Nitromethane

Endpoint summary

Description of key information

OECD considered nitromethane, nitroethane and 1-nitropropane to be a category at SIAM.  Therefore data from the analogue 1-nitropropane is used. Oral administration of the analogue, 1-Nitropropane, to rats for a period of twenty-eight consecutive days at dose levels of up to 100 mg active ingredient/kg/day resulted in toxicologically significant effects at 100 mg active ingredient/kg/day. No such effects were detected at 30 or 10 mg active ingredient/kg/day and, the "No Observed Effect, Level " (NOEL) is, therefore, considered to be 30 mg active ingredient/kg/day.

Rabbits exposed to 98 ppm (244.5 mg/m3) for 6 months had decreased serum T4 levels.  In the same study rats did not have decreased T4 levels at 6 months.  In a more recent study, the NOEL in rats and mice was 94 ppm following 13 weeks of exposure.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP, guideline study (Note- The exposure period was only 28 days.)

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Health and Welfare Guidelines (1986)

Deviations:

no

Remarks:

Not specified in report

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals: Animals (male and female Sprague-Dawley CD rats) were acclimated for 8 days before use. A total of 30/sex were accepted into the study. At the beginning of the study, males weighed 121 - 161 g and females weighed 121-159 g, and were approximately 5-6 weeks old. The animals were allowed free access to food (except for the night prior to urine collection, when it was withdrawn) and water. The diet and drinking water did not contain any contaminants that might have influenced the study. Rats were maintained on a 12 hour light/dark cycle. Animals were randonly allocated to 6 groups (5/sex/group).

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on oral exposure:

The test material was administered daily, for up to twenty-eight consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 2 ml/kg/day of Arachis oil B.P. Animals from satellite groups 5 and 6 were maintained for a further fourteen days treatment-free period following termination of treatment. The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at weekly intervals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability and homogeneity of the test material formulations were determined by Safepharm Analytical Laboratory. Samples were taken of each test material formulation and were analysed for concentration of 1-nitropropane at Safepharm Analytical Laboratory.

Duration of treatment / exposure:

28 days, post exposure 14 days

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5/sex/group

Control animals:

yes, concurrent vehicle

Details on study design:

Test conduct: four groups of animals were dosed with 0 (control), 10, 30 or 100 mg/kg/day active material for 28 consecutive days by gavage, and then were terminated. Controls were dosed with 2 ml/kg/day Arachis oil BP. The two additional groups (satellite animals) were dosed with 0 or 100 mg/kg/day test material for 28 days and then were allowed to recover for 14 days before termination.

Positive control:

No

Observations and examinations performed and frequency:

All animals were examined for signs of toxicity before dosing and 1 and 5 hours after dosing on weekdays and before dosing and 1 hour after treatment on weekends. During the treatment-free period, satellite animals were observed twice daily on weekdays and once daily on weekends.

Body weights were recorded on day 0 and on days 7, 14, 21 and 28. Satellite animals also were weighed on days 35 and 42 (at termination). Food consumption was recorded for each cage (5 animals) at weekly intervals. Water intake was visually inspected.

Clinical chemistry (urea, total protein, albumin, albumin/globulin ratio, sodium , potassium, chloride, calcium, inorganic phosphorus, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, glucose, gamma glutamyl transpeptidase, triglycerides, total chloesterol, total bilirubin and creatinine) and hematological analyses (hematocrit, hemoglobin, erythrocyte count, total and differential leukocyte count, platelet count, mean corpuscular hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin concentration, methemoglobin concentration, reticulocyte count and clotting time) were performed on blood collected from the lateral tail vein of all survivors from the control and high dose groups (both main study and satellite animals) at termination. Blood for hematological and blood chemistry analyses was collected into tubes containing potassium EDTA or lithium heparin, respectively (with the exception of blood for clotting time analysis, which was collected into sodium citrate). Animals were not fasted prior to blood collection. Urinalysis (volume, specific gravity, pH, protein, glucose, ketones, bilirubin, urobilinogen, reducing substances and blood) was performed on control and high dose main study animals during week 4 and from satellite animals during the final week of the study. Urine samples were collected over a period of approximately 16 hours, by housing the rats in metabolism cages. Animals did not have access to food during the collection period.

Sacrifice and pathology:

At termination, all surving animals were euthanized and were subjected to a full external and internal examination. The adrenals, brain, testes, ovaries, heart, kidneys, liver, pituitary and spleen were weighed. The organs, plus the aorta (thoracic), bone and bone marrow (femur and sternum), cecum, colon, duodenum, eyes, gross lesions, ileum, jejunum, lungs, lymph nodes (cervial and mesenteric), muscle (skeletal), esophagus, pancreas, prostate, rectum, salivary glands, sciatic nerve, seminal vesicles, skin (hind limb), stomach, thymus, thyroid/parathyroid, trachea, urinary bladder and uterus were fixed in 10% buffered formalin. Microscopic examinations were performed on tissues from control and high dose animals. All lesions from other groups were also examined microscopically.

Other examinations:

None

Statistics:

Statistical analyses: Absolute and relative organ weights, hematological, blood chemistry, weekly body weight gain and quantitative urinalysis data were analyzed by one was analysis of variance (ANOVA) incorporating the Fmax test for homogeneity of variance. Data with heterogenous variances were analyzed with the Kruskal Wallis non-parametric analysis of variance and the Mann Whitney U-Test. The critical level of significance was P < 0.05.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

Effects at 100 mg/kg: One high dose male was killed in extremis on day 27. This animal had exhibited hunched posture, lethargy and ataxia on day 16. This animal exhibited dark kidneys, thickening of the forestomach and sloughing of the glandular gastric epithelium at necropsy. Animals showed signs of increased salivation one hour after dosing on day 15. Sporadic incidents were then noted approximately 5 minutes after dosing from day 16 on. This lasted for up to one hour after dosing. Red/brown staining around the mouth was apparent from day 15 on. Two females developed hunched posture on day 28. Body weight gain was reduced in main study males during weeks 2 and 4. This was not observed in females or satellite males. Food consumption of main study males was reduced by 13% during the last week of the study. A reduction in food intake was not observed in satellite males. Water consumption did not appear to be altered.

Hemoglobin, hematocrit and erythrocyte counts were reduced and white blood cells (lymphocytes) and clotting time were increased in main study females (with respect to the study control). However, hemoglobin, hematocrit and erythrocyte counts were within the normal ranges for rats of the same strain and age. Mean corpuscular hemoglobin concentration was reduced in main study males. Methemoglobin and lymphocyte counts were increased in satellite males and hematocrit was decreased in satellite females. Plasma urea was increased in main study males with respect to study controls, but not to historical controls. The albumin/globulin ratio and triglycerides were elevated and alkaline phosphatase was less than control in main study females. Urine volume was increased and urine specific gravity was decreased in main study females.

Absolute and relative brain weights were increased in main study animals, with only female relative weights not achieving statistical significance. Main study females also had increased absolute and relative kidney weights and decreased relative ovary weights. The absolute weight of the pituitary was decreased and the relative heart weight was increased in main study males with respect to the study control (but not historical controls).

There was no pathological evidence of brain toxicity. The incidences of all morphological changes in the organs that were examined were similar to control.

Effects at 30 mg/kg: There were no deaths or clinical signs of toxicity. Males had increased body weight gains during weeks 2 and 4. There was no effect of treatment on food consumption. Water consumption did not appear to be altered. Urine volume was increased and urine specific gravity was decreased in main study females. Changes in these parameters were dose-dependent. Absolute brain and liver weights were increased in main study females and males, respectively.

Effects at 10 mg/kg: There were no deaths or clinical signs of toxicity. Body weight gains and food consumption were unaffected by treatment. Water consumption did not appear to be altered. There was an increase in methemoglobin in main study males. Alkaline phosphatase was less than control in main study females. Urine specific gravity was decreased in main study females. The absolute weights of the adrenals and heart were increased in main study females.

Controls: A dark accessory lobe of the liver was found in one female at necropsy.

Key result

Dose descriptor:

NOEL

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no significant effects observed at this dose level

Key result

Dose descriptor:

LOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

System:

central nervous system

Organ:

brain

Treatment related:

yes

Dose response relationship:

no

Relevant for humans:

not specified

The dose levels were chosen based on a 14-day study with 10, 50, 150 and 250 mg/kg test material.  All animals treated with 250 mg/kg and one animal treated with 150 mg/kg were killed in extremis. No effects were noted at 10 mg/kg. A detailed justification for setting the NOAEL (actually a NOEL) at 30 mg/kg was provided by the investigators.  Effects considered to be related to treatment with 100 mg/kg/day were hunched posture, lethargy, ataxia and death. Effects considered possibly related to treatment with 100 mg/kg/day were reduced body weight gain and increased brain weight.  The increase in the absolute brain weight of females treated with 30 mg/kg/day was not considered to be biologically significant since an increase in the relative weight was not observed. Changes in clincial chemistries, hematologies, urinalysis and weights of organs other than the brain were not considered to be related to treatment since there were no corresponding morphologic changes and/or the changes were not dose-dependent.

Conclusions:

Clinical findings in a few animals dosed with 100 mg/kg were consistent with toxicity to the nervous system. Organ weight data revealed significant increases in brain weights in high dose animals. However, there were no treatment-related morphologic changes in the brain. Kidney weights were increased in high dose animals and urinalyses suggested that females treated with 30 and 100 mg/kg produced increased amounts of dilute urine. However, blood chemistries and histopathology revealed no evidence of kidney toxicity. Therefore, there was "no convincing evidence of treatment-related renal effects." None of the other changes observed were considered to be related to treatment since they were not dose-dependent.

Oral administration of the test material, 1-nitropropane, to rats for a period of twenty-eight consecutive days at dose levels of up to 100 mg active ingredient/kg/day resulted in toxicologically significant effects at 100 mg active ingredient/kg/day. No such effects were detected at 30 or 10 mg active ingredient/kg/day and, the "No Observed Effect, Level " (NOEL) is, therefore, considered to be 30 mg active ingredient/kg/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

30 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Good

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

GLP compliance:

not specified

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system 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).

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

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.

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).

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.

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.

Critical effects observed:

not specified

No additional information available.

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEC

936.2 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

Good

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

GLP compliance:

not specified

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system 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).

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

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.

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).

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.

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.

Critical effects observed:

not specified

No additional information available.

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

188 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

Good

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Oral Exposure

No reliable animal studies were found for nitromethane exposure through the oral route. Instead 1 -nitropropane was used as a surrogate for read across. In a 28 -day (sub acute) oral gavage study in male and female Spraque-Dawley rats exposed to 0, 10, 30 and 100 mg/kg/day of 1 -nitropropane, no treatment related effects were seen at the 10 and 30 mg/kg/d dose levels (NOEL = 30 mg/kg/d). At the 100 mg/kg/day level (LOAEL), the treatment related effects were hunched posture, lethargy, and axatia. Death occurred in one high dose male. Also, observed in the high dose group was a slight decrease in body weight gain in males and an increased in brain weight.

Inhalation studies

Groups of 10 male and 10 female rats and 10 male and 10 female mice 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 and mice 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. The final mean body weights and weight gains of exposed mice were generally similar to those of the controls. Clinical findings included hindlimb paralysis in rats in the 750 and 1,500 ppm groups. There were no treatment-related clinical findings in mice.

 

In rats, inhalation exposure 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. The NOAEL for systemic effects was 94 ppm in rats.

 

In mice, the absolute right kidney weights of all groups of exposed male mice except the 1500 ppm group and of females exposed to 188 ppm or greater and the relative right kidney weights of all groups of exposed males and of females in the 750 and 1500 ppm groups were significantly greater than those of the controls. The absolute liver weights of male mice in the 750 ppm group and the relative liver weights of males exposed to 375 ppm or greater were significantly greater than those of the controls. Olfactory epithelial degeneration and respiratory epithelial hyaline droplets were observed microscopically in all male and female mice exposed to 375 ppm or greater. Degeneration also occurred in females in the 188 ppm group, and hyaline droplets occurred in females in the 94 and 188 ppm groups. The average severity of the nasal lesions ranged from minimal to mild in males. In females, the average severity of olfactory epithelial degeneration ranged from minimal to mild and the severity of respiratory epithelial hyaline droplets ranged from minimal to moderate. All males and nine females in the 1500 ppm groups also had minimal extramedullary hematopoiesis of the spleen. The NOAEL for systemic effects was 94 ppm in mice.

 

During 6 months of exposure to 98 or 745 ppm nitromethane, only mild to moderate symptoms of toxicity were observed in rats. A reduction in body weight gain was observed in rats exposed to 745 ppm nitromethane. Hematocrit and hemoglobin levels in rats were slightly depressed from 10 days through 6 months of exposure to 745 ppm nitromethane. Other hematologic parameters such as prothrombin time and methemoglobin concentration were unaffected in rats. Weights of all organs evaluated were comparable to controls in rats. Histopathologic evaluation indicated no exposure-related abnormalities in rats due to exposure to nitromethane at 98 or 745 ppm for up to 6 months. The NOEC was 98 ppm following 6 months exposure to nitromethane in rats.

 

During 6 months of exposure to 98 or 745 ppm NM, only mild to moderate symptoms of toxicity were observed in rabbits. Rabbits provided a suggestion of depression in hemoglobin levels. Other hematologic parameters such as prothrombin time and methemoglobin concentration were unaffected in rabbits. Ornithine carbanyl transferase in rabbits was elevated after 1 and 3 months, but not 6 months of exposure to 745 ppm NM. No apparent effects on glutamic-pyruvic transaminase in rabbits were observed. Serum T4, however, was statistically significantly depressed in rabbits exposed at either 98 or 745 ppm NM at the 6-month testing period as well as at the 1-month sacrifice for rabbits exposed to 745 ppm. Weights of all organs evaluated were comparable to controls in rabbits except for thyroid weights in rabbits after 6 months of exposure at 745 ppm NM. The increased thyroid weights after 6 months and decreased thyroxin levels at all testing intervals, for both concentrations, indicated an effect on the thyroid in rabbits by NM. Some evidence of pulmonary edema and other pulmonary abnormalities was observed in rabbits exposed to both levels of NM for 1 month. The LOAEC was 98 ppm in rabbits based on decreased serum T4 levels.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

GLP, guideline study

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:

Comparable to a GLP/Guideline study

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:

Comparable to a GLP/Guideline study

Repeated dose toxicity: inhalation - systemic effects (target organ) cardiovascular / hematological: other

Justification for classification or non-classification

According to the GHS and EU DSD criteria the results of the repeat dose toxicity data do not warrant classification for chronic toxicity