the males in the 20 ppm group. One female in the 10 ppm group and 2 females in the 20 ppm group showed spongiosis hepatic. These effects were considered to be degenerative in nature.

Nasal cavity – The incidence and severity of focal atrophy of the olfactory epithelium, focal hyperplasia of the submucosal gland and the olfactory epithelium were all increase in dose related fashion. In addition, minimal to moderate mucopurulant inflammation, focal metaplasia and minimal to slight focal hyperplasia of the basal cell layer were observed in exposed animals though the incidence did not always increase in a dose related fashion.

18-month exposure period and 6 month post exposure

Liver- Foci of eosinophilic, clear cell and non-classified hyperplasia were observed only in exposed animals. Spongiosis hepatis was observed in 6 contr males, 6 males at 5 ppm, 7 males at 10 ppm and all males exposed to 20 ppm. In females, spongiosis hepatis was observed in one control animal, 2 animals at 5 ppm, 4 animals at 10 ppm and 4 animals at 20 ppm.

Nasal cavity – The incidence of mucopurulent inflammation, focal atrophy of the olfactory epithelium, hyperplasia of the submucosal glands all increased in a dose related manner. The incidence of hyperplasia of the olfactory epithelium while low only occurred in exposed animals.

24-month exposure period

Liver – Focal hepatocellular hyperplasia was found in 3 control males, 8 males from the 5 ppm group, 14 males from the 10 ppm group and 21 males in the 20 ppm group. In females, hepatocellular hyperplasia occurred in 5 control females, 15 females in the 5 ppm group, 20 females in the 10 ppm group and 28 females in the 20 ppm group. The number of animals with eosinophilic foci was dose-dependently increased in exposed animals of both sexes, compared to controls.

Spongiosis hepatis occurred in 37 control males, 36 males in the 5 ppm group, 45 males in the 10 ppm group, and 55 males in the 20 ppm group. In females, spongiosis hepatis was observed in 7 control animals, 19 animals in the 5 ppm group, 28 animals in the 10 ppm group, and 42 animals in the 20 ppm group. The incidence of this finding increased in a dose dependent manner in females and in males from 10 ppm and up.

Nasal cavity – The incidence and severity of mucopurulent inflammation of the nasal cavity, focal atrophy of the olfactory epithelium, focal hyperplasia of the submucosal gland and the olfactory epithelium was increased in a dosed related manner in both sexes. Focal metaplasia of the respiratory epithelium or olfactory epithelium occurred more often in exposed animals.

Larynx - Minimal to moderate focal epithelial hyperplasia was observed in 3 males in the 10 ppm group, and 6 males in the 20 ppm group. Mucopurulent inflammation was noted in 6 control males, and 4 males in each of the other exposure groups. Mucoppurulent inflammation was only observed in a few exposed females.

Effect levels

Dose descriptor:
LOAEC
Effect level:
5 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Liver and respiratory effects

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The effects of exposure to NVP via the inhalation route, for up to 2 years, has been evaluated in the Sprague Dawley rat. A sufficient number of animals of both sexes survived until termination of the study for the purpose of drawing valid conclusions with regard to both non-neoplastic and neoplastic effects.

The inhalation of NVP vapor 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 affects in the liver and respiratory tract.