Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Effects on fertility

Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

According to Annex IX, an extended 1-generation reproductive study is only required when evidence of potential reproductive toxicity exists. As there is no such evidence, a study is not proposed.

Effects on developmental toxicity

Description of key information

The test substance, N-Butyronitrile, in the vehicle (corn oil) was administered orally by gavage to 3 groups of 25 bred female Crl:CD(SD) rats once daily from gestation days 6 through 19. Dosage levels were 10, 25, and 50 mg/kg/day administered at a dose volume of 10 mL/kg. A concurrent control group composed of 25 bred females received the vehicle on a comparable regimen. The females were approximately 14 weeks of age at the initiation of dose administration. All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. On gestation day 20, clinical pathology (haematology and serum chemistry) evaluations were performed on 10 females/group and a laparohysterectomy was performed on all females. The uteri, placentae, and ovaries were examined, and the numbers of foetuses, early and late resorptions, total implantations, and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The liver, brain, kidneys, and adrenal glands from each female were removed, weighed, and preserved in 10% neutral-buffered formalin for possible future histopathological examination. The foetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and developmental variations.

 

All females survived to the scheduled necropsy on gestation day 20. Clear and/or red material around the mouth was noted in a dose-related manner for females in all test substance-treated groups approximately 1 hour following dose administration primarily during gestation days 12-19. The aforementioned material observations did not persist to the daily examinations and were considered nonadverse in the absence of other signs of systemic toxicity (e.g.body weight decrements). No test substance-related clinical observations were noted at the daily examinations at any dosage level. Test substance-related lower mean food consumption was noted in the 50 mg/kg/day group during gestation days 6-9. Mean food consumption in this group was generally similar to the control group for the remainder of the treatment period. In the absence of any effects on mean body weight gain or body weight at 50 mg/kg/day, this transient decrement in food consumption was not considered adverse. Mean food consumption at 10 and 25 mg/kg/day and mean body weights, body weight gains, net body weights, net body weight gains, and gravid uterine weights at 10, 25, and 50 mg/kg/day were unaffected by test substance administration.

 

Test substance-related alterations in haematology parameters were observed in the 50 mg/kg/day group and included lower mean red blood cell count and lower mean haemoglobin and corpuscular haemoglobin concentrations, and higher mean corpuscular volume, platelet count, and percentage and absolute reticulocyte counts compared to the control group. Higher mean red blood cell distribution widths were noted in the 25 and 50 mg/kg/day groups; however, the difference at 25 mg/kg/day was considered non-adverse in the absence of any other effects on hematology parameters at this dosage level. Lower mean phosphorus, potassium, and sodium concentrations were also noted at 50 mg/kg/day and these alterations in serum chemistry were considered test substance-related. There were no test substance-related changes in haematology or serum chemistry parameters at 10 mg/kg/day. There were no test substance-related macroscopic findings in the test substance-treated groups at the scheduled necropsy on gestation day 20. Test substance-related higher mean adrenal gland and liver weights (absolute and relative to brain weight) were noted in the 50 mg/kg/day group. There were no test substance-related effects on organ weights at 10 and 25 mg/kg/day. Test substance-related, lower (7.5% to 7.9%) mean foetal body weights were noted in the 50 mg/kg/day group when compared to the control group. An increased occurrence of the skeletal developmental variation sternebra(e) nos. 5 and/or 6 unossified was noted in the 50 mg/kg/day group and was considered indicative of developmental delay and secondary to the test substance-related effect on foetal growth at this dosage level. There were no test substance-related effects on intrauterine growth or fetal morphology (external, visceral, or skeletal) at 10 or 25 mg/kg/day or intrauterine survival at 10, 25, and 50 mg/kg/day.

 

Based on test substance-related alterations in haematology and serum chemistry parameters and higher mean adrenal gland and liver weights at 50 mg/kg/day, a dosage level of 25 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity. Based on lower mean foetal weights and an increased occurrence of the skeletal developmental variation sternebra(e) nos. 5 and/or 6 unossified at 50 mg/kg/day, a dosage level of 25 mg/kg/day was considered to be the NOAEL for embryo/foetal development when n-Butyronitrile was administered orally by gavage to bred Crl:CD(SD) rats.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
n-Butyronitrile
Lot no. TXTXOL
CAS No.:109-74-0
Clear, colorless liquid
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
Sexually mature, virgin female Sprague Dawley [Crl:CD(SD)] rats were used as the test system on this study. The number of animals selected for this study(25 females/group) was based on the United States EPA Health Effects Test Guidelines:OPPTS 870.3700, Prenatal Developmental Toxicity Study, Aug-1998 and the OECD Guidelines for the Testing of Chemicals Guideline 414, Prenatal Developmental Toxicity Study, 22-Jan-2001, which recommend evaluation of approximately 20 females per treatment group with implantation sites at necropsy.
Crl:CD(SD) rats (125 females) were received in good health from Charles River Laboratories, Inc., Raleigh, NC, on 24-Nov-2015. The animals were approximately 79 days old upon receipt. Each female was examined by a qualified biologist on the day of receipt. The day following receipt, all animals were weighed and clinical observations were recorded. Each animal was uniquely identified using a programmable microchip (BMDS system) which was implanted subcutaneously in the dorsoscapular region during the acclimation period. The animals were housed for a minimum of 14 days for acclimation purposes. During the acclimation period, the rats were observed twice daily for mortality and changes in general appearance and behaviour.
Upon arrival, all rats were housed 2-3 per cage in clean, solid-bottom cages with bedding material (Bed-O'Cobs®; The Andersons, Cob Products Division, Maumee, OH). The rats were paired for mating in the home cage of the male. Following positive evidence of mating, the females were individually housed in clean, solid-bottom cages with bedding material.

Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). The animal facilities at WIL Research are fully accredited by AAALAC International. Enrichment devices were provided to all animals as appropriate throughout the study for environmental enrichment and to aid in maintaining the animals’ oral health, and were sanitized weekly.
The basal diet used in this study, PMI Nutrition International, LLC Certified Rodent LabDiet® 5002, was a certified feed with appropriate analyses performed by the manufacturer and provided to WIL Research. Feed lots used during the study were documented in the study records. The feeders were changed and sanitized once per week.
Municipal water supplying the facility was sampled for contaminants according to WIL Research SOPs. The results of the diet and water analyses are maintained at WIL Research. No contaminants were present in animal feed or water at concentrations sufficient to interfere with the objectives of this study. Reverse osmosis-purified (on-site) drinking water, delivered by an automatic watering system, and the basal diet were provided ad libitum throughout the acclimation period and during the study

All rats were housed throughout the acclimation period and during the study in an environmentally controlled room. The room temperature and relative humidity controls were set to maintain environmental conditions of 71°F ± 5°F (22°C ± 3°C) and 50% ± 20%, respectively. Room temperature and relative humidity data were monitored continuously and were scheduled for automatic collection on an hourly basis.
Actual mean daily temperature ranged from 70.8°F to 71.8°F (21.6°C to 22.1°C) and mean daily relative humidity ranged from 39.2% to 50.2% during the study. Fluorescent lighting provided illumination for a 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod. The light status (on or off) was recorded once every 15 minutes. Air handling units were set to provide a minimum of 10 fresh air changes per hour.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
The vehicle and test substance formulations were administered orally by gavage, via an appropriately sized flexible, Teflon®-shafted, stainless steel ball-tipped dosing cannula, once daily during gestation days 6-19. The dose volume for all groups was 10 mL/kg. Individual dosages were based on the most recently recorded body weights to provide the correct mg/kg/day dose. All animals were dosed at approximately the same time each day.
The following table presents the study group assignment:

Group Number Treatment Dosage Level (mg/kg/day) Dose Volume (mL/kg) Number of Animals
1 Vehicle control 0 10 25
2 n-Butyronitrile 10 10 25
3 n-Butyronitrile 25 10 25
4 n-Butyronitrile 50 10 25


Dosage levels were based on the results of previous studies including a dose range-finding prenatal developmental toxicity study (Tanner, 2016, WIL-387066) and were provided by the Sponsor after consultation with the Study Director. In that study,
the test substance was administered orally by gavage to bred female rats from gestation days 6 through 19 at dosage levels of 25, 50, 100, and 150 mg/kg/day. Moribundity, adverse clinical observations, body weight losses, and corresponding reductions in food consumption were noted at 100 and 150 mg/kg/day, resulting in the early euthanasia of all females at 150 mg/kg/day. Marked reductions in embryo-fetal survival occurred at 100 mg/kg/day, and lower mean fetal weights were noted in the 50 and 100 mg/kg/day group litters. Based on the maternal and/or fetal toxicity observed at 100 and 150 mg/kg/day, dosage levels of 10, 25, and 50 mg/kg/day were selected for the current study. The selected route of administration for this study was oral (gavage) because this is a potential route of exposure for humans. Historically, this route has been used extensively for studies of this nature.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability and homogeneity of the test substance in the vehicle at concentrations of 1 and 50 mg/mL was established for at least 10 days of room temperature and refrigerated storage (Freer, 2015, WIL-387069). Therefore, stability analyses were not performed on this study. Samples for homogeneity and/or concentration determinations were collected from the top, middle, and bottom strata of the first 1 and 5 mg/mL dosing formulations and from the middle stratum of the first control and 2.5 mg/mL dosing formulations. In addition, samples for resuspension homogeneity determination were collected from the top and bottom strata of an aliquot taken from the first 1 and 5 mg/mL dosing suspensions following room temperature storage for 10 days; aliquots were mixed for a minimum of 30 minutes prior to sample collection. Samples for concentration analysis were also collected from the middle stratum of the last dosing formulations (including the control group) prepared during the study. One set of samples from each collection was subjected to the appropriate analyses. All remaining samples were stored at room temperature as back-up. All analyses were conducted by the WIL Research Analytical Chemistry Department using a validated gas chromatography method with flame ionization detection (Freer, 2015, WIL-387069).
Details on mating procedure:
At the conclusion of the acclimation period, all available females were weighed and examined in detail for physical abnormalities. At the discretion of the Study Director, each animal judged to be in good health and meeting acceptable body weight requirements was placed in a solid-bottom cage with bedding material with a resident male from the same strain and source for breeding. Resident males were untreated, sexually mature rats utilized exclusively for breeding. These rats were maintained under similar laboratory conditions as the females. A breeding record containing the male and female identification numbers and the dates of cohabitation was maintained. The selected females were approximately 13 weeks old when paired for breeding. Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm in a vaginal lavage and verified by a second biologist. Each mating pair was examined daily. The day on which evidence of mating was identified was termed gestation day 0 and the animals were separated. The experimental design consisted of 3 test substance-treated groups and 1 control group, composed of 25 rats per group. The bred females were assigned to groups using a WTDMS™ computer program which randomized the animals based on stratification of the gestation day 0 body weights in a block design. Animals not assigned to study were transferred to the WIL Research colony or euthanized by carbon dioxide inhalation and discarded. Body weight values ranged from 233 g to 282 g on gestation day 0.
Duration of treatment / exposure:
14 days
Frequency of treatment:
Once daily
Duration of test:
21 days
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
The test substance, n-Butyronitrile, in the vehicle (corn oil) was administered orally by gavage to 3 groups of 25 bred female Crl:CD(SD) rats once daily from gestation days 6 through 19. Dosage levels were 10, 25, and 50 mg/kg/day administered at a dose volume of 10 mL/kg. A concurrent control group composed of 25 bred females received the vehicle on a comparable regimen. The females were approximately 14 weeks of age at the initiation of dose administration. All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. On gestation day 20, clinical pathology (hematology and serum chemistry) evaluations were performed on 10 females/group and a laparohysterectomy was performed on all females. The uteri, placentae, and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations, and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The liver, brain, kidneys, and adrenal glands from each female were removed, weighed, and preserved in 10% neutral-buffered formalin for possible future histopathological examination. The fetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and developmental variations.

A scheme describing the stduy design is attached below. Furthermore, the parameters evaluated are attached for the purpose of completeness as well.
Maternal examinations:
All rats were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality. Individual clinical observations were recorded daily from gestation days 0 through 20 (prior to dose administration during the treatment period). Animals were also observed for signs of toxicity approximately 1 hour following dose administration. The absence or presence of findings was recorded for all animals. In addition, the presence of findings at the time of dose administration was recorded for individual animals.

Individual maternal body weights were recorded on gestation days 0 and 6-20 (daily). Group mean body weights were calculated for each of these days. Mean body weight changes were calculated for each corresponding interval and also for gestation days 6-9, 9-12, 12-15, 15-20, and 6-20.

Individual food consumption was recorded on gestation days 0 and 6-20 (daily). Food intake was reported as g/animal/day and g/kg/day for the corresponding body weight change intervals. When food consumption could not be determined for an animal during a given interval (due to a weighing error, food spillage, etc.), group mean values were calculated for that interval using the available data. The time periods when food consumption values were unavailable for a given animal were designated as “NA” on the individual report tables.

Blood samples for clinical pathology evaluations (hematology and serum chemistry) were collected from 10 females/group at the time of scheduled necropsy on gestation day 20. The animals were not fasted overnight prior to blood collection. Blood was collected from the retro-orbital sinus under isoflurane anesthesia into tubes containing K2EDTA (hematology) or tubes without anticoagulant (serum chemistry). The following parameters were evaluated:

Total leukocyte count (WBC) Erythrocyte count (RBC)
Hemoglobin (HGB) Hematocrit (HCT)
Mean corpuscular volume (MCV) Mean corpuscular hemoglobin(MCH)
Mean corpuscular hemoglobin concentration (MCHC) Platelet count (PLATELET)
Reticulocyte count Percent (RETIC)
Absolute (RETIC ABSOLUTE) Mean platelet volume (MPV)
Differential leukocyte count -
Percent and absolute
-Neutrophil (NEU)
-Lymphocyte (LYMPH)
-Monocyte (MONO)
-Eosinophil (EOS)
-Basophil (BASO)
-Large unstained cell (LUC) Red cell distribution width(RDW)
Hemoglobin distribution width (HDW) Platelet estimate
Red cell morphology Bile acids
Albumin Total protein
Globulin [by calculation] Albumin/globulin ratio (A/G Ratio)
Total bilirubin (Total Bili) Urea nitrogen
Creatinine Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT) Aspartate aminotransferase (AST)
Gamma glutamyltransferase (GGT) Glucose
Total cholesterol (Cholesterol) Calcium
Chloride Phosphorus
Potassium Sodium
Triglycerides (Triglyceride) Appearance
Ovaries and uterine content:
Gravid uterine weight was collected and net body weight (the gestation day 20 body weight exclusive of the weight of the uterus and contents) and net body weight change (the gestation day 0-20 body weight change exclusive of the weight of the uterus and contents) were calculated and presented for each gravid female at the scheduled laparohysterectomy.

Laparohysterectomies and macroscopic examinations were performed blind to treatment group. All rats were euthanized on gestation day 20 by carbon dioxide inhalation. The cranial, thoracic, abdominal, and pelvic cavities were opened by a ventral mid-line incision, and the contents were examined. In all instances, the postmortem findings were correlated with the antemortem observations, and any abnormalities were recorded. The uterus and ovaries were then exposed and excised. The number of corpora lutea on each ovary was recorded. The trimmed uterus was weighed and opened, and the number and location of all fetuses, early and late resorptions, and the total number of implantation sites were recorded. The placentae were also examined. The individual uterine distribution of implantation sites was documented using the following procedure. All implantation sites, including resorptions, were numbered in consecutive order beginning with the left distal to the left proximal uterine horn, noting the position of the cervix, and continuing from the right proximal to the right distal uterine horn.

Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss (Salewski, 1964). The adrenal glands, brain, kidneys, and liver from all females were weighed and retained in 10% neutral-buffered formalin for possible future histopathologic examination. Paired organs were weighed together and absolute weights and organ to brain weight ratios were reported, as applicable. Other maternal tissues were preserved for possible future histopathologic examination only as indicated by the gross findings; representative sections of corresponding organs from a sufficient number of control animals were retained for comparison. The carcass of each female was then discarded.
Fetal examinations:
Fetal examinations were performed blind to treatment group. Each viable fetus was examined externally, individually sexed, weighed, euthanized by a subcutaneousinjection of sodium pentobarbital in the scapular region, and tagged for identification Fetal tags contained the WIL Research study number, the female number, and the fetus number. The detailed external examination of each fetus included, but was not limited to, an examination of the eyes, palate, and external orifices, and each finding was recorded. Crown-rump measurements and degrees of autolysis were recorded for late resorptions, a gross external examination was performed (if possible), and the tissues were discarded.

Each viable fetus was subjected to a visceral examination using a modification of the Stuckhardt and Poppe fresh dissection technique to include the heart and major blood vessels (Stuckhardt and Poppe, 1984). The sex of each fetus was confirmed by internal examination. Fetal kidneys were examined and graded for renal papillae development (Woo and Hoar, 1972). Heads from approximately one-half of the fetuses in each litter were placed in Harrison’s fixative for subsequent soft-tissue examination by the Wilson sectioning technique (Wilson, 1965). The heads from the remaining one-half of the fetuses were examined by a midcoronal slice. All carcasses were eviscerated and fixed in 100% ethyl alcohol.

Following fixation in alcohol, each fetus was stained with Alizarin Red S (Dawson,1926) and Alcian Blue (Inouye, 1976). Fetuses were then examined for skeletal malformations and developmental variations.

External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or occur at high incidence, representing slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).

The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison and calculating the number of affected fetuses in a litter on a proportional basis as follows:

Summation per Group (%) = Sum of Viable Fetuses Affected per Litter (%)/No. Litters/Group

Where:

Viable Fetuses Affected/Litter (%) = No. Viable Fetuses Affected per Litter/No. Viable Fetuses/Litter x 100
Statistics:
Due to a low character count limit in this entry box, please see "Any other information on materials and methods incl tables" section for details on statistics
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
All females in the control, 10, 25, and 50 mg/kg/day groups survived to the scheduled necropsy on gestation day 20. Test substance-related increased incidences of clear and/or red material around the mouth were noted in the 10, 25, and 50 mg/kg/day group females approximately 1 hour following dose administration. These observations were noted in a dose-responsive manner primarily during gestation days 12-19 but did not persist to the daily examinations, and therefore were not considered adverse. There were no test substance-related clinical observations noted at the daily examinations at any dosage level. Findings noted in the treated groups, including hair loss on various body surfaces and red material around the nose, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean maternal body weights, body weight gains, net body weights, net body weight gains, and gravid uterine weights in the 10, 25, and 50 mg/kg/day groups were unaffected by test substance administration. Differences from the control group were slight and not statistically significant.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 50 mg/kg/day group was significantly (p<0.05 or p<0.01) lower than the control group during gestation days 6-9. Mean food consumption in this group was similar to the control group for the remainder of the treatment period (gestation days 9-12, 12-15, and 15-20) and when the entire treatment period (gestation days 6-20) was evaluated, with the exception of a transient, significantly (p<0.05) lower mean food consumption value (g/kg/day only) during gestation day 12-13. The initial transient decrements in mean food consumption in this group were considered test substance-related but nonadverse in the absence of any effects on mean body weight gain or body weight at 50 mg/kg/day.

Mean maternal food consumption in the 10 and 25 mg/kg/day groups was unaffected by test substance administration. Differences from the control group were slight and not statistically significant, with the following exception. Significantly (p<0.05) higher mean food consumption was noted in the 10 mg/kg/day group during gestation day 7-8 compared to the control group. This difference was transient, not dose-related, and therefore was not considered test substance-related.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related alterations in hematology parameters included lower mean red blood cell count and hemoglobin and mean corpuscular hemoglobin concentrations, and higher mean corpuscular volume, platelet count, and percentage and absolute reticulocyte counts; differences from the control group were significant (p<0.01). Test substance-related significantly (p<0.05 or p<0.01) higher mean red blood cell distribution widths (RDW) were noted in the 25 and 50 mg/kg/day groups (12.2% and 12.3%, respectively) compared to the concurrent control group (11.5%). The RDWs in both groups were within the WIL Research historical control data (version 3.6) range for nonpregnant Crl:CD(SD) rats (11.1% to 13.6%). In the absence of any other effects on hematology parameters at 25 mg/kg/day, the difference in RDW was considered nonadverse at this dosage level. No other test substance-related changes in hematology parameters were noted at any dosage level.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related lower mean phosphorus, potassium, and sodium concentrations were noted in the 50 mg/kg/day group; differences from the control group were generally significant (p<0.05). No other test substance-related changes in serum chemistry parameters were noted at any dosage level. Mean cholesterol concentrations in the 25 and 50 mg/kg/day groups were lower (18.9% and 17.6%, respectively) than the control group; the difference was significant (p<0.05) at 25 mg/kg/day. The lower mean cholesterol concentrations did not occur in a dose-related manner, and therefore were not considered test substance-related.
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
The mean litter proportions of postimplantation loss in the 10, 25, and 50 mg/kg/day groups (7.3%, 8.4%, and 8.1% per litter, respectively) were slightly higher than the concurrent control group (5.0% per litter). However, differences from the concurrent control group were not statistically significant and there was no dose-response relationship; therefore, the increases in postimplantation loss in the test substance-treated groups were not considered test substance-related.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
not specified
Key result
Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
clinical signs
Abnormalities:
not examined
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean fetal body weights (male, female, and combined) were noted in the 50 mg/kg/day group (3.7 g, 3.5 g, and 3.6 g, respectively) were significantly (p<0.01) lower (7.5% to 7.9%) when compared to the concurrent control group (4.0 g, 3.8 g, and 3.9 g,respectively). The mean fetal body weights in this group were within the WIL Research historical control data ranges (3.475 g to 4.138 g, 3.354 g to 3.901 g, and 3.411 g to 4.026 g for male, female, and combined, respectively). In addition, the mean fetal weights in the concurrent control group were at the higher end of the WIL Research historical control data ranges. However, given the magnitude of change from the concurrent control group, the lower mean fetal body weights in the 50 mg/kg/day group were considered test substance-related. No test substance-related effects on intrauterine growth were noted at dosage levels of 10 and 25 mg/kg/day.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Mean fetal body weights (male, female, and combined) were noted in the 50 mg/kg/day group (3.7 g, 3.5 g, and 3.6 g, respectively) were significantly (p<0.01) lower (7.5% to 7.9%) when compared to the concurrent control group (4.0 g, 3.8 g, and 3.9 g, respectively). The mean fetal body weights in this group were within the WIL Research historical control data ranges (3.475 g to 4.138 g, 3.354 g to 3.901 g, and 3.411 g to 4.026 g for male, female, and combined, respectively). In addition, the mean fetal weights in the concurrent control group were at the higher end of the WIL Research historical control data ranges. However, given the magnitude of change from the concurrent control group, the lower mean fetal body weights in the 50 mg/kg/day group were considered test substance-related. No test substance-related effects on intrauterine growth were noted at dosage levels of 10 and 25 mg/kg/day.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Mean fetal body weights (male, female, and combined) were noted in the 50 mg/kg/day group (3.7 g, 3.5 g, and 3.6 g, respectively) were significantly (p<0.01) lower (7.5% to 7.9%) when compared to the concurrent control group (4.0 g, 3.8 g, and 3.9 g, respectively). The mean fetal body weights in this group were within the WIL Research historical control data ranges (3.475 g to 4.138 g, 3.354 g to 3.901 g, and 3.411 g to 4.026 g for male, female, and combined, respectively). In addition, the mean fetal weights in the concurrent control group were at the higher end of the WIL Research historical control data ranges. However, given the magnitude of change from the concurrent control group, the lower mean fetal body weights in the 50 mg/kg/day group were considered test substance-related. No test substance-related effects on intrauterine growth were noted at dosage levels of 10 and 25 mg/kg/day.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
External malformations were noted for 1 fetus each in the 10 and 50 mg/kg/day groups. Fetus no. 8521-01 in the 50 mg/kg/day group was noted with anal atresia. In the 10 mg/kg/day group, fetus no. 8477-13 was noted with a cleft palate (entire length); skeletally, this finding consisted of palatine plates not joined. This fetus was noted with a very low body weight (2.4 g). The external malformations noted in the 10 and 50 mg/kg/day groups were not attributed to test substance administration because they occurred in single fetuses, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant when compared to the concurrent control group, and/or were within the WIL Research historical control data ranges. No external developmental variations were observed in fetuses in this study
Skeletal malformations:
no effects observed
Description (incidence and severity):
Skeletal malformations were noted for 1 fetus each in the 10 and 50 mg/kg/day groups. Fetus no. 8521-01 in the 50 mg/kg/day group was noted with bent limb bones (both humeri and right femur) and a costal cartilage anomaly (consisting of fused and malpositioned costal cartilage). This same fetus was noted with the external malformation anal atresia. In the 10 mg/kg/day group, fetus no. 8477-13 was noted with a vertebral anomaly with associated rib anomaly (misshapen centra; fused centra and arches; absent, interrupted and malpositioned costal cartilage; and only 10 pairs of ribs present). This fetus was also observed with a cleft palate. The skeletal malformations noted in the 10 and 50 mg/kg/day groups were not attributed to the test substance because they occurred infrequently, in single fetuses, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the WIL Research historical control data ranges.

The mean litter proportion of sternebra(e) nos. 5 and/or 6 unossified (23.5% per litter) in the 50 mg/kg/day group was significantly (p<0.01) higher than the concurrent control group (8.5% per litter). The mean litter proportion for this skeletal variation was near the upper end of the WIL Research historical control range (0.00% to 26.06% per litter) and was considered indicative of developmental delay and secondary to the test substance-related effect on fetal growth at this dosage level.

No other test substance-related skeletal variations were noted. The mean litter proportion of cervical centrum no. 1 ossified in the 50 mg/kg/day group was lower (not statistically significant) than the control group. However, this variation was not considered test substance-related because this finding is the most common skeletal variation in the WIL Research historical control database and the mean litter proportion of this finding for this group (13.5% per litter) was within the WIL Research historical control range (1.39% to 35.78% per litter). Other findings observed in the test substance-treated groups were noted infrequently, similarly in the control group, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the WIL Research historical control data.
Visceral malformations:
no effects observed
Description (incidence and severity):
The only visceral malformation (diaphragmatic hernia) in this study was noted for a single fetus (no. 8457-11) in the 10 mg/kg/day group. This malformation consisted of a portion of the right lobe of the liver protruding into the thoracic cavity through an opening in the diaphragm. Based on the occurrence in a single fetus and the absence of the dose-response relationship, this malformation was not considered test substance-related.

No test substance-related visceral developmental variations were noted. Findings observed in the test substance-treated groups were noted infrequently, similarly in the control group, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the WIL Research historical control data.

Three fetuses (nos. 8442-04, 8510-02, and 8510-05) in the 25 mg/kg/day group and 2 fetuses (nos. 8447-03 and 8491-08) in the 50 mg/kg/day group were observed with renal papilla(e) not fully developed (Woo and Hoar Grade 1). This finding was not classified as either a malformation or developmental variation, was not included on the summary tables, and was not considered to be test substance-related because it occurred infrequently and in a manner that was not dose-related.
Other effects:
not specified
Key result
Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
fetal/pup body weight changes
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
25 mg/kg bw (total dose)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
not specified
Relevant for humans:
not specified

The table S1- S19 depicted below show the summary data of the study results.

Table S1

Project No.: WIL-387067

Oral Prenatal Developmental Toxicity Study Of N-Butylronitrile In Rats

Sponsor: EASTMAN CHEMICAL Co.

Summary of Maternal Survival and Pregnancy Status

Dose Group:

1

2

3

4

 

No.

%

No.

%

No.

%

No.

%

Females on Study

25

25

25

25

Females That Aborted OR DeLivered

0

0.0

0

0.0

0

0.0

0

0.0

Females That Died

0

0.0

0

0.0

0

0.0

0

0.0

Females That Aborted

0

0.0

0

0.0

0

0.0

0

0.0

Nongravid

0

0.0

0

0.0

0

0.0

0

0.0

Gravid

0

0.0

0

0.0

0

0.0

0

0.0

Females That were Euthanized

0

0.0

0

0.0

0

0.0

0

0.0

Nongravid

0

0.0

0

0.0

0

0.0

0

0.0

Gravid

0

0.0

0

0.0

0

0.0

0

0.0

Females Examined at Scheduled Necropsy

25

100.0

25

100.0

25

100.0

25

100.0

Nongravid

1

4.0

1

4.0

0

0.0

2

8.0

Gravid

24

96.0

24

96.0

25

100.0

23

92.0

with Resorptions only

0

0.0

0

0.0

0

0.0

0

0.0

with viable Fetuses

24

100.0

24

100.0

25

100.0

23

100.0

Total Females Gravid

24

96.0

24

96.0

25

100.0

23

92.0

1-    0 mg/kg/Day

2-  10 mg/kg/Day

3-  25 mg/kg/Day

4-  50 mg/kg/Day

Table S2  (Daily Examinations)

Project No.: WIL-387067

Oral Prenatal Developmental Toxicity Study Of N-Butylronitrile In Rats

Sponsor: EASTMAN CHEMICAL Co.

Clinical Findings: Total Occurrence/No. of Animals

Female

Table Range:

12-08-15 To 01-01-16

Group:

1

2

3

4

Normal

 -No Significant Clinical Observations

52l/25

508/25

458/25

494/25

Disposition

 -Scheduled Euthanasia; Gestation Day 20

25/25

25/25

25/25

25/25

Body/Integ.ument

 -Hair Loss Forelimbs

1/ 1

16/ 2

64/ 8

25/ 4

-Hair Loss Ventral Trunk

0/ 0

0/ 0

4/ 2

0/ 0

Eyes/Ears/Nose

-Dried Red Material Around Nose

2/ 2

1/ 1

2/ 2

4/ 1

Excreta

 -Decreased Defecation

0/ 0

0/ 0

0/ 0

2/ 1

Body/Integ. II

-Scabbing Ventral Trunk

1/ 1

0/ 0

0/ 0

0/ 0

1-    0 mg/kg/Day

2-  10 mg/kg/Day

3-  25 mg/kg/Day

4-  50 mg/kg/Day

Table S3

Project No.: WIL-387067

Oral Prenatal Developmental Toxicity Study Of N-Butylronitrile In Rats

Sponsor: EASTMAN CHEMICAL Co.

Summary of Post-Dose Findings: Total Occurrence/No. Of Animals

Female

Table Range:

12-14-15 To 12-31-15

Group:

1

2

3

4

Normal

1 Hour Post-Dosing

 -No Significant Clinical Observations

349/25

345/25

344/25

315/25

Eyes/Ears/Nose

1 Hour Post-Dosing

-Dried Red Material Around Nose

0/0

0/0

0/0

1/1

Body/Integ. II

1 Hour Post-Dosing

-Wet Red Material Urogenital Area

0/0

0/0

0/0

1/1

Oral/Dental

1 Hour Post-Dosing

-Wet Clear Material Around Mouth

1/1

2/2

4/3

19/8

-Dried Clear Material Around Mouth

0/0

1/1

0/0

12/9

-Dried Red Material Around Mouth

0/0

2/2

2/2

2/2

Special II

Time of Dose

 -Undetermined Amount of Dose Expelled

1/1

0/0

1/1

0/0

1-    0 mg/kg/Day

2-  10 mg/kg/Day

3-  25 mg/kg/Day

4-  50 mg/kg/Day

Table S4

Project No.: WIL-387067

Oral Prenatal Developmental Toxicity Study Of N-Butylronitrile In Rats

Sponsor: EASTMAN CHEMICAL Co.

Summary of Body Weights During Gestation [g]

Group (mg/kg bw/Day)

0

10

25

50

Day 0

Mean

254.

252.

254.

254.

% D% Difference

-0.8

0.0

0.0

S.D.

11.2

11.1

12.3

10.9

S.E.

2.3

2.3

2.5

2.3

N

24

24

25

23

Day 6

Mean

289.

288.

288.

288.

% D% Difference

-0.3

-0.3

-0.3

S.D.

14.0

12.5

13.3

13.3

S.E.

2.9

2.6

2.7

2.8

N

24

24

25

23

Day 7

Mean

287.

288.

288.

287.

% D% Difference

0.3

0.3

0.0

S.D.

15.4

12.9

13.4

14.7

S.E.

3.1

2.6

2.7

3.1

N

24

24

25

23

Day 8

Mean

291.

293.

293.

293.

% D% Difference

0.7

0.7

0.7

S.D.

15.4

12.8

14.1

14.1

S.E.

3.1

2.6

2.8

2.9

N

24

24

25

23

Day 9

Mean

295.

296.

297.

295.

% D% Difference

0.3

0.7

0.0

S.D.

15.8

14.4

15.3

13.8

S.E.

3.2

2.9

3.1

2.9

N

24

24

25

23

Day 10

Mean

299.

303.

301.

301.

% D% Difference

1.3

0.7

0.7

S.D.

15.6

14.4

15.1

14.5

S.E.

3.2

2.9

3.0

3.0

N

24

24

25

23

Day 11

Mean

305.

309.

308.

309.

% D% Difference

1.3

1.0

1.3

S.D.

16.1

15.5

15.1

14.5

S.E.

3.3

3.2

3.0

3.0

N

24

24

25

23

Day 12

Mean

310.

314.

313.

314.

% D% Difference

1.3

1.0

1.3

S.D.

16.3

17.2

15.9

15.7

S.E.

3.3

3.5

3.2

3.3

N

24

24

25

23

Day 13

Mean

315.

320.

318.

319.

% D% Difference

1.6

1.0

1.3

S.D.

15.8

17.1

15.6

15.9

S.E.

3.2

3.5

3.1

3.3

N

24

24

25

23

Day 14

Mean

321.

325.

323.

326.

% D% Difference

1.2

0.6

1.6

S.D.

16.2

17.2

15.9

17.6

S.E.

3.3

3.5

3.2

3.7

N

24

24

25

23

Day 15

Mean

329.

332.

332.

333.

% D% Difference

0.9

0.9

1.2

S.D.

17.0

18.3

16.4

17.1

S.E.

3.5

3.7

3.3

3.6

N

24

24

25

23

Day 16

Mean

339.

342.

340.

341.

% D% Difference

0.9

0.3

0.6

S.D.

17.7

18.1

18.7

17.3

S.E.

3.6

3.7

3.7

3.6

N

24

24

25

23

Day 17

Mean

352.

357.

354.

354.

% D% Difference

1.4

0.6

0.6

S.D.

19.2

20.2

19.2

19.6

S.E.

3.9

4.1

3.8

4.1

N

24

24

25

23

Day 18

Mean

368.

375.

370.

369.

% D% Difference

1.9

0.5

0.3

S.D.

19.1

20.0

20.5

20.1

S.E.

3.9

4.1

4.1

4.2

N

24

24

25

23

Day 19

Mean

386.

391.

385.

385.

% D% Difference

1.3

-0.3

-0.3

S.D.

21.0

22.5

22.0

22.0

S.E.

4.3

4.6

4.4

4.6

N

24

24

25

23

Day 20

Mean

399.

405.

400.

395.

% D% Difference

1.5

0.3

-1.0

S.D.

21.2

24.6

25.4

25.7

S.E. N

4.3 24

5.0 24

5.1 25

5.4 23

None Significantly different from control Group

Nongravid Weight (s) Not Included in Calculation of Mean

Table S5

Project No.: WIL-387067

Oral Prenatal Developmental Toxicity Study Of N-Butylronitrile In Rats

Sponsor: EASTMAN CHEMICAL Co.

Summary of Body Weight Changes During Gestation [g]

Group (mg/kg bw/Day)

0

10

25

50

Day       0- 6

Mean

34.

36.

34.

34.

S.D.

6.4

6.2

7.2

6.3

S.E.

1.3

1.3

1.4

1.3

N

24

24

25

23

Day       6- 7

Mean

-1.

0.

-1.

-1.

S.D.

3.2

4.0

3.7

4.1

S.E.

0.7

0.8

0.7

0.9

N

24

24

25

23

Day     7- 8

Mean

3.

5.

5.

6.

S.D.

3.6

2.6

4.7

3.8

S.E.

0.7

0.5

0.9

0.8

N

24

24

25

23

Day       8- 9

Mean

4.

3.

4.

2.

S.D.

3.6

3.9

3.6

3.7

S.E.

0.7

0.8

0.7

0.8

N

24

24

25

23

Day       9- 10

Mean

4.

7.

5.

6.

S.D.

4.0

4.0

4.2

2.9

S.E.

0.8

0.8

0.8

0.6

N

24

24

25

23

Day   10- 11

Mean

6.

6.

6.

8.

S.D.

3.7

4.0

3.1

2.8

S.E.

0.8

0.8

0.6

0.6

N

24

24

25

23

Day   11- 12

Mean

6.

5.

5.

5.

S.D.

3.2

4.1

2.7

3.5

S.E.

0.7

0.8

0.5

0.7

N

24

24

25

23

Day   12- 13

Mean

5.

6.

5.

5.

S.D.

4.0

3.3

4.0

3.2

S.E.

0.8

0.7

0.8

0.7

N

24

24

25

23

Day   13- 14

Mean

5.

6.

6.

6.

S.D.

4.0

4.0

3.1

3.5

S.E.

0.8

0.8

0.6

0.7

N

24

24

25

23

Day   14- 15

Mean

8.

7.

8.

7.

S.D.

5.7

5.8

4.0

3.0

S.E.

1.2

1.2

0.8

0.6

N

24

24

25

23

Day   15- 16

Mean

10.

10.

8.

9.

S.D.

3.8

4.0

5.2

4.7

S.E.

0.8

0.8

1.0

1.0

N

24

24

25

23

Day   16- 17

Mean

13.

15.

14.

12.

S.D.

4.9

3.5

4.2

5.4

S.E.

1.0

0.7

0.8

1.1

N

24

24

25

23

Day   17- 18

Mean

17.

18.

16.

15.

S.D.

4.9

4.9

5.2

4.8

S.E.

1.0

1.0

1.0

1.0

N

24

24

25

23

Day   18- 19

Mean

18.

16.

16.

16.

S.D.

4.9

5.9

5.1

5.0

S.E.

1.0

1.2

1.0

1.0

N

24

24

25

23

Day   19- 20

Mean

13.

15.

15.

10.

S.D.

5.2

6.3

6.5

7.3

S.E.

1.1

1.3

1.3

1.5

N

24

24

25

23

Day     6- 9

Mean

6.

9.

8.

7.

S.D.

4.7

4.9

4.5

4.7

S.E.

1.0

1.0

0.9

1.0

N

24

24

25

23

Day     9- 12

Mean

15.

18.

17.

19.

S.D.

4.3

6.2

4.2

4.4

S.E.

0.9

1.3

0.8

0.9

N

24

24

25

23

Day   12- 15

Mean

19.

18.

19.

19.

S.D.

5.1

6.3

6.1

4.4

S.E.

1.0

1.3

1.2

0.9

N

24

24

25

23

Day   15- 20

Mean

70.

73.

69.

62.

S.D.

8.5

13.8

13.7

16.4

S.E.

1.7

2.8

2.7

3.4

N

24

24

25

23

Day     6- 20

Mean

111.

118.

112.

107.

S.D.

12.6

18.8

18.8

21.6

S.E

2.6

3.8

3.8

4.5

N

24

24

25

23

None Significantly different from control Group

Mean D% Differences Calculated from individual D% Differences

Nongravid Weight (s) Not Included in Calculation of Mean

Table S6

Project No.: WIL-387067

Oral Prenatal Developmental Toxicity Study Of N-Butylronitrile In Rats

Sponsor: EASTMAN CHEMICAL Co.

Summary of Gravid Uterine weights and Net Body weight Changes [g]

Group (mg/kg bw/Day)

0

10

25

50

Initial Body WT.

Mean

254.

252.

254.

254.

S.D.

11.2

11.1

12.3

10.9

S.E.

2.3

2.3

2.5

2.3

N

24

24

25

23

Terminal Body WT.

Mean

399.

405.

400.

395.

S.D.

21.2

24.6

25.4

25.7

S.E.

4.3

5.0

5.1

5.4

N

24

24

25

23

Gravid Uterine WT.

Mean

88.1

87.5

82.8

80.0

S.D.

8.18

11.87

13.48

15.67

S.E.

1.67

2.42

2.70

3.27

N

24

24

25

23

NET Body WT.

Mean

311.3

317.9

317.6

314.8

S.D.

17.64

17.38

20.10

18.04

S.E.

3.60

3.55

4.02

3.76

N

24

24

25

23

NET Body WT. Change

Mean

57.1

66.0

63.7

61.2

S.D.

12.17

14.40

14.72

14.57

S.E.

2.49

2.94

2.94

3.04

N

24

24

25

23

None Significantly different from control Group

Table S7

Project No.: WIL-387067

Oral Prenatal Developmental Toxicity Study Of N-Butylronitrile In Rats

Sponsor: EASTMAN CHEMICAL Co.

Summary of Food Consumption During Gestation [g/Animal/Day]

Group (mg/kg bw/Day)

0

10

25

50

Day     0- 6

Mean

20.

21.

20.

21.

S.D.

1.7

1.5

1.4

2.0

S.E.

0.3

0.3

0.3

0.4

N

24

24

25

23

Day     6- 7

Mean

15.

16.

15.

14.

S.D.

2.4

2.2

2.8

4.0

S.E.

0.5

0.4

0.6

0.8

N

24

24

25

23

Day     7- 8

Mean

17.

19.*

17.

16.

S.D.

3.0

2.0

2.6

2.7

S.E.

0.6

0.4

0.5

0.6

N

23

24

25

23

Day     8- 9

Mean

17.

18.

17.

15.*

S.D.

2.3

2.5

2.1

2.5

S.E.

0.5

0.5

0.4

0.5

N

24

24

25

23

Day     9- 10

Mean

16.

18.

17.

17.

S.D.

2.3

2.0

2.5

3.4

S.E.

0.5

0.4

0.5

0.7

N

23

24

25

23

Day   10- 11

Mean

16.

18.

17.

17.

S.D.

2.8

2.7

2.1

2.3

S.E.

0.6

0.5

0.4

0.5

N

23

24

25

23

Day   11- 12

Mean

18.

19.

18.

17.

S.D.

3.2

3.3

3.1

2.5

S.E.

0.6

0.7

0.6

0.5

N

24

24

25

23

Day   12- 13

Mean

18.

19.

17.

17.

S.D.

3.3

3.1

2.3

2.7

S.E.

0.7

0.6

0.5

0.6

N

24

24

25

23

Day   13- 14

Mean

19.

18.

18.

18.

S.D.

3.5

3.7

2.3

3.1

S.E.

0.7

0.8

0.5

0.6

N

24

24

25

23

Day   14- 15

Mean

17.

17.

17.

17.

S.D.

6.2

4.0

6.4

2.4

S.E.

1.3

0.8

1.3

0.5

N

24

24

25

23

Day   15- 16

Mean

19.

20.

18.

18.

S.D.

2.2

5.0

2.7

2.2

S.E.

0.5

1.0

0.5

0.5

N

24

24

25

23

Day   16- 17

Mean

21.

20.

21.

19.

S.D.

4.0

4.1

3.0

3.0

S.E.

0.8

0.8

0.6

0.6

N

24

24

25

23

Day   17- 18

Mean

21.

21.

20.

20.

S.D.

2.6

3.6

3.2

2.9

S.E.

0.5

0.7

0.6

0.6

N

24

24

25

23

Day   18- 19

Mean

19.

19.

19.

19.

S.D.

4.7

4.8

2.9

3.7

S.E.

1.0

1.0

0.6

0.8

N

24

24

25

23

Day   19- 20

Mean

18.

20.

18.

18.

S.D.

3.1

6.6

5.2

8.5

S.E.

0.6

1.3

1.0

1.8

N

24

24

25

23

Day     6- 9

Mean

16.

17.

16.

15.*

S.D.

2.2

1.7

1.7

2.4

S.E.

0.5

0.3

0.3

0.5

N

24

24

25

23

Day     9- 12

Mean

17.

18.

17.

17.

S.D.

2.3

2.4

2.0

2.3

S.E.

0.5

0.5

0.4

0.5

N

24

24

25

23

Day   12- 15

Mean

18.

18.

17.

17.

S.D.

2.8

1.8

2.8

2.2

S.E.

0.6

0.4

0.6

0.5

N

24

24

25

23

Day   15- 20

Mean

20.

20.

19.

19.

S.D.

2.0

2.8

2.6

2.6

S.E.

0.4

0.6

0.5

0.5

N

24

24

25

23

Day     6- 20

Mean

18.

19.

18.

17.

S.D.

1.8

1.9

1.8

2.0

S.E.

0.4

0.4

0.4

0.4

N

24

24

25

23

*   = Significantly different from the control Group at 0.05 using Dunnett's test

Nongravid Weight (s) Not Included in Calculation of Mean

Table S8

Project No.: WIL-387067

Oral Prenatal Developmental Toxicity Study Of N-Butylronitrile In Rats

Sponsor: EASTMAN CHEMICAL Co.

Summary of Food Consumption During Gestation [g/kg/Day]

Group (mg/kg bw/Day)

0

10

25

50

Day     0- 6

Mean

73.

76.

74.

76.*

S.D.

4.7

4.5

5.5

5.5

S.E.

1.0

0.9

1.1

1.2

N

24

24

25

23

Day     6- 7

Mean

52.

55.

52.

47.

S.D.

7.2

7.2

10.0

13.4

S.E.

1.5

1.5

2.0

2.8

N

24

24

25

23

Day     7- 8

Mean

58.

64.*

58.

55.

S.D.

8.3

6.2

7.6

8.1

S.E.

1.7

1.3

1.5

1.7

N

23

24

25

23

Day     8- 9

Mean

59.

59.

57.

52.**

S.D.

6.4

6.8

6.1

8.0

S.E.

1.3

1.4

1.2

1.7

N

24

24

25

23

Day     9- 10

Mean

55.

62.

58.

58.

S.D.

6.9

6.8

8.0

11.4

S.E.

1.4

1.4

1.6

2.4

N

23

24

25

23

Day   10- 11

Mean

54.

57.

55.

54.

S.D.

8.3

8.0

6.1

7.8

S.E.

1.7

1.6

1.2

1.6

N

23

24

25

23

Day   11- 12

Mean

57.

60.

59.

54.

S.D.

8.9

9.8

9.6

6.7

S.E.

1.8

2.0

1.9

1.4

N

24

24

25

23

Day   12- 13

Mean

59.

58.

54.

53.*

S.D.

9.9

8.5

6.6

7.7

S.E.

2.0

1.7

1.3

1.6

N

24

24

25

23

Day   13- 14

Mean

59.

57.

55.

56.

S.D.

10.4

12.1

8.4

7.4

S.E.

2.1

2.5

1.7

1.5

N

24

24

25

23

Day   14- 15

Mean

51.

51.

50.

53.

S.D.

18.8

11.7

18.9

6.2

S.E.

3.8

2.4

3.8

1.3

N

24

24

25

23

Day   15- 16

Mean

58.

59.

54.

52.

S.D.

6.3

13.9

7.2

5.5

S.E.

1.3

2.8

1.4

1.1

N

24

24

25

23

Day   16- 17

Mean

60.

58.

61.

56.

S.D.

10.6

11.0

7.8

7.4

S.E.

2.2

2.2

1.6

1.5

N

24

24

25

23

Day   17- 18

Mean

60.

58.

55.

55.

S.D.

6.7

9.9

8.0

7.8

S.E.

1.4

2.0

1.6

1.6

N

24

24

25

23

Day   18- 19

Mean

51.

49.

51.

51.

S.D.

11.4

12.3

7.0

8.9

S.E.

2.3

2.5

1.4

1.9

N

24

24

25

23

Day   19- 20

Mean

45.

49.

46.

46.

S.D.

7.3

16.2

12.7

21.6

S.E.

1.5

3.3

2.5

4.5

N

24

24

25

23

Day     6- 9

Mean

56.

60.

55.

51.**

S.D.

5.8

4.7

4.9

7.3

S.E.

1.2

1.0

1.0

1.5

N

24

24

25

23

Day     9- 12

Mean

56.

59.

57.

56.

S.D.

6.0

7.2

6.4

6.9

S.E.

1.2

1.5

1.3

1.4

N

24

24

25

23

Day   12- 15

Mean

56.

55.

53.

54.

S.D.

8.1

4.8

8.4

5.0

S.E.

1.6

1.0

1.7

1.0

N

24

24

25

23

Day   15- 20

Mean

54.

54.

54.

52.

S.D.

4.4

6.9

5.8

6.0

S.E.

0.9

1.4

1.2

1.2

N

24

24

25

23

Day     6- 20

Mean

56.

56.

55.

53.

S.D.

4.2

4.8

4.6

4.9

S.E.

0.9

1.0

0.9

1.0

N

24

24

25

23

*   = Significantly different from the control Group at 0.05 using Dunnett's test

** = Significantly different from the control Group at 0.01 using Dunnett's test

Nongravid Weight (s) Not Included in Calculation of Mean

Table S9

Project No.: WIL-387067

Oral Prenatal Developmental Toxicity Study Of N-Butylronitrile In Rats

Sponsor: EASTMAN CHEMICAL Co.

Summary of Hematology Values

Females

Analysis

Group (mg/kg bw/day):

0

10

25

50

WBC (thous/µL)

Day 20

Mean

13.31

12.71

11.75

12.49

% Difference

-4.5

-11.7

-6.2

S.D.

2.596

3.197

2.354

1.960

S.E.

0.865

1.011

0.744

0.653

N

9

10

10

9

RBC (mil/µL)

Day 20

Mean

6.41

6.32

6.20

5.52**

% Difference

-1.4

-3.3

-13.9

S.D.

0.265

0.263

0.370

0.503

S.E.

0.088

0.083

0.117

0.168

N

9

10

10

9

HGB (g/dL)

Day 20

Mean

12.2

12.0

11.6

10.6**

% Difference

-1.6

-4.9

-13.1

S.D.

0.42

0.66

0.62

0.80

S.E.

0.14

0.21

0.20

0.27

N

9

10

10

9

HCT (%)

Day 20

Mean

36.9

36.3

35.5

34.8

% Difference

-1.6

-3.8

-5.7

S.D.

1.31

1.67

1.85

1.98

S.E.

0.44

0.53

0.58

0.66

N

9

10

10

9

MCV (fL)

Day 20

Mean

57.6

57.5

57.4

63.2**

% Difference

-0.2

-0.3

9.7

S.D.

2.00

1.92

1.97

3.48

S.E.

0.67

0.61

0.62

1.16

N

9

10

10

9

MCH (pg)

Day 20

Mean

19.1

18.9

18.6

19.3

% Difference

-1.0

-2.6

1.0

S.D.

0.70

0.73

0.68

0.66

S.E.

0.23

0.23

0.22

0.22

N

9

10

10

9

MCHC (g/dL)

Day 20

Mean

33.1

33.0

32.5

30.6**

 

% Difference

-0.3

-1.8

-7.6

S.D.

0.42

0.81

0.61

0.92

S.E.

0.14

0.26

0.19

0.31

N

9

10

10

9

PLATELET (thous/µL)

Day 20

Mean

1087.

1051.

1169.

1326.**

 

% Difference

-3.3

7.5

22.0

S.D.

123.3

190.7

122.2

147.6

S.E.

41.1

60.3

38.6

49.2

N

9

10

10

9

RETIC (%)

Day 20

Mean

2.2

2.3

2.9

5.9**

 

% Difference

4.5

31.8

168.2

S.D.

0.66

0.57

0.86

1.85

S.E.

0.22

0.18

0.27

0.62

N

9

10

10

9

RETIC Absolute (thous/µL)

Day 20

Mean

143.7

143.0

178.4

319.6**

 

% Difference

-0.5

24.1

122.4

S.D.

39.88

39.41

48.94

79.24

S.E.

13.29

12.46

15.48

26.41

N

9

10

10

9

MPV (fL)

Day 20

Mean

7.03

7.20

7.16

7.34

 

% Difference

2.4

1.8

4.4

S.D.

0.150

0.533

0.337

0.450

S.E.

0.050

0.169

0.107

0.150

N

9

10

10

9

NEU (%)

Day 20

Mean

30.1

27.9

29.9

31.9

 

% Difference

-7.3

-0.7

6.0

S.D.

4.60

6.08

7.28

11.80

S.E.

1.53

1.92

2.30

3.93

N

9

10

10

9

LYMPH (%)

Day 2G

Mean

66.0

68.1

66.2

63.7

 

% Difference

3.2

0.3

-3.5

S.D.

4.59

6.13

7.52

12.15

S.E.

1.53

1.94

2.38

4.05

N

9

10

10

9

MONO (%)

Day 2G

Mean

2.6

2.4

2.7

3.0

 

% Difference

_7.7

3.8

15.4

S.D.

0.92

0.46

0.53

1.13

S.E.

0.31

0.15

0.17

0.38

N

9

10

10

9

EOS (%)

Day 2G

Mean

0.4

0.5

0.4

0.4

 

% Difference

25.0

0.0

0.0

S.D.

0.14

0.19

0.18

0.19

S.E.

0.05

0.06

0.06

0.06

N

9

10

10

9

BASO (%)

Day 2G

Mean

0.1

0.1

0.1

0.1

 

% Difference

0.0

0.0

0.0

S.D.

0.05

0.06

0.07

0.05

S.E.

0.02

0.02

0.02

0.02

N

9

10

10

9

LUC (%)

Day 20

Mean

0.8

1.0

0.7

0.8

 

% Difference

25.0

-12.5

0.0

S.D.

0.32

0.48

0.09

0.21

S.E.

0.11

0.15

0.03

0.07

N

9

10

10

9

NEU Absolute (thous/µL)

Day 20

Mean

3.93

3.49

3.44

3.92

 

% Difference

-11.2

-12.5

-0.3

S.D.

0.552

0.968

0.766

1.256

S.E.

0.184

0.306

0.242

0.419

N

9

10

10

9

LYMPH Absolute (thous/µL)

Day 20

Mean

8.86

8.71

7.85

8.02

 

% Difference

-1.7

-11.4

-9.5

S.D.

2.221

2.506

2.171

2.171

S.E.

0.740

0.792

0.687

0.724

N

9

10

10

9

MONO Absolute (thous/µL)

Day 20

Mean

0.34

0.31

0.31

0.37

 

% Difference

-8.8

-8.8

8.8

S.D.

0.135

0.092

0.070

0.136

S.E.

0.045

0.029

0.022

0.045

N

9

10

10

9

EOS Absolute (thous/µL)

Day 2 0

Mean

0.06

0.06

0.05

0.05

 

% Difference

0.0

-16.7

-16.7

S.D.

0.018

0.026

0.024

0.024

S.E.

0.006

0.008

0.008

0.008

N

9

10

10

9

BASO Absolute (thous/µL)

Day 20

Mean

0.02

0.01

0.01

0.01

 

% Difference

-50.0

-50.0

-50.0

S.D.

0.011

0.008

0.009

0.006

S.E.

0.004

0.003

0.003

0.002

N

9

10

10

9

LUC Absolute (thous/µL)

Day 20

Mean

0.10

0.12

0.09

0.10

 

% Difference

20.0

-10.0

0.0

S.D.

0.058

0.063

0.022

0.037

S.E.

0.019

0.020

0.007

0.012

N

9

10

10

9

RDW (%)

Day 20

Mean

11.5

11.8

12.2*

12.3**

 

% Difference

2.6

6.1

7.0

S.D.

0.48

0.58

0.40

0.52

S.E.

0.16

0.18

0.13

0.17

N

9

10

10

9

HDW (g/dL)

Day 20

Mean

2.57

2.53

2.46

2.67

 

% Difference

-1.6

-4.3

3.9

S.D.

0.134

0.131

0.164

0.348

S.E.

0.045

0.042

0.052

0.116

N

9

10

10

9

thous/µL = thousands/microliter

mil/µL = millions/microliter

fL = femtoliters

pg = picograms

g/dL = grams/deciliter

*   = significantly different from the control Group at 0.05   using Dunnett's test

** = significantly different from the control Group at 0.01   using Dunnett's test

Table S10

Project No.: WIL-387067

Oral Prenatal Developmental Toxicity Study Of N-Butylronitrile In Rats

Sponsor: EASTMAN CHEMICAL Co.

Summary of Serum Chemistry Values

Females

Analysis

Group (mg/kg bw/day):

0

10

25

50

Albumin (g/dL)

Day 20

Mean

3.2

3.3

3.2

3.3

 

% Difference

3.1

0.0

3.1

S.D.

0.18

0.32

0.24

0.24

S.E.

0.06

0.10

0.08

0.08

N

9

10

10

9

Total Protein (g/dL)

Day 20

Mean

5.2

5.2

5.2

5.2

 

% Difference

0.0

0.0

0.0

S.D.

0.30

0.48

0.35

0.36

S.E.

0.10

0.15

0.11

0.12

N

9

10

10

9

Globulin (g/dL)

Day 20

Mean

2.0

2.0

2.0

1.9

 

% Difference

0.0

0.0

-5.0

S.D.

0.15

0.19

0.13

0.13

S.E.

0.05

0.06

0.04

0.04

N

9

10

10

9

A/G Ratio

Day 20

Mean

1.59

1.62

1.61

1.69

 

% Difference

1.9

1.3

6.3

S.D.

0.093

0.092

0.074

0.105

S.E.

0.031

0.029

0.023

0.035

N

9

10

10

9

Total BILI (mg/dL)

Day 20

Mean

0.01

0.00

0.00

0.04

% DIE

% Difference

-100.0

-100.0

300.0

S.D.

0.033

0.000

0.000

0.053

S.E.

0.011

0.000

0.000

0.018

N

9

10

10

9

Urea Nitrogen (mg/dL)

Day 20

Mean

13.4

13.4

12.7

12.2

% DIF

% Difference

0.0

-5.2

-9.0

S.D.

1.06

2.44

1.62

1.63

S.E.

0.35

0.77

0.51

0.54

N

9

10

10

9

Creatinine (mg/dL)

Day 20

Mean

0.31

0.30

0.30

0.31

% DIF

% Difference

-3.2

-3.2

0.0

S.D.

0.036

0.030

0.061

0.058

S.E.

0.012

0.010

0.019

0.019

N

9

10

10

9

ALP (U/L)

Day 20

Mean

264.

201.

253.

279.

% DIF

% Difference

-23.9

-4.2

5.7

S.D.

90.0

59.7

106.1

151.5

S.E.

30.0

18.9

33.5

50.5

N

9

10

10

9

ALT (U/L)

Day 20

Mean

66.

61.

68.

69.

% DIF

% Difference

-7.6

3.0

4.5

S.D.

8.6

9.7

9.7

8.4

S.E.

2.9

3.1

3.1

2.8

N

9

10

10

9

AST (U/L)

Day 20

Mean

63.

69.

66.

72.

% DIF

% Difference

9.5

4.8

14.3

S.D.

8.1

10.8

7.2

10.6

S.E.

2.7

3.4

2.3

3.5

N

9

10

10

9

GGT (U/L)

Day 20

Mean

0.0

0.0

0.0

0.1

% DIF

% Difference

NA

NA

NA

S.D.

0.00

0.00

0.00

0.33

S.E.

0.00

0.00

0.00

0.11

N

9

10

10

9

GLUCOSE (mg/dL)

Day 20

Mean

104.

103.

103.

94.

% DIF

% Difference

-1.0

-1.0

-9.6

S.D.

7.4

8.5

6.0

15.0

S.E.

2.5

2.7

1.9

5.0

N

9

10

10

9

CHOLESTEROL (mg/dL)

Day 20

Mean

74.

68.

60.*

61.

% DIFF

% Difference

-8.1

-18.9

-17.6

S.D.

14.4

13.7

8.4

8.0

S.E.

4.8

4.3

2.7

2.7

N

9

10

10

9

CALCIUM (mg/dL)

Day 20

Mean

10.5

10.3

10.4

10.2

% DIFF

% Difference

-1.9

-1.0

-2.9

S.D.

0.32

0.54

0.46

0.66

S.E.

0.11

0.17

0.14

0.22

N

9

10

10

9

CHLORIDE (mEq/L)

Day 20

Mean

100.

102.

101.

101.

% DIFF

% Difference

2.0

1.0

1.0

S.D.

1.5

1.3

1.5

1.8

S.E.

0.5

0.4

0.5

0.6

N

9

10

10

9

PHOSPHORUS (mg/dL)

Day 20

Mean

6.0

5.7

5.7

4.9*

% DIFF

% Difference

-5.0

-5.0

-18.3

S.D.

0.40

0.85

0.73

1.10

S.E.

0.13

0.27

0.23

0.37

N

9

10

10

9

POTASSIUM (mEq/L)

Day 2 0

Mean

4.66

4.80

4.47

4.26

% DI

% Difference

3.0

-4.1

-8.6

S.D.

0.357

0.449

0.162

0.449

S.E.

0.119

0.142

0.051

0.150

N

9

10

10

9

SODIUM (mEq/L)

Day 20

Mean

138.

138.

139.

136.*

% DI

% Difference

0.0

0.7

-1.4

S.D.

1.5

1.3

1.4

1.9

S.E.

0.5

0.4

0.4

0.6

N

9

10

10

9

TRIGLYCERIDE (mg/dL)

Day 20

Mean

412.

412.

360.

298.

% DI

% Difference

0.0

-12.6

-27.7

S.D.

178.9

270.5

130.9

120.7

S.E.

59.6

85.5

41.4

40.2

N

9

10

10

9

mg/dL = milligrams/deciliter

U/L = international Unit/Liter

 g/dL = grams/deciliter

mEq/L = milliequivalents/Liter

*   = Significantly different from the control Group at 0.05 using Dunnett's test

NA = not applicable

Table S11

Project No.: WIL-387067

Oral Prenatal Developmental Toxicity Study Of N-Butylronitrile In Rats

Sponsor: EASTMAN CHEMICAL Co.

Summary of Other Chemistry Values

Females

Analysis

Group (mg/kg bw/day):

0

10

25

50

Bile Acid (umol/L)

Day 20

Mean

54.4

34.7

30.8

62.0

% Difference

-36.2

-43.4

14.0

S.D.

29.77

27.27

16.34

29.68

S.E.

9.92

8.62

5.17

9.89

N

9

10

10

9

µmol/L = micromoles/Liter

None Significantly different from control Group

Table S12

Project No.: WIL-387067

Oral Prenatal Developmental Toxicity Study Of N-Butylronitrile In Rats

Sponsor: EASTMAN CHEMICAL Co.

Summary of Maternal Macroscopic Findings

Group :

1

2

3

4

Number Examined

25

25

25

25

No Significant Changes Observed

22

22

21

20

Nongravid -- Ammonium Sulfide Negative

1

1

0

2

Uterus: Contents,  Clear Fluid

0

0

0

1

Placentae: enlarged

0

0

1

2

Kidneys: Area(S), depressed

1

0

0

0

Spleen: Accessory

0

0

1

1

Placentae: Fused

1

0

1

0

Uterus: Contents, Dark red

0

2

0

2

Liver: Accessory Lobule (s)

0

0

1

0

1-    0 mg/kg/Day

2-  10 mg/kg/Day

3-  25 mg/kg/Day

4-  50 mg/kg/Day

Table S13

Project No.: WIL-387067

Oral Prenatal Developmental Toxicity Study Of N-Butylronitrile In Rats

Sponsor: EASTMAN CHEMICAL Co.

Summary of Organ Weights and Organ Weights relative to Brain Weights

Females

Group (mg/kg bw/day):

0

10

25

50

Adrenal Glands (g)

Mean

0.0763

0.0761

0.0752

0.0870*

% Difference

-0.3

-1.4

14.0

S.D.

0.00962

0.01118

0.01107

0.01794

S.E.

0.00196

0.00228

0.00221

0.00374

N

24

24

25

23

Adrenal Glands (g/100 g Brain)

Mean

3.906

3.901

3.839

4.408*

S.D.

0.4897

0.5508

0.5522

0.8933

S.E.

0.1000

0.1124

0.1104

0.1863

N

24

24

25

23

Brain (g)

Mean

1.95

1.95

1.96

1.97

% Difference

0.0

0.5

1.0

S.D.

0.064

0.093

0.072

0.060

S.E.

0.013

0.019

0.014

0.013

N

24

24

25

23

Kidneys (g)

Mean

2.05

2.03

2.04

2.07

% Difference

-1.0

-0.5

1.0

S.D.

0.184

0.206

0.139

0.196

S.E.

0.038

0.042

0.028

0.041

N

24

24

25

23

Kidneys (g/100 g Brain)

Mean

104.587

103.974

103.947

104.791

S.D.

8.3348

9.4695

6.8994

8.9884

S.E.

1.7013

1.9329

1.3799

1.8742

N

24

24

25

23

Liver (g)

Mean

16.58

16.92

16.96

18.07**

% Difference

2.1

2.3

9.0

S.D.

1.229

1.589

1.459

1.552

S.E.

0.251

0.324

0.292

0.324

N

24

24

25

23

Liver (g/100 g Brain)

848.262

869.241

866.052

914.587**

57.8310

87.0126

69.6214

68.1823

11.8047

17.7614

13.9243

14.2170

N

24

24

25

23

*   = Significantly different from the control Group at 0.05 using Dunnett's test

** = Significantly different from the control Group at 0.01   using Dunnett's test

Nongravid Weights not included in calculation of the mean

Table S14

Project No.: WIL-387067

Oral Prenatal Developmental Toxicity Study Of N-Butylronitrile In Rats

Sponsor: EASTMAN CHEMICAL Co.

Summary of Fetal Data at Scheduled Necropsy

Group:

Sex

Viable Fetuses

Dead Fetuses

Resorptions

Post Implantation Loss

Implantation

Corpora Lutea

Pre Implantation Loss

Fetal Weights IN GRAMS

No. of Gravid Females

M

F

early

late

Sites

1) Total

178

178

356

0

19

1

20

376

395

19

NA

24

Mean

7.4

7.4

14.8

0.0

0.8

0.0

0.8

15.7

16.5

0.8

3.9

S.D.

1.93

1.53

1.27

0.00

0.93

0.20

0.92

1.76

2.28

1.69

0.22

S.E.

0.39

0.31

0.26

0.00

0.19

0.04

0.19

0.36

0.47

0.35

0.05

2) Total

167

184

351

0

28

0

28

379

408

29

NA

24

Mean

7.0

7.7

14.6

0.0

1.2

0.0

1.2

15.8

17.0

1.2

3.9

S.D.

2.18

2.44

2.08

0.00

1.37

0.00

1.37

1.96

2.43

2.25

0.19

S.E.

0.44

0.50

0.42

0.00

0.28

0.00

0.28

0.40

0.50

0.46

0.04

3) Total

187

162

349

0

30

0

30

379

424

45

NA

25

Mean

7.5

6.5

14.0

0.0

1.2

0.0

1.2

15.2

17.0

1.8

3.9

S.D.

2.06

3.02

2.56

0.00

1.44

0.00

1.44

1.75

2.86

2.33

0.26

S.E.

0.41

0.60

0.51

0.00

0.29

0.00

0.29

0.35

0.57

0.47

0.05

4) Total

155

175

330

0

30

0

30

360

392

32

NA

23

Mean

6.7

7.6

14.3

0.0

1.3

0.0

1.3

15.7

17.0

1.4

3.6**

S.D.

2.43

2.78

2.92

0.00

2.70

0.00

2.70

1.77

2.67

1.85

0.26

S.E.

0.51

0.58

0.61

0.00

0.56

0.00

0.56

0.37

0.56

0.39

0.05

 

** = Significantly different from the control Group at 0.01

NA = Not Applicable

Mean Number of viable Fetuses

Mean Number of Implantation Sites

Mean Number of Corpora Lutea

Fetal Weights Compared Using Dunnett's Test

1 - 0 mg/kg/Day

2 - 10 mg/kg/Day

3 - 25 mg/kg/Day

4 - 50 mg/kg/Day

Table S15

Project No.: WIL-387067

Oral Prenatal Developmental Toxicity Study Of N-Butylronitrile In Rats

Sponsor: EASTMAN CHEMICAL Co.

Summary of Fetal Data AT Scheduled Necropsy  [% Per Litter]

Group (mg/kg bw/day):

0

10

25

50

Corpora Lutea

Mean

16.5

17.0

17.0

17.0

S.D.

2.28

2.43

2.86

2.67

S.E.

0.47

0.50

0.57

0.56

N

24

24

25

23

Implantation Sites

Mean

15.7

15.8

15.2

15.7

S.D.

1.76

1.96

1.75

1.77

S.E.

0.36

0.40

0.35

0.37

N

24

24

25

23

Viable Fetuses (%)

Mean

95.1

92.8

91.6

91.9

S.D.

5.34

8.26

11.33

16.00

S.E.

1.09

1.69

2.27

3.34

N

24

24

25

23

Dead Fetuses (%)

Mean

0.0

0.0

0.0

0.0

S.D.

0.00

0.00

0.00

0.00

S.E.

0.00

0.00

0.00

0.00

N

24

24

25

23

Early Resorptions (%)

Mean

4.7

7.3

8.4

8.1

S.D.

5.43

8.26

11.32

15.99

S.E.

1.11

1.69

2.26

3.34

N

24

24

25

23

LATE Resorptions (%)

Mean

0.3

0.0

0.0

0.0

S.D.

1.29

0.00

0.00

0.00

S.E.

0.26

0.00

0.00

0.00

N

24

24

25

23

Total Resorptions (%)

Mean

5.0

7.3

8.4

8.1

S.D.

5.34

8.26

11.32

15.99

S.E.

1.09

1.69

2.26

3.34

N

24

24

25

23

PRE-Implantation Loss (%)

Mean

4.1

6.2

9.3

7.2

S.D.

8.27

10.92

10.46

9.00

S.E.

1.69

2.23

2.09

1.88

N

24

24

25

23

Post-Implantation Loss (%)

Mean

5.0

7.3

8.4

8.1

S.D.

5.34

8.26

11.32

15.99

S.E.

1.09

1.69

2.26

3.34

N

24

24

25

23

MALES (%)

Mean

49.7

47.8

55.1

46.6

S.D.

11.13

14.16

15.85

14.66

S.E.

2.27

2.89

3.17

3.06

N

24

24

25

23

Females (%)

Mean

50.3

52.2

44.9

53.4

S.D.

11.13

14.16

15.85

14.66

S.E.

2.27

2.89

3.17

3.06

N

24

24

25

23

MALE Fetal Weights (g)

Mean

4.0

4.0

3.9

3.7 **

% Difference

0.0

 -2.5

 -7.5

S.D.

0.21

0.20

0.29

0.28

S.E.

0.04

0.04

0.06

0.06

N

24

24

25

23

FEMALE Fetal Weights (g)

Mean

3.8

3.8

3.8

3.5 **

% Difference

0.0

0.0

 - 7.9

S.D.

0.25

0.21

0.23

0.26

S.E.

0.05

0.04

0.05

0.05

N

24

24

25

23

Combined Fetal Weights (g)

Mean

3.9

3.9

3.9

3.6**

% Difference

0.0

0.0

 - 7.7

S.D.

0.22

0.19

0.26

0.26

S.E.

0.05

0.04

0.05

0.05

N

24

24

25

23

Proportional(%) Data Compared Using Dunn's Test

Fetal Weigts Compared Using Dunnett's Test

Modified Statistics used.

 * Indicates Parametric Analysis and

 + Indicates Non-Parametric Analysis.

** = Significantly different from the control Group at 0.01

Table S16

Project No.: WIL-387067

Oral Prenatal Developmental Toxicity Study Of N-Butylronitrile In Rats

Sponsor: EASTMAN CHEMICAL Co.

Summary of Fetuses and Litters with Malformations  [Absolute No.]

Day 20

 

Fetuses

Litters

Dose Group:

1

2

3

4

1

2

3

4

Number Examined Externally

356

351

349

330

24

24

25

23

Anal Atresia

0

0

0

1

0

0

0

1

Cleft Palate

0

1

0

0

0

1

0

0

Number Examined Viscerally

356

351

349

330

24

24

25

23

Diaphragmatic Hernia

0

1

0

0

0

1

0

0

Number Examined Skeletally

356

351

349

330

24

24

25

23

Bent Limb bone(s)

0

0

0

1

0

0

0

1

Costal cartilage Anomaly

0

0

0

1

0

0

0

1

Vertebral Anomaly with or without Associated Rib Anomaly

0

1

0

0

0

1

0

0

Total Number with Malformations

External :

0

1

0

1

0

1

0

1

Soft tissue :

0

1

0

0

0

1

0

0

Skeletal :

0

1

0

1

0

1

0

1

Combined :

0

2

0

1

0

2

0

1

1 - 0 mg/kg/Day

2 - 10 mg/kg/Day

3 - 25 mg/kg/Day

4 - 50 mg/kg/Day

Table S17

Project No.: WIL-387067

Oral Prenatal Developmental Toxicity Study Of N-Butylronitrile In Rats

Sponsor: EASTMAN CHEMICAL Co.

Summary of Litter Proportions of Malformations

% Per Litter                                                                            Day 20

Dose Group:

1

2

3

4

Number of Litters Examined Externally

24

24

25

23

Anal Atresia

Mean

0.0

0.0

0.0

1.1

S.D.

0.00

0.00

0.00

5.21

S.E.

0.00

0.00

0.00

1.09

Cleft Palate

Mean

0.0

0.4

0.0

0.0

S.D.

0.00

1.86

0.00

0.00

S.E.

0.00

0.38

0.00

0.00

Diaphragmatic Hernia

Mean

0.0

0.3

0.0

0.0

S.D.

0.00

1.57

0.00

0.00

S.E.

0.00

0.32

0.00

0.00

Bent Limb bone(s)

Mean

0.0

0.0

0.0

1.1

S.D.

0.00

0.00

0.00

5.21

S.E.

0.00

0.00

0.00

1.09

Costal cartilage Anomaly

Mean

0.0

0.0

0.0

1.1

S.D.

0.00

0.00

0.00

5.21

S.E.

0.00

0.00

0.00

1.09

Vertebral Anomaly with or without Associated RIB Anomaly

Mean

0.0

0.4

0.0

0.0

S.D.

0.00

1.86

0.00

0.00

S.E.

0.00

0.38

0.00

0.00

Total Malformations

Percent Per Litter with External Malformations

Mean

0.0

0.4

0.0

1.1

S.D.

0.00

1.86

0.00

5.21

S.E.

0.00

0.38

0.00

1.09

Percent Per Litter with Soft tissue Malformations

Mean

0.0

0.3

0.0

0.0

S.D.

0.00

1.57

0.00

0.00

S.E.

0.00

0.32

0.00

0.00

Percent Per Litter with Skeletal Malformations

Mean

0.0

0.4

0.0

1.1

S.D.

0.00

1.86

0.00

5.21

S.E.

0.00

0.38

0.00

1.09

Total Percent Per Litter with Malformations

Mean

0.0

0.7

0.0

1.1

S.D.

0.00

2.38

0.00

5.21

S.E.

0.00

0.49

0.00

1.09

1 - 0 mg/kg/Day

2 - 10 mg/kg/Day

3 - 25 mg/kg/Day

4 - 50 mg/kg/Day

Modified Statistics used.

None Significantly different from control Group

Table S18

Project No.: WIL-387067

Oral Prenatal Developmental Toxicity Study Of N-Butylronitrile In Rats

Sponsor: EASTMAN CHEMICAL Co.

Summary of Fetuses and Litters with Variations [Absolute No.]

Day   20

 

Fetuses

Litters

Dose Group:

1

2

3

4

1

2

3

4

Number Examined Externally

356

351

349

330

24

24

25

23

Number with Findings

0

0

0

0

0

0

0

0

Number Examined Viscerally

356

351

349

330

24

24

25

23

Major Blood Vessel Variation

2

0

1

1

2

0

1

1

Renal Papilla(e) Not Developed And/Or Distended Ureter(s)

0

4

1

0

0

2

1

0

Liver- Accessory Lobule(s)

11

5

3

6

4

4

3

3

Brachiocephalic Trunk- Short

0

1

0

0

0

1

0

0

Liver- Pale

0

0

1

0

0

0

1

0

Hemorrhagic Ring Around The Iris

0

0

0

1

0

0

0

1

Number Examined Skeletally

356

351

349

330

24

24

25

23

Sternebra(e)  #5 And/Or #6 Unossified

32

26

32

77

10

12

11

21

Cervical Centrum #1 Ossified

104

114

91

47

21

22

23

14

14Th Rudimentary Rib(s)

29

26

21

35

11

13

13

12

7Th Cervical Rib(s)

0

0

4

4

0

0

4

2

Sternebra(e)  #1,#2,#3 And/Or #4 Unossified

1

1

1

2

1

1

1

2

Sternebra(e) Malaligned(Slight Or Moderate)

4

3

4

3

3

3

3

3

Reduced Ossification Of The Vertebral Arches

3

1

0

1

2

1

0

1

Reduced Ossification Of The Skull

0

1

3

2

0

1

2

2

Reduced Ossification Of The 13Th Rib(S)

2

0

0

7

2

0

0

1

Bent Rib(S)

0

0

1

1

0

0

1

1

Reduced Ossification Of The Rib(s)

0

0

1

0

0

0

1

0

25 Presacral Vertebrae

0

0

1

3

0

0

1

1

Sternebrae With Thread-Like Attachment

1

0

0

0

1

0

0

0

Hyoid Unossified

1

0

0

0

1

0

0

0

Bent Scapula(e)

0

0

0

1

0

0

0

1

Vertebral Centra Unossified

0

0

0

1

0

0

0

1

Number Examined Skeletally

356

351

349

330

24

24

25

23

pubis unossified

0

0

0

1

0

0

0

1

14th full rib(s)

0

0

0

1

0

0

0

1

1 - 0 mg/kg/Day

2 - 10 mg/kg/Day

3 - 25 mg/kg/Day

4 - 50 mg/kg/Day

Table S19

Project No.: WIL-387067

Oral Prenatal Developmental Toxicity Study Of N-Butylronitrile In Rats

Sponsor: EASTMAN CHEMICAL Co.

Summary of Litter Proportions of Variations % Per Litter

Day 20

Dose Group:

1

2

3

4

Number of Litters Examined Externally

24

24

25

23

Number of Litters with Findings

0

0

0

0

Number of Litters Examined Viscerally

24

24

25

23

Major Blood Vessel Variation

Mean

0.6

0.0

0.4

0.4

S.D.

1.88

0.00

1.82

1.74

S.E.

0.38

0.00

0.36

0.36

Renal Papilla(e) Not Developed And/Or Distended Ureter(s)

Mean

0.0

1.0

0.3

0.0

S.D.

0.00

3.96

1.43

0.00

S.E.

0.00

0.81

0.29

0.00

Liver- Accessory Lobule(s)

Mean

3.4

1.4

0.9

1.7

S.D.

9.95

3.43

2.41

4.98

S.E.

2.03

0.70

0.48

1.04

Brachiocephalic Trunk- Short

Mean

0.0

0.3

0.0

0.0

S.D.

0.00

1.28

0.00

0.00

S.E.

0.00

0.26

0.00

0.00

Liver- Pale

Mean

0.0

0.0

0.3

0.0

S.D.

0.00

0.00

1.43

0.00

S.E.

0.00

0.00

0.29

0.00

Hemorrhagic Ring Around The Iris

Mean

0.0

0.0

0.0

1.1

S.D.

0.00

0.00

0.00

5.21

S.E.

0.00

0.00

0.00

1.09

Number of Litters Examined Skeletally

 

24

24

25

23

Sternebra(e)  #5 And/Or #6 Unossified

Mean

8.5

7.5

8.9

23.5**

S.D.

14.65

12.34

15.49

22.14

S.E.

2.99

2.52

3.10

4.62

Cervical Centrum #1 Ossified

Mean

29.5

32.0

28.0

13.5

S.D.

24.03

22.09

25.59

17.24

S.E.

4.90

4.51

5.12

3.59

14Th Rudimentary Rib(s)

Mean

8.2

7.6

6.0

10.4

S.D.

12.25

11.71

7.23

14.30

S.E.

2.50

2.39

1.45

2.98

7Th Cervical Rib(s)

Mean

0.0

0.0

1.0

1.0

S.D.

0.00

0.00

2.45

3.46

S.E.

0.00

0.00

0.49

0.72

Sternebra(e)  #1,#2,#3 And/Or #4 Unossified

Mean

0.2

0.4

0.3

1.3

S.D.

1.13

1.86

1.33

5.28

S.E.

0.23

0.38

0.27

1.10

Sternebra(e) Malaligned(Slight Or Moderate)

Mean

1.1

0.9

1.1

0.9

S.D.

3.25

2.36

3.31

2.35

S.E.

0.66

0.48

0.66

0.49

Reduced Ossification Of The Vertebral Arches

Mean

0.8

0.3

0.0

1.1

S.D.

3.10

1.36

0.00

5.21

S.E.

0.63

0.28

0.00

1.09

Reduced Ossification Of The Skull

Mean

0.0

0.3

1.0

1.3

S.D.

0.00

1.36

3.85

5.30

S.E.

0.00

0.28

0.77

1.11

Reduced Ossification Of The 13Th Rib(s)

Mean

0.5

0.0

0.0

2.0

S.D.

1.81

0.00

0.00

9.73

S.E.

0.37

0.00

0.00

2.03

Bent Rib(s)

Mean

0.0

0.0

0.3

1.1

S.D.

0.00

0.00

1.43

5.21

S.E.

0.00

0.00

0.29

1.09

Reduced Ossification Of The Rib(s)

Mean

0.0

0.0

0.3

0.0

S.D.

0.00

0.00

1.43

0.00

S.E.

0.00

0.00

0.29

0.00

25 Presacral Vertebrae

Mean

0.0

0.0

0.3

0.9

S.D.

0.00

0.00

1.33

4.17

S.E.

0.00

0.00

0.27

0.87

Sternebrae With Thread-Like Attachment

Mean

0.3

0.0

0.0

0.0

S.D.

1.28

0.00

0.00

0.00

S.E.

0.26

0.00

0.00

0.00

Hyoid Unossified

Mean

0.2

0.0

0.0

0.0

S.D.

1.20

0.00

0.00

0.00

S.E.

0.25

0.00

0.00

0.00

Bent Scapula(e)

Mean

0.0

0.0

0.0

1.1

S.D.

0.00

0.00

0.00

5.21

S.E.

0.00

0.00

0.00

1.09

Vertebral Centra Unossified

Mean

0.0

0.0

0.0

1.1

S.D.

0.00

0.00

0.00

5.21

S.E.

0.00

0.00

0.00

1.G9

Pubis Unossified

Mean

0.0

0.0

0.0

1.1

S.D.

0.00

0.00

0.00

5.21

S.E.

0.00

0.00

0.00

1.G9

14Th Full Rib(s)

Mean

0.0

0.0

0.0

1.1

S.D.

0.00

0.00

0.00

5.21

S.E.

0.00

0.00

0.00

1.G9

Number of Litters Examined

24

24

25

23

Total Variations

Percent Per Litter with External Variations

Mean

0.0

0.0

0.0

0.0

S.D.

0.00

0.00

0.00

0.00

S.E.

0.00

0.00

0.00

0.00

Percent Per Litter with Soft tissue Variations

Mean

3.9

2.7

1.8

3.2

S.D.

9.93

5.31

4.22

6.99

S.E.

2.03

1.08

0.84

1.46

Percent Per Litter with Skeletal Variations

Mean

45.3

42.1

42.0

44.9

S.D.

20.73

22.47

22.60

21.64

S.E.

4.23

4.59

4.52

4.51

Total Percent Per Litter with Variations

Mean

46.8

43.7

43.5

46.6

S.D.

20.78

21.32

23.96

21.15

S.E.

4.24

4.35

4.79

4.41

1 - 0 mg/kg/Day

2 - 10 mg/kg/Day

3 - 25 mg/kg/Day

4 - 50 mg/kg/Day

Modified Statistics used.

** = Significantly different from the control Group at 0.01

Further a table shows a summary

 Dose  0 mg/kg  10 mg/kg  25 mg/kg  50 mg/kg

 Pregnant/non-pregnant dams

 24/1

24/1

25/0

 23/2

 Dams with abortions, early delivery, stillbirth or dead fetus

 0

 0

 0

 0

 Pre-implant loss #/%

19/4.1

29/6.2

45/9.3

 32/7.2

 Post implant loss #/%

20/5.0

28/7.3

30/8.4

30/8.1

 Body weight (g) 399 405  400 395
 Body weight change (g) 57.1 66  63.7 61.2
 gravid uterine weight (g) 88.1 87.5 82.8 80
 #/% live offspring 356/95 351/92 349/91 330/91
 fetal body weight M/F/combined (g)  3.9 3.9 3.9  3.6
 #/% fetuses with malformation   Variations/Marformations were given as #/% per type of incidence, rather than absolute #/% with ANY finding
 #/% litter with malformation   Variations/Marformations were given as #/% per type of incidence, rather than absolute #/% with ANY finding
   #/% fetuses with variations  Variations/Marformations were given as #/% per type of incidence, rather than absolute #/% with ANY finding
   #/% litter with variations  Variations/Marformations were given as #/% per type of incidence, rather than absolute #/% with ANY finding
             
Conclusions:
Based on test substance-related alterations in hematology and serum chemistry parameters and higher mean adrenal gland and liver weights at 50 mg/kg/day, a dosage level of 25 mg/kg/day was considered to be the no-observed-adverse-effect level
(NOAEL) for maternal toxicity. Based on lower mean fetal weights and an increased occurrence of the skeletal developmental variation sternebra(e) nos. 5 and/or 6 unossified at 50 mg/kg/day, a dosage level of 25 mg/kg/day was considered to be the NOAEL for
embryo/fetal development when n-Butyronitrile was administered orally by gavage to bred Crl:CD(SD) rats.
Executive summary:

The test substance, n-Butyronitrile, in the vehicle (corn oil) was administered orally by gavage to 3 groups of 25 bred female Crl:CD(SD) rats once daily from gestation days 6 through 19. Dosage levels were 10, 25, and 50 mg/kg/day administered at a dose volume of 10 mL/kg. A concurrent control group composed of 25 bred females received the vehicle on a comparable regimen. The females were approximately 14 weeks of age at the initiation of dose administration. All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. On gestation day 20, clinical pathology (hematology and serum chemistry) evaluations were performed on 10 females/group and a laparohysterectomy was performed on all females. The uteri, placentae, and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations, and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The liver, brain, kidneys, and adrenal glands from each female were removed, weighed, and preserved in 10% neutral-buffered formalin for possible future histopathological examination. The fetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and developmental variations.

 

All females survived to the scheduled necropsy on gestation day 20. Clear and/or red material around the mouth was noted in a dose-related manner for females in all test substance-treated groups approximately 1 hour following dose administration primarily during gestation days 12-19. The aforementioned material observations did not persist to the daily examinations and were considered nonadverse in the absence of other signs of systemic toxicity (e.g.body weight decrements). No test substance-related clinical observations were noted at the daily examinations at any dosage level. Test substance-related lower mean food consumption was noted in the 50 mg/kg/day group during gestation days 6-9. Mean food consumption in this group was generally similar to the control group for the remainder of the treatment period. In the absence of any effects on mean body weight gain or body weight at 50 mg/kg/day, this transient decrement in food consumption was not considered adverse. Mean food consumption at 10 and 25 mg/kg/day and mean body weights, body weight gains, net body weights, net body weight gains, and gravid uterine weights at 10, 25, and 50 mg/kg/day were unaffected by test substance administration.

 

Test substance-related alterations in hematology parameters were observed in the 50 mg/kg/day group and included lower mean red blood cell count and lower mean hemoglobin and corpuscular hemoglobin concentrations, and higher mean corpuscular volume, platelet count, and percentage and absolute reticulocyte counts compared to the control group. Higher mean red blood cell distribution widths were noted in the 25 and 50 mg/kg/day groups; however, the difference at 25 mg/kg/day was considered non-adverse in the absence of any other effects on hematology parameters at this dosage level. Lower mean phosphorus, potassium, and sodium concentrations were also noted at 50 mg/kg/day and these alterations in serum chemistry were considered test substance-related. There were no test substance-related changes in hematology or serum chemistry parameters at 10 mg/kg/day. There were no test substance-related macroscopic findings in the test substance-treated groups at the scheduled necropsy on gestation day 20. Test substance-related higher mean adrenal gland and liver weights (absolute and relative to brain weight) were noted in the 50 mg/kg/day group. There were no test substance-related effects on organ weights at 10 and 25 mg/kg/day. Test substance-related, lower (7.5% to 7.9%) mean fetal body weights were noted in the 50 mg/kg/day group when compared to the control group. An increased occurrence of the skeletal developmental variation sternebra(e) nos. 5 and/or 6 unossified was noted in the 50 mg/kg/day group and was considered indicative of developmental delay and secondary to the test substance-related effect on fetal growth at this dosage level. There were no test substance-related effects on intrauterine growth or fetal morphology (external, visceral, or skeletal) at 10 or 25 mg/kg/day or intrauterine survival at 10, 25, and 50 mg/kg/day.

 

Based on test substance-related alterations in hematology and serum chemistry parameters and higher mean adrenal gland and liver weights at 50 mg/kg/day, a dosage level of 25 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity. Based on lower mean fetal weights and an increased occurrence of the skeletal developmental variation sternebra(e) nos. 5 and/or 6 unossified at 50 mg/kg/day, a dosage level of 25 mg/kg/day was considered to be the NOAEL for embryo/fetal development when n-Butyronitrile was administered orally by gavage to bred Crl:CD(SD) rats.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
25 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

Significant effects on maternal toxicity has been limited to haematological changes, serum chemistry variations and slight effects on organ weights in the highest dose group, and are considered to be of minor importance. The information from the general repeated dose toxicity study (90-day study) has also been taken into account, and has shown that maternal toxicity could be anticipated at the dose of 50 mg/kg bw.

As maternal toxicity may, depending on severity, influence development via non-specific secondary mechanisms, producing effects such as depressed foetal weight, retarded ossification, and possibly resorptions and certain malformations in some strains of certain species, these effects have to be taken into account. However, the adverse effects in the embryo/foetus shall be first considered.

First of all, no death, structural abnormality or functional deficiency of the developing organism has been observed. Classification is in most cases based on severe effects like malformation or death of the foetus for developmental toxicants. In this prenatal developmental toxicity study only altered growth has been observed (reduced foetal body weight in the 50 mg/kg/day group (3.7 g, 3.5 g, and 3.6 g, for males, females and combined, respectively) and were significantly (p<0.01) lower (7.5% to 7.9%) when compared to the concurrent control group (4.0 g, 3.8 g, and 3.9 g, respectively), which might be indicative of developmental toxicity. The unossified sternebrae also observed in the highest dose group, were considered indicative of developmental delay and secondary to the test substance-related effect on foetal growth at this dosage level.

In an attempt to further evaluate the observed reduction of foetal body weights, historical control data has been taken into account. For foetal body weights (male, female, and combined) a mean value of 3.703 g is reported (S.D. 0.1251 and SEM 0.0080) for both sexes combined. The historical data ranges are 3.475 g to 4.138 g, 3.354 g to 3.901 g, and 3.411 g to 4.026 g for male, female, and combined, respectively. In addition, the mean foetal weights in the concurrent control group were at the higher end of the WIL Research historical control data ranges.

This clearly shows that the observed foetal body weights in the high dose group are still within the historical control range data.

These conclusions are further supported by the fact, that there were no test substance-related effects on intrauterine growth or foetal morphology (external, visceral, or skeletal) at 10 or 25 mg/kg/day or intrauterine survival at 10, 25, and 50 mg/kg/day.

Furthermore, the observed effects on maternal haematology - significant but of low order of magnitude – may have attributed to this reduction of foetal body weight, as the haematological competence of the dams has been slightly affected, which increased the foetal body weight variability.

Concerning the possibility of cyanide formation from n-Butyronitrile in vivo, which might contribute to the toxicity to the embryo/foetus, it needs to be considered that the foetal body weights were only slightly reduced (-7.5 to -7.9%), but still within the range of historical controls. Therefore, any cyanide that potentially could have been formed, may have influenced the foetal body weight only on a limited scale. Nevertheless, a literature review is ongoing to evaluate the possible range of any cyanide formed and its relevance on the toxicity potential of n-Butyronitrile. If anything relevant will be identified, the dossier will be updated again accordingly.

In summary, an increased incidence of significant haematological signs of toxicity has been observed in treated dams, which has influenced secondarily slightly the development of the embryo/foetus. The observed foetal body weight changes are still within the range reported by historical control data.

The findings are not considered to be adverse and not severe enough to trigger classification as a developmental toxicant. Based on these data, N-Butyronitrile does not need to be classified and labelled as reproductive or developmental toxicant according to Regulation (EC) 1272/2008.

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