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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP, followed guideline OECD 422 (Note- Fetuses were not examined for skeletal and visceral abnormalities.)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline 422
Deviations:
no
Remarks:
Not specified in report
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
A final purity of 99.69% +/- 0.01% [determined by gas chromatography with thermal conductivity (GC/TCD)] corrected for water, was reported for the sample. The water content of the sample was 0.05%. Infrared spectroscopy (FTIR) and gas chromatographic mass spectroscopy (GC/MS) were used to confirm the proposed structure.

Test animals

Species:
rat
Strain:
Sprague-Dawley
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.

Administration / exposure

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.
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.
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.
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
Duration of test:
Females were necropsied on post-partum day 5. The males were exposed for two weeks prior to breeding and continuing through breeding (two weeks) until necropsy (test day 29). Duration of exposure is to gestation day 19.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 25, 50 and 100 ppm
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0, 24, 48 and 96 ppm
Basis:
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.

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.

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. 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.

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.

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).

Examinations

Maternal 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.
Ovaries and uterine content:
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 (Kopf et al., 1964).
Fetal examinations:
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. All pups surviving to LD 4 were euthanized by oral administration of sodium pentobarbital solution, examined for gross external alterations, and then discarded.
Statistics:
See below
Indices:
The female and male mating, conception, and fertility indices, gestation index, gestation survival index, Day 1 or 4 pup survival index and post implatation loss were calculated.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
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.

Effect levels (maternal animals)

Dose descriptor:
NOEC
Remarks:
general toxicity
Effect level:
25 ppm (nominal)
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Effect levels (fetuses)

Dose descriptor:
NOEC
Effect level:
100 ppm (nominal)
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

This study is described in detail in Section 7.8.1.  The only effect that was considered to be related to exposure was reduced mean litter size of rats exposed to 48 ppm.  There is no mention of any effect of exposure on external examinations of pups.

Applicant's summary and conclusion

Conclusions:
The no-observed-effect concentration for maternal toxicity is 25 ppm based on histologic changes in nasal tissues. The no-observed-effect concentration for teratogenicity is 100 ppm (the highest concentration tested).