Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP, guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
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 and environmental conditions:
Animals: CD rats (Crl: CD(SD) IGSBR) were approximately eight weeks when treatment was initiated. Examinations performed on all animals prior to the study start revealed that all animals were in good health for study purposes. The animals were acclimated to the laboratory for at least one week prior to the start of the study. Animals had free access to food and water (except during exposure, when both were withheld). Food and water had no contaminants at levels that would interfere with the conduct of this study or interpretation of the results.
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
Test material atmosphere: Exposure chambers were 2 cubic meter stainless steel and glass Rochester-type whole-body exposure chambers [1.3 meters (m) x 1.2 m wide x 1.2 m deep with a pyramidal top and bottom]. The various concentrations of 1-nitropropane were generated using the glass J-tube method (Miller et al., Am Ind Hyg Assoc J, 41:84-846,1980). Liquid test material was pumped into the glass J-tube assemblies (1 per exposure chamber) and vaporized by the flow of nitrogen gas passing through the bead bed of the glass J-tube. The nitrogen was heated as needed with a flameless heat torch to the minimum extent necessary to vaporize the test material. All chambers (including the filtered air control) received the same amount (20 liters per minute) of supplemental nitrogen. The minimum amount of nitrogen necessary to reach the desired chamber concentrations was used. The generation system was electrically grounded and the J-tubes were changed as needed. The vaporized test material and carrier gas were mixed and diluted with supply air to achieve a total flow of 450 liters per minute at the desired test chamber concentration. This flow rate was sufficient to provide the normal concentration of oxygen to the animals and 12-15 calculated air changes per hour. The chamber temperature and relative humidity were controlled by a system designed to maintain values of approximately 22 +/- 3 degrees C and 30 to 70%, respectively. The chambers were operated at a slightly negative pressure, relative to the surrounding area.
Details on mating procedure:
Animals were bred after approximately two weeks of treatment. Each female was placed with a single male from the same dose level (1:1 mating) until pregnancy occurred or two weeks had elapsed. During the breeding period, daily vaginal lavage samples were evaluated for the presence of sperm as an indication of mating. The day on which sperm were detected or a vaginal copulatory plug was observed in situ was considered gestation day (GD) 0. The sperm- or plug-positive (presumed pregnant) females were then separated from the males and returned to their home cages. If mating did not occur after two weeks, the animals were separated without further opportunity for mating.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The chamber concentrations of 1-nitropropane, measured approximately in the center of the breathing zone of the animals, were determined at least once per hour with a Miran 1A infrared (IR) spectrophotometer and reported by a strip chart recorder. The IR spectrophotometer was calibrated and a standard curve was compiled prior to and at the midpoint of the study, using air standards prepared by vaporizing measured volumes of 1-nitropropane into Tedlar sample bags along with the metered volumes of dry, compressed air. Analytical concentrations during the exposures were interpolated using the standard curve. The analytical system was checked prior to each exposure with a 1-nitropropane standard gasbag of known concentration. The nominal concentration of the test material in each chamber was estimated based on the amount of test material used and the total airflow through the chamber. Prior to the start of the study, each of the chambers was checked to ensure that a uniform distribution of vapor was present throughout the breathing zone of the animals.
Duration of treatment / exposure:
14 days prior to mating, during mating and to gestation day 19 (females), 14 days prior to mating and during mating (males)
Frequency of treatment:
6 hours/day, 7 days/week
Details on study schedule:
Groups of 12 male and 12 femaleCD rats were whole-body exposed to target concentrations of 0, 25, 50, and 100 ppm vaporized 1-nitropropane for six hours/day, seven days/week. Female rats were exposed daily for two weeks priorto breeding, through breeding (two weeks), and continuing through gestation day 19. Females werenecropsied on post-partum day 5. The males were exposed for two weeks prior to breeding andcontinuing through breeding (two weeks) until necropsy (test day 29). Effects on reproductive andneurological function as well as general toxicity were evaluated. In addition, post-mortemexaminations included a gross necropsy of the adults with collection of organ weights and extensivehistopathologic examination of tissues. Litter size, pup survival, sex, body weight, and the presence ofgross external abnormalities were also assessed.
Remarks:
Doses / Concentrations:0, 25, 50 and 100 ppmBasis:nominal conc.
Remarks:
Doses / Concentrations:0, 24, 48 and 96 ppmBasis:other: actual mean chamber concentrations
No. of animals per sex per dose:
12/sex/dose
Control animals:
yes, concurrent no treatment
Details on study design:
Study contact: Groups of 12 male and 12 female CD rats were whole-body exposed to target concentrations of 0, 25, 50, or 100 ppm vaporized 1-nitropropane for six hours/day, seven days per week. The concentrations used were based on the results of a previous range-finding study. The females were exposed daily for approximately two weeks prior to breeding, continuing through breeding (two weeks) and continuing through gestation day 19. The males were exposed beginning approximately two weeks prior to breeding and continuing through breeding (two weeks) for a minimum exposure period of 28 days. Animals were bred after approximately two weeks of treatment. Each female was placed with a single male from the same dose level (1:1 mating) until pregnancy occurred or two weeks had elapsed. During the breeding period, daily vaginal lavage samples were evaluated for the presence of sperm as an indication of mating. The day on which sperm were detected or a vaginal copulatory plug was observed in situ was considered gestation day (GD) 0. The sperm- or plug-positive (presumed pregnant) females were then separated from the males and returned to their home cages. If mating did not occur after two weeks, the animals were separated without further opportunity for mating.
Positive control:
No
Parental animals: Observations and examinations:
Once each day, a clinical examination was conducted at approximately the same time each afternoon following exposure. This examination included a careful hand-held evaluation of skin, respiration, nervous system function (including tremors and convulsions), swelling, masses, and animal behavior. Cage-side examinations was conducted twice daily and the following parameters were evaluated (if possible): skin, fur, mucous membranes, respiration, nervous system function (including tremors and convulsions), animal behavior, moribundity, mortality, and the availability of feed and water. Detailed clinical observations (DCO) were conducted on all rats pre-exposure, and then weekly throughout the study. Mated females were given detailed examinations on gestation days 0, 7, 14, and 20. Functional tests were conducted pre-exposure and during the last week of the treatment period. For male rats, this took place on test day 26. For female rats, this took place on lactation day (LD) 4. The functional tests included a sensory evaluation, rectal temperature, grip performance, and motor activity. All rats were weighed at least once during the pre-exposure period and on the first day of exposure. Body weights for males were recorded weekly throughout the course of the study. Females were weighed weekly during the premating and mating periods. During gestation, females were weighed on days 0, 7, 14, and 20. Females that delivered litters were weighed on days 1 and 4 post-partum. Females that failed to mate or deliver a litter were not weighed during the gestation or lactation phases. For males and females, feed consumption was determined weekly during the two week pre-breeding period by weighing feed containers at the start and end of a measurement cycle. During breeding, feed consumption was not measured in males or females due to co-housing. Following breeding, feed consumption was not measured for males. During gestation, feed consumption was measured for females on days 0-7, 7-14, and 14-20. After parturition, feed consumption was measured on LD 1 and 4. Feed consumption was not recorded for females that failed to mate or deliver a litter. Females were observed for signs of parturition beginning on or about GD 20. Insofar as possible, parturition was observed for signs of difficulty or unusual duration. The day of parturition was recorded as the first day the presence of the litter was noted and was designated as lacation day (LD) 0. Litters were examined as soon as possible after delivery. The following information was recorded on each litter: the date of parturition, litter size on the day of parturition (LD 0), the number of live and dead pups on days 0, 1, and 4 postpartum, and the sex and the weight of each pup on LD 1 and 4. Any visible physical abnormalities or demeanor changes in the neonates were recorded as they were observed during the lactation period. Any pups found dead were sexed and examined grossly, if possible, for external and visceral defects and were discarded.
Oestrous cyclicity (parental animals):
No data
Sperm parameters (parental animals):
The histopathological examination of the testes included a qualitative assessment of stages of spermatogenesis. A cross section through the approximate center of both testes of control and high-dose males were embedded in paraffin, sectioned at 5 microns and stained with modified periodic acid-Schiffs-hematoxylin. The presence and integrity of the 14 stages of spermatogenesis were qualitatively evaluated. Microscopic evaluation included a qualitative assessment of the relationships between spermatogonia, spermatocytes, spermatids, and spermatozoa seen in cross sections of the seminiferous tubules. The progression of these cellular association were defined the cycle of spermatogenesis. In addition, sections of both testes were examined for the presence of degenerative changes (e.g., vacuolation of the germinal epithelium, multinucleated giant cells, a decrease in the thickness of the germinal epithelium, a preponderance of Sertoli cells, sperm stasis, inflammatory changes, mineralization, and fibrosis)
Litter observations:
The following information was recorded on each litter: the date of parturition, litter size on the day of parturition (LD 0), the number of live and dead pups on days 0, 1, and 4 postpartum, and the sex and the weight of each pup on LD 1 and 4. Any visible physical abnormalities or demeanor changes in the neonates were recorded as they were observed during the lactation period. Any pups found dead were sexed and examined grossly, if possible, for external and visceral defects and were discarded.
Postmortem examinations (parental animals):
Males were euthanized on test day 29, while females that delivered litters were euthanized on post-partum day 5. Females that did not deliver a litter were euthanized at least 24 days after the last day of the mating period. All pups surviving to LD 4 were euthanized by oral administration of sodium pentobarbital solution, examined for gross external alterations, and then discarded. Necropsies were conducted on adult animals by a veterinary pathologist assisted by a team of trained individuals. The necropsy included an examination of the external tissues, and all orifices. The head was removed, the cranial cavity opened and the brain, pituitary, and adjacent cervical tissues were examined. The eyes were examined in situ by application of a moistened microscope slide to each cornea. The nasal cavity was flushed via the nasopharyngeal duct, and the lungs were distended to an approximately normal inspiratory volume with neutral, phosphate-buffered 10% formalin using a hand-held syringe and blunt needle. The thoracic and abdominal cavities were opened and the viscera examined. All visceral tissues were dissected from the carcass, re-examined and selected tissues were incised. The uteri of all females were examined and the number of implantation sites were recorded. The uteri of females that did not deliver litters were stained with a 10% solution of sodium sulfide in order to verify pregnancy status. The following tissues were trimmed and weighed: testes, epididymides, ovaries, liver, kidneys, adrenals, thymus, spleen, brain, thyroid/parathyroid (after fixation), and heart. The organ to body weight ratios were calculated. Similar necropsy procedures were followed for animals found dead or moribund with the exception that terminal body weights and organ weights were not collected. Representative samples of tissues [adrenals, aorta, auditory sebaceous glands, bone (including joint), bone marrow, brain (cerebrum, brainstem and cerebellum), cecum, cervix, coagulating glands, colon, cranial nerve (optic), duodenum, epididymides, esophagus, eyes, gross lesions, heart, ileum (with Peyer's Patch), jejunum, kidneys, lacrimal/Harderian glands, larynx, liver, lungs, mammary gland, mediastinal lymph node, mediastinal tissues, mesenteric lymph node, mestenteric tissues, nasal tissues/pharynx, oral tissues, ovaries, oviducts, pancreas, parathroid glands, peripheral nerve (tibial), pituitary, prostate, rectum, salivary glands, seminal vesicles, skeletal muscle, skin and subcutis, spinal acord (cervical, thoracic, lumbar), spleen, stomach, testes, thymus, thryoid gland, tongue, trachea, urinary bladder, uterus and vagina] were collected and preserved in neutral, phosphate-buffered 10% formalin, except that the testes and epididymides were preserved by immersion in Bouin's fixative. Histopathologic examination of these tissues was conducted on all adult rats from the control and high-dose groups. Examination of tissues from the remaining groups was limited to nasal tissues/pharynx and relevant gross lesions. Selected histopathologic findings were graded to reflect the severity of specific lesions to evaluate: 1) the contribution of a specific lesion to the health status of an animal, 2) exacerbation of common naturally occurring lesions as a result of the test material, and 3) dose-response relationships for treatment related effects. Very slight and slight grades were used for conditions that were altered from the normal textbook appearance of an organ/tissue, but were of minimal severity and usually with less than 25% involvement of the parenchyma. This type of change was not expected to significantly affect the function of the specific organ/tissue or have a significant effect on the overall health of the animal. A moderate grade was used for conditions that were of sufficient severity and/or extent (up to 50% of the parenchyma) that the function of the organ/tissue may have been adversely affected, but not to the point of organ failure. The health status of the animal may or may not have been affected, depending on the organ/tissue involved, but generally lesions graded as moderate were not life threatening. A severe grade was used for conditions that were extensive enough to cause significant organ/tissue dysfunction or failure. This degree of change in a critical organ/tissue may have been life threatening.The histopathological examination of the testes included a qualitative assessment of stages of spermatogenesis. A cross section through the approximate center of both testes of control and high-dose males were embedded in paraffin, sectioned at 5 microns and stained with modified periodic acid-Schiffs-hematoxylin. The presence and integrity of the 14 stages of spermatogenesis were qualitatively evaluated. Microscopic evaluation included a qualitative assessment of the relationships between spermatogonia, spermatocytes, spermatids, and spermatozoa seen in cross sections of the seminiferous tubules. The progression of these cellular association were defined the cycle of spermatogenesis. In addition, sections of both testes were examined for the presence of degenerative changes (e.g., vacuolation of the germinal epithelium, multinucleated giant cells, a decrease in the thickness of the germinal epithelium, a preponderance of Sertoli cells, sperm stasis, inflammatory changes, mineralization, and fibrosis).
Postmortem examinations (offspring):
Any pups found dead were sexed and examined grossly, if possible, for external and visceral defects and were discarded.
Statistics:
Statistical analyses of body weights and body weight gains during gestation were performed using data collected on days 0, 7, 14, and 20. Statistical analyses of body weight and body weight gains during the post-partum period were performed using data collected on days 1 and 4.Statistics continued below.
Reproductive indices:
The female and male mating, conception, and fertility indices and gestation index were calculated.
Offspring viability indices:
The gestation survival index, Day 1 or 4 pup survival index and post implatation loss were calculated.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Test material: Mean chamber concentration values for the study were 0, 24.4 +/- 1.8, 48.4 +/- 1.8, and 96.3 +/- 2.6 ppm. Actual mean chamber concentration values deviated 2-4% from the targeted values of 0, 25, 50, and 100 ppm. Chamber airflow in all four chambers was maintained throughout the study duration. Mean chamber temperatures for all chambers were maintained between 20 and 23 C. Mean chamber relative humidity was maintained in the range of 33-44% for all exposure chambers. On one exposure day the mean relative humidity of the 25, 50, and 100 ppm chambers was below 30% (29.4, 29.4, and 28.1%, respectively). This single, minor excursion from the protocol-specified range of 30-70% is not considered to have affected the integrity of the study.Effects at 96 ppm: All animals survived until termination. There were no treatment-related effects at any exposure level on reproductive indices, time to mating, gestation length, post-implantation loss, pup survival, pup sex ratio or histological examination of reproductive organs. Although conception and fertility indices of males and females exposed to 96 ppm 1-nitropropane (83.3%) were numerically (but not statistically) lower than those of the controls, this was most likely due to the fact that the control group values were quite high (100%). The conception and fertility indices in the 96 ppm group were within the range of historical control values from four OECD 422 studies conducted by the laboratory from 2000 - 2004 (83.3 -100%). The mean number of pups born live (11.9/litter) and the mean number of pups on day 1 and 4 postpartum (both 11.8/litter) were less than controls (14.0/litter and 13.8/litter, respectively). Although these differences were not statistically significant, each of these values was outside the range of the historical control data from recently completed OECD 422 studies (13.3 - 15.6 fetuses born live/litter, 12.8 - 15.5 alive on day 1/litter and 12.5 - 15.5 alive on day 4/litter). Inspection of the individual animal data revealed that 3 of 10 dams in this group had litter sizes of fewer than 12 pups, whereas in the control, 24 and 48 ppm groups the number of dams producing fewer than 12 pups was 1/12, 1/12 and, 0/10, respectively. Mean male and female pup weights were increased relative to the controls on days 1 (7.3 and 6.9 g in exposed males and females compared to 6.7 and 6.3 g in control males and females) and 4 postpartum (10.4 and 9.7 g in exposed males and females compared to 9.2 and 8.8 g in control males and females) in dams exposed to 96 ppm 1-nitropropane. The mean pup weight values at 96 ppm were within the historical control range from recently completed OECD 422 studies, while the control values were outside the historical range. Therefore, the changes in pup body weights were not considered adverse.Feed consumption was reduced during the pre-breeding phase on test days 1-7 and 7-14 in males and on days 1-7 in females. Average body weight of males was decreased (6.9%) on test day 7. Males exposed to 96 ppm had higher relative brain and testes weights. There were no treatment related gross pathologic observations. Treatment-related histopathologic effects were noted in the nasal tissues of one male and one to seven females exposed to 96 ppm (see Section 5.4 for additional details. The severity of all lesions was graded very slight to slight. There was no effect of treatment on histopathology of any reproductive organ examined. Effects at 48 ppm: Treatment-related histopathologic effects were noted in the nasal tissues of a few females exposed to 48 ppm (see Section 5.4 for additional details). There was no effect of exposure on histopathology of reproductive organs. Although conception and fertility indices of males and females exposed to 48 ppm 1-nitropropane (83.3%) were numerically (but not statistically) lower than those of the controls, this was most likely due to the fact that the control group values were quite high (100%). The conception and fertility indices in the 96 ppm group were within the range of historical control values from four OECD 422 studies conducted by the laboratory from 2000 - 2004 (83.3 -100%). Effects at 24 ppm: Very slight, chronic, active, multifocal or focal inflammation of the squamous epithelium of the nasal tissue was noted in one female. There were no effects on any reproductive parameter.Effects in controls: Very slight, chronic, active, multifocal or focal inflammation of the squamous epithelium of the nasal tissue was noted in one and two females, respectively. There were no effects on any reproductive parameter.
Key result
Dose descriptor:
NOEC
Remarks:
general toxicity
Effect level:
50 ppm (nominal)
Sex:
male
Basis for effect level:
other: Based on histologic changes in the nasal tissues.
Key result
Dose descriptor:
NOEC
Remarks:
general toxicity
Effect level:
25 ppm (nominal)
Sex:
female
Basis for effect level:
other: Based on histologic changes in the nasal tissues.
Key result
Dose descriptor:
NOEC
Remarks:
reproductive effects
Effect level:
50 ppm (nominal)
Sex:
female
Basis for effect level:
other: Based on differences in litter size between high-dose animals and controls.
Key result
Dose descriptor:
NOEC
Remarks:
neurologic function
Effect level:
100 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: The highest concentration tested.
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
In the high-exposure group, the mean number of pups born live and the mean number of pups on day 1 and 4 postpartum were less than controls. Although these differences were not statistically significant, each of these values was outside the range of the historical control data from recently completed OECD 422 studies. Inspection of the individual animal data revealed that 3 of 10 dams in this group had litter sizes of fewer than 12 pups, whereas in the control, 25 and 50 ppm groups the number of dams producing fewer than 12 pups was 1/12, 1/12 and, 0/10, respectively. The toxicological significance of this finding is equivocal, as there were no apparent effects on any other reproductive parameter examined, nor were there any corroborating findings revealed at gross or histopathological examination. When considered together, these results suggest that the slightly reduced litter size in animals exposed to 100 ppm 1-nitropropane was likely secondary to maternal toxicity and/or stress associated with nasal irritation. Mean Litter Size Exposure Level (ppm)Parameter (mean values) 0 25 50 100No. Born Live 14.0 14.3 15.1 11.9Day 1 13.8 14.3 15.1 11.8Day 4 13.8 14.1 15.1 11.8 Historical Control Data for Mean Litter SizeStudy #Year 1 2 3 4 Range 2000 2003 2004 2004 (Average) No. Born Live 13.6 15.1 15.6 13.3 13.3-15.6 (14.4)Day 1 13.4 15.1 15.5 12.8 12.8-15.5 (14.2)Day 4 13.4 14.9 15.5 12.5 12.5-15.5 (14.1)Mean male and female pup weights were statistically identified as increased relative to the controls on days 1 and 4 postpartum in dams exposed to 100 ppm 1-nitropropane. The increase in mean pup weights at 100 ppm was most likely secondary to the smaller litter size in the 100 ppm group, as pup weight varies inversely with litter size. The mean pup weight values at 100 ppm were within the historical control range from recently completed OECD 422 studies, while the control values were outside the historical range. Therefore, the changes in pup body weights were not considered adverse. No significant differences were noted at the lower exposure levels. Mean Pup Weights Exposure Level (ppm)Parameter (mean values) 0 25 50 1001 Female (g) 6.3 6.5 6.2 6.9*1 Male (g) 6.7 6.9 6.6 7.3*4 Female (g) 8.8 9.2 8.6 9.7*4 Male (g) 9.2 9.7 9.2 10.4** Statistically different from control mean by Dunnett’s test, alpha = 0.05. Historical Control Data for Mean Pup WeightsStudy # 1 2 3 4 RangeYear 2000 2003 2004 2004 (Average)1 Female (g) 6.9 6.5 6.6 7.0 6.5-7.0 (6.8)1 Male (g) 7.3 7.0 7.0 7.4 7.0-7.4 (7.2)4 Female (g) 9.8 9.1 9.1 10.1 9.1-10.1 (9.5)4 Male (g) 10.2 9.6 9.7 10.7 9.6-10.7 (10.1)
Reproductive effects observed:
not specified

This summary only describes procedures and results related to fertility.  Repeated dose and developmental data are discussed in Sections 5.4 and 5.8.2, respectively.

The authors considered the toxicological significance of reduced litter sizes at 96 ppm as equivocal, as there were no apparent effects on any other reproductive parameter examined, nor were there any corroborating findings revealed at gross or histopathological examination.
  The authors suggested that the slightly reduced litter size in animals exposed to 96 ppm 1-nitropropane was likely secondary to maternal toxicity and/or stress associated with nasal irritation. The authors also stated that the increases in mean pup weights at 96 ppm were most likely secondary to the smaller litter size in the 96 ppm group, as pup weight varies inversely with litter size.

Conclusions:
The no-observed-effect concentration (NOEC) for general toxicity, based on histologic changes in the nasal tissues, was 50 ppm for males and 25 ppm for females. The NOEC for reproductive effects, based on differences in litter size between high-dose animals and controls, was 50 ppm. The NOEC for neurologic function was 100 ppm, the highest concentration tested.
Executive summary:

None

Effect on fertility: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
182 mg/m³
Species:
rat
Quality of whole database:
Good
Additional information

The subchronic toxicity of nitroethane was examined in mice and rats. In the 13-week study mice and rats were exposed to 0, 100, 350, or 1000 ppm (0, 0.3, 1.0 or 3.0 mg/L) of NE for 6 hr/day, 5 days/wk for a total of 64-65 exposures with an interim sacrifice of mice and rats after 20-21 exposures. Parameters monitored were clinical observations, body weights, organ weights, hematologic characteristics including methemoglobin (MetHb) determination, clinical chemistries, (urinalysis – rats) gross pathology and histopathology.

Reproduction related alterations due to nitroethane exposure occurred in the testes of most male mice of the 1000 ppm group and consisted of the presence of multinucleated spermatids. There was no gross or histopathologic changes noted in male or female rats exposed to concentrations as high as 1000 ppm nitroethane for 6 hours/day, 5 days/week for 13 weeks.


Short description of key information:
Read across from 1-nitropropane (see Section 13): No treatment effects were seen in reproductive performance, pup survival and growth, neurologic function, clinical chemistry, or hematology. A very slight decrease in litter size was seen in animals exposed to 100 ppm 1-nitropropane. However, given the lack of other effects on reproductive performance or gross/histologic findings in the reproductive organs, the slight decrease in litter size may have been secondary to maternal toxicity and/or stress in the high-dose females. Nevertheless the NOEC for reproductive effects, based on differences in litter size between high-dose animals and controls assigned by the author, was 50 ppm.

Reproduction related alterations due to nitroethane exposure occurred in the testes of most male mice of the 1000 ppm group and consisted of the presence of multinucleated spermatids. There was no gross or histopathologic changes noted in male or female rats exposed to concentrations as high as 1000 ppm nitroethane for 6 hours/day, 5 days/week for 13 weeks.

Justification for selection of Effect on fertility via inhalation route:
Read across from 1-nitropropane: GLP, guideline study

Effects on developmental toxicity

Description of key information

Read-across from study with nitromethane

A developmental toxicity study according OECD 414 study was conducted to study the possible effects of nitromethane on pregnant female rats (Wistar Han IGS), and on the development of the embryo and fetus. The test material was administered by inhalation via whole body exposure to groups of 24 mated females from gestation day (GD) 6 up to and including GD 20. The overall average actual concentrations of the test material in the test atmospheres as determined by total carbon analysis were 303 (± 3.3), 601 (± 12.4) and 1178 (± 43.9) ppm for the low-, mid- and high concentration, respectively and close to the respective target concentrations of 0, 300, 600 or 1200 ppm. At gestation day 21 caesarean section was performed, dams were examined macroscopically and reproductive organs, liver and kidney were weighed. Fetuses and placentas were weighed and fetuses were examined externally. Half of the fetuses of each litter were subjected to visceral examination, the other half to skeletal examination.Based on the lower body weight and body weight gain at the end of gestation and the lower feed intake at the start of exposure and the end of gestation in the high concentration group, the No Observed Adverse Effect Concentration (NOAEC) for maternal toxicity was considered to be 600 ppm. Based on the effects on post-implantation loss (as evidenced by an increase in late resorptions), fetus weight, the delay in ossification and skeletal and visceral observations in the high concentration group, the NOAEC for developmental toxicity was considered to be 600 ppm.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April - August 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
An inhalation developmental toxicity with nitromethane in rats has been conducted. This study is used as read-acrcoss in this dossier. A document justifying the category/read-across approach has been attached.
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
The study was conducted with albino rats. The rat was used because this species is considered suitable for this type of study, and is usually required by regulatory agencies. Male and female Wistar Han IGS rats (Crl:WI(Han)) were obtained from a colony maintained under SPF-conditions at Charles River Deutschland, Sulzfeld, Germany. This rat strain was used because it is routinely used at the test facility for this type of studies. In the study the female rats were about 10 weeks old and the male rats about 11 weeks old at arrival. The age difference between males and females was deliberate, to avoid mating of siblings. Male animals were used for mating only and were not part of the study otherwise. Upon arrival, the animals were housed in quarantine and checked for overt signs of ill health and anomalies. During the quarantine period, serological investigation of the microbiological status were carried out in a few randomly chosen rats of the lot delivered. Upon satisfactory serology results, the animals were transferred to their definitive animal room (or the quarantine room will be cleared for use as experimental room). The rats were acclimatized to the laboratory conditions for at least 5 days prior to the start of the exposure.

The animals used for the study were housed under conventional conditions in one animal room. The room was ventilated with about 10 air changes per hour and was maintained at a temperature of 20-24oC and a relative humidity of at least 45-65%. Lighting was artificial with a sequence of 12 hours light and 12 hours dark.
Route of administration:
inhalation
Type of inhalation exposure (if applicable):
whole body
Vehicle:
air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
The inhalation equipment was designed to expose the animals to a continuous supply of fresh test atmosphere. To generate the test atmospheres, a liquid flow of test material, controlled by a peristaltic pump (Watson-Marlow type 502s, Bredel Pumps Limited, Falmouth, Cornwall, England) was allowed to evaporate in a mass flow controlled (Bronkhorst Hi Tec, Ruurlo, the Netherlands) stream of dry compressed air, by directing it through a glass evaporator which was kept at a constant temperature of 37˚C by circulating heated water. For safety reasons, this was performed in a safety cabinet. The resulting test atmosphere was mixed with the main air stream (available as a laboratory provided source of non-pressurized, HEPA-filtered air), which was subsequently directed to the inlets at the top of the exposure chamber; the atmosphere was exhausted at the bottom. The exposure unit for the control animals was supplied with a controlled flow of HEPA-filtered air only.

The flow of the test atmosphere was controlled using a constant volume controller and was measured in the exhaust of the exposure chamber using a KIMO air velocity sensor (type CTV110-AOD150; KIMO, Emerainville, France). The air flow was continuously measured and recorded on a PC every minute, using a CAN transmitter (G. Lufft Mess- und Regeltechnik GmbH, 70719 Felbach, Germany). The animals were placed in the exposure chamber prior to the start of the test atmosphere generation. Test atmosphere generation was stopped six hours after the start of generation. The animals were removed from the exposure chamber after the concentration had dropped below a level of 1% of the target concentration.

Prior to the first exposure of the animals, homogeneous distribution of the test material in the exposure chambers was confirmed by analysis of samples taken at five different locations in each exposure chamber (deviation of individual values from the mean of all five samples should not exceed 10%; the actual maximum deviation was 4.8% for the low-concentration group).

TEST ATMOSPHERE
The actual concentration of the test material in the test atmospheres was measured by total carbon analysis (Sick Maihak GMS 810 EuroFID Total Hydrocarbon Analyzer; Sick Instruments Benelux, Hedel, the Netherlands). Test atmosphere samples were taken continuously from the exposure chamber at the animals’ breathing zone and were passed to the total carbon analyzer (TCA) through a sample line. The response of the analyzers was recorded on a PC at one minute intervals using a CAN transmitter (G. Lufft Mess- und Regeltechnik GmbH, 70719 Felbach, Germany). The responses of the analyzers were converted to concentrations by means of calibration graphs (the formulas used to convert responses into concentrations are given below). For each exposure day, the mean concentration was calculated from the values determined every minute. The average concentration during exposure was corrected for the duration of the animals’ stay in the exposure chamber and the number of measurements during exposure.

TOTAL AIR FLOW, TEMPERATTURE, RELATIVE HUMIDITY, OXYGEN AND CARBON DIOXIDE CONCENTRATION
The total flow of test atmosphere during exposure was continuously measured using air velocity sensors (type CTV110-AOD150; KIMO, Emerainville, France) in the chamber exhaust. The measurements of the air velocity sensors were recorded on a PC every minute using CAN transmitters (G. Lufft Mess- und Regeltechnik GmbH, 70719 Felbach, Germany). The responses of the sensors were converted to air flows by means of calibration graphs. Prior to the start of the exposure, the air velocity sensors were calibrated by measuring the response at a range of air flows (encompassing the flows used during the study) generated using mass flow controlled streams of clean dry air.

Temperature and relative humidity of the test atmospheres were measured continuously and recorded every minuteat one minute intervals using a CAN transmitter with temperature and relative humidity probes (G.Lufft Mess- und Regeltechnik GmbH, 70719 Fellbach, Germany).

The concentrations of oxygen (Oxygen analyzer type PMA-10, M&C Products Analysentechnik GmbH, Ratingen-Lintorf, Germany) and carbon dioxide (GM70 probe with MI70 read-out unit, Vaisala, Helsinki, Finland) in the test atmosphere were measured two times for each group, once during the first and once during the last week of the exposure period.


Prior to the first exposure, the output of the flame ionization detector of each TCA was dynamically calibrated. To this end, test atmospheres were generated at known concentrations by evaporating a flow of liquid test material, controlled using by a motor-driven syringe pump (WPI Type SPLG110, World Precision Instruments, Sarasota FL, USA) in a mass flow controlled (Bronkhorst Hi Tec) stream of compressed dry air, by directing it through a stainless steel tube which was kept at an above-ambient temperature to facilitate the evaporation (25-37°C) using a water mantle. The resulting test atmosphere was led directly to the TCA; excess air was exhausted. The mass flow controller was calibrated using a volumetric flow meter (DryCal, Bios International Corporation, Butler, NJ, USA) at the flow settings used for calibrating the TCA. The calibration settings were selected to generate about 80%, 100% and 120% of each target concentration. A zero calibration was included for each TCA, using clean dry air only. Linear relations were found between the response Y of the analyzers (in % of full scale) and the concentration X of the test material (in ppm; see below).

The calibrations were checked weekly during the study. To this end, a test atmosphere was generated at each target concentration as described above. The concentration as calculated from the measured flow of the mass flow controller and the settings of the pump, was compared to the concentration as measured by the analyzers. If the measured concentration deviated more than 5% from the calculated concentration, the calibration check was repeated. If the deviation was more than 5% at the re-check, a complete re-calibration was carried out.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The overall average actual concentrations (± standard deviation) of nitromethane in the test atmospheres, as determined by total carbon analysis, were 303 (± 3.3), 601 (± 12.4) and 1178 (± 43.9) ppm for the low-, mid-, and high concentration, respectively (Table 1.1). These concentrations were close to the respective target concentrations of 300, 600 and 1200 ppm.
Details on mating procedure:
Upon arrival, the animals were housed in quarantine and checked for overt signs of ill health and anomalies. During the quarantine period, serological investigation of the microbiological status were carried out in a few randomly chosen rats of the lot delivered. Upon satisfactory serology results, the animals were transferred to their definitive animal room. The rats were acclimatized to the laboratory conditions for at least 5 days prior to the start of the exposure. At the end of the acclimatisation period, males and females were mated (two females were caged with one male). Animals were caged together until mating occurred. Every consecutive morning during the mating period, vaginal smears were made for determination of the presence of sperm. The day on which sperm was detected in the vaginal smear was considered as gestation day 0 (GD 0). Upon evidence of copulation the females were caged individually. Ninety-six mated females out of 104 females were distributed in an unbiased manner by manual randomization over the 4 experimental groups (24 mated females/group) in such a way that the animals from the same day of pregnancy were equally distributed over all groups. Females mated by the same male were placed in different groups. The remaining females were discarded after mating.
Duration of treatment / exposure:
The study comprised 4 groups of 24 mated females each, viz. one control group and three test groups receiving different concentrations of test material from gestation day (GD) 6 up to and including GD 20 (weekend and holidays included). The animals of the control group were handled in the same manner as those of the other groups, except for exposure to the test substance.
Frequency of treatment:
Animals were exposed for 6 hours per day from GD 6 up to and including GD 20.
Duration of test:
Exposure from gestation day (GD) 6 up to and including GD 20.
Dose / conc.:
0 ppm (nominal)
Dose / conc.:
300 ppm (nominal)
Dose / conc.:
600 ppm (nominal)
Dose / conc.:
1 200 ppm (nominal)
No. of animals per sex per dose:
In each group 24 females were used.
Control animals:
yes, sham-exposed
Details on study design:
DOSE SELECTION RATIONALE
In a dose range finding study for the OECD 414 study, mated females were exposed to concentrations of 25, 100 and 300 ppm nitromethane from gestation day 6 to gestation day 20. No treatment-related effects were observed in this study. In a subsequent main prenatal development study, mated females were exposed to concentrations of 0, 100, 300 and 600 ppm nitromethane from gestation day 6 to gestation day 20. No treatment-related effects were observed in this study on maternal parameters (body weight gain, food consumption, clinical observations) and litter parameters (number and distribution of live and dead fetuses, resorptions).

In a sub-chronic inhalation study with nitromethane, Fischer 344 rats were exposed to concentrations of 94, 188, 375, 750 and 1500 ppm, 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. 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. 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.

Following Haber’s rule, based on an No-Effect-Level of 94 ppm in the 90-day study, the No-Effect-Level in the 14-day exposure period in the OECD 414 study in rats would be expected around 600 ppm. Exposure to 600 ppm did not result in maternal effects in terms of body weight, food consumption or clinical signs. Taking into account both these results and Haber’s rule, a concentration of 600 ppm was selected as mid concentration level. As high concentration a level of 1200 ppm was selected, whereas 300 ppm was selected as low concentration level.
Maternal examinations:
CAGE SIDE OBSERVATIONS
Each animal was observed daily in the morning hours (pre-dosing) by cage-side observations and, if necessary, handled to detect signs of toxicity. The animals were also observed about halfway through the 6-hour exposure period, in particular to monitor any breathing abnormalities and restlessness. All animals were also thoroughly checked again after exposure. All abnormalities, signs of ill health, reaction to treatment and mortality were recorded.

BODY WEIGHT
Body weights of the parental female animals was recorded on gestation days (GD) 0, 6, 9, 12, 15, 18 and 21.

FOOD CONSUMPTION AND COMPOUND INTAKE
The food consumed for each mated female was measured over the periods: GD 0-6, GD 6-9, GD 9-12, GD 12-15, GD 15-18 and GD 18-21. The results are expressed in gram per animal per day.

POST-MORTEM EXAMINATIONS:
The females were killed by exsanguination after isofluorane anaesthesia on GD 21 and examined for gross abnormalities. The female showing signs of premature delivery was also examined macroscopically, and the number of fetuses, corpora lutea and implantations were recorded.
Blood was collected from all dams at necropsy and plasma (using heparin as anticoagulant) and serum samples were stored in a freezer (≤-18°C) for possible future analysis. The samples will be discarded after authorization of the report.

From all animals the following organs were preserved in formalin for possible future histopathological examination:
- nose
- larynx
- trachea
- lungs
- liver
- sciatic nerve
- gross lesions

All females killed on GD 21 were examined for the following parameters:
- number of corpora lutea
- number of implantation sites
- number of early and late resorptions
- number of live and dead fetuses
- sex of the fetuses
- number of grossly visible malformed fetuses and fetuses with external abnormalities
- gross evaluation of placentas.
- abnormal tissues or organs in dams

The following organs were weighed:
- kidneys
- liver
- uterus, containing placentas and fetuses
- uterus, empty
- ovaries
- live fetuses (individually) with corresponding placentas

If necessary the implantation sites were made visible following Salewski E. (1964).
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: yes
Examinations included:
- Gravid uterus weight: yes
- Number of corpora lutea: yes
- Number of implantations: yes
- Number of early resorptions: yes
- Number of late resorptions: yes
Fetal examinations:
Fetuses were examined for external alterations and sacrificed by hypothermia Subsequently, approximately half of the fetuses of each litter was fixed in Bouin's fixative, examined for soft tissue anomalies according to a method modified after Barrow and Taylor (1969)5 and then discarded. Abnormal tissues were preserved in Bouin’s fixative. The other half of the fetuses were fixed in 70% alcohol, subsequently partly eviscerated, and then cleared in potassium hydroxide and stained with Alizarin Red S modified after Dawson (1926)6. They were examined for skeletal abnormalities and then retained. During the fetopathological examination, the observer was unaware of the dose group of the fetuses.

Indices:
The following parameters were calculated:
- pre-implantation loss = [(number of corpora lutea – number of implantation sites) / number of corpora lutea] x 100
- post-implantation loss = [(number of implantation sites- number of live fetuses)/number of implantation sites] x 100
- gestation index = (number of females with live fetuses / number of females pregnant) x 100
- sex ratio = [(number of live male fetuses / number of live fetuses)] x 100

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical abnormalities were noted during exposure. There were no treatment-related clinical abnormalities directly after exposure.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weight was statistically significantly lower in the high concentration group at the end of gestation (e.g. gestation days 18 and 21) as compared to the control group (13% reduction on day 21). Mean body weight changes were statistically significantly lower in the high concentration group in the intervals from gestation day 6-9, 15-18, 18-21 and also in the overall gestation period (38% reduction) as compared to the control group. No effects on body weight or body weight changes were observed in the low and mid concentration groups.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Mean food consumption was statistically significantly lower in the high concentration group as compared to the control group at the start of exposure (gestation days 6-8) and at the end of gestation (18-21). The reduction in food consumption was 11% on day 21. No effects on food consumption were observed in the low and mid concentration groups.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Mean terminal body weight was statistically significantly lower in the high concentration group as compared to the control group. The mean weight of the full uterus (including fetal and placental tissues) and empty uterus were statistically significantly lower in the high concentration group as compared to the control group. This was considered to be related to the increased number of late resorptions observed in this group. No effects on uterus weight were observed in the low and mid concentration groups.

Mean absolute ovary weight in the high concentration group was comparable to the control group, whereas relative ovary weight was statistically significantly increased. Mean absolute liver weight in the high concentration group was comparable to the control group, whereas relative liver weight was statistically significantly increased. The statistically significant differences in relative ovary weight and liver weight in the high concentration group are related to the lower terminal body weight in that group and are not considered to be treatment-related effects.

Mean absolute kidney weight was statistically significantly increased in all treatment groups and the effect was considered concentration-dependent. Therefore, the kidney was considered to be a target organ. Considering the limited increase in mean absolute kidney weight and the absence of statistically significant changes in relative kidney weight, the effect was not considered adverse in the low and mid concentration groups . In the high concentration group relative kidney weight was statistically significantly increased. This was considered related to the lower mean terminal body weight in this group. Therefore, the effect was considered not adverse.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
no effects observed
Description (incidence and severity):
No effects were observed on the mean number of corpora lutea and the mean number of implantation sites.
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
No effects were observed on the mean pre-implantation loss. The mean post-implantation loss was statistically significantly increased in the high concentration group.
Total litter losses by resorption:
effects observed, treatment-related
Description (incidence and severity):
Total litter loss was observed in 5 out of 22 pregnant females of the high concentration group.
Early or late resorptions:
effects observed, treatment-related
Description (incidence and severity):
The mean number of early resorptions and dead fetuses was comparable in all groups. The mean number of late resorptions was statistically significantly increased in the high concentration group.
Dead fetuses:
no effects observed
Description (incidence and severity):
The mean number of dead fetuses was comparable in all groups.
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Changes in number of pregnant:
not examined
Other effects:
not examined
Key result
Dose descriptor:
NOAEC
Effect level:
600 ppm (nominal)
Based on:
test mat.
Basis for effect level:
other: no effects observed at this level
Key result
Dose descriptor:
LOAEC
Effect level:
1 200 ppm (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
uterus
Description (incidence and severity):
The mean weight of the full uterus (including fetal and placental tissues) and empty uterus were statistically significantly lower in the high concentration group as compared to the control group. This was considered to be related to the increased number of late resorptions observed in this group. No effects on uterus weight were observed in the low and mid concentration groups.
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean fetus weight was statistically significantly decreased in fetuses in the high concentration group, for both male and females fetuses. This effect is considered to be related to treatment. No effects on fetus weight were observed in the low and mid concentration groups.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
The reduction in the number of live offspring was statistically significantly decreased in fetuses in the high concentration group.
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
effects observed, treatment-related
Changes in postnatal survival:
not examined
External malformations:
effects observed, treatment-related
Description (incidence and severity):
One fetus in the high concentration group showed sub-cutaneous oedema, which is classified as a malformation. One fetus in the control group and one in the high concentration group showed subcutaneous hemorrhageshaemorrhages. In addition, 13 fetuses in the high concentration group were pale. This was a statistically significant increased number as compared to the control group. No external malformations and variations were observed in the low and mid concentration groups.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Observations in skeletal examinations indicated a delay in ossification, which was considered to be related to the smaller fetus size. In addition, the number of fetuses showing wavy ribs or bent radius and ulna bones were increased in the high concentration group. Wavy ribs and bending of the long bones are commonly observed skeletal variations in this strain of rats and are considered to be skeletal observations that are secondary to developmental toxicity.
Visceral malformations:
no effects observed
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEC
Effect level:
600 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed at this level
Key result
Dose descriptor:
LOAEC
Effect level:
1 200 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
fetal/pup body weight changes
changes in litter size and weights
changes in postnatal survival
external malformations
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: sternum
skeletal: hindlimb
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
1 200 ppm (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
no
Relevant for humans:
not specified
Conclusions:
Based on the lower body weight and body weight gain at the end of gestation and the lower feed intake at the start of exposure and the end of gestation in the high concentration group, the No Observed Adverse Effect Concentration (NOAEC) for maternal toxicity was considered to be 600 ppm. Based on the effects on post-implantation loss (as evidenced by an increase in late resorptions), fetus weight, the delay in ossification and skeletal and visceral observations in the high concentration group, the NOAEC for developmental toxicity was considered to be 600 ppm.
Executive summary:

The objective of this study was to provide data on the possible effects of the test material nitromethane on pregnant female rats (Wistar Han IGS), and on the development of the embryo and fetus. The test material was administered by inhalation (via whole body exposure) to groups of 24 mated females from gestation day (GD) 6 up to and including GD 20. The overall average actual concentrations of the test material in the test atmospheres as determined by total carbon analysis were 303 (± 3.3), 601 (± 12.4) and 1178 (± 43.9) ppm for the low-, mid- and high concentration, respectively and close to the respective target concentrations of 0, 300, 600 or 1200 ppm. At gestation day 21 caesarean section was performed, dams were examined macroscopically and reproductive organs, liver and kidney were weighed. Fetuses and placentas were weighed and fetuses were examined externally. Half of the fetuses of each litter were subjected to visceral examination, the other half to skeletal examination.

 

Daily whole body exposure (6 hours per day) to nitromethane from gestation day 6 up to and including gestation day 20 at concentrations of 0, 300, 600 and 1200 ppm resulted in:

-     No treatment related clinical signs or macroscopic changes in the dams;

-     Lower food consumption at the onset of treatment and at the end of gestation in the 1200 ppm group;

-     Lower body weight and body weight gain at the end of gestation in the 1200 ppm group;

-     Lower mean uterus weight in the 1200 ppm group, which was related to the increased post-implantation loss;

-     The kidney was identified as the target organ, as evidenced by a slight increase in kidney weight in all treatment groups. In view of the limited increase, this was not considered an adverse effect;

-     No effects on the mean number of corpora lutea or the mean number of implantation sites;

-     A marked, statistically significant, increase in post-implantation loss in the 1200 ppm group, due to a statistically significant increase in late resorptions and the complete litter loss of 5 females;

-     Statistically significantly decreased mean fetus weight in the 1200 ppm group.

-     Delayed ossification in the fetuses in the 1200 ppm group. In addition, the visceral observations and skeletal observations in the 1200 ppm group, were indicative for fetal toxicity or considered secondary to the fetal toxicity observed in this group.

-     No effects on fetal weight, visceral observations and skeletal observations were observed in the 300 and 600 ppm groups.

 

Based on the lower body weight and body weight gain at the end of gestation and the lower feed intake at the start of exposure and the end of gestation in the high concentration group, the No Observed Adverse Effect Concentration (NOAEC) for maternal toxicity was considered to be 600 ppm. Based on the effects on post-implantation loss (as evidenced by an increase in late resorptions), fetus weight, the delay in ossification and skeletal and visceral observations in the high concentration group, the NOAEC for developmental toxicity was considered to be 600 ppm.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
1 500 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
Study according to OECD 414 guideline and under GLP.

Justification for classification or non-classification

In the developmental toxicity study (OECD 414) with nitromethane, daily whole body exposure (6 hours per day) to nitromethane from gestation day 6 up to and including gestation day 20 at concentrations of 0, 300, 600 and 1200 ppm resulted in the following effects:lower food consumption (11% reduction) in the 1200 ppm group; lower body weight (13% reduction) and body weight gain at the end of gestation in the 1200 ppm group; lower mean uterus weight in the 1200 ppm group, which was related to the increased post-implantation loss; amarked, statistically significant, increase in post-implantation loss in the 1200 ppm group, due to a statistically significant increase in late resorptions and the complete litter loss of 5 females; statistically significantly decreased mean fetus weight in the 1200 ppm group; delayed ossification in the fetuses in the 1200 ppm group. In addition, visceral observations and skeletal observations in the 1200 ppm group, which were indicative for fetal toxicity or considered secondary to the fetal toxicity observed in this group.Based on the reduction in body weight and body weight gain and the lower feed intake n the high concentration group, the No Observed Adverse Effect Concentration (NOAEC) for maternal toxicity was considered to be 600 ppm. Based on the effects on post-implantation loss (as evidenced by an increase in late resorptions), fetus weight, the delay in ossification and skeletal and visceral observations in the high concentration group, the NOAEC for developmental toxicity was considered to be 600 ppm.

 

In view of theabove results of the developmental toxicity study,it is proposed to self-classify nitroethane as a Repro tox cat. 2 according to the CLP/GHS Regulation.