Isobutyric acid, monoester with 2,2,4-trimethylpentane-1,3-diol

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Additional information

The potential for isobutyric acid, monoester with 2,2,4 -trimethylpentane-1,3 -diol to cause reproductive toxicity is well understood. In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test conducted according to OECD Guideline 422, both sexes were exposed to up to 1000 mg/kg bw/day by oral gavage with no gross or microscopic effects on reproductive organs of either sex. 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 1000 mg/kg bw/day when the test material was administered by oral gavage.

In a prenatal developmental toxicity study, the test substance, Texanol™, was administered orally by gavage to 3 groups of 25 bred female Crl:CD(SD) rats once daily from gestation days 1 through 19. Dosage levels were 100, 300, and 1000 mg/kg/day administered at dosage volumes of 0.11, 0.32, and 1.05 mL/kg, respectively. A concurrent control group composed of 25 bred females received the vehicle (deionized water) on a comparable regimen at a dosage volume of 1.05 mL/kg. The females were approximately 13 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 daily. On gestation day 20, a laparohysterectomy was performed on each female. 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 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. Test substance-related increased incidences of red and/or clear material around the mouth were noted for females in the 100, 300, and 1000 mg/kg/day groups approximately 1 hour following dose administration. In addition, low incidences of red material around the nose and yellow material around the urogenital area were noted for females in the 100, 300, and/or 1000 mg/kg/day groups at the daily examinations and/or approximately 1 hour following dose administration. However, the post-dose findings did not generally persist to the daily examinations the following day, and in the absence of other signs of systemic toxicity at these dosage levels, these clinical findings were considered non-adverse. Test substance-related reductions in mean food consumption values with corresponding decreases in mean body weight gains were noted in the 1000 mg/kg/day group during gestation days 1-3 and 3-6 compared to the control group. Mean body weight gains and food consumption in this group were similar to the control group during the remainder of the dosing period (gestation days 6-20). The initial lower mean body weight gains in this group resulted in a lower mean body weight gain when the overall dosing period (gestation days 1-20) was evaluated compared to the control group; however, this change occurred in the absence of reduced food consumption. Mean net body weight gain in this group was also lower than the control group, while mean net body weight and gravid uterine weight were similar to the control group. The test substance-related changes in mean body weight gain and food consumption at 1000 mg/kg/day during gestation days 1-6 were considered adverse. There were no test substance-related effects on mean maternal body weights, body weight gains, food consumption, net body weights, net body weight gains, and gravid uterine weights at 100 and 300 mg/kg/day. No test substance-related macroscopic findings were noted at any dosage level. Intrauterine growth and survival were unaffected by test substance administration at 100, 300, and 1000 mg/kg/day. In addition, there were no test substance-related external, visceral, or skeletal fetal malformations or developmental variations observed at any dosage level. Based on reduced mean food consumption values with corresponding decreases in mean body weight gains at 1000 mg/kg/day during the first week of treatment, a dosage level of 300 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity. Intrauterine growth, survival, and external, visceral, and skeletal fetal morphology were unaffected by test substance administration at all dosage levels. Based on these results, a dosage level of 1000 mg/kg/day (the highest dosage level tested) was considered to be the no-observed-adverse-effect level (NOAEL) for embryo/fetal developmental toxicity when Texanol™ was administered orally by gavage to bred Crl:CD(SD) rats.

Effects on developmental toxicity

Description of key information

The test substance, Texanol™, was administered orally by gavage to 3 groups of 25 bred female Crl:CD(SD) rats once daily from gestation days 1 through 19. Dosage levels were 100, 300, and 1000 mg/kg/day administered at dosage volumes of 0.11, 0.32, and 1.05 mL/kg, respectively. A concurrent control group composed of 25 bred females received the vehicle (deionized water) on a comparable regimen at a dosage volume of 1.05 mL/kg. The females were approximately 13 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 daily. On gestation day 20, a laparohysterectomy was performed on each female. 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 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. Test substance-related increased incidences of red and/or clear material around the mouth were noted for females in the 100, 300, and 1000 mg/kg/day groups approximately 1 hour following dose administration. In addition, low incidences of red material around the nose and yellow material around the urogenital area were noted for females in the 100, 300, and/or 1000 mg/kg/day groups at the daily examinations and/or approximately 1 hour following dose administration. However, the post-dose findings did not generally persist to the daily examinations the following day, and in the absence of other signs of systemic toxicity at these dosage levels, these clinical findings were considered non-adverse. Test substance-related reductions in mean food consumption values with corresponding decreases in mean body weight gains were noted in the 1000 mg/kg/day group during gestation days 1-3 and 3-6 compared to the control group. Mean body weight gains and food consumption in this group were similar to the control group during the remainder of the dosing period (gestation days 6-20). The initial lower mean body weight gains in this group resulted in a lower mean body weight gain when the overall dosing period (gestation days 1-20) was evaluated compared to the control group; however, this change occurred in the absence of reduced food consumption. Mean net body weight gain in this group was also lower than the control group, while mean net body weight and gravid uterine weight were similar to the control group. The test substance-related changes in mean body weight gain and food consumption at 1000 mg/kg/day during gestation days 1-6 were considered adverse. There were no test substance-related effects on mean maternal body weights, body weight gains, food consumption, net body weights, net body weight gains, and gravid uterine weights at 100 and 300 mg/kg/day. No test substance-related macroscopic findings were noted at any dosage level. Intrauterine growth and survival were unaffected by test substance administration at 100, 300, and 1000 mg/kg/day. In addition, there were no test substance-related external, visceral, or skeletal fetal malformations or developmental variations observed at any dosage level.

Based on reduced mean food consumption values with corresponding decreases in mean body weight gains at 1000 mg/kg/day during the first week of treatment, a dosage level of 300 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity. Intrauterine growth, survival, and external, visceral, and skeletal fetal morphology were unaffected by test substance administration at all dosage levels. Based on these results, a dosage level of 1000 mg/kg/day (the highest dosage level tested) was considered to be the no-observed-adverse-effect level (NOAEL) for embryo/fetal developmental toxicity when Texanol™ was administered orally by gavage to bred Crl:CD(SD) rats.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

yes

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST SYSTEM, ANIMAL RECEIPT, AND ACCLIMATION
Sexually mature, virgin female Sprague Dawley [Crl:CD(SD)] rats were used as the test system on this study. This species and strain of animal is recognized as appropriate for developmental toxicity studies. WIL Research has historical control 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. The number of animals selected for this study 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 with implantation sites at necropsy. Given the possibility of nongravid animals, unexpected deaths, or treatment-related moribundity and/or mortality, this was an appropriate number of animals to obtain a sample size of 20 females at termination. Crl:CD(SD) rats (125 females) were received in good health from Charles River Laboratories, Inc., Raleigh, NC, on 08-APR-2014. 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 rat was uniquely identified by a Monel® metal ear tag displaying the animal number and 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 behavior.

ANIMAL HOUSING
Upon arrival and until pairing, all rats were individually housed in clean, stainless steel wire-mesh cages suspended above cage-board. The cage-board was changed at least 3 times per week. The rats were paired for mating in the home cage of the male. Following positive evidence of mating, the females were returned to individual suspended wire-mesh cages; nesting material was not required as the females were euthanized prior to the date of expected parturition. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996). 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.

DIET, DRINKING WATER, AND MAINTENANCE
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’s 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.

ENVIRONMENTAL CONDITIONS
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 70.2°F to 70.7°F (21.2°C to 21.5°C) and mean daily relative humidity ranged from 44.3% to 52.2% 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.

ASSIGNMENT OF ANIMALS TO TREATMENT GROUPS AND BREEDING PROCEDURES
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 suspended wire-mesh cage 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-dosed 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 stock colony or euthanized by carbon dioxide inhalation
and discarded. Body weight values ranged from 222 g to 298 g on gestation day 0.

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on exposure:

ORGANIZATION OF TEST GROUPS, DOSAGE LEVELS, AND TREATMENT REGIMEN
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 1-19. The dosage volumes for the control, 100, 300, and 1000 mg/kg/day groups were 1.05, 0.11, 0.32, and 1.05 mL/kg, respectively. 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.

Dosage levels were determined from the results of a previous combined repeated-dose and reproductive/developmental toxicity study in the rat (Faber and Hosenfeld, 1992). In that study, the no-observed-adverse-effect level (NOAEL) for subchronic toxicity was 1000 mg/kg/day and the no-observed-effect level (NOEL) for reproductive toxicity was 1000 mg/kg/day. The selected route of administration for this study was oral (gavage) because this is a potential route of human exposure. Historically, this route has been used extensively for studies of this nature.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Gestation days 1-19

Frequency of treatment:

once daily

Duration of test:

20 days

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Maternal examinations:

CLINICAL OBSERVATIONS AND SURVIVAL
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 dosing 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.

BODY WEIGHTS AND GRAVID UTERINE WEIGHTS
Individual maternal body weights were recorded on gestation days 0-20 (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 1-3, 3-6, 6-9, 9-12, 12-16, 16-20, and 1-20.
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.

FOOD CONSUMPTION
Individual food consumption was recorded on gestation days 0-20 (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.

GESTATION DAY 20 LAPAROHYSTERECTOMY
Laparohysterectomies and macroscopic examinations were performed blind to treatment group. All females were euthanized on gestation day 20 by carbon dioxide inhalation. The 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). Maternal tissues were preserved in 10% neutral-buffered formalin for possible future histopathologic examination only as indicated by the gross findings. The carcass of each female was then discarded.

Fetal examinations:

FETAL MORPHOLOGICAL EXAMINATION
Fetal examinations were performed blind to treatment group. Each viable fetus was examined externally, individually sexed, weighed, euthanized by a subcutaneous injection 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. Nonviable fetuses (if the degree of autolysis was minimal or absent) were examined, the crown-rump length measured, weighed, and tagged individually. 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 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 Bouin'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).

Statistics:

All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-dosed group to the control group. Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean. Data obtained from nongravid animals were excluded from statistical analyses. Due to the use of significant figures and the different rounding conventions inherent in the types of software used, the means, standard deviations, and standard errors on the summary and individual tables may differ slightly. Therefore, the use of reported individual values to calculate subsequent parameters or means will, in some instances, yield minor variations from those listed in the report data tables. Where applicable, the litter was used as the experimental unit. Maternal body weights (absolute and net), body weight changes (absolute and net), and food consumption, gravid uterine weights, numbers of corpora lutea, implantation sites, and viable fetuses, and fetal body weights (separately by sex and combined) were subjected to a parametric one-way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test substance-dosed groups to the control group. Mean litter proportions (percent per litter) of prenatal data (viable and nonviable fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and fetal sex distribution), total fetal malformations and developmental variations (external, visceral, skeletal, and combined) and each particular external, visceral, and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test (Kruskal and Wallis, 1952) to determine intergroup differences.

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
MATERNAL CLINICAL OBSERVATIONS AND SURVIVAL
All females in the control, 100, 300, and 1000 mg/kg/day groups survived to the scheduled necropsy on gestation day 20. Test substance-related clinical findings were observed at all dosage levels at the daily examinations and/or approximately 1 hour following dose administration during the dosing period. Limited occurrences of red material around the nose for females in the 100, 300, and 1000 mg/kg/day groups and yellow material around the urogenital area, primarily for females in the 1000 mg/kg/day group, were noted at the daily examinations and/or approximately 1 hour following dose administration. These findings were noted in single females as early as gestation day 2 and continued sporadically through the end of the dosing period. In addition, red and/or clear material around the mouth was noted for the majority of females in the 300 and 1000 mg/kg/day groups, and to a lesser extent in the 100 mg/kg/day group, approximately 1 hour following dose administration during the dosing period. These material findings were noted in a dose-dependent manner beginning as early as gestation day 1 and continued through the end of the dosing period. However, as all of the aforementioned findings occurred in single animals, in a manner that was not dose related, and/or did not generally persist to the daily examinations the following day, they were considered non-adverse. Other findings noted in the treated groups, including hair
loss on various body surfaces, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.

MATERNAL BODY WEIGHTS AND GRAVID UTERINE WEIGHTS
Test substance-related, significantly (p<0.05 or p<0.01) lower mean body weight gains were noted in the 1000 mg/kg/day group during gestation days 1-3 and 3-6 compared to the control group. Mean body weight gains in this group were generally similar to the control group during the remainder of the dosing period (gestation days 6-9, 9-12, 12-16, and 16-20). However, mean body weight gain in the 1000 mg/kg/day group was significantly (p<0.01) lower than the control group when the entire dosing period (gestation days 1-20) was evaluated due to the initial lower mean body weight gains. Although not of sufficient magnitude to result in statistically significantly lower mean body weights during the dosing period, the initial lower mean body weight gains noted in the 1000 mg/kg/day group were considered adverse. A significantly (p<0.01) lower mean net body weight gain was noted in the 1000 mg/kg/day group compared to the control group, while mean net body weight and gravid uterine weight in this group were unaffected by test substance administration. Mean maternal body weights, body weight gains, net body weights, net body weight gains, and gravid uterine weights in the 100 and 300 mg/kg/day groups were unaffected by test substance administration. Differences from the control group were slight and not statistically significant.

MATERNAL FOOD CONSUMPTION
Maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 1000 mg/kg/day group was significantly (p<0.01) lower than the control group during gestation days 1-3 and 3-6, corresponding to the lower mean body weight gains observed during these intervals. The lower mean food consumption was considered to be test substance-related and adverse. Mean food consumption in the 1000 mg/kg/day group was similar to the control group throughout the remainder of the dosing period (gestation days 6-9, 9-12, 12-16, and 16-20) and when the overall dosing period (gestation days 1-20) was evaluated. Significantly (p<0.05) lower mean food consumption (g/animal/day only) was also noted in the 1000 mg/kg/day group on gestation day 17-18 compared to the control group. This transient difference was not attributed to the test substance because there was no corresponding effect on body weight. Maternal food consumption in the 100 and 300 mg/kg/day groups was unaffected by test substance administration. Significantly (p<0.05 or p<0.01) higher mean food consumption was noted in the 300 mg/kg/day group on gestation days 13-14 (g/kg/day only), 19-20, and 16-20 (g/kg/day only) and significantly (p<0.05) lower mean food consumption was noted in this group during gestation days 1-3 (g/kg/day only). These transient differences were not attributed to the test substance because there were no corresponding effects on body weight at this dosage level.

MATERNAL NECROPSY DATA
At the scheduled necropsy on gestation day 20, no test substance-related internal findings were observed at dosage levels of 100, 300, and 1000 mg/kg/day. Macroscopic findings observed in the test substance-treated groups occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related. Three, 4, and 1 females in the control, 100, and 300 mg/kg/day groups, respectively, were determined to be nongravid.

GESTATION DAY 20 LAPAROHYSTERECTOMY DATA
Intrauterine growth and survival were unaffected by test substance administration at dosage levels of 100, 300, and 1000 mg/kg/day. Parameters evaluated included postimplantation loss, live litter size, mean fetal body weights, and fetal sex ratios. Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups. Differences from the control group were slight and not statistically significant.

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Remarks on result:

not determinable

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Remarks on result:

not determinable

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
FETAL MORPHOLOGICAL DATA
The numbers of fetuses (litters) available for morphological evaluation were 340(22), 320(21), 352(24), and 376(25) in the control, 100, 300, and 1000 mg/kg/day groups, respectively. Malformations were observed in 3(2), 2(2), 3(3), and 2(2) fetuses (litters) in these same respective dosage groups and were considered spontaneous in origin.

EXTERNAL MALFORMATIONS AND VARIATIONS
There were no test substance-related external malformations noted for fetuses at any dosage level. Microphthalmia or anophthalmia was noted for 1(1), 2(2), and 1(1) fetuses (litters) in the control, 300, and 1000 mg/kg/day groups, respectively. Fetus no. 14043-02 in the 1000 mg/kg/day group had anophthalmia (left) and fetus nos. 13992-02 and 14037-10 in the 300 mg/kg/day group had microphthalmia (right or left; no apparent skeletal origin). Because these findings were noted in single fetuses, did not occur in a dose-related manner, the mean litter proportions were not statistically
significantly different from the concurrent control group and were within the WILResearch historical control data range, and/or were also noted in a control group fetus (no. 14015-14, anophthalmia [bilateral]; skeletally, orbit smaller than normal), they were not considered test substance-related. No other external developmental malformations were noted at any dosage level. There were no external developmental variations noted in fetuses at any dosage level.

VISCERAL MALFORMATIONS AND VARIATIONS
There were no test substance-related soft tissue malformations noted for fetuses at any dosage level. Soft tissue malformations were noted for 1 fetus each in the control, 100, and 1000 mg/kg/day groups. Lobular dysgenesis of the lungs (1 lobe present, bilateral) was noted for fetus no. 13986-12 in the 1000 mg/kg/day group. Because this finding was noted for a single fetus and the mean litter proportion (0.3% per litter) was within the
WIL Research historical control data range (0.0% to 0.7% per litter) and was not statistically significantly different from the concurrent control group, it was not considered test substance-related. Fetus no. 14005-06 in the 100 mg/kg/day group had situs inversus (lateral transposition of the trachea, esophagus, heart, great and major vessels, lungs, liver, stomach, pancreas, spleen, kidneys, adrenals, and intestine). Because this finding was observed in a single fetus and was not noted in the higher dosage levels, it was not considered test substance-related. In the control group, fetus no.
13989-07 had a malpositioned kidney (left). No test substance-related soft tissue developmental variations were noted for fetuses in the 100, 300, and 1000 mg/kg/day groups. An increased mean litter proportion of renal papilla(e) not developed and/or distended ureter(s) was noted in the 1000 mg/kg/day group (6.9% per litter) compared to the control group (1.3% per litter). Although the difference from the concurrent control group was not statistically significant, the value exceeded the maximum mean value in the WIL Research historical control data (4.0% per litter). However, this is the most common soft tissue developmental variation in the WIL Research historical control database and the majority (13 of 25 occurrences) of this finding was noted in fetuses from a single litter (no. 14023). Therefore, this soft tissue developmental variation was not attributed to maternal test substance administration. Other soft tissue developmental variations, including pale spleen, accessory lobule(s) of the liver, and major blood vessel variation (right subclavian artery coursed retroesophageal and joined aortic arch adjacent to ductus arteriosis [no brachiocephalic trunk]), were noted infrequently, at similar frequencies in the concurrent control group, and in a manner that was not dose-related. One, 3, and 2 fetuses in the control, 100, and 1000 mg/kg/day groups, respectively, were noted 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 in a manner that was not dose-related.

SKELETAL MALFORMATIONS AND VARIATIONS
There were no test substance-related skeletal malformations noted for fetuses at any dosage level. Fetus no. 14004-05 in the 300 mg/kg/day group was noted with a costal cartilage anomaly (fused costal cartilages) and fetus no. 14071-16 in the 100 mg/kg/day group had a skull anomaly (a 1 mm opening in the right parietal bone). Because these malformations were noted in single fetuses, in a non-dose-related manner, and the mean litter proportions were not statistically significantly different from the control group, the findings were not considered test substance-related. No skeletal malformations were noted in the 1000 mg/kg/day group. In the control group, fetus no. 14015-02 had a rib anomaly (extra and forked rib).
No test substance-related skeletal developmental variations were noted for fetuses in the 100, 300, and 1000 mg/kg/day groups. A significantly (p<0.05) lower mean litter proportion of cervical centrum no. 1 ossified was noted for fetuses in the 1000 mg/kg/day group (8.4% per litter) compared to the concurrent control group (26.1% per litter). However, this is the most common skeletal developmental variation in the WIL Research historical control database and the value was within the WIL Research historical control data range (1.4% to 35.8% per litter); therefore, in the absence of any other signals of developmental toxicity or effects on fetal weight, this finding was not attributed to maternal test substance administration. Other skeletal developmental variations observed in the test substance-treated groups were noted in single fetuses, occurred infrequently, did not occur in a dose-related manner, and/or the mean litter proportions were within the WIL Research historical control data ranges. Therefore, these findings were not
considered test substance-related.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no changes

Remarks on result:

not determinable

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

Based on reduced mean food consumption values with corresponding decreases in mean body weight gains at 1000 mg/kg/day during the first week of treatment, a dosage level of 300 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity. Intrauterine growth, survival, and external, visceral, and skeletal fetal morphology were unaffected by test substance administration at all dosage levels. Based
on these results, a dosage level of 1000 mg/kg/day (the highest dosage level tested) was considered to be the no-observed-adverse-effect level (NOAEL) for embryo/fetal developmental toxicity when Texanol™ was administered orally by gavage to bred Crl:CD(SD) rats.

Executive summary:

The test substance, Texanol™, was administered orally by gavage to 3 groups of 25 bred female Crl:CD(SD) rats once daily from gestation days 1 through 19. Dosage levels were 100, 300, and 1000 mg/kg/day administered at dosage volumes of 0.11, 0.32, and 1.05 mL/kg, respectively. A concurrent control group composed of 25 bred females received the vehicle (deionized water) on a comparable regimen at a dosage volume of 1.05 mL/kg. The females were approximately 13 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 daily. On gestation day 20, a laparohysterectomy was performed on each female. 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 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. Test substance-related increased incidences of red and/or clear material around the mouth were noted for females in the 100, 300, and 1000 mg/kg/day groups approximately 1 hour following dose administration. In addition, low incidences of red material around the nose and yellow material around the urogenital area were noted for females in the 100, 300, and/or 1000 mg/kg/day groups at the daily examinations and/or approximately 1 hour following dose administration. However, the post-dose findings did not generally persist to the daily examinations the following day, and in the absence of other signs of systemic toxicity at these dosage levels, these clinical findings were considered non-adverse. Test substance-related reductions in mean food consumption values with corresponding decreases in mean body weight gains were noted in the 1000 mg/kg/day group during gestation days 1-3 and 3-6 compared to the control group. Mean body weight gains and food consumption in this group were similar to the control group during the remainder of the dosing period (gestation days 6-20). The initial lower mean body weight gains in this group resulted in a lower mean body weight gain when the overall dosing period (gestation days 1-20) was evaluated compared to the control group; however, this change occurred in the absence of reduced food consumption. Mean net body weight gain in this group was also lower than the control group, while mean net body weight and gravid uterine weight were similar to the control group. The test substance-related changes in mean body weight gain and food consumption at 1000 mg/kg/day during gestation days 1-6 were considered adverse. There were no test substance-related effects on mean maternal body weights, body weight gains, food consumption, net body weights, net body weight gains, and gravid uterine weights at 100 and 300 mg/kg/day. No test substance-related macroscopic findings were noted at any dosage level. Intrauterine growth and survival were unaffected by test substance administration at 100, 300, and 1000 mg/kg/day. In addition, there were no test substance-related external, visceral, or skeletal fetal malformations or developmental variations observed at any dosage level.

Based on reduced mean food consumption values with corresponding decreases in mean body weight gains at 1000 mg/kg/day during the first week of treatment, a dosage level of 300 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity. Intrauterine growth, survival, and external, visceral, and skeletal fetal morphology were unaffected by test substance administration at all dosage levels. Based on these results, a dosage level of 1000 mg/kg/day (the highest dosage level tested) was considered to be the no-observed-adverse-effect level (NOAEL) for embryo/fetal developmental toxicity when Texanol™ was administered orally by gavage to bred Crl:CD(SD) rats.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION

- Pre-existing data:
There are no pre-existing data available that would address the pre-natal developmental toxicity study endpoint (GLP or non-GLP). A rat pre-natal developmental toxicity study is not adequate according to the regulation. There are no historical human data that could be used in place of this study.

- (Q)SAR:
There are no accepted QSAR approaches for predicting the outcome of this endpoint. However, there were no Reproductive Toxicity alerts for Texanol.

- In vitro methods
There are no accepted in vitro approaches for predicting the outcome of this endpoint

- Weight of evidence
WoE is not possible as there are not similar substances that could be used for such a comparison.

- Grouping and read-across
The Registrant has not identified any suitable analogues that could be used to provide data for this endpoint. Other similar substances are quite data poor.

- Substance-tailored exposure driven testing
Based on the use patterns of the registered substance in coatings, exposure-based waiving is not possible.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Test Substance

Identification: 2,2,4-trimethyl-1,3-pentanediol monoisobutyrate (TMPD-MIB)
Alternate Identification: Texanol
Batch/Lot No.: TD19034470 TD21007237
Expiration/Retest Date: 11 Dec 2022 19 Apr 2023
Physical Description: Colorless clear liquid
Purity: 99.3% - 99.3%
Correction Factor: -
Density: 0.9464 g/mL
Storage Conditions: 18°C to 24°C
Provided by: Sponsor

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Test System
Receipt
On 16 Apr 2021, time-mated female New Zealand White rabbits were received from Envigo Global Services Inc., Denver, PA on Gestation Day 1, 2, or 3. The animals were approximately 6 months old at receipt and weighed between 2825 and 3690 g at the initiation of dosing.

Justification for Test System and Number of Animals
At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models that do not use live animals currently do not exist.
The number of animals was based on the US EPA Health Effects Test Guidelines OPPTS 870.3700, Prenatal Development Toxicity Study, Aug 1998 and the OECD Guidelines for the Testing of Chemicals: Guideline 414, Prenatal Developmental Toxicity Study, Jan 2018, which recommend evaluation of approximately 20 females/group with implantation sites at necropsy. Given the possibility of nongravid animals, unexpected deaths, or treatment-related moribundity and/or mortality, 24 females/group was an appropriate number of animals to obtain a sample size of 20 females/group at termination.

Animal Identification
Each animal was identified using a subcutaneously implanted electronic identification chip.

Quarantine
After receipt at the Testing Facility, the New Zealand White rabbits were acclimated prior to the initiation of dosing. During acclimation, animals were acclimated to the handling and restraint procedures required for dosing.

Selection, Assignment, and Disposition of Animals
Animals were assigned to groups by a stratified randomization scheme designed to achieve similar group mean body weights. The disposition of all animals was documented in the Study Records.

Husbandry

Housing
Housing: Single/Individual
Caging: Stainless steel cages with perforated flooring elevated above ground corncob bedding (Pel-O’Cobs® or other suitable material).

Cage Identification: Individual (color-coded) cage cards were affixed to each cage and displayed at least the animal number(s), group number, dose level, study number, and sex of the animal.
Housing set-up was as specified in the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). Cages were arranged on the racks in group order. Where possible, control group animals were housed on a separate rack from the test substance-treated animals.

Animal Enrichment
Method: For enrichment, animals were provided with items such as a gnawing device, except when interrupted by study procedures/activities.

Environmental Conditions
The targeted conditions for animal room environment were as follows:
Temperature: 61°F to 70°F (16°C to 21°C)
Humidity: 30% to 70%
Light Cycle: 12 hours light and 12 hours dark.
Food
Diet: PMI Nutrition International, LLC Certified Rabbit LabDiet® 5322.
Type: Pellets
Frequency: Gradual increments during the period prior to dosing according to Charles River SOPs and approximately 180 g/day during the study.
Analysis: Results of analysis for nutritional components and environmental contaminants are provided by the supplier and are on file at the Testing Facility. It was considered that there were no known contaminants in the feed that would interfere with the objectives of the study.


Water
Type: Municipal tap water, treated by reverse osmosis and ultraviolet irradiation.
Frequency/Ration: Ad libitum, via an automatic watering system. Water bottles were provided, if required.
Analysis: Periodic analysis of the water was performed, and results of these analyses are on file at the Testing Facility. It was considered that there were no known contaminants in the water that could interfere with the outcome of the study.

Veterinary Care
Veterinary care was available throughout the course of the study, and animals were examined by the veterinary staff as warranted by clinical signs or other changes. All veterinary examinations and recommended therapeutic treatments, if any, were documented in the study records and reviewed by the Study Director.

Route of administration:

oral: gavage

Details on exposure:

The test substance and vehicle were administered as a single daily oral gavage dose during Gestation Days 7 through 28. All animals were dosed at approximately the same time each day. The oral route of exposure was selected because this a possible route of human exposure.
The dose levels were selected based on the results from a dose range finding prenatal developmental toxicity study (Humes, 2021, 00387134). Time-mated New Zealand White rabbits were dosed with 100, 300, 700, or 1000 mg/kg/day of TMPD-MIB. There were no remarkable clinical observations nor effects on mean body weight or food consumption during the treatment period. There were also no effects on mean fetal weight nor were any external malformations or variations noted in the fetuses. Therefore, the limit dose of 1000 mg/kg/day was selected as the high dose for the current definitive prenatal developmental toxicity study. The lower dose levels were selected at intervals that are predicted to be narrow enough to reveal any dose-related trends.

The test substance and vehicle were administered as a single daily oral gavage dose during Gestation Days 7 through 28. All animals were dosed at approximately the same time each day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses described below were performed by gas chromatography (GC) with flame ionization detection (FID) using a validated analytical procedure (Engda, 2021, 00387132).

Concentration and Homogeneity Analysis

Storage Conditions: Temperature set to maintain a target of 5°C
Acceptance Criteria: For concentration: Mean sample concentration within 100% ± 15% of theoretical concentration. Individual sample concentration of ± 20%.
For homogeneity: Relative standard deviation (RSD) of concentrations of  10% for each group.

Resuspension Homogeneity and Stability Analysis
Test substance formulations have been previously shown to be stable and homogeneous over the range of concentrations used on this study for at least 24 hours at 18°C to 24°C and at least 8 days at a target of 5°C (Engda, 2021, 00387132). Therefore, stability and resuspension homogeneity of test substance formulations were not assessed on this study.

Details on mating procedure:

On 16 Apr 2021, time-mated female New Zealand White rabbits were received from Envigo Global Services Inc., Denver, PA on Gestation Day 1, 2, or 3. The animals were approximately 6 months old at receipt and weighed between 2825 and 3690 g at the initiation of dosing.

Duration of treatment / exposure:

The test substance and vehicle were administered as a single daily oral gavage dose during Gestation Days 7 through 28. All animals were dosed at approximately the same time each day.

Frequency of treatment:

daily

Duration of test:

21 days (gestation days 7-28)

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

75 mg/kg bw/day

Dose / conc.:

175 mg/kg bw/day

Dose / conc.:

250 mg/kg bw/day

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

Timed pregnant does were treated with the test article from GS 7-28 at which point a cessarian section was performed. Necropsy data was gathered from the dams and from the pups. Pup measures included litter data, visceral and skeletal exams.

Maternal examinations:

See Text Table 7 in the "other information" box below

Ovaries and uterine content:

Laparohysterectomies and macroscopic examinations were performed blind to treatment group. All surviving females were euthanized and subjected to a gross necropsy. The cranial, thoracic, abdominal, and pelvic cavities were opened and the contents examined. The uterus of each dam was excised and its adnexa trimmed. Corpora lutea were also counted and recorded. Gravid uterine weights were obtained and recorded. The uterus of each dam was opened and the number of viable and nonviable fetuses, early and late resorptions, and total number of implantation sites were recorded, and the placentae were examined. The individual uterine distribution was documented using the following procedure: all implantation sites, including early and late resorptions, were 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 were opened and placed in a 10% ammonium sulfide solution for detection of early implantation loss (Salewski, 1964). The carcasses were discarded.

Blood sampling:

not conducted

Fetal examinations:

Fetal Examinations
Fetal examinations were conducted without knowledge of treatment group. External, internal, and skeletal fetal findings were recorded as either 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), malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life), or incidental (minor changes in coloration, mechanical damage to specimen, etc.). Representative photographs of all malformations, as appropriate, were included in the Study Records (for exceptions, see Appendix 1). Corresponding low magnification photographs, depicting both the malformed fetus and a comparison control fetus or normal littermate, were also included in the Study Records as needed and as appropriate for comparison, when possible.

External
Each viable fetus was examined in detail, weighed, tagged, and euthanized by a subcutaneous injection of sodium pentobarbital in the scapular region. The crown-rump length of late resorptions (advanced degree of autolysis) was measured, the degree of autolysis recorded, a gross external examination performed (if possible), and the tissue was discarded.

Visceral (Internal)
All fetuses were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected using a technique described by Stuckhardt and Poppe (1984). This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development (Woo and Hoar, 1972). The sex of all fetuses was determined by internal examination. The heads from all fetuses were examined by a mid coronal slice.
All carcasses were eviscerated, skinned, and fixed in 100% ethyl alcohol for subsequent examination of skeletons.

Skeletal
Following fixation in alcohol, each eviscerated fetus was macerated in potassium hydroxide and stained with Alizarin Red S (Dawson, 1926) and Alcian Blue (Inouye, 1976). The skeletal examination was made following this procedure.

Statistics:

Statistics are presented below in the "other information" box due to a severe character limit in this box

Clinical signs:

no effects observed

Description (incidence and severity):

All females in the control, 100, 300, 700, and 1000 mg/kg/day groups survived to the scheduled necropsy on Gestation Day 29. Clinical observations noted at the daily examinations or approximately 1 hour following dose administration occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related. With the exception of Female No. 2501 in the 100 mg/kg/day group, all females were gravid.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no test substance-related effects on survival at any dose level. In the 1000 mg/kg/day group, 1 female (No. 4501) was found dead on Gestation Day 26 in the absence of any clinical findings or effects on body weight or food consumption. At necropsy, this female was noted with a perforated esophagus, and therefore this death was the result of a dosing error. In the control group, Female No. 1508 was euthanized in extremis on Gestation Day 9 due to limited usage of a hindlimb and was noted with a fractured tibia internally. All other animals survived to the scheduled necropsy.
No test substance related clinical observations were noted at the daily examinations or approximately 1 hour following dose administration at any dose level. Observations noted in the test substance-treated groups occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Test substance-related lower mean body weight gains were noted in the 700 and 1000 mg/kg/day groups during Gestation Days 7–24; the difference from the control group was statistically significant during Gestation Days 7–10 at 1000 mg/kg/day. Mean body weight gains in these group were generally comparable to the control group for the remainder of the gestation dosing period (Gestation Days 24–29). The initial decrements in mean body weight gain in the 700 and 1000 mg/kg/day groups were not of sufficient magnitude to impact mean absolute body weights in these groups, and therefore the effects were considered nonadverse. A statistically significantly lower mean adjusted body weight change was noted in the 1000 mg/kg/day group compared to the control group and was attributed to the nonadverse lower maternal body weight gains in this group. Mean adjusted body weights and gravid uterine weights in the 700 and 1000 mg/kg/day groups were comparable to the control group.
Mean body weights, body weight gains, adjusted body weight, adjusted body weight change, and gravid uterine weight in the 300 mg/kg/day group 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, non-treatment-related

Description (incidence and severity):

Test substance-related lower mean food consumption, evaluated as g/animal/day, was noted in the 700 and 1000 mg/kg/day groups during Gestation Days 7–24; differences from the control group were generally statistically significant. The effects on food consumption corresponded with the lower initial mean body weight gains noted in these groups. Although mean food consumption in the 700 and 1000 mg/kg/day groups was comparable to the control group during late gestation (Gestation Days 24–29), the initial decrements resulted in statistically significantly lower mean food consumption when the overall gestation dosing period (Gestation Days 7 29) was evaluated. In the absence of any effects on mean absolute body weights in these groups, the lower mean food consumption at 700 and 1000 mg/kg/day was considered nonadverse.
Mean food consumption in the 300 mg/kg/day group was unaffected by test substance administration. Differences from the control group were slight and not statistically significant.

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

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

At the scheduled necropsy, there were no test substance-related alterations in maternal liver, kidney, and adrenal gland weights at any dose level. Differences from the control group were slight and not statistically significant.

Gross pathological findings:

no effects observed

Description (incidence and severity):

At the scheduled necropsy on Gestation Day 29, no test substance-related internal findings were observed at dose levels of 300, 700, and 1000 mg/kg/day. Macroscopic findings observed in the test substance treated groups occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose related.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Details on results:

Intrauterine growth and survival were unaffected by test substance administration at dose levels of 300, 700, and 1000 mg/kg/day. Parameters evaluated included mean litter proportions of postimplantation loss, mean number of live fetuses, mean fetal body weights, and fetal sex ratios. Differences from the control group were slight and not statistically significant.
Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre implantation loss were similar across all groups.

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

Changes in number of pregnant:

no effects observed

Other effects:

not specified

Description (incidence and severity):

Intrauterine growth and survival were unaffected by test substance administration at dose levels of 300, 700, and 1000 mg/kg/day. Parameters evaluated included mean litter proportions of postimplantation loss, mean number of live fetuses, mean fetal body weights, and fetal sex ratios. Differences from the control group were slight and not statistically significant.
Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre implantation loss were similar across all groups.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

changes in number of pregnant

changes in pregnancy duration

clinical signs

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Intrauterine growth and survival were unaffected by test substance administration at dose levels of 300, 700, and 1000 mg/kg/day. Parameters evaluated included mean litter proportions of postimplantation loss, mean number of live fetuses, mean fetal body weights, and fetal sex ratios. Differences from the control group were slight and not statistically significant.
Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre implantation loss were similar across all groups.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Intrauterine growth and survival were unaffected by test substance administration at dose levels of 300, 700, and 1000 mg/kg/day. Parameters evaluated included mean litter proportions of postimplantation loss, mean number of live fetuses, mean fetal body weights, and fetal sex ratios. Differences from the control group were slight and not statistically significant.
Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre implantation loss were similar across all groups.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Intrauterine growth and survival were unaffected by test substance administration at dose levels of 300, 700, and 1000 mg/kg/day. Parameters evaluated included mean litter proportions of postimplantation loss, mean number of live fetuses, mean fetal body weights, and fetal sex ratios. Differences from the control group were slight and not statistically significant.
Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre implantation loss were similar across all groups.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Intrauterine growth and survival were unaffected by test substance administration at dose levels of 300, 700, and 1000 mg/kg/day. Parameters evaluated included mean litter proportions of postimplantation loss, mean number of live fetuses, mean fetal body weights, and fetal sex ratios. Differences from the control group were slight and not statistically significant.
Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre implantation loss were similar across all groups.

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

Intrauterine growth and survival were unaffected by test substance administration at dose levels of 300, 700, and 1000 mg/kg/day. Parameters evaluated included mean litter proportions of postimplantation loss, mean number of live fetuses, mean fetal body weights, and fetal sex ratios. Differences from the control group were slight and not statistically significant.
Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre implantation loss were similar across all groups.

Changes in postnatal survival:

not specified

Description (incidence and severity):

Not relevant as postnatal survival is not part of the study

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

External malformations were noted for 1(1), 1(1), and 3(2) fetuses (litters) in the control, 300, and 700 mg/kg/day groups, respectively. Additionally, 1 late resorption was noted with external malformations in the 300 mg/kg/day group. In the 700 mg/kg/day group, Fetus No. 3502-7 was noted with an extra fat pad, Fetus No. 3522-2 was noted with a short tail, and Fetus No. 3522 5 was noted with a small eye bulge. In the 300 mg/kg/day group, Fetus No. 2509-6 was noted with a cleft palate. A late resorption (No. 2518-04) at 300 mg/kg/day was noted with thoracogastroschisis, brachydactyly, and absent head and forelimbs. These malformations were noted in single fetuses or litters and were not observed in the high-dose group, and therefore were not considered test substance-related. In the control group, Fetus No. 1506-1 was noted with an omphalocele (portion of the intestines protruded, remnants of a membranous sac).
No external developmental variations were observed in fetuses in this study.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal developmental malformations were noted for 3(3), 2(2), 3(2), and 2(2) fetuses (litters) in the control, 300, 700, and 1000 mg/kg/day groups, respectively. One animal in the 1000 mg/kg/day group (No. 4524-5) was noted with absent rib, branched rib, supernumerary lumbar arch, and thoracic hemivertebra. A fetus in a different litter (No. 4521-9) was noted with a supernumerary lumbar vertebra. Supernumerary lumbar vertebrae were also noted in two fetuses in the same litter in the 700 mg/kg/day group (Nos. 3513-2 and 3513-6). One fetus in another litter in the 700 mg/kg/day group (No. 3522-2) was noted with fused caudal vertebrae. In the 300 mg/kg/day group, No. 2503-7 was noted with a branched rib, supernumerary rib, an absent lumbar vertebra, and a supernumerary thoracic arch. Another fetus in a different litter (No. 2509 6) was noted with fused rib, split palatine (corresponding to external finding of cleft palate), and fused thoracic arch. The malformations in the test substance-treated groups were limited to single fetuses/litters and/or did not occur in a dose-related manner, and therefore were not considered test substance-related. In the control group, a fused thoracic arch and fused rib were noted in Fetus No. 1517-11. Fused ribs were also noted in two other control group fetuses (Nos. 1506-1 and 1510-9). Fetus No. 1506-1 was also noted with a lumbar hemivertebra. Fetus No. 1510-9 was also noted with branched rib and absent lumbar vertebra.
No test substance-related skeletal 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 Charles River Ashland historical control data.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The numbers of fetuses (litters) available for morphological evaluation were 205(22), 189(22), 213(23), and 213(22) in the control, 300, 700, and 1000 mg/kg/day groups, respectively. Malformations were observed in 5(4), 6(5), 5(3), and 3(3) fetuses (litters) in these same respective dose groups and were considered spontaneous in origin. In addition, 1 late resorption in the 300 mg/kg/day group was noted with multiple malformations. When the total malformations and developmental variations were evaluated on a proportional basis, no statistically significant differences from the control group were noted. 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 Charles River Ashland historical control data ranges. Based on these data, no fetal malformations or developmental variations were attributed to the test substance.

Other effects:

not specified

Details on embryotoxic / teratogenic effects:

There were no significant effects observed that were significant compared to the control.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

changes in litter size and weights

changes in postnatal survival

external malformations

Abnormalities:

no effects observed

Developmental effects observed:

no

Summary data tables are provided as a single .PDF file attached below.

Conclusions:

There were no test substance-related effects on intrauterine growth and survival or fetal morphology (external, visceral, and skeletal) at any dose level.

Executive summary:

The objectives of this study were to determinethe potential of 2,2,4-trimethyl-1,3-pentanediol monoisobutyrate (TMPD-MIB) to induce developmental toxicity after maternal exposure (via oral gavage) from implantation to 1 day prior to expected parturition, to characterize maternal toxicity at the exposure levels tested, and to determine a no‑observed-adverse-effect level (NOAEL) for maternal and developmental toxicity.

Experimental Design

Animals were dosed via oral gavage once daily during Gestation Days 7–28.

The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, gravid uterine weights, food consumption, organ weights, macroscopic examinations, intrauterine growth and survival, and fetal morphology.

The analyzed dosing formulations were within the protocol-specified range of target concentrations for suspensions and were homogeneous.

There were no test substance-related effects on survival. No test substance-related clinical observations were noted at the daily examinations or approximately 1 hour postdose at any dose level.

Test substance-related lower mean body weight gains with correspondingly lower mean food consumption were noted in the 700 and 1000 mg/kg/day groups during Gestation Days 7–24. Mean body weight gains and food consumption in these groups were comparable to the control group for the remainder of the dosing period (Gestation Days 24–29). In addition, a lower mean adjusted body weight change was noted in the 1000 mg/kg/day group compared to the control group. In the absence of effects on mean absolute body weights in the 700 and 1000 mg/kg/day groups, the aforementioned decrements were considered nonadverse. Mean body weights, body weight gains, and food consumption at 300 mg/kg/day, mean adjusted body weight changes at 300 and 700 mg/kg/day, and mean adjusted body weights and gravid uterine weights at 300, 700, and 1000 mg/kg/day were unaffected by test substance administration.

At the scheduled necropsy on Gestation Day 29, there were no test substance-related macroscopic findings or effects on maternal organ weights.

There were no test substance-related effects on intrauterine growth and survival or fetal morphology (external, visceral, and skeletal) at any dose level.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Additional information

The potential for isobutyric acid, monoester with 2,2,4 -trimethylpentane-1,3 -diol to cause developmental toxicity is well understood. In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test conducted according to OECD Guideline 422, both sexes were exposed to up to 1000 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, pup body weights increased until Day 4 of lactation, and pups surviving until Day 4 of lactation did not demonstrate any treatment-related effects. The NOEL for embryo/fetal toxicity was 1000 mg/kg bw/day when the test material was administered by oral gavage.

To obtain conclusive evidence that the test material is not a developmental/reproductive toxicant, a prenatal developmental toxicity study (OECD 414) will be conducted using the related substance 2,2,4 -trimethyl-1,3 -pentanediol diisobutyrate (TXIB) in accordance with US Good Laboratory Practices (GLPs).

Justification for classification or non-classification

There were no adverse effects on reproductive organs in either sex of rats 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 1000 mg/kg bw/day isobutyric acid, monoester with 2,2,4 -trimethylpentane-1,3 -diol by oral gavage from 2 weeks prior to mating through early lactation (Day 4).  Based on a weight-of-the-evidence assessment, isobutyric acid, monoester with 2,2,4 -trimethylpentane-1,3 -diol is not selectively toxic to the fetus and is not classified for “Reproductive/Developmental Toxicity” according to EU GHS.