Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Additional information

The potential for 1 -isopropyl-2,2 -dimethyltrimethylene diisobutyrate to cause reproductive toxicity is well understood. In a developmental/reproductive screening study conducted according to OECD Guideline 421, several endpoints of reproductive toxicity were observed in high-dose males and females receiving 905 and 1135 mg/kg bw/day, respectively, of 1 -isopropyl-2,2 -dimethyltrimethylene diisobutyrate in the diet throughout pre-mating, mating, gestation, and lactation. In addition, isolated endpoints of reproductive function were also statistically significantly different in the low and mid-dose groups (91-120 and 276-359 mg/kg bw/day, respectively). In the high-dose group, there was a statistically significant reduction in the mean number of implantation sites, reduced mean number of live pups on postnatal Day 4, reduced mean litter weights on postnatal Days 0 and 4, decreased mean absolute epididymal sperm counts, and reduced absolute and relative (to body weight) testicular sperm counts. There was also a non-significant reduction in the mean number of live pups per litter on postnatal Day 0. Effects observed in the mid-dose group were limited to decreased mean absolute epididymal sperm counts while the low-dose group had decreased mean absolute epididymal sperm counts and reduced absolute and relative (to body weight) testicular sperm counts. Absolute and relative (to body weight) testicular sperm counts for the mid-dose group were slightly higher than the mean control group value. The lack of effect in the mid-dose group suggests that the effect noted for testicular sperm counts in the low-dose group was not related to 1 -isopropyl-2,2 -dimethyltrimethylene diisobutyrate exposure. There was no effect on absolute or relative (to body weight) testes or epididymides weights in any of the treatment groups. There were also no changes in the histopathology of the testes or epididymides in the high-dose group animals.

The male rats in the developmental/reproductive screening study were exposed for 50 days prior to collection of the sperm parameters. One entire cycle of spermatogenesis in the rat takes approximately 58 days with another 5 days for maturation of spermatid to spermatozoa within the epididymides. Therefore, the sperm count collected from the epididymides in this study was determined by testicular cell division that occurred from 7-13 days prior to exposure to 1 -isopropyl-2,2 -dimethyltrimethylene diisobutyrate. Testicular sperm counts reflect cell division that occurred from 2-7 days prior to 1 -isopropyl-2,2 -dimethyltrimethylene diisobutyrate exposure. Since the stages of spermatogenesis responsible for these counts occurred prior to 1 -isopropyl-2,2 -dimethyltrimethylene diisobutyrate exposure, it is highly unlikely that the reductions in these counts were related to altered cell division within the testes. While a chemical could theoretically affect sperm maturation within the testes leading to increased phagocytosis of damaged immature sperm by the Sertoli cells within the seminiferous tubules, there was no evidence in this study of damaged immature sperm observed histologically nor was there any effect on sperm motility, a function certain to be affected in damaged sperm. In addition, the epididymal counts were similar to control values when the mass of the organ was considered in the analysis.

The findings of decreased number of implantation sites, reduced mean litter weights on postnatal Days 0 and 4, and decreased live pups on postnatal Day 4 in the high-dose group suggest a treatment-related effect of 1 -isopropyl-2,2 -dimethyltrimethylene diisobutyrate exposure. At this dose level, systemic toxicity, including decreases in mean body weights and/or body weight gains and mean feed consumption and/or feed utilization were observed in both sexes of the parental generation. There were no adverse effects on any other parameters including reproductive performance, fertility index, fecundity index, precoital interval, gestation duration, percent survival, pre- and post-implantation loss, live and dead pups on postnatal Day 0, percentage of male and female pups, and mean pup body weights. There was a clear NOAEL for reproductive toxicity in the mid-dose group in which the parental generation received the equivalent of 276 mg/kg bw/day for males and 359 mg/kg bw/day for females in the diet.  

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test conducted by a method similar to OECD Guideline 422, both sexes were exposed to up to 750 mg/kg bw/day by oral gavage with no gross or microscopic effects on reproductive organs of either sex. Although estrous cycle length was significantly shorter in high-dose females (4.1 days versus 4.6 days in the control), values fell within the historical control range for the testing laboratory. There were no treatment-related effects on sexual function, fertility, or any reproductive parameters of parental animals exposed continuously during premating, mating, gestation, and through lactation Day 4. The gestation period, numbers of corpora lutea, implantation sites, pups born and live pups born, sex ratio, number of live pups on Day 4 after birth, and number of stillborns were similar across groups. All pregnant animals delivered normally and there were no adverse effects during the lactation period. The NOEL for reproductive toxicity was 750 mg/kg bw/day when the test material was administered by oral gavage.

In addition, there were no test-substance-related effects in the reproductive organs of either sex in a USFDA guideline subchronic toxicity study in which male and female rats were administered up to 750 mg/kg bw/day in the diet for 13 weeks.  

TXIB also was evaluated in a dietary prenatal developmental toxicity study (OECD 414) in groups of Sprague Dawley rats that were given either 0, 0.15, 0.45, or 1.50 % TXIB in the diet. Data indicate that the NOAEL for maternal toxicity was determined to be 1.5 mg/g of food, due to significantly lower body weight gain and/or body weight loss which was observed at 4.5 mg/g of food or above. The NOAEL for fetal growth and development was determined to be 4.5 mg/g of food due to the significantly lower mean fetal body weights noted in the high dose (15 mg/g of food) group. Importantly, effects on fetal body weight were only observed following administration of TXIB that produced evidence of maternal toxicity, and therefore TXIB is not considered to produce adverse developmental effects in rats.

Effects on developmental toxicity

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
100 females (maximum of 125 purchased). A sufficient number of sexually mature untreated resident males of the same strain and source will be used to induce pregnancies. Animals not assigned to the study will be transferred to the stock animal colony or will be euthanized by carbon dioxide inhalation and the carcasses discarded. The number of animals is based on the US EPA Health Effects Test Guidelines OPPTS 870.3700, Prenatal Development Toxicity Study, August 1998 and the OECD Guidelines for Testing of Chemicals: Guideline 414, Prenatal Developmental Toxicity Study, January 2001, which recommend evaluation of approximately 20 females with implantation sites at necropsy. Given the possibility of nongravid animals, unexpected deaths, or treatment-related moribundity and/or mortality, this is an appropriate number of animals to obtain a sample size of 20 at termination.

Body Weight Range: A minimum of 220 g at initiation ofbreeding.

Approximate Age: 80 to 120 days at the initiation of breeding.

Justification for Selection: This species and strain of rat has been recognized as appropriate for developmental toxicity studies. WIL Research has historical data on the background incidence of fetal malformations and developmental variations in the Crl:CD(SD) rat. This animal model has been proven to be susceptible to the effects of developmental toxicants.

Animal Housing: Female rats will be individually housed (except during mating) in clean suspended wire-mesh cages in an environmentally controlled room during the study. The cages will be elevated above cage-board or other suitable material, which will be changed at least three times each week. Nesting material will not be provided, as euthanasia is scheduled prior to anticipated parturition. Nylabone® (or a similar enrichment device) will be provided to each animal for environmental enrichment and to aid in maintaining the animal's oral health, beginning during acclimation and continuing throughout the course of the study. The cages will be subjected to routine cleaning at a frequency consistent with maintaining good animal health and WIL Standard Operating Procedures. The facilities at WIL Research are accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International).

Environmental Conditions: Controls will be set to maintain temperature at 71 ± 5°F (22 ± 3°C) and relative humidity at 50 ± 20%. Temperature and relative humidity will be monitored continuously. Data for these two parameters will be scheduled for automatic collection on an hourly basis. Fluorescent lighting controlled by light timers will provide illumination for a 12-hour light/dark photoperiod. The ventilation rate will be set at a minimum of 10 room air changes per hour, 100% fresh air.

Drinking Water: Reverse osmosis-purified water will be available ad libitum. Filters servicing the automatic watering system are changed regularly according to WIL Standard Operating Procedures. The municipal water supplying the laboratory is analyzed according to WIL Standard Operating Procedures on a routine basis to ensure that contaminants are not present in concentrations that would be expected to affect the outcome of the study.

Basal Diet: PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 will be offered ad libitum during the study. Periodic analyses of the certified feed are performed by the manufacturer to ensure that heavy metals and pesticides are not present at concentrations that would be expected to affect the outcome of the study. Results of the analyses are provided to WIL Research by the manufacturer. Feeders will be changed and sanitized once per week.

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
Four groups of rats were used in these studies. Rats in each group were fed diets containing either 0, 0.15, 0.45, or 1.50% TXIB. The test substance was added to PMI Nutrition International, Inc. Certified Rodent LabDiet® 5002 (meal) on a weight/weight basis and blended in a Hobart blender. The resulting premix was mixed thoroughly with additional PMI Nutrition International, Inc. Certified Rodent LabDiet® 5002 (meal) in a twin-shell blender to obtain the appropriate dietary concentrations.


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
All diets were analyzed for TXIB concentrations, and all were determined to contain the target amount of TXIB in a homogenous distribution throughout the diets.

Homogeneity and Stability of Test Diet: Stability of the test substance in rodent diet has been established for at least 35 days at room temperature (Analytical method AM-777-006-A-05. 2005). For homogeneity assessments, test substance formulations of sufficient volume for dosing a group of animals for up to one week (length dependent on stability results) were prepared for the lowest and highest concentrations. One set of 1 00-g samples will be collected from the top, middle and bottom of the formulations. Samples were analyzed for homogeneity and any remaining sample were stored at room temperature as backup. Any backup samples kept at WIL Research will be discarded following acceptance of the analytical results by the Study Director.

An approximate 1 00-g sample of diet from each dosage level, including
controls, will be collected weekly throughout the study and stored room
temperature. Additional samples of diet from each dosage level,
including controls, will be analyzed for test substance concentration for
the first and last preparations. If stability permits, analytical verification
will be performed prior to introduction of the test diets to the animals.
The approximate 1 00-g weekly retention samples will be discarded at the end of the in-life phase following acceptance of the analytical results
by the Study Director.
Details on mating procedure:
At the conclusion of the quarantine period, female rats judged to be suitable test subjects and meeting acceptable body weight requirements will be cohabitated with untreated resident male rats (1 : 1) of the same strain and source in suspended wire-mesh cages for mating (home cage of the male). Detection of mating will be confirmed by the appearance of a vaginal copulatory plug or by evidence of sperm in a vaginal lavage. Vaginal lavages will be performed daily during the mating period until evidence of mating is observed. After confirmation of mating, the female will be returned to an individual suspended wire-mesh cage (assigned to a group), and the ·day will be designated as day 0 of gestation.

Randomization: Mated females will be assigned to groups using a WIL Toxicology Data Management System (WTDMS™) computer program which assigns animals based on stratification of gestation day 0 body weights into a block design to one control group and three test substance groups of 25 rats each. Any animal assigned to the study that is found dead, euthanized in extremis or exhibits abnormal clinical signs, reduced food consumption or body weight losses prior to the start of dosing may be replaced by an animal of appropriate gestation age when possible. Replacement animals will be arbitrarily assigned (not computer randomized) to the study based on comparable body weights (if possible) with respect to the animal that was replaced.
Duration of treatment / exposure:
The route of administration will be dietary as this is a potential route of exposure for humans and has been the route used on previous animal studies
with this test substance. Historically, this route has been used extensively for studies of this nature.
Frequency of treatment:
daily
Duration of test:
Gestation days 0 through 20
No. of animals per sex per dose:
25
Control animals:
yes, plain diet
Details on study design:
Appearance and Behavior: Each rat will be observed twice daily for moribundity and mortality, once in the morning and once in the afternoon from gestation day 0 until euthanasia. Clinical observations will be recorded daily. Mortality and all signs of overt toxicity will be recorded on the day observed. The observations shall include, but are not limited to, evaluation for changes in appearance of skin and fur, eyes, mucous membranes, respiratory and circulatory system, autonomic and central nervous systems, somatomotor activity and behavior. All animals will also be observed
on the day of necropsy and any findings will be recorded.

Body Weights: Individual body weights will be recorded on gestation days 0 and 6-20 (daily).

Food Consumption: Individual food consumption will be recorded on gestation days 0 and 6-20 (daily). Food intake will be reported as g/animal/day and g/kg/day for each corresponding body weight interval of gestation.

Deaths and Animals Euthanized in Extremis: Females not surviving until the scheduled euthanasia will be necropsied and cause of death recorded, if possible. Rats not expected to survive to the next observation period (moribund) will be euthanized by carbon dioxide inhalation. The cranial, thoracic, abdominal and pelvic cavities will be opened and the organs examined. The number and location of implantation sites and viable fetuses will be recorded. Corpora lutea will also be counted and recorded. Uteri which appear nongravid by macroscopic examination will be opened and placed in a 10% ammonium sulfide solution (Salewski, 1964) for detection of early implantation loss. Gross lesions will be preserved in 10% neutral-buffered formalin for possible future histopathologic examination. Carcasses from adult animals will be discarded. Viable fetuses will be euthanized by a subcutaneous injection of sodium pentobarbital in the scapular region. Recognizable fetuses will be examined externally and preserved in 10% neutral-buffered formalin.

Premature Deliveries: Females that deliver prematurely will be euthanized by carbon dioxide inhalation that day. The cranial, thoracic, abdominal and pelvic cavities will be opened and the organs examined. The number and location of former implantation sites and viable fetuses will be recorded. Corpora lutea will also be counted and recorded. Gross lesions will be preserved in 10% neutral-buffered formalin for possible future histopathologic examinations. Carcasses from adult animals will be discarded. Viable fetuses or pups will be euthanized by a subcutaneous (scapular region) or intraperitoneal injection of sodium pentobarbital (as appropriate). Recognizable fetuses or pups will be examined externally and preserved
in 10% neural buffered formalin. Recognizable fetuses or pups aborted on GD 20 will be examined according to Section 7.8.2, if possible.

Laparohysterectomy and Macroscopic Examination on gestation day 20: Laparohysterectomy and macroscopic examinations will be performed
blind to treatment group. All surviving rats will be euthanized by carbon dioxide inhalation on gestation day 20. The cranial, thoracic, abdominal and pelvic cavities will be opened and the organs examined. The uterus of each dam will be excised and its adnexa trimmed. Corpora lutea will be counted and recorded. Gravid uterine weights will be obtained and recorded. The uterus of each dam will be opened and the number of viable and nonviable fetuses, early and late resorptions and total number of implantation sites will be recorded, and the placentae will be examined. The individual uterine distribution will be documented using the following procedure: all implantation sites, including early and late resorptions, will be numbered in consecutive fashion beginning with the left distal uterine horn, noting the position of the cervix and continuing from the proximal to the distal right uterine
horn. Uteri which appear nongravid by macroscopic examination will be opened and placed in a 10% ammonium sulfide solution as described
by Salewski (Salewski, 1964) for detection of early implantation loss. Maternal tissues will be preserved for future histopathologic examination in 10% neutral-buffered formalin only as deemed necessary by the gross findings. Representative sections of corresponding organs from a sufficient number of controls will be retained for comparison, if possible. The carcasses will be discarded.

Fetal Examination: Fetal examinations will be conducted without knowledge of treatment. group. External, internal and skeletal fetal findings will be recorded as developmental variations or malformations. Representative photographs of all malformations, as appropriate, will be included in the study
records. Corresponding low magnification photographs, depicting both the malformed fetus and a comparison control fetus, or normal littermate, will also be included in the study records as needed and as appropriate for comparison, when possible. Prenatal data (viable and nonviable fetuses, early and late resorptions, pre- and post-implantation loss and the fetal sex distribution) will be presented on a group mean basis and additionally as proportional data (%per litter).

External examinations: Each viable fetus will be examined in detail, sexed, weighed, euthanized by a subcutaneous injection of sodium pentobarbital
in the scapular region and tagged. Nonviable fetuses (the degree of autolysis is minimal or absent) will be examined, crown-rump length measured, weighed, sexed and tagged individually. The crown-rump length of late resorptions (advanced degree of autolysis) will be measured, the degree of autolysis recorded, a gross external examination performed (if possible) and the tissue will be discarded.

Visceral (Internal) examinations: Fetuses will be examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities will be opened and dissected using a technique described by Stuckhardt and Poppe (1984). Fetal kidneys will be examined and graded for renal papillae development (Woo and Hoar, 1972). This examination will include the heart and major vessels. The sex of all fetuses will be confirmed by internal examination. The heads will be removed from approximately one-half of the fetuses in each litter and placed in Harrison's fixative for subsequent processing and soft-tissue examination using the Wilson sectioning technique (Wilson, 1965). The heads from the remaining one-half of the fetuses in each litter will be examined by a mid-coronal slice. All carcasses, including the carcasses without heads, will be eviscerated, skinned and fixed in 100% ethyl alcohol for subsequent examination of skeletons.

Skeletal examination: Each eviscerated fetus, following fixation in alcohol, will be stained with Alizarin Red S and Alcian Blue by a method similar
to that described by Dawson (1926) and Inouye (1976). The skeletal examination will be made following this procedure.

Maternal examinations:
All females survived to scheduled euthanasia. No adverse clinical observationswere noted at any dosage level. Mean body weights in Group 4 (15 mg/g of food) were significantly lower compared to the control group through the entire exposure period. A mean loss of body weight was noted in Group 4 following the first day of TXIB administration. Significantly lower mean body weight gains were noted on gestation days 7-8 for both Group 3 (4.5 mg/g of food) and Group 4. This resulted in significantly lower mean body weight gain and a mean body weight loss for the gestation day 6-9 interval for Group 3 and Group 4, respectively. Mean body weight gain was significantly higher than the control group for Group 4 during the gestation day 9-10 interval and resulted in a significantly higher mean body weight gain for the gestation day 9-12 interval. Despite the increase in mean body weight gain, mean body weights were significantly lower for the entire dosing period (gestation day 6-20) for Group 4. All other TXIB groups were comparable to the control.

Mean food consumption (g/animal/day) was significantly lower on gestation days 6-7. 7-8 and 8-9 resulting in significantly lower mean food consumption for the gestation day 6-9 interval and for the entire exposure period (gestation days 6-20) for Group 4. Mean food consumption (g/animal/day) was significantly lower for Group 3 on gestation days 7-8. All other TXIB groups were comparable to the control. Due to the lower food consumption, the target doses in Group 4 were not met until gestation day 9-10, and the target doses were slightly lower for both Group 3 and Group 4. The dose levels for all TXIB groups were slightly lower than target (0, 118, 343 and 1077 mg/kg/day) for all TXIB groups during the exposure period (gestation days 6-20), but did appear to be consistent with a previous OECD 422 study that was conducted (TX-2001-031) where dosing at with the same mg/g of food resulted in 0, 120, 359 and 1135 mg/kg/day.
Ovaries and uterine content:
There was one female in each Group 2, 3 and 4 that was not gravid. All otherfemales were gravid. Net body weight gain and net body weight change for Group 4 was significantly lower than the control group. There were no noted macroscopic observations related TXIB exposure.
Fetal examinations:
There were no adverse effects on corpora lutea, implantation sites, viable fetuses or early/late resorptions. There were no dead fetuses. There was a
dose dependent significantly lower mean male, female and combined fetal weight in Group 4 compared to the control group. These weights were within the WIL Historical Control Data range for these study types:

Males: Mean 3.8 g (range 3.5 to 4.1 g); this study mean 3.7 g
Females: Mean 3.6 g (range (3.5 to 4.1 g); this study mean 3.5 g
Combined: Mean 3.7 g (range 3.4 to 4.0g); this study mean 3.6 g

There were no noteworthy external or visceral malformations or variations noted in any of the dose groups.
Statistics:
All analyses will be two-tailed for significance levels of 5% and 1%. All statistical tests will be performed using a computer with appropriate programming as referenced below. Data from nongravid females will be excluded from calculation of means and from comparative statistics. The litter, rather than the fetus, will be considered as the experimental unit.

Maternal In-Life Data: Continuous data variables [mean body weights (absolute and net), body weight gains (absolute and net) and food consumption of each interval] will be subjected to a parametric one-way analysis of variance (ANOV A) (Snedecor and Cochran, 1980) to determine intergroup difference. If the results of the AN 0 VA are significant (p
Laparohysterectomy Data: The group mean numbers of corpora lutea, implantation sites, viable fetuses, maternal gravid uterine weights and mean fetal weight (separately by sex, and combined) were subjected to a parametric one-way analysis of variance (ANOV A) (Snedecor and Cochran, 1980) and Dunnett's test (1964) as described above. The mean litter proportions of prenatal data (% per litter of viable and nonviable fetuses, early and lateresorptions, total resorptions, pre- and post_implantation loss and the fetal sex distribution) were subjected to the Kruskal-Wallis nonparametric ANOV A test (Kruskal and Wallis, 1952) to determine intergroup difference. If the results of the ANOV A are significant (p<0.05), Dunn's Test (Dunn, 1964) will be applied to the data.

Fetal Morphology Data: The mean litter proportion (% per litter) of total fetal malformations and developmental variations (external, visceral, skeletal and combined) and of each particular external, visceral and skeletal malformation or variation were tabulated. The mean litter proportions of fetal malformations and developmental variations were subjected to the Kruskal-Wallis nonparametric ANOVA test (1952) followed by Dunn's T-Test (1964).
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A significan t decrease was found in the high dose group
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A significan t decrease was found in the high dose group
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
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:
no effects observed
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
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:
no effects observed
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
In the 15 mg/g group, a test substance-related significant (p<0.01) mean body weight loss was noted compared to the control group during gestation days 6-9. Mean body weight gain was higher in this group during gestation days 9-12; the difference from the control group was significant (p<0.01). Mean body weight gains in the 15 mg/g group were similar to the control group for the remainder of the treatment period (gestation days 12-15 and 15-20). However, a significantly (p<0.01) lower mean body weight gain was noted in this group when the overall exposure period (gestation days 6-20) was evaluated. As a result of the initial mean body weight loss in the 15 mg/g group, mean body weights were lower (6.9% to 11.4%) than the control group during gestation days 7-20; differences were significant (p<0.01). Lower mean net body weight gain and mean net body weight were noted in this group; differences from the control group were significant (p<0.01). The aforementioned changes in mean body weight, net body weight, and net body weight gain were attributed to the test substance. The mean gravid uterine weight in the 15 mg/g group was unaffected by the test substance exposure. In the 4.5 mg/g group, a lower mean body weight gain was noted following the initiation of test substance exposure (gestation days 7-8), resulting in a lower mean body weight gain during gestation days 6-9; differences from the control group were significant (p<0.01). Mean body weight gains in this group were similar to the control group for the remainder of the exposure period (gestation days 9-12, 12-15, and 15-20) and when the entire exposure period (gestation days 6-20) was evaluated. The initial lower mean body weight gain in the 4.5 mg/g group was considered test substance-related, but not adverse because the changes were transient and did not affect mean body weights. Mean body weights, body weight gains in the 1.5 mg/g group and net body weights, net body weight gains, and gravid uterine weights in the 1.5 and 4.5 mg/g groups were unaffected by test substance exposure. Differences from the control group were slight and not statistically significant.

Mean food consumption, evaluated as g/animal/day and g/kg/day, in the 15 mg/g group was significantly (p<0.01) lower than the control group during gestation day 6-9. Mean food consumption in this group was similar to the control group during gestation days 9-12 and 12-15. However, lower mean food consumption was again noted in the 15 mg/g group during gestation days 15-20 (on a g/animal/day basis) and when the entire exposure period was evaluated (gestation days 6-20). The differences from the control group were generally significant (p<0.01), corresponded with the lower mean body weights noted in the 4.5 mg/g group (see Section 6.3.), and were therefore considered test substance-related. In the 4.5 mg/g group, mean food consumption was significantly (p<0.01) lower than the control group during gestation days 6-9 and corresponded to the lower initial mean body weight gain in this group (see Section 6.3.). However, mean food consumption in this group was similar to the control group for the remainder of the exposure period (gestation days 9-12, 12-15, and 15-20) and when the entire exposure period (gestation days 6-20) was evaluated. The initial lower mean food consumption in the 4.5 mg/g group was transient and considered test substance-related but not adverse because there were no
corresponding effects on mean body weights. Food consumption in the 1.5 mg/g group was unaffected by test substance exposure. Differences from the control group were slight and generally not statistically significant.
Dose descriptor:
NOAEL
Effect level:
343 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
343 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Significant decrease observed in high dose group
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
The numbers of fetuses (litters) available for morphological evaluation were 358(25), 360(24), 350(24), and 363(24) in the control, 1.5, 4.5, and 15 mg/g groups, respectively. Malformations were observed in 1(1), 0(0), 1(1), and 1(1) fetuses (litters) in the same respective groups. Test substance-related skeletal variations noted in the 15 mg/g group included increased occurrences of bent scapula (unilateral) and bent rib(s). Although bent scapulae and bent rib(s) in this group were considered test substance-related, studies have demonstrated that remodeling of bent ribs and scapulae occurs postnatally; consequently, these findings are transient alterations that resolve postnatally and are not considered to be adverse. A test substance-related higher mean litter proportion of the skeletal developmental variation sternebra(e) nos. 5 and/or 6 unossified was noted in the 15 mg/g group; the difference from the control group was significant (p<0.05). When the total malformations and developmental variations were evaluated on a proportional basis, no statistically significant differences from the control group were noted. Other fetal malformations and developmental variations, when
observed in the test substance-treated groups, occurred infrequently or at a frequency similar to that in the control group, did not occur in a dose-related manner, and/or were within the WIL Research historical control data ranges.



Dose descriptor:
NOAEL
Effect level:
343 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
fetal/pup body weight changes
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
2,2,4-trimethyl-1,3-pentanediol diisobutyrate (TXIB) was administed via diet to groups of Sprague Dawley rats at concentrations of 0, 0.15, 0.45, and 1.50 %. The NOAEL for maternal toxicity was determined to be 4.5 mg/g of food, equivalent to 343 mg/kg body weight/day. This determination was due to significantly lower body weight gain and/or body weight loss which was observed at 15 mg/g of food. The NOAEL for fetal growth and development was determined to be 4.5 mg/g of food due to the significantly lower mean fetal body weights noted in the high dose (15 mg/g of food) group. Importantly, effects on fetal body weight were only observed following administration of TXIB that produced evidence of maternal toxicity. There were no testsubstance related external and visceral malformations or developmental variations noted. Test substance-related skeletal malformations (bent scapula) were noted in one fetus from the 4.5 mg/g group and in 4 fetuses (3 litters) from the 15 mg/g group. A test substance-related higher mean litter proportion of the skeletal developmental variation sternebra(e) nos. 5 and/or 6 unossified was noted in the 15 mg/g group; the difference from the control group was significant (p<0.05). A higher mean litter proportion of bent rib(s) was noted in the 15 mg/g group compared to the concurrent control group (not statistically significant); however, the findings were considered to represent skeletal variations rather than malformations per se. Based on lower mean fetal body weights at 15 mg/g, an exposure level of 4.5 mg/g was also considered to be the NOAEL for embryo/fetal development when TXIB was offered in the diet to bred Crl:CD(SD) rats.
Executive summary:

The test article, 2,2,4-trimethyl-1,3-pentanediol diisobutyrate (TXIB) was administered via diet to each of 4 groups of Sprague Dawley rats, at concentrations of 0, 0.15, 0.45, and 1.50 percent, respectively. There was one female in each Group 2, 3 and 4 that was not gravid. All other females were gravid. Net body weight gain and net body weight change for Group 4 was significantly lower than the control group. There were no noted macroscopic observations related TXIB exposure. There were no adverse effects on corpora lutea, implantation sites, viable fetuses or early/late resorptions. There were no dead fetuses. There was a dose dependent significantly lower mean male, female and combined fetal weight in Group 4 compared to the control group. These weights were within the WIL Historical Control Data range for these study types: Due to significantly lower body weight gain and/or body weight loss, the NOAEL for maternal toxicity using these unaudited data is considered to be 4.5 mg/g of food, equivalent to 343 mg/kg body weight/day. There were no testsubstance related external and visceral malformations or developmental variations noted. When the total malformations and developmental variations were evaluated on a proportional basis, no statistically significant differences from the control group were noted. Test substance-related skeletal malformations (bent scapula) were noted in one fetus from the 4.5 mg/g group and in 4 fetuses (3 litters) from the 15 mg/g group. A test substance-related higher mean litter proportion of the skeletal developmental variation sternebra(e) nos. 5 and/or 6 unossified was noted in the 15 mg/g group; the difference from the control group was significant (p<0.05); A higher mean litter proportion of bent rib(s) was noted in the 15 mg/g group compared to the concurrent control group (not statistically significant); however, the findings were considered to represent skeletal variations rather than malformations per se. Based on lower mean fetal body weights at 15 mg/g, an exposure level of 4.5 mg/g (343 mg/kg body weight/day) was also considered to be the NOAEL for embryo/fetal development when TXIB was offered in the diet to bred Crl:CD(SD) rats.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
Test item: TXIB.
Test item identity (including alternative names): TXIB.
2,2,4-trimethyl-1,3-pentanediol diisobutyrate.
Intended use: Industrial chemical.
Appearance: Clear colorless liquid.
Storage conditions: At ambient temperature (15 to 25C).
Supplier: Sponsor
Batch number: TD17015102
Retest date: February 2018 (or within 2 years of manufacture).
Purity: 99.2%.
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
Rabbit facility Limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.
Air supply Filtered fresh air which was passed to atmosphere and not recirculated.
Temperature and relative humidity Monitored and maintained within the range of 15-21°C and 45-70%. (There were no deviations from these ranges.)
Lighting Artificial lighting, 14 hours light : 10 hours dark.
Alarm systems Activated on ventilation failure and when temperature/humidity limits exceeded.
Electricity supply Public supply with automatic stand-by generators.

Animal Accommodation
Cages Suspended cages fitted with perforated floor panels and mounted in batteries. Undertrays lined with absorbent paper were changed at least three times a week. Cages were also
fitted with a plastic resting platform.
Cage distribution The cages constituting each group were blocked by group and mounted in batteries.
Number of animals per cage Females were individually housed.

Environmental Enrichment
Aspen chew block A soft white untreated wood block; provided to each cage throughout the study and replaced when necessary.
Stainless steel key ring Attached to the cage.

Diet Supply Teklad 2930 Diet. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Diet Availability Restricted (initially 150 g/animal/day during acclimatization up to one week prior to the onset of mating and 200 g/animal/day thereafter). Should an individual show a significant non-treatment related reduced food consumption, moistened diet (50 g pelleted diet moistened with 50 mL of water) was offered, the consumption was recorded.
In addition to this diet, a small supplement of autoclaved hay was given on a daily basis to promote gastric motility and a small amount of chopped fresh vegetables were given twice weekly. Consumption of hay and vegetables were monitored qualitatively but not quantitatively.

Water Supply Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals. Water bowls were also used when necessary.
Availability Non-restricted.

Supplier Certificates of Analysis
Certificates of analysis for the diet were scrutinized and approved before any batch of diet was released for use. Certificates of analysis are provided by the water supplier.
Certificates of analysis were also received from the suppliers of the Aspen chew blocks. No specific contaminants were known that may have interfered with or prejudiced the
outcome of the study and therefore no special assays were performed.
Route of administration:
oral: gavage
Vehicle:
other: 1% methylcellulose
Details on exposure:
Route Oral gavage using a suitably graduated syringe and a rubber catheter inserted via the mouth.
Treated at Constant doses in mg/kg/day.
Volume dose 5 mL/kg body weight.
Individual dose volume Calculated from the most recently recorded scheduled body weight.
Control (Group 1) Vehicle at the same volume dose as treated groups.
Frequency Females were treated from Day 1 to Day 28 (inclusive) after mating, once daily at approximately the same time each day.
Formulation A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration. No significant discrepancy was found.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Approximately 30% of the required amount of vehicle was added to the pre-weighed quantity of test item and magnetically stirred until uniformly mixed. It was made up to volume with the remaining vehicle and magnetically stirred for a minimum of 20 minutes, until homogeneous. A series of emulsions at the required concentrations were prepared by dilution of individual weighings of the test item. Dosing material was prepared weekly and stored under refrigeration (2-8 degrees C.). Detailed records of compound usage were maintained. The amount of test item necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.

Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 10 and 200 mg/mL were analyzed to assess the stability and homogeneity of the test item in the liquid matrix.Samples of each formulation prepared for administration on Days 1 and 28 of treatment were analyzed for achieved concentration of the test item.

Details on mating procedure:
Male/female ratio 1:1 using identified stock New Zealand White bucks.
Checks Natural mating observed.
After mating Each female was injected intravenously with 25 i.u. luteinizing hormone.
Day 0 of gestation On the day of mating.

A colony of stud males was maintained specifically for the purpose of mating; these animals were not part of the study and were maintained as stock animals.

Allocation and Identification
Allocation On the day of mating. Where possible only females mating at least twice were allocated.
Method To group and cage position in the sequence of mating. Females mating on any one day were evenly distributed amongst the groups. Allocation was controlled to prevent any stock male from providing more than one mated female in each treated group and to prevent more than one sibling female in each group, where possible.

Identification of animals Each animal was assigned a number and identified uniquely within the study by an ear tag.
Identification of cages Each cage label was color-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupant.
Duration of treatment / exposure:
Test subjects were treated from gestation days 1 to 28
Frequency of treatment:
daily
Duration of test:
29 days
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
22
Control animals:
yes, concurrent vehicle
Details on study design:
Mortality
A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary. A complete necropsy was performed in all cases.

Clinical Observations
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages and cage-trays were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate. During the acclimatization period, observations of the animals and their cages were recorded at least once per day. Signs Associated with Dosing Detailed observations were recorded daily during the treatment period at the following times
in relation to dose administration:

Pre-dose observation
One to two hours after completion of dosing As late as possible in the working day

Clinical Signs
A detailed physical examination was performed on each animal on Days 0, 6, 12, 18, 23 and 29 after mating to monitor general health.

Body Weight
The weight of each adult was recorded weekly during acclimatization and daily from Day 0-29 after mating.

Food Consumption
The weight of food supplied to each animal, that remaining and an estimate of any spilled was recorded daily from Day 1-28 after mating.Method of Kill

Method of kill for all adult animals: Intravenous injection of sodium pentobarbitone.
Method of kill for fetuses: Subcutaneous injection of sodium pentobarbitone.
Maternal examinations:
Necropsy
All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

Schedule Animals surviving until the end of the scheduled study period were killed on Day 29 after mating.

Sequence To allow satisfactory inter-group comparison. The tissue samples fixed are detailed as follows:
Abnormalities
Adrenals
Esophagus
Kidneys
Liver
Stomach
Animal ID retained

Tissues were routinely preserved in 10% Neutral Buffered Formalin. This list of tissues preserved was intended to satisfy any possible future requirement for further examination of tissues.
Ovaries and uterine content:
For females surviving to term, the following was recorded: Uterus Gravid uterine weight (including cervix and ovaries). The following were recorded for all animals (including those prematurely sacrificed, where possible):
Corpora lutea.
Implantation sites.
Intrauterine deaths (classified as early or late resorptions).
Fetuses (live and dead).

The absence or number of uterine implantation sites was confirmed.
Fetal examinations:
Examination of all viable fetuses and placentae Dissected from the uterus, individually weighed and identified within the litter using a coding system based on their position in the uterus. Examined externally with
abnormalities recorded, sampled as appropriate and retained in appropriate fixative. All fetuses were subject to a gross internal examination of the viscera of the neck, thorax and abdominal cavities and the sex of each fetus was also recorded.

Fixation Nominally one half of fetuses were decapitated; heads were initially stored in Bouin’s fluid.

All fetuses and torsos (intact ad decapitated) were eviscerated and fixed in Industrial Methylated Spirit.

Processing Bouin’s fixed fetal heads were subject to free-hand serial sectioning.

Industrial Methylated Spirit fixed fetuses and torsos were processed and stained with Alizarin Red.

Fetal Pathology Examination
Bouin’s fixed heads Serial sections were examined for soft tissue abnormalities.
Alizarin Red stained fetuses and torsos Assessed for skeletal development and abnormalities
Statistics:
The following data types were analyzed at each timepoint separately:
Body weight, using absolute values and gains over appropriate study periods
Gravid uterine weight and adjusted body weight
Food consumption, over appropriate study periods
Litter size and survival indices
Fetal, placental and litter weight

The following comparisons were performed: Group 1 vs 2, 3 and 4

A parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. For pretreatment data, analysis of variance was used to test for any group differences. Where this was significant (p<0.05) inter group comparisons using t-tests, with the error mean square from the one-way analysis of variance, were made. For all other comparisons the F1
approximate test was applied. This test is designed to detect significant departure from monotonicity of means when the main test for the comparison of the means is a parametric monotonic trend test, such as Williams’ test (Williams 1971, 1972). The test statistic compares the mean square, NMS, for the deviations of the observed means from the maximum likelihood means, calculated under a constraint of
monotonicity with the usual error mean square, EMS. The null hypothesis is that the true means are monotonically ordered. The test statistic is F1 = NMS/EMS which can be compared with standard tables of the F-distribution with 1 and error degrees of freedom. If the F1 approximate test for monotonicity of dose-response was not significant at the 1% level, Williams' test for a monotonic trend was applied. If the
F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test (Dunnett 1955, 1964) was performed instead.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no adverse clinical signs observed at the detailed physical examination that were attributable to treatment with TXIB.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
1F, 8 (control): On Day 10 of gestation this female was found dead in her home cage. No signs of ill health had been observed prior to her death. Macroscopic examination revealed a
small spleen. The female was also found to be pregnant with 7 embryos. This death was considered to be incidental.

1F, 20 (control): On Day 10 of gestation this female was killed for reasons of animal welfare. Clinical signs for this animal consisted of little diet and hay eaten, little water drunk, fast breathing, thin build, pallor whole body and reduced faeces and urine output. At macroscopic examination lungs and bronchi were dark, mass(es) in the thorax, and fluid filled area and soft material adhering to the lung lobes. This female was pregnant with 10 embryos. This death was considered to be incidental.

2F, 37 (100 mg/kg/day): On the morning of Day 8 of gestation (at the first am check), this female was found dead in her home cage. No signs of ill health had been observed prior to her death. Macroscopic examination revealed abnormal contents in the trachea (Dark congealed fluid); in addition, all lung lobes were congested and were unable to collapse fully. This death is considered to be incidental and likely due to an underlying respiratory problem.

4F, 73 (1000 mg/kg/day): On Day 22 of gestation this female was found dead in her home cage. No signs of ill health had been observed prior to her death. Macroscopic examination revealed trauma to the trachea and bruising to the oesophagus, the right lobes of the lungs and bronchi were also dark in appearance. The female was also found to be pregnant with 7 normal fetuses. This death was considered to be incidental and not related to treatment.

4F, 74 (1000 mg/kg/day): On Day 8 of gestation, this female was despatched following a prolonged period of in-appetence and significant body weight loss during the gestation period despite offerings of moistened diet and additional hay. Furthermore, this female produced little faecal and urine output and was noted from Day 4 of gestation, to have red/brown staining on the cage tray paper and to be of thin build and was subsequently despatched due to poor clinical condition. At macroscopic examination, there were no abnormalities observed. This female was pregnant with 9 implantations.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Group mean body weight gain (Day 1 to 28 of gestation) was slightly reduced across all TXIB treated groups (100, 300 or 1000 mg/kg/day) when compared with Controls; the differences did not attain statistical significances.

On Day 29 of gestation, group mean gravid uterine weight was low in females treated with TXIB when compared with Controls however the magnitude of difference was slight and showed no relationship to dose level. Overall body weight change, when adjusted for the weight of the gravid uterus was comparable to controls at all dose levels (100, 300 or 1000 mg/kg/day).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Group mean food consumption was slightly reduced in females treated at 1000 mg/kg/day, until day 24 of gestation; thereafter it was comparable to, or greater than Controls.

In females receiving 100 or 300 mg/kg/day food intake were comparable to Controls.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
On Day 29 of gestation, six females were confirmed not pregnant however the distribution showed no correlation to treatment.

Mean numbers of corpora lutea and implantations were considered to be unaffected by treatment with TXIB.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
effects observed, treatment-related
Description (incidence and severity):
Mean numbers of early resorptions and thus post-implantation losses, were high in females treated at 1000 mg/kg/day when compared with Controls and when compared with historical control data ranges; as a result, the mean live litter size was slightly low compared with controls. A relationship to treatment cannot be ruled out.
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
gross pathology
maternal abnormalities
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
At 100, 300 or 1000 mg/kg/day, an increase in the incidence of short supernumerary cervical ribs was observed when compared with Controls however the distribution showed no relationship to treatment. The numbers of litters involved in the 300 mg/kg/day or 1000 mg/kg/day groups were within the historical control data ranges so no effect of treatment is inferred.

At 1000 mg/kg/day or 300 mg/kg/day there was also a slight increase in incidence of delayed
ossification of 1st to 4th and 6th sternebrae when compared with control litters however the
incidence and distribution were found to be within historical control data ranges and as such a
relation to treatment is not inferred.
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
changes in postnatal survival
external malformations
skeletal malformations
visceral malformations
Abnormalities:
not specified
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other:
Description (incidence and severity):
Higher numbers of early resorptions and post-implantation losses were observed in females treated at 1000 mg/kg/day and consequently total live young were marginally reduced however in surviving fetuses examined on Day 29 of gestation, there were no effects on fetal growth or development at any level.
Developmental effects observed:
not specified

Litter Data
    Corpora Lutea implantations Early Resorptions  Late Resorptions  Total Resorptions  Live Male Live female Total Sex% Male Preimplantation loss (%) Post implantation loss (%)
1F
0 mg/kg
mean 10.1 9 0.6 0.3 0.8 4.6 3.5 8.2 52.6 13.7 8.9
SD 1.94 2.81       2.29 1.35 2.65      
  N 19 19 19 19 19 19 19 19 19 19 19
                         
2F
100 mg/kg
mean 8.9 7.5 0.7 0.2 0.9 3.6 3.1 6.6 58.1 18.5 11.6
SD 2.01 2.77       1.82 2.26 2.7      
  N 18 18 18 18 18 18 18 18 18 18 18
                         
3F
300 mg/kg
mean 10.7 8.8 1.2 0.3 1.5 3.8 3.5 7.3 54.3 18.1 16.4
SD 1.8 2.68       1.63 1.91 2.67      
  N 21 21 21 21 21 21 21 21 21 21 21
                         
4F
1000 mg/kg
mean 10.2 8.8 1.5* 0.4 1.9* 4.2 2.7 6.9 61.3 13.8 22.5**
SD 2.18 2.59       2.14 1.87 2.61      
  N 20 20 20 20 20 20 20 20 20 20 20

Placental, litter and fetal weights - group mean values (g) on Day 29 of gestation
Group/Sex Placental Weight Litter Weight Litter Size Male fetal weight Female fetal weight Male fetal weight Overall Fetal Weight
1F
0 mg/kg
Mean 5.24 338.25 8.16 41 42.67 42.28
SD 0.94 103.025 2.651 3.42 5.2 4.114
  N 19 19 19 18 19 19
               
1F
100 mg/kg
Mean 5.45 277.98 6.61 44.17 41.3 43.21
SD 0.981 103.79 2.704 5.636 4.564 5.152
  N 18 18 18 18 16 18
               
1F
300 mg/kg
Mean 5.2 293.77 7.33 41.58 40.15 41.15
SD 0.752 97.042 2.671 5.546 3.964 4.754
  N 21 21 21 21 20 21
               
1F
1000 mg/kg
Mean 5.39 280.43 6.9 41.27 41.9 41.56
SD 0.746 95.252 2.614 3.804 5.229 4.159
  N 20 20 20 20 20 20

Fetal examinations - major abnormality findings - group incidences
              Fetuses       Litters
Group 1 2 3 4 1 2 3 4
Number Examined 155 119 154 138 19 18 21 20
Total number Affected             0 2 0 1 0 2 0 1
                                   
Head
Visceral
Partially open eye lid(s) 0 0 0 1 0 0 0 1
Cervical/Thoracic
Skeletal
      Multiple thoracic vertebral/rib abnormalities     0 2 0 0       0 2 0 0
Note: Individual fetuses/litters may occur in more than one category.                              

Fetal examinations - minor skeletal abnormality findings - group incidences                      
                           
      Fetuses       Litters
Group 1 2 3 4 1 2 3 4
Number Examined 155 119 154 138 19 18 21 20
Number Intact     73 55 69 64       19 18 21 20
Minor skeletal abnormalities
Cranial
sutural bone(s)   0 1 0 0       0 1 0 0
  fissure(s) 0 1 0 0 0 1 0 0
  additional suture(s) 1 1 1 1 1 1 1 1
  bent cornu(a) of hyoid 0 0 3 0 0 0 3 0
Vertebral element abnormalities thoracic 0 2 1 0 0 2 1 0
Ribs branched 0 1 2 1 0 1 2 1
  absent 0 2 0 0 0 2 0 0
  partially fused 0 1 0 0 0 1 0 0
  additional 0 0 1 0 0 0 1 0
  absent articulating surface 0 0 1 0 0 0 1 0
Sternebrae partially fused 1 2 1 2 1 2 1 2
  bipartite ossified 0 0 1 0 0 0 1 0
  misaligned hemicentres 0 1 0 0 0 1 0 0
  supernumerary site 1 2 4 1 1 2 3 1
  misshapen 0 2 1 0 0 2 1 0
Costal cartilage partially fused 0 0 1 1 0 0 1 1
  misaligned 0 1 1 1 0 1 1 1
  branched 0 2 0 0 0 2 0 0
  additional 0 1 2 1 0 1 2 1
  7th not connected to sternum 15 16 12 15 7 6 7 10
Minor skeletal abnormalities  
Costal cartilage hole in xiphoid 0 0 1 1 0 0 1 1
Total affected by one or more of the above 17 24 23 22 9 11 13 12
Rib and vertebral configuration  

Cervical rib
short supernumerary 2 10 6 7 1 7 3 4
  full supernumerary 0 0 1 1 0 0 1 1
12th rib short/with costal cartilage 0 0 0 1 0 0 0 1
Number of 13th ribs short supernumerary 55 32 55 35 16 12 19 15
  full supernumerary 26 31 34 29 10 10 13 16
  total 70 54 78 52 17 13 19 18
Thoracolumbar vertebrae 18 1 1 1 0 1 1 1 0
  20 8 4 7 10 6 2 5 7
Pelvic girdle unilateral caudal shift 4 2 4 3 3 1 4 3
Delayed/incomplete ossification/unossified  
Cranial large anterior fontanelle 0 1 0 0 0 1 0 0
  large posterior fontanelle 1 1 0 1 1 1 0 1
Sternebrae 5th 18 21 18 21 8 8 9 12
  other 7 8 11 15 3 6 5 8
  total 25 25 25 32 9 11 10 15
Vertebrae cervical (includes odontoid process) 0 0 2 1 0 0 2 1
  thoracic 2 0 0 0 2 0 0 0
Appendicular pubes 0 0 0 1 0 0 0 1
  epiphyses 9 7 10 9 3 5 6 6
  talus 0 0 0 1 0 0 0 1
  metacarpals/phalanges 7 6 5 8 4 4 4 5
  metatarsals/ phalanges 1 1 1 4 1 1 1 3
Increased ossification  
Cranial partially fused jugal to maxilla   2 2 2 0       1 2 2 0
Note: Individual fetuses/litters may occur in more than one category.

Fetal examinations - minor visceral abnormality and necropsy findings - group incidences
            Fetuses       Litters
Group 1 2 3 4 1 2 3 4
Number Examined 155 119 154 138 19 18 21 20
Number of Heads Examined at Detailed Visceral Examination         82 64 85 74       18 17 21 20
   
Head abnormalities (fixed visceral)  
Brain                                                subdural haemorrhage 2 0 0 2 2 0 0 2
Head                                                supernumerary minor upper incisor 0 0 0 1 0 0 0 1
Total affected by one or more of the above 2 0 0 3 2 0 0 3
Necropsy observations (fresh visceral)  
Heart                                                pericardium contains fluid 0 1 0 0 0 1 0 0
Total affected by one or more of the above                                                                            0 1 0 0 0 1 0 0
Necropsy observations (external)  
Limb(s) flexure forepaw(s) 0 1 0 3 0 1 0 3
Total affected by one or more of the above                                                                                      0 1 0 3       0 1 0 3
Conclusions:
Oral administration of TXIB at dose levels of 100, 300 or 1000 mg/kg/day from Day 1 to 28 of gestation was generally well tolerated with no adverse effects on general condition body weight gain, food consumption or macropathology in the maternal New Zealand White rabbit.

Higher numbers of early resorptions and post-implantation losses were observed in females treated at 1000 mg/kg/day and consequently total live young were marginally reduced however in surviving fetuses examined on Day 29 of gestation, there were no effects on fetal growth or development at any level.

Based on these results, it is concluded that dose levels up to and including 1000 mg/kg/day did not adversely affect maternal performance or fetal development and growth, however a reduction in embryo-fetal survival at 1000 mg/kg/day cannot be discounted as an effect of treatment. Therefore, the No observed adverse effect level (NOAEL), based on embryo-fetal survival is considered to be 300 mg/kg/day.
Executive summary:

This study was designed to assess the influence of TXIB (an industrial chemical) on embryo-fetal survival and development when administered during the organogenesis and fetal

growth phases of pregnancy (Days 1-28 after mating) in the New Zealand White rabbit. Three groups of 22 females received TXIB at doses of 100, 300 or 1000 mg/kg/day by oral

gavage administration, from Day 1 to 28 after mating. A similarly constituted Control group received the vehicle, 1% methyl cellulose, at the same volume dose as the treated groups.

Animals were killed on Day 29 after mating for reproductive assessment and fetal examination. Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 29 after mating and the gravid uterus weight recorded. All fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination of the head or skeletal examination.

Results

On Day 8 of gestation, one female (4F, 74) treated at 1000 mg/kg/day was despatched following a prolonged period of in-appetence, significant body weight loss and reduced

faecal and urine output; at macroscopic examination, there were no abnormalities observed. as this premature death was of an isolated incidence, there is no clear correlation to treatment with TXIB. All other premature deaths were incidental and not associated with treatment. There were no signs observed that were attributable to treatment and macroscopic examination revealed no treatment related findings. Group mean body weight gain during the treatment period (Day 1 to 28 of gestation) was slightly low in all treated groups when compared with Controls and food consumption was slightly reduced until Day 24 of gestation in females treated at 1000 mg/kg/day. On Day 29 of gestation, group mean gravid uterine weight was low in females treated with TXIB when compared with Controls however the magnitude of difference was slight and showed no relationship to dose level. Overall body weight change, when adjusted for the weight of the gravid uterus was comparable to controls at all dose levels (100, 300 or 1000 mg/kg/day).

At macroscopic examination, six females were confirmed not pregnant however the distribution showed no correlation to treatment. Mean numbers of corpora lutea and

implantations were considered to be unaffected by treatment with TXIB. Mean numbers of early resorptions and thus post-implantation losses, were high in females treated at 1000 mg/kg/day when compared with Controls and when compared with historical control data ranges; as a result the mean live litter size was slightly low compared with Controls. A relationship to treatment cannot be ruled out. Group mean placental, fetal and litter weights were unaffected by treatment. At 100, 300 or 1000 mg/kg/day, an increase in the incidence of short supernumerary cervical ribs was observed when compared with Controls however the distribution showed no relationship to treatment. At 1000 mg/kg/day or 300 mg/kg/day there was also a slight increase in incidence of delayed ossification of 1st to 4th and 6th sternebrae when compared with control litters however the incidence and distribution were found to be within historical background data ranges and as such a relation to treatment is not inferred.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Additional information

The potential for 1 -isopropyl-2,2 -dimethyltrimethylene diisobutyrate to cause developmental toxicity is well understood. In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test conducted by a method similar to OECD Guideline 422, both sexes were exposed to up to 750 mg/kg bw/day by oral gavage. There was no evidence of embryo/fetal toxicity at any dose level tested. There was no increase in the incidence of pup mortality across dose groups, no treatment-related effects detected on external examination of pups born, and pup body weights increased until Day 4 of lactation. Necropsy of stillborns, dead pups and pups surviving until Day 4 of lactation did not demonstrate any treatment-related effects. The NOEL for embryo/fetal toxicity was 750 mg/kg bw/day when the test material was administered by oral gavage.

In a developmental/reproductive screening feeding study conducted according to OECD Guideline 421, there was no clear evidence of an adverse effect on pre- or post-natal development in the absence of toxicity in the parental generation. Reduced litter weights on postnatal Days 0 and 4 and increased mortality on postnatal Day 4 were observed in the offspring of animals administered 750 mg/kg bw/day in the diet from premating through lactation Day 4. In addition, mean litter weights were 19-20% lower on Days 0 and 4 in this dose group. These effects suggest a treatment-related effect of 1 -isopropyl-2,2 -dimethyltrimethylene diisobutyrate exposure. At this dose level, parental systemic toxicity, including decreases in mean body weights and/or body weight gains and mean feed consumption and/or feed utilization were observed in both sexes of the parental generation. There was a clear NOAEL for embryo/fetal toxicity in the mid-dose group in which the parental generation received the equivalent of 276 mg/kg bw/day for males and 359 mg/kg bw/day for females.

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TXIB also was evaluated in a dietary prenatal developmental toxicity study (OECD 414) in groups of Sprague Dawley rats that were given either 0, 0.15, 0.45, or 1.50 % TXIB in the diet. There was one female in each Group 2, 3 and 4 that was not gravid. All other females were gravid. Net body weight gain and net body weight change for Group 4 was significantly lower than the control group. There were no noted macroscopic observations related TXIB exposure. There were no adverse effects on corpora lutea, implantation sites, viable fetuses or early/late resorptions. There were no dead fetuses. There was a dose dependent significantly lower mean male, female and combined fetal weight in Group 4 compared to the control group. These weights were within the WIL Historical Control Data range for these study types: Due to significantly lower body weight gain and/or body weight loss, the NOAEL for maternal toxicity using these unaudited data is considered to be 4.5 mg/g of food, equivalent to 343 mg/kg body weight/day. There were no testsubstance related external and visceral malformations or developmental variations noted. When the total malformations and developmental variations were evaluated on a proportional basis, no statistically significant differences from the control group were noted. Test substance-related skeletal malformations (bent scapula) were noted in one fetus from the 4.5 mg/g group and in 4 fetuses (3 litters) from the 15 mg/g group. A test substance-related higher mean litter proportion of the skeletal developmental variation sternebra(e) nos. 5 and/or 6 unossified was noted in the 15 mg/g group; the difference from the control group was significant (p<0.05); A higher mean litter proportion of bent rib(s) was noted in the 15 mg/g group compared to the concurrent control group (not statistically significant); however, the findings were considered to represent skeletal variations rather than malformationsper se. Based on lower mean fetal body weights at 15 mg/g, an exposure level of 4.5 mg/g (343 mg/kg body weight/day) was also considered to be the NOAEL for embryo/fetal development when TXIB was offered in the diet to bred Crl:CD(SD) rats.

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A study was designed to assess the influence of TXIB (an industrial chemical) on embryo-fetal survival and development when administered during the organogenesis and fetal

growth phases of pregnancy (Days 1-28 after mating) in the New Zealand White rabbit. Three groups of 22 females received TXIB at doses of 100, 300 or 1000 mg/kg/day by oral

gavage administration, from Day 1 to 28 after mating. A similarly constituted Control group received the vehicle, 1% methyl cellulose, at the same volume dose as the treated groups.

Animals were killed on Day 29 after mating for reproductive assessment and fetal examination. Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 29 after mating and the gravid uterus weight recorded. All fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination of the head or skeletal examination.

Results

On Day 8 of gestation, one female (4F, 74) treated at 1000 mg/kg/day was despatched following a prolonged period of in-appetence, significant body weight loss and reduced

faecal and urine output; at macroscopic examination, there were no abnormalities observed. as this premature death was of an isolated incidence, there is no clear correlation to treatment with TXIB. All other premature deaths were incidental and not associated with treatment. There were no signs observed that were attributable to treatment and macroscopic examination revealed no treatment related findings. Group mean body weight gain during the treatment period (Day 1 to 28 of gestation) was slightly low in all treated groups when compared with Controls and food consumption was slightly reduced until Day 24 of gestation in females treated at 1000 mg/kg/day. On Day 29 of gestation, group mean gravid uterine weight was low in females treated with TXIB when compared with Controls however the magnitude of difference was slight and showed no relationship to dose level. Overall body weight change, when adjusted for the weight of the gravid uterus was comparable to controls at all dose levels (100, 300 or 1000 mg/kg/day).

At macroscopic examination, six females were confirmed not pregnant however the distribution showed no correlation to treatment. Mean numbers of corpora lutea and

implantations were considered to be unaffected by treatment with TXIB. Mean numbers of early resorptions and thus post-implantation losses, were high in females treated at 1000 mg/kg/day when compared with Controls and when compared with historical control data ranges; as a result the mean live litter size was slightly low compared with Controls. A relationship to treatment cannot be ruled out. Group mean placental, fetal and litter weights were unaffected by treatment. At 100, 300 or 1000 mg/kg/day, an increase in the incidence of short supernumerary cervical ribs was observed when compared with Controls however the distribution showed no relationship to treatment. At 1000 mg/kg/day or 300 mg/kg/day there was also a slight increase in incidence of delayed ossification of 1st to 4th and 6th sternebrae when compared with control litters however the incidence and distribution were found to be within historical background data ranges and as such a relation to treatment is not inferred.

Justification for classification or non-classification

There were no adverse effects on reproductive organs in either sex of rats exposed to 1 -isopropyl-2,2 -dimethyltrimethylene diisobutyrate by the oral route for up to 13 weeks in the diet and no adverse effects on any reproductive or developmental parameters in a combined repeat dose and reproductive/developmental toxicity screening test in which both sexes were exposed to up to 750 mg/kg bw/day by oral gavage from 2 weeks prior to mating through early lactation (Day 4).  In a developmental/reproductive screening feeding study, there was no clear evidence of an adverse effect on pre- or post-natal development in the absence of toxicity in the parental generation. In an OECD 414 developmental toxicity study, skeletal variations, but not malformations, were observed at doses that also produced evidence of maternal toxicity.  Based on a weight-of-the-evidence assessment, 1 -isopropyl-2,2 -dimethyltrimethylene diisobutyrate is not selectively toxic to the fetus and is not classified for reproductive toxicity but sufficient information is available in the rabbit pre-natal developmental toxicity test to support category 2 developmental classification according to EU CLP.