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Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3 October 1988 - 5 October 1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1997

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
GLP compliance:
not specified

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
>98% nitromethane

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
The rats were 4 weeks old on receipt from the supplier. They were quarantined for 13-14 days before use. Five animals per sex were randomly selected for parasite evaluation and gross examination for evidence of disease. At the end of the study, serologic analyses were performed on 5 sentinel rats/sex. Water and food were available ad libitum (except during exposure, when food was withheld).

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: Not applicable.
Details on inhalation exposure:
The test material was held in a stainless-steel reservoir under a nitrogen blanket. The material was pumped through a liquid distribution manifold of stainless steel tubing to heated-wick vaporizers.One set of dual vaporizers supplied vapor to all chambers. The vapor-laden air was transferred through the distribution line and diluted with HEPA- and charcoal-filtered air. Three-way valves in the chamber inlet ducts allowed nitromethane vapors to be diverted to the exhaust until a stable concentration of test material was built up in the distribution line. At each chamber, vapor moving through the inlet duct was further diluted with filtered air to the appropriate concentration of test material with a metered three-way valve. A small particle detector was placed in the chambers to measure concentrations of aerosol. No particle counts above the minimum resolvable level (200 particles/cm3) were detected.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber concentrations were monitored with an on-line gas chromatograph (GC). The monitor was coupled with the inhalation chanbers by a computer-controlled 12-port stream select valve. The GC was calibrated comparing chamber concentration data to data from grab samples analyzed by an off-line GC. The grab samples were collected in bubblers containing dimethylformamide. The off-line GC was calibrated with gravimetrically prepared nitromethane standards. Chamber concentration uniformity was maintained throughout the study.
Duration of treatment / exposure:
6 hrs/day
Frequency of treatment:
5 days/week for 13 weeks
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 94, 188, 375, 750, or 1,500 ppm
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0, 0.235, 0.47, 0.938, 1.88 or 3.75 mg/L
Basis:
nominal conc.
No. of animals per sex per dose:
10 males and 10 females/dose level (core study). Additional groups of 10 animals/sex designated for clinical pathology examinations
Control animals:
yes, concurrent no treatment
Details on study design:
Groups of 10 animals/sex (core study animals) were exposed to 0, 94, 188, 375, 750 or 1500 ppm test material by inhalation, 6 hours and 12 minutes per day, 5 days per week for 13 weeks. Additional groups of 10 animals/sex designated for clinical pathology examinations were also exposed. Clinical observations were recorded weekly. The core study animals were weighed initially, weekly, and at the end of the study.

Neurobehavior tests including forelimb and hindlimb strength measurements, response to stimulus (tail flick latency) and startle response wre performed on all male and female rats in the core study over a 2-day period during week 11. Rats were allowed to acclimate to the testing room for at least 2 hours.

Clnical pathology analyses were performed on rats designated for this purpose on days 3 and 23 and on core study rats at termination. Rats were anesthtized and blood was withdrawn from the retroorbital plexus. Blood for hematology determinations (erythrocyte, platelet and total and differential leukocyte counts, hematocrit, hemoglobin concentration, mean cell volume, mean cell hemoglobin (and hemoglobin concentration), morphologica evaluation of blood cells, nucleated erythrocyte counts and methemoglobin concentration) was placed in tubes containing the anticoagulant potassium EDTA and blood for clinical chemistry analyses was allowed to clot. Serum was obtained from the latter blood samples and analyzed for urea nitrogen, creatinine, total protein, ablumin and globulin concentrations, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase, bile acid, triiodothyronine, total and free thyroxine and thyroid-stimulating hormone.

For 7 consecutive days before termination, the vaginal vaults of all females in the 0, 375, 750 and 1000 ppm groups were moistened with saline (if necessary) and samples of vaginal fluid and cells were stained. Relative numbers of leukocytes, nucleated epithelial cells and large squamous epithelial cells were enumerated and used to ascertain estrous cycle stage. At the end of the study samples were collected for sperm motility from all males in the same groups. The left epidiymis and testis were isolated and weighed. The tail of the epidiymis (cauda) was removed and weighed. Test yolk was applied to slides, and a small incision was made at the distal border of the cauda epidiymis. Sperm effluxing from the incision were dispersed on the slides and the numbers of motile and nonmotile spermatozoa were counted (5 fields/slide) by 2 observers. Caudae were then finely minced and fixed at 65 degrees C. Sperm density was then determined using a hemacytometer and microscope. The testicular spermatid head count was determined by removing the tunica albuginea and homogenizing the left testis in phosphate-buffered saline containing 10% dimethyl sulfoxide. Homogenization-resistant spermatied nuclei were counted using a hemacytometer and microscope.

Necropsies were performed on all core study animals. The heart, right kidney, liver, lungs, right testis, thymus and thyroid were weighed. These tissues plus the adrenal gland, bone and marrow, brain, clitoral gland, epididymis, esophagus, eyes (if grossly abnormal), left kidney, large intestine, larynx, lymph nodes, mammary gland, nose, ovary, parcreas, parathyroid, pharynx (if grossly abnormal), pituitary gland, preputial gland, prostate, salivary gland, seminal vesicle, skin, small intestine, spinal cord and sciatic nerve, spleen, stomach, left testis, thigh muscle, trachea, urinary bladder, uterus and vagina were fixed and preserved in 10% neutral buffered formalin. All tissues collected from rats in the control and 1500 ppm groups were processed for microscopic examination. The bone marrow, lungs, nose and all gross lesions and tissue masses were examined in rats from the other exposure groups.
Positive control:
Not applicable.

Examinations

Observations and examinations performed and frequency:
Clinical observations were recorded weekly. The core study animals were weighed initially, weekly, and at the end of the study.

Neurobehavior tests including forelimb and hindlimb strength measurements, response to stimulus (tail flick latency) and startle response wre performed on all male and female rats in the core study over a 2-day period during week 11. Rats were allowed to acclimate to the testing room for at least 2 hours.
Sacrifice and pathology:
Clnical pathology analyses were performed on rats designated for this purpose on days 3 and 23 and on core study rats at termination. Rats were anesthtized and blood was withdrawn from the retroorbital plexus. Blood for hematology determinations (erythrocyte, platelet and total and differential leukocyte counts, hematocrit, hemoglobin concentration, mean cell volume, mean cell hemoglobin (and hemoglobin concentration), morphologica evaluation of blood cells, nucleated erythrocyte counts and methemoglobin concentration) was placed in tubes containing the anticoagulant potassium EDTA and blood for clinical chemistry analyses was allowed to clot. Serum was obtained from the latter blood samples and analyzed for urea nitrogen, creatinine, total protein, ablumin and globulin concentrations, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase, bile acid, triiodothyronine, total and free thyroxine and thyroid-stimulating hormone.

For 7 consecutive days before termination, the vaginal vaults of all females in the 0, 375, 750 and 1000 ppm groups were moistened with saline (if necessary) and samples of vaginal fluid and cells were stained. Relative numbers of leukocytes, nucleated epithelial cells and large squamous epithelial cells were enumerated and used to ascertain estrous cycle stage. At the end of the study samples were collected for sperm motility from all males in the same groups. The left epidiymis and testis were isolated and weighed. The tail of the epidiymis (cauda) was removed and weighed. Test yolk was applied to slides, and a small incision was made at the distal border of the cauda epidiymis. Sperm effluxing from the incision were dispersed on the slides and the numbers of motile and nonmotile spermatozoa were counted (5 fields/slide) by 2 observers. Caudae were then finely minced and fixed at 65 degrees C. Sperm density was then determined using a hemacytometer and microscope. The testicular spermatid head count was determined by removing the tunica albuginea and homogenizing the left testis in phosphate-buffered saline containing 10% dimethyl sulfoxide. Homogenization-resistant spermatied nuclei were counted using a hemacytometer and microscope.

Necropsies were performed on all core study animals. The heart, right kidney, liver, lungs, right testis, thymus and thyroid were weighed. These tissues plus the adrenal gland, bone and marrow, brain, clitoral gland, epididymis, esophagus, eyes (if grossly abnormal), left kidney, large intestine, larynx, lymph nodes, mammary gland, nose, ovary, parcreas, parathyroid, pharynx (if grossly abnormal), pituitary gland, preputial gland, prostate, salivary gland, seminal vesicle, skin, small intestine, spinal cord and sciatic nerve, spleen, stomach, left testis, thigh muscle, trachea, urinary bladder, uterus and vagina were fixed and preserved in 10% neutral buffered formalin. All tissues collected from rats in the control and 1500 ppm groups were processed for microscopic examination. The bone marrow, lungs, nose and all gross lesions and tissue masses were examined in rats from the other exposure groups.

Other examinations:
No additional information available.
Statistics:
Organ and body weight and neurobehavioral data were analyzed using the parametric multiple comparison procedures of Dunnett (J Am Stat Assoc 50:1096-1121, 1955) and Williams (Biometrics 27:103-117, 1971 and Biometrics 28:519-531, 1972). Hematology, clinical chemistry, spermatid and epididymal spermatozoal data were analyzed with the nonparametric multiple comparison methods of Shirley (Biometrics 33:386-389, 1977) and Dunn (Technometrics 6:241-252, 1964). Jonckheere's test (Biometrika 41:133-145, 1954) was used to assess the significance of dose-related trends and to determine whether a trend-sensitive test (Williams' or Shirley's test) was more appropriate for pariwise comparisons than a test that does not assume a monotonic dose-related trend (Dunnett's or Dunn's test). Prior to analysis, extreme values were identified by the outlier test of Dixon and Massey (Introduction to Statistical Analysis, McGraw-Hill, 1951, p. 145-147) and implausible values were eliminated from the analyses. Average severity values were analyzed for significance with the Mann-Whitney U test (Nonparametric Statistical Methods, John Wiley and Sons, 1973, p.120-123). Vaginal cytology data were transformed using the arcsine test before analysis. Treatment effects were determined by applying a multivariate analysis of variance to the transformed data. The Fisher's exact test was used to analyze histopathological data (with p < 0.05 as the level of significance).

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
1500 ppm: There were no deaths at this or any other concentration. Hindlimb paralysis was noted in all male and female rats beginning on day 21.
Mortality:
mortality observed, treatment-related
Description (incidence):
1500 ppm: There were no deaths at this or any other concentration. Hindlimb paralysis was noted in all male and female rats beginning on day 21.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
1500 ppm: The final mean body weight and weight gain of male rats in the high dose group were significantly less than those of controls. Final body weights of males were 88% of control.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
1500 ppm: Increased methemoglobin concentration and decreased RBCs
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
1500 ppm: Decreased triiodothyronine, thyroxine and free thyroxine was observed in males and females at day 23
Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
1500 ppm: Forelimb and hindlimb grip strength of males and hindlimb grip strength of females were less than control.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
1500 ppm: Several organ weights were affected.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
1500 ppm: Minimal to mild effects noted in a number of tissues.
Histopathological findings: neoplastic:
not specified
Details on results:
Test material concentrations: The average (+/- SD) concentrations of test material analyzed in chambers containing nominal concentrations of 94, 188, 375, 750 and 1500 ppm were 94 +/- 6, 187 +/- 10, 373 +/- 19, 748 +/- 37 and 1500 +/- 58 ppm. The total number of samples taken from each chamber ranged from 938-974. Since analytical concentrations were very close to nominal concentrations, results are reported according to nominal concentrations.

Effects at 1500 ppm: There were no deaths at this or any other concentration. The final mean body weight and weight gain of male rats in the high dose group were significantly less than those of controls. Final body weights of males were 88% of control. Hindlimb paralysis was noted in all male and female rats beginning on day 21. Forelimb and hindlimb grip strength of males and hindlimb grip strength of females were less than control. Hematocrit and hemoglobin concentrations were less than controls for treated males and females at all time points. Erythrocyte counts in males and females were depressed on day 3 and greater than control in treated males at termination. Number of nucleated erythrocytes were greater than control in males and females on day 23 and at termination. Decreased mean cell volume was noted at all time points measured in males and females. Mean cell hemoglobin was decreased at all time points measured in females and on days 23 and at termination in males. Mean cell hemoglobin concentration was increased at all time points measured in males and females. Platelet counts were increased at all time points measured in males and females (with the exception of day 3 in females). Lymphocytes were increased at termination in males. Segmented neutrophil numbers were decreased in females on day 3. Methemoglobin concentration was increased at all time points measured in males and females (with the exception of day 3 in females). On day 3, minimal numbers of Heinz bodies were noted in red blood cells of males. A hypothyroid state characterized by decreased triiodothyronine, thyroxine and free thyroxine was observed in males and females at day 23. Relative heart and right kidney weights of males and females were greater than control. Relative weight of the thryoid gland was increased in males and relative liver weights were increased in females. Absolute weight of the lungs, testis and thymus were lower than control. Epididymal spermatozoal motility and weights of the left cauda, epidiymis and testis were lower than control. Hyperplasia of the bone marrow, spinal cord degeneration, sciatic nerve degeneration, degeneration of the olfactory epithelium and goblet cell hyperplasia in the nose/turbinates were observed in all males and females. Hyaline droplets were observed in the respiratory epithelium of the nose/turbinates in 8/10 males and all females. The average severity of all lesions ranged from minimal to mild.

EFfects at 750 ppm: Hindlimb paralysis was noted in one male and four female rats beginning on day 63. Hindlimb grip strength of females was less than control. Hematocrit and hemoglobin concentrations were less than controls for treated males and females at all time points. Erythrocyte counts in males and females were depressed on day 3 and greater than control at termination. Decreased mean cell volume was noted at all time points measured in males and females. Mean cell hemoglobin was decreased at all time points measured in males and females. Mean cell hemoglobin concentration was increased at all time points measured in males. Platelet counts were increased at all time points measured in males and females (with the exception of day 3 in females). Methemoglobin concentration was increased at all time points measured in males and females (with the exception of day 3 in females). On day 3, minimal numbers of Heinz bodies were noated in red blood cells of males. A hypothyroid state characterized by decreased triiodothyronine, thyroxine and free thyroxine was observed in males and females at day 23. Epididymal spermatozoal motility of treated males was lower than control. Spinal cord degeneration, sciatic nerve degeneration and degeneration of the olfactory epithelium were observed in all males and females. Bone marrow hyperplasia was noted in 9/10 males and 7/10 females. Hyaline droplets were observed in the respiratory epithelium of the nose/turbinates in one male and 4 females. Goblet cell hyperplasia in the nose/turbinates were observed in one male and two females. The average severity of all lesions ranged from minimal to mild.

Effects at 375 ppm: Hematocrit and hemoglobin concentrations were less than controls for treated males and females at all time points. Erythrocyte counts in males were depressed on day 3 and greater than control on day 23 and at termination. Erythrocyte counts in females were greater than control on day 23 and at termination. Decreased mean cell volume was noted at all time points measured in males and females. Mean cell hemoglobin was decreased at all time points measured in males and females. Platelet counts were increased at day 3 and at termination in males and at termination in females. Methemoglobin concentration was increased on day 3 and at termination in males and on day 23 in females. A hypothyroid state characterized by decreased triiodothyronine, thyroxine and free thrroxine was observed in males on day 23. Females had decreased thyroxine. Absolute liver weight was increased in females. Degeneration of the olfactory epithelium was observed in 9/10 males and all females. Bone marrow hyperplasia was found in 6/10 females. Sciatic nerve degeneration was noted in 5/10 males and 8/10 females. Spinal cord degeneration was noted in 9/10 males and 2/10 females. The average severity of all lesions was minimal.

Effects at 188 ppm: Hematocrit and hemoglobin concentrations were less than controls for treated males on day 3 and for treated females on day 23 and at termination. Erythrocyte counts in males were depressed on day 3 and greater than control at termination. Decreased mean cell volume was noted at day 23 and at termination in males and at all time points measured in females. Mean cell hemoglobin was decreased at day 23 and at termination in males and females. Platelet counts were increased in males on day 3. Leukocyte counts were increased in females on day 3. Methemoglobin concentration was increased at termination in males and and on day 23 in females. Females had decreased thyroxine. Absolute thymus weights were increased in females.

Effects at 94 ppm: Hematocrit and hemoglobin concentrations were less than controls for treated females on day 23. Erythrocyte counts in males were greater than control on day 23 and at termination. Decreased mean cell volume was noted at termination in males and at all time points measured in females. Mean cell hemoglobin was decreased at termination in males and at day 23 and at termination in females. Platelet counts were increased in males and decreased in females on day 3.

Effect levels

open allclose all
Dose descriptor:
LOAEC
Remarks:
Systemic
Effect level:
375 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: According to NTP effects observed at 375 ppm were minimal and this was used for the highest concentration in the 2 year study.
Dose descriptor:
NOAEC
Remarks:
Systemic
Effect level:
94 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
LOAEC
Remarks:
Local
Effect level:
375 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Lesions observed in the upper respiratory tract at concentrations >/=375 ppm.
Dose descriptor:
NOEC
Remarks:
Local
Effect level:
188 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No lesions noted in the upper respiratory tract at a concentration of 188 ppm.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

No additional information available.

Applicant's summary and conclusion

Conclusions:
Groups of 10 male and 10 female rats were exposed to 0, 94, 188, 375, 750, or 1,500 ppm nitromethane by inhalation, 6 hours per day, 5 days per week, for 13 weeks. All rats survived to the end of the study. The final mean body weight and weight gain of male rats in the 1,500 ppm group were significantly less than those of the controls. Clinical findings included hindlimb paralysis in rats in the 750 and 1,500 ppm groups.

Inhalation exposure of rats to nitromethane resulted in an exposure concentration-dependent, microcytic, responsive anemia; anemia was most pronounced in males and females exposed to 375 ppm or greater. The presence of schistocytes, Heinz bodies, and spherocytes and increased mean cell hemoglobin concentration and methemoglobin concentration were evidence that a hemolytic process was occurring; this hemolytic process could have accounted, in part, for the anemia. Thrombocytosis accompanied the anemia and would be consistent with a reactive bone marrow or could have been due to the erroneous inclusion of small erythrocyte fragments as part of the platelet count. On day 23, transient decreases in serum triiodothyronine, thyroxine, and free thyroxine were observed in male rats exposed to 375 ppm or greater and female rats exposed to 750 or 1,500 ppm. There was little or no pituitary response to the thyroid hormone decreases, as evidenced by the lack of significantly increased concentrations of thyroid-stimulating hormone in exposed rats.

No biologically significant differences in organ weights were observed. The forelimb and hindlimb grip strengths of males in the 1,500 ppm group were significantly less than those of the controls. The hindlimb grip strengths of females in the 750 and 1,500 ppm groups were also significantly less than the control value.

Minimal to mild hyperplasia of the bone marrow was observed microscopically in male rats in the 750 and 1,500 ppm groups and in females exposed to 188 ppm or greater. Nasal lesions in exposed males and females included olfactory epithelial degeneration in males and females exposed to 375 ppm or greater and in one female exposed to 188 ppm and respiratory epithelial hyaline droplets and goblet cell hyperplasia in males and females in the 750 and 1,500 ppm groups; the severity of nasal lesions in males and females was minimal to mild. Males and females exposed to 375 ppm or greater had minimal to mild degeneration of the sciatic nerve and the lumbar spinal cord.
Executive summary:

Groups of 10 male and 10 female rats were exposed to 0, 94, 188, 375, 750, or 1,500 ppm nitromethane by inhalation, 6 hours per day, 5 days per week, for 13 weeks. All rats survived to the end of the study. The final mean body weight and weight gain of male rats in the 1,500 ppm group were significantly less than those of the controls. Clinical findings included hindlimb paralysis in rats in the 750 and 1,500 ppm groups.

Inhalation exposure of rats to nitromethane resulted in an exposure concentration-dependent, microcytic, responsive anemia; anemia was most pronounced in males and females exposed to 375 ppm or greater. The presence of schistocytes, Heinz bodies, and spherocytes and increased mean cell hemoglobin concentration and methemoglobin concentration were evidence that a hemolytic process was occurring; this hemolytic process could have accounted, in part, for the anemia. Thrombocytosis accompanied the anemia and would be consistent with a reactive bone marrow or could have been due to the erroneous inclusion of small erythrocyte fragments as part of the platelet count. On day 23, transient decreases in serum triiodothyronine, thyroxine, and free thyroxine were observed in male rats exposed to 375 ppm or greater and female rats exposed to 750 or 1,500 ppm. There was little or no pituitary response to the thyroid hormone decreases, as evidenced by the lack of significantly increased concentrations of thyroid-stimulating hormone in exposed rats.

No biologically significant differences in organ weights were observed. The forelimb and hindlimb grip strengths of males in the 1,500 ppm group were significantly less than those of the controls. The hindlimb grip strengths of females in the 750 and 1,500 ppm groups were also significantly less than the control value.

Minimal to mild hyperplasia of the bone marrow was observed microscopically in male rats in the 750 and 1,500 ppm groups and in females exposed to 188 ppm or greater. Nasal lesions in exposed males and females included olfactory epithelial degeneration in males and females exposed to 375 ppm or greater and in one female exposed to 188 ppm and respiratory epithelial hyaline droplets and goblet cell hyperplasia in males and females in the 750 and 1,500 ppm groups; the severity of nasal lesions in males and females was minimal to mild. Males and females exposed to 375 ppm or greater had minimal to mild degeneration of the sciatic nerve and the lumbar spinal cord.