Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 September 2015 to 22 April 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted at a GLP facility according to GLP guidelines

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
Lot no. TXTXOL
Exp. date: 19-May-2016
White, crystalline powder

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
Crl:CD(SD) rats were used as the test system for this study. A minimum of 10 animals/sex/group was necessary for repeated-dose rodent studies in order to provide information on the possible health hazards likely to arise from prolonged exposure. Rats (45 males and 45 females) were received in good health from Charles River Laboratories, Inc., Raleigh, NC. The animals were approximately 35 days old at receipt. Each animal was examined by a qualified technician on the day of receipt and weighed on the following day. Each animal was uniquely identified with a subcutaneous microchip (BMDS system) implanted in the dorsoscapular area. All animals were housed for a 16 day acclimation. During this period, each animal was observed twice daily for mortality and changes in general appearance or behavior.
Upon arrival, all animals were housed 2 to 3 per cage by sex in clean, solid bottom cages containing ground corncob bedding material (Bed O’Cobs®; The Andersons, Cob Products Division, Maumee, OH). Following placement into treatment groups, animals were housed in groups of 2 of the same sex per cage. The cages were cleaned and changed routinely at a frequency consistent with maintaining good animal health. The bedding material is periodically analyzed by the manufacturer for contaminants. Analyses of the bedding material were provided by the manufacturer. No contaminants were present in the bedding at concentrations sufficient to interfere with the objectives of this study. The animals were temporarily separated as necessary to allow for the performance of protocol specified activities. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals. Enrichment devices were provided to all animals as appropriate throughout the study for environmental enrichment and to aid in maintaining the animals’ oral health, and were sanitized weekly.
The basal diet used in this study, PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002 (meal), is a certified feed with appropriate analyses performed by the manufacturer and provided to WIL Research. Reverse osmosis treated (on site) drinking water, delivered by an automatic watering system, and the basal diet were provided ad libitum throughout the study, except during the period of fasting prior to clinical pathology blood collection and during the conduct of FOBs and motor activity assessments when food, but not water, was withheld. Municipal water supplying the facility was analyzed for contaminants according to SOPs. No contaminants were present in animal feed or water at concentrations sufficient to interfere with the objectives of this study.
All animals were housed throughout acclimation and during the study in an environmentally controlled room. The room temperature and relative humidity controls were set to maintain environmental conditions of 71 ± 5°F (22 ± 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 69.8°F to 70.7°F (21.0°C to 21.5°C) and mean daily relative humidity ranged from 40.2% to 48.2% 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. The 12 hour light/12 hour dark photoperiod was interrupted as necessary to allow for the performance of protocol specified activities. Air handling units were set to provide a minimum of 10 fresh air changes per hour.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
The vehicle and test item formulations were administered orally by gavage, via an appropriately sized flexible, Teflon® shafted, stainless steel ball-tipped dosing cannula, once daily during gestation days 6-19. The dosage volume for all groups was 5 mL/kg. Individual dosages were based on the most recently recorded body weights to provide the correct mg/kg/day dose. All animals were dosed at approximately the same time each day.
The following table presents the study group assignment:
Group
Number Treatment Dosage Level (mg/kg/day) Dosage Volume (mL/kg) Number of Females
1 Vehicle Control 0 5 25
2 TMPD™ 350 5 5 25
3 TMPD™ 700 5 5 25
4 TMPD™ 1000 5 5 25

Dosage levels were selected based on results of a previous range finding study in pregnant rats. In that study, TMPD™ was administered by oral gavage to bred Crl:CD(SD) rats once daily from gestation days 6 19 at dosage levels of 0, 350, 700, and 1000 mg/kg/day. All females survived to schedule euthanasia. In the 700 and 1000 mg/kg/day groups, clonic convulsions were noted from around mid gestation and also prior to euthanasia approximately 10 15 minutes after dose administration. These convulsions did not persist to the scheduled 1 hour post-dose observation. Based on these data and the lack of mortality at 1000 mg/kg/day, the same dosage levels of 350, 700, and 1000 mg/kg/day were selected for this current study following consultation between the Sponsor and the Study Director.
The selected route of administration for this study was oral (gavage) because this is a potential route of exposure for humans. Historically, this route has been used extensively for studies of this nature.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item in the vehicle has been shown to be homogenous and stable at concentrations of 10 and 200 mg/mL after at least 12 hours of room temperature or at least 4 or 10 days of refrigerated storage (Haubenstricker, 2015, WIL 387072).
Samples for homogeneity and/or concentration determination were collected from the middle stratum of the first control group dosing formulation and from the top, middle, and bottom strata of the first test item dosing formulations. In addition, samples for resuspension homogeneity determination were collected from the top and bottom strata of an aliquot taken from the first test item dosing suspensions following refrigerated (2°C to 8°C) storage for 10 days; aliquots were mixed for a minimum of 30 minutes prior to sample collection. Samples for concentration analysis were also collected from the middle stratum of the last dosing formulations (including the control group) prepared during the study. One set of samples from each collection was subjected to the appropriate analysis. All remaining samples were stored refrigerated (2°C to 8°C) as back-up. All analyses were conducted by the WIL Research Analytical Chemistry Department using a validated gas chromatography method with flame ionization detection (Haubenstricker, 2015, WIL 387072).

Details on mating procedure:
At the conclusion of the acclimation period, all available females were weighed and examined in detail for physical abnormalities. At the discretion of the Study Director, each animal judged to be in good health and meeting acceptable body weight requirements was placed in a solid-bottom cage with bedding material with a resident male from the same strain and source for breeding. Resident males were untreated, sexually mature rats utilized exclusively for breeding. These rats were maintained under similar laboratory conditions as the females. 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 item treated groups and 1 control group, composed of 25 rats per group. The bred females were assigned to groups using a WTDMS™ computer program which randomized the animals based on stratification of the gestation day 0 body weights in a block design. Animals not assigned to study were transferred to the WIL Research colony. Body weight values ranged from 247 g to 319 g on gestation day 0.

Duration of treatment / exposure:
14 days
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
1 000 mg/kg bw/day
Dose / conc.:
700 mg/kg bw/day
Dose / conc.:
350 mg/kg bw/day
Dose / conc.:
0 mg/kg bw/day
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
Laparohysterectomies and macroscopic examinations were performed blind to treatment group. All surviving 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. Maternal tissues were preserved in 10% neutral buffered formalin for possible future histopathologic examination only as indicated by the gross findings. Representative sections of corresponding organs from a sufficient number of control animals were retained for comparison. The carcass of each female was then discarded.
Ovaries and uterine content:
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.
Fetal examinations:
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. The detailed external examination of each fetus included, but was not limited to, an examination of the eyes, palate, and external orifices, and each finding was recorded. Crown rump measurements and degrees of autolysis were recorded for late resorptions, a gross external examination was performed (if possible), and the tissues were discarded.
Each viable fetus was subjected to a visceral examination using a modification of the Stuckhardt and Poppe fresh dissection technique to include the heart and major blood vessels. The sex of each fetus was confirmed by internal examination. Fetal kidneys were examined and graded for renal papillae development. Heads from approximately one half of the fetuses in each litter were placed in Harrison’s fixative for subsequent soft tissue examination by the Wilson sectioning technique. 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 and Alcian Blue. 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 item treated group to the control group. Data obtained from nongravid animals were excluded from statistical analyses. 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 to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test was used to compare the test item treated 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 to determine intergroup differences. If the nonparametric ANOVA revealed significant (p<0.05) intergroup variance, Dunn’s test was used to compare the test item treated groups to the control group.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Test item-related mortality was noted at 1000 mg/kg/day, as 2 females (nos. 4005 and 3986) were found dead on gestation days 19 and 20, respectively. Both females were noted with clonic convulsions and clear material around the mouth approximately 10 minutes after dose administration during gestation days 7-18; clonic convulsions were also noted as early as 4 and 1 minutes following dose administration on gestation days 12 and 19, respectively, for female no. 3986. Limited occurrences of these findings were also noted for these females approximately 1 hour following dose administration (clonic convulsions on gestation days 8 and 16 for female no. 3986; clear material around the mouth for both females on gestation days 13, 16 and/or 17). Female no. 4005 was also noted with rales approximately 10 minutes after dose administration on gestation day 7 and ataxia and/or red material around the mouth approximately 1 hour after dose administration during gestation days 16-18. No remarkable changes in body weight or food consumption were noted for female no. 4005. Female no. 3986 was noted with hyper reactivity to touch and partial closure of both eyes at the daily examinations, and had ataxia, piloerection, cool and pale body, and shallow respiration at approximately 10 minutes and/or 1 hour following dose administration during gestation days 17-19; single occurrences of ataxia and cool extremities were also noted following dosing on gestation day 6. Female no. 3986 was also noted with red material around the nose and mouth, yellow material around the urogenital, anogenital, and ventral trunk areas, and decreased defecation at the daily examinations on gestation days 17-19, and single occurrences of rales (gestation day 9) and salivation (gestation day 16) at approximately 1 hour following dose administration. Severe body weight loss (20.8%) and reduced food consumption (0 to 5 g/day) were noted during gestation days 16-19 for female no. 3986. The following table presents the clinical observations noted for the 2 females that were found dead.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
Test item-related mortality was noted at 1000 mg/kg/day, as 2 females (nos. 4005 and 3986) in the 1000 mg/kg/day were found dead on gestation days 19 and 20, respectively.
Description (incidence and severity):
Mean body weight gain in the 1000 mg/kg/day group was significantly (p<0.05) lower compared to the control group during the gestation days 6-9 interval due to a mean body weight loss (significant at p<0.01) and absence of mean body weight gain following the first 2 days of dosing (gestation days 6-8), and despite a significantly (p<0.01) higher compensatory mean body weight gain on the third day of dosing. Mean body weight gains in this group were comparable
to the control group during the gestation days 9-12 and 12-15 intervals, but significantly (p<0.05) lower during the gestation days 15-20 interval primarily due to lower body weight gains during gestation day 16-18. As a result, mean body weight gain in the 1000 mg/kg/day was significantly (p<0.05) lower for the overall treatment period (gestation days 6-20) and mean body weights were slightly lower (3.2% to 3.8%) during gestation days 18-20 (significant [p<0.05] on gestation day 19) compared to the control group. In addition, mean net body weight change in the 1000 mg/kg/day group was significantly (p<0.05) lower than the control group. Mean net body weight and gravid uterine weight in the 1000 mg/kg/day group were comparable to the control group. A significant (p<0.01) mean body weight loss was noted for the 700 mg/kg/day group following the first dose (gestation day 6-7). Mean body weight gains in 700 mg/kg/day group were generally comparable to the control group throughout the remainder of the treatment period with the following exceptions. Mean body weight gain in the 700 mg/kg/day group was significantly (p<0.01) higher on gestation day 13-14 and significantly (p<0.05) lower on gestation day 17-18 compared to the control group. The initial body weight loss in the 700 mg/kg/day group was considered test item-related, but not adverse because mean body weight gain in this group was comparable to the control group when the entire treatment period (gestation days 6-20) was evaluated and mean body weights were comparable to the control group throughout the treatment period. Mean net body weight change in the 700 mg/kg/day group was lower than the control group. Mean net body weight and gravid uterine weight in the 700 mg/kg/day group were comparable to the control group. Mean maternal body weights, body weight gains, net body weight, net body weight gain, and gravid uterine weight in the 350 mg/kg/day group were unaffected by test item administration. Differences from the control group were slight and not statistically significant, with the following exception. A significantly (p<0.05) higher mean body weight gain was noted in the
350 mg/kg/day group on gestation day 13-14 compared to the control group and was considered transient and not test item-related.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Mean 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 the gestation days 6-9 interval due to significantly (p<0.01) lower mean food consumption following the first 2 days of dosing (gestation days 6-8). These changes corresponded with mean body weight loss and lower mean body weight gain noted in this group. Mean food consumption in the 1000 mg/kg/day group was comparable to the control group throughout the remainder of the treatment period (gestation days 9-12, 12-15, and 15-20) with the exception of significantly (p<0.05) lower mean food consumption (g/animal/day only) on gestation day 17-18. Due to the initial decrements in food consumption at 1000 mg/kg/day, mean food consumption in this group was significantly (p<0.01) lower than the control group when the entire treatment period (gestation days 6-20) was evaluated. These decrements in food consumption were considered test item-related. Mean food consumption in the 700 mg/kg/day group was intermittently lower than the control group during gestation day 6-13; differences were significant (p<0.05 or p<0.01) during gestation day 6-7, 9-10, 12-13, and the 6-9 and 9-12 cumulative intervals. Mean food consumption in the 700 mg/kg/day group was comparable to the control group throughout the remainder of the treatment period (gestation days 13-20). Due to the initial decrements in food consumption at 700 mg/kg/day, mean food consumption in this group was significantly (p<0.01) lower than the control group when the entire treatment period (gestation days 6-20) was evaluated. These decrements in food consumption were considered test item-related, but not adverse (as there were no effects on absolute mean body weights). Mean food consumption in the 350 mg/kg/day group was unaffected by test item administration. Differences from the control group were slight and not statistically significant, with the following exception. Significantly (p<0.05) lower mean food consumption was noted in the 350 mg/kg/day group on gestation day 10-11, resulting in significantly (p<0.05) lower mean food consumption for the gestation days 9-12 interval. This change was transient, not doseresponsive, and therefore not test item-related.
Food efficiency:
not specified
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:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Female nos. 4005 and 3986 in the 1000 mg/kg/day group were found dead on gestation days 19 and 20, respectively. At necropsy, female no. 4005 was noted with an enlarged spleen and placentae, brown vaginal contents, and dark red uterine contents surrounding all sites of the uterus, and was noted with 15 dead fetuses and 2 late resorptions with no apparent malformations, and 4 early resorptions in utero. Female no. 3986 was noted with a small spleen, red skin mating around both eyes and forelimbs and the nasal area, and had 7 dead fetuses and 11 late resorptions with no apparent malformations in utero. These deaths were considered test item-related. Female no. 4007 in the 700 mg/kg/day aborted 1 early resorption (based on the degree of autolysis) on gestation day 11 and was noted with an enlarged and swollen spleen and liver, enlarged and pale adrenal glands, and green discoloration of the kidneys at necropsy. This abortion was not considered test item-related because no abortions were noted at 1000 mg/kg/day, the highest dose level tested. At the scheduled necropsy on gestation day 20, no test item-related internal findings were observed at dosage levels of 350, 700, and 1000 mg/kg/day. Macroscopic findings observed in the test item-treated groups occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related. One female each in the 350 and 700 mg/kg/day groups were nongravid.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
One female in the 700 mg/kg/day group aborted 1 early resorption (based on the degree of autolysis) on gestation day 11 after being noted with a cool and pale body, cool extremities, red material around the urogenital area and mouth, and clear material around the mouth during gestation days 9-11. This abortion was not considered test item-related because no abortions were noted at 1000 mg/kg/day, the highest dose level tested.
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:
effects observed, non-treatment-related
Description (incidence and severity):
There was a single non-gravid female in the 350 and 700 mg/kg groups.
Other effects:
no effects observed
Details on maternal toxic effects:

Test item-related mortality was noted at 1000 mg/kg/day, as 2 females (nos. 4005 and 3986) were found dead on gestation days 19 and 20, respectively. Both females were noted with clonic convulsions and clear material around the mouth approximately 10 minutes after dose administration during gestation days 7-18; clonic convulsions were also noted as early as 4 and 1 minutes following dose administration on gestation days 12 and 19, respectively, for female no. 3986. Limited occurrences of these findings were also noted for these females approximately 1 hour following dose administration (clonic convulsions on gestation days 8 and 16 for female no. 3986; clear material around the mouth for both females on gestation days 13, 16 and/or 17).

All other females in the control, 350, 700, and 1000 mg/kg/day groups survived to the scheduled necropsy on gestation day 20. Test item-related clinical observations were noted for surviving females in all treated groups. Clonic convulsions were noted in 3, 13, and 12 females in the 350, 700, and 1000 mg/kg/day groups, respectively, approximately 10 minutes after dose administration throughout the treatment period and generally did not persist 1 hour after the dose administration with the exception of 1 female in the 700 mg/kg/day group on gestation day 13. Clonic convulsions did not persist to the following day’s examination for any female in the test item treated groups. Ataxia was noted for 1 female each in the 700 and 1000 mg/kg/day groups approximately 10 minutes after dose administration on gestation days 10 and 7, respectively, and in 2 females in the 1000 mg/kg/day group approximately 1 hour after dose administration on gestation days 6 and 16. With the exception of 1 female in the 1000 mg/kg/day group, this finding occurred in females that were also noted with clonic convulsions, however, not simultaneously.


Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
< 350 mg/kg bw/day (actual dose received)
Basis for effect level:
other: maternal toxicity
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Basis for effect level:
other: developmental toxicity

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
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:
not examined
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
not specified
Details on embryotoxic / teratogenic effects:
None found

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
not specified
Sex:
male/female
Basis for effect level:
other: No adverse effects at any dose

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

 Dose  0 mg/kg  350 mg/kg  700 mg/kg  1000 mg/kg

 Pregnant/non-pregnant dams

 25/0

 24/1

 24/1

 25/0

 Dams with abortions, early delivery, stillbirth, resorptions or dead fetus

 0

 0

 1

 0

 Pre-implant loss #/%

33 /7

 31/6.9

 46/8.5

 36/9.3

 Post implant loss #/%

 35/8.7

16 /4.0

 23/5.9

 18/8.3

 Body weight  442  445  437  428
 Body weight change  350.2  350.4  343.1  341.8
 gravid uterine weight  91.4  94.5  94.2  86.4
 #/% live offspring  379/100  375/100  365/100  342/100
 fetal body weight M/F/combined  3.9/3.8/3.9  4.0/3.8/3.9  3.9/3.7/3.8  3.8/3.6/3.7
 #/% fetuses with malformation   Variations/Marformations were given as #/% per type of incidence, rather than absolute #/% with ANY finding
 #/% litter with malformation   Variations/Marformations were given as #/% per type of incidence, rather than absolute #/% with ANY finding
   #/% fetuses with variations  Variations/Marformations were given as #/% per type of incidence, rather than absolute #/% with ANY finding
   #/% litter with variations  Variations/Marformations were given as #/% per type of incidence, rather than absolute #/% with ANY finding
         

Applicant's summary and conclusion

Conclusions:
There was no developmental toxicity seen at any dose level. Brief, minor, transient convulsions were observed throughout the treatment period immediately after dosing, usually resolving after a few minutes.
Executive summary:

The objectivesof the studywereto determine the potentialof the test item, 2, 2, 4‑Trimethyl-1, 3-Pentanediol, to induce developmentaltoxicityafter maternal exposure from implantation to one 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 toxicity and developmental toxicity.

The testitem,TMPD™, in the vehicle (corn oil) was administered orally by gavage to3 groups of25bred female Crl:CD(SD) rats once daily from gestation days6through19. Dosage levels were350,700, and1000 mg/kg/day administered at a dosage volume of5 mL/kg. A concurrent control group composed of25bred females received the vehicle on a comparable regimen. The females were approximately14weeks of age at the initiation of dose administration. All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. On gestation day 20, a laparohysterectomy was performed on each surviving 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.

Test item-related mortality was noted for 2 females in the 1000 mg/kg/day group on gestation days 19 and 20. Both females were noted with test-item related clinical observations including clonic convulsions, ataxia, rales, and clear and/or red material around the mouth at approximately 10 minutes and/or 1 hour after dose administration. The female that died on gestation day 20 was also noted with hyper reactivity to touch, piloerection, cool and pale body, cool extremities, shallow respiration, salivation, decreased defecation, partially closed eyelids, and red and yellow material findings at the daily examinations, and/or approximately 10 minutes and/or 1 hour following dose administration; this female was also noted with body weight loss (20.8%) and reduced food consumption (≤5 g/day) during gestation days 16‑19.

One female in the 700 mg/kg/day group aborted 1 early resorption on gestation day 11 after being noted with a cool and pale body, cool extremities, red material around the urogenital area and mouth, and clear material around the mouth during gestation days 9‑11. This abortion was not considered test item-related because no abortions were noted at 1000 mg/kg/day, the highest dose level tested.

All other females survived to the scheduled necropsy on gestation day 20. Test item‑related clinical observations were noted for surviving females in the 350, 700, and 1000 mg/kg/day groups following dose administration. Clonic convulsions were noted for 3, 13, and 12 surviving females at 350, 700, and 1000 mg/kg/day, respectively, and ataxia was noted for 1 and 3 females in the 700 and 1000 mg/kg/day groups, respectively. Clear material around the mouth was noted in all females in all test item‑treated groups. Red material around the nose and/or mouth was noted in increased incidences in all test item‑treated groups. Other test item‑related findings included single occurrences of cool body or extremities at 350 and 1000 mg/kg/day and rales for 5 and 8 females at 700 and 1000 mg/kg/day, respectively. All of the aforementioned findings were generally noted throughout the treatment period and were predominately observed at approximately 10 minutes following dose administration, with limited occurrences at 1 hour following dose administration.

Test item-related lower mean body weight gains and food consumption were noted in the 1000 mg/kg/day group during the gestation days 6-9 interval due to a mean body weight loss or absence of mean body weight gain and reduced food consumption following the first 2 days of dosing. Mean body weight gains and food consumption for the 1000 mg/kg/day group were comparable to the control group during gestation days 9‑12 and 12-15. Mean body weight gain was again lower in this group compared to the control group during the gestation days 15-20 cumulative interval, primarily due to lower body weight gains during gestation days 16-18, with a corresponding effect on food consumption on gestation day 17-18. As a result, mean body weights in the 1000 mg/kg/day group were slightly lower (3.2% to 3.8%) compared to the control group during gestation days 18‑20. Mean net body weight change in the 1000 mg/kg/day group was also lower than the control group.

Test item-related mean body weight loss and reduced food consumption were noted for the 700 mg/kg/day group following the first dose (gestation day 6-7). Mean body weight gains in the 700 mg/kg/day were generally comparable to the control group for the remainder of the treatment period. Lower mean food consumption was intermittently noted for the 700 mg/kg/day group through gestation day 13, but was generally comparable to the control group thereafter. The mean body weight loss and lower food consumption in the 700 mg/kg/day group were not considered adverse because mean body weights were comparable to the control group throughout the treatment period. Mean net body weight change in the 700 mg/kg/day group was also lower than the control group.

There were no testitem-related effects on body weights, body weight gains, or food consumption in the350 mg/kg/day group. Mean net body weight change in the 350 mg/kg/day group and mean gravid uterine weights and net body weights in the 350, 700, and 1000 mg/kg/day groups were comparable to the control group. 

At the scheduled necropsy on gestation day 20, no test item-related internal findings were noted at dosage levels of 350, 700, and 1000 mg/kg/day.

Intrauterine growth and survival in the 350, 700, and 1000 mg/kg/day groups were unaffected by test item administration.

No test item-related external, visceral, or skeletal fetal malformations and developmental variations were noted at any dosage level.

Based ontest item-related mortality and adverse effects on body weight gain and food consumption at 1000 mg/kg/day and adverse clinical observations, includingclonic convulsions, at 350, 700, and 1000 mg/kg/day,theno‑observed‑adverse‑effect level (NOAEL) for maternal toxicitywas determined to be <350 mg/kg/day. Based on the absence of effects on fetal growth and survival, and fetal morphology,a dosage level of1000 mg/kg/day was considered to be the NOAEL for embryo/fetal development whenTMPD™was administered orally by gavage to bred Crl:CD(SD) rats.