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Toxicological information

Toxicity to reproduction: other studies

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Administrative data

toxicity to reproduction: other studies
Type of information:
experimental study
Adequacy of study:
key study
Study period:
circa 1997
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: The study was not conducted according to guidelines but was conducted according to GLPs and the report contains sufficient data for interpretation of study results.

Data source

Reference Type:
Toxicology and carcinogenesis studies of nitromethane (CAS No. 75-52-5) in F344/N rats and B6C3F1 mice
National Toxicology Program
Bibliographic source:
National Toxicology Program Technical Report Series No. 461. U.S. Department of Health and Human Services (USDHHS), Public Health Service, National Institute of Health (NIH), NIH Publication No. 97-3377, dated February 1997

Materials and methods

Principles of method if other than guideline:
Special attention was paid to the reproductive organs in the NTP 13 week inhalation study.
GLP compliance:
Type of method:
in vivo

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
>98% nitromethane

Test animals

Details on test animals or test system and environmental conditions:
The mice 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. The kidneys of 5 animals/sex were screened to ensure genetic integrity. At the end of the study, serologic analyses were performed on 5 control mice/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 (if applicable):
whole body
Details on 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:
Details on analytical verification of doses or concentrations:
Chamber concentrations were monitored with an one-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.

Buildup and decay rates for chamber concentrations were determined with and without animals in the chambers. The time to achieve 90% of the target concentration was 6-13 minutes and the time for decay to 10% of the target concentration was 11-15 minutes. Studies of nitromethane degradation and monitoring for impurities were conducted throughout the studies by comparing bubbler samples to a reference sample. No significant degradation was observed during the studies.
Duration of treatment / exposure:
6 hours/day
Frequency of treatment:
5 days/week for 13 weeks
Duration of test:
13 weeks
Doses / concentrations
Doses / Concentrations:
94, 188, 375, 750 or 1500 ppm
nominal conc.
No. of animals per sex per dose:
10 male and 10 female mice/concentration
Control animals:
yes, concurrent vehicle
Details on study design:
Groups of 10 animals/sex 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. Clinical observations were recorded weekly. The animals were weighed initially, weekly, and at the end of the study.

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. Modified Tyrode's buffer 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 animals. The heart, right kidney, liver, lungs, right testis, and thymus were weighed. These tissues plus the adrenal gland, bone and marrow, brain, clitoral gland, epididymis, esophagus, eyes (if grossly abnormal), gallbladder, 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, 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 nose, spleen and all gross lesions and tissue masses were examined in mice from the other exposure groups.

Organ and body weight 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). 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

Effect levels

Dose descriptor:
Effect level:
>= 375 ppm
Based on:
test mat.
Basis for effect level:
other: A dose-related decrease in sperm mobility was noted in male mice exposed to 375, 750 or 1500 ppm. Females had a dose-dependent increase in estrous cycle length over the same concentrations.

Observed effects

A dose-related decrease in sperm mobility was noted in male mice exposed to 375, 750 or 1500 ppm for 6 hrs/day, 5 days/week for 13 weeks. Females had a dose-dependent increase in estrous cycle length when exposed to 375, 750 or 1500 ppm for the same time interval.

Any other information on results incl. tables

Summary of Reproductive Tissue Evaluations and Estrous Cycle Characterization for Mice in the 13 -Week Inhalation Study of Nitromethane

   0 ppm  375 ppm  750 ppm  1500 ppm
n  10  10  10  10
 Epididymal spermatozoal measurementsMotility (%)  93.50 + 0.46  85.09 + 1.21**  86.47 + 1.17**  82.42 + 1.30**
 n  10  10  10  10
 Estrous cycle length (days)  4.00 + 0.00c  4.33 + 0.14*d  4.50 + 0.21*  4.71 + 0.26**e

* Significantly different (P<0.05) from the control group by Shirley's test.

** P<0.01

c Estrous cycle was longer than 12 days or unclear in 2 of 10 animals.

d Estrous cycle was longer than 12 days or unclear in 1 of 10 animals.

e Estrous cycle was longer than 12 days or unclear in 3 of 10 animals.

Applicant's summary and conclusion

The lowest adverse effect concentration based on reproductive endpoints in the mouse 13 week NTP inhalation study was 375 ppm. In males, the percentage of epididymal spermatozoal motility was decreased in all three exposure groups. In females, the estrous cycle length was increased.
Executive summary:

The lowest adverse effect concentration based on reproductive endpoints in the mouse 13 week NTP inhalation study was 375 ppm. In males, the percentage of epididymal spermatozoal motility was decreased in all three exposure groups. In females, the estrous cycle length was increased.

The NTP study authors did not consider the findings related to sperm motility to have biologicial significance. Addditionally these effects were not considered toxiocological relevant because: 1) there was significant systemic toxicity particularly at the 1500 ppm concentration, which was clearly causing secondary effects in a number of organs,2)     the sample size was low (n=10) for this end point,3)    motility determined via hemacytometer can introduce technical variability as compared to automated systems, 4) there was a lack of dose response in the mice and 5)   there were no male reproductive organ histopathologic effects in the 2 -year studies.