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Diss Factsheets

Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline GLP study. Study was conducted on structural analogue and suitable for read across.
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
Phosphorodithioic acid, mixed O,O-bis(iso-Bu and pentyl) esters, zinc salts
EC Number:
270-608-0
EC Name:
Phosphorodithioic acid, mixed O,O-bis(iso-Bu and pentyl) esters, zinc salts
Cas Number:
68457-79-4
IUPAC Name:
Phosphorodithioic acid, mixed O,O-bis(iso-Bu and pentyl) esters, zinc salts

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
Each rat was uniquely identified by a Monel® metal ear tag displaying the animal number.

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: approximately 11 weeks old at initiation of treatment; the 10 rats/sex/group used for pairing were approximately 13 weeks old when paired on study day 13.
- Weight at study initiation: 335 g to 429 g (males) and 227 g to 279 g (females); female body weights ranged from 238 g to 319 g on gestation day 0.
- Housing: Following receipt and until pairing for 10 animals/sex/group, all animals were housed individually in clean, stainless steel wire mesh cages suspended above cage board. The cage-board was changed at least 3 times per week. During cohabitation, rats were paired for mating in the home cage of the male. Following positive evidence of mating, the males were housed in suspended wire mesh cages until the scheduled necropsy, and the females were transferred to plastic maternity cages with nesting material, ground corncob bedding (Bed O'Cobs®; The Andersons, Cob Products Division, Maumee, OH). The dams and their litters were housed in these cages until euthanasia on lactation day 4. Females that failed to deliver were housed in plastic maternity cages until post-mating day 25. Males and females not used for pairing remained in suspended stainless steel wire mesh cages until euthanasia.
- Diet: PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 ad libitum except during the period of fasting of all males and post-treatment phase females prior to clinical pathology blood collection Feeders were changed and sanitized once per week.
- Water: Reverse osmosis-purified (on site) drinking water, delivered by an automatic watering system ad libitum
- Acclimation period: 13 days prior to the first day of treatment. During the acclimation period, the animals were observed twice daily for mortality and general changes in appearance and behavior.

ENVIRONMENTAL CONDITIONS
- Temperature: 71.1 to 72.3 deg F (21.7 to 22.4 deg C)
- Humidity: 36.3% to 63.6%
- Air changes: 10 fresh air changes per hour
- Photoperiod: 12 hour light (0600 hours to 1800 hours)/12 hour dark photoperiod

IN-LIFE DATES: From:8 December 2009 To: 12 February 2010 (Last female necropsy)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Mineral Oil USP
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored at room temperature. The test item formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures. The vehicle and test item formulations were administered orally by gavage, via an appropriately sized flexible, Teflon®-shafted, stainless steel ball-tipped dosing cannula (Natume, Japan) once daily.

VEHICLE
- mineral oil, USP
- Supplier: Spectrum Chemical Manufacturing Corporation, New Brunswick, NJ and Gardena, CA locations.
- The vehicle was dispensed into glass container approximately weekly for administration to the control group (Group 1) and for preparation of the test item formulations; aliquots were prepared for daily dispensation to the control group and stored at room temperature. The vehicle was mixed throughout preparation, sampling, and dose administration procedures.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability of the formulated test item, at concentrations between 0.5 and 20 mg/mL in mineral oil, USP, was established by the Sponsor. Formulations are stable after 10 and 30 days of stationary storage under room temperature conditions. Therefore, stability analyses were not conducted as part of this study.
Quadruplicate samples for homogeneity and concentration analyses were collected from the middle of the vehicle formulation and from the top, middle, and bottom strata of each test item batch formulation. One set of duplicate samples from each batch was shipped under ambient conditions to the sponsor for dose formulation analyses. The remaining set of duplicate samples was stored under ambient conditions as back-up. The analyzed dosing formulations were within WIL Research’s SOP range for solutions (90% to 110%) and were homogeneous.
Details on mating procedure:
The 10 rats/sex/group selected for evaluation of reproductive toxicity were paired on a 1:1 basis within each treatment group following 14 days of treatment for the males and females. Each female was housed in the home cage of the male. Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm following a vaginal lavage and verified by a second biologist. Each mating pair was examined daily. The day when evidence of mating was identified was termed gestation day 0.

For the purpose of calculating pre-coital intervals, rats paired over a 12-hour dark cycle were considered to have been paired for 1 day.
Duration of treatment / exposure:
Males: 10/group selected for pairing were dosed for 14 days prior to mating through 1 day prior to euthanasia for a total of 28 doses.
Females: 10/group selected for pairing were dosed for 14 days prior to mating through lactation day 3 for a total of 40-52 doses; females that failed to deliver were dosed through the day prior to euthanasia (post-mating day 25) for a total of 40 doses.

The extra 5 males and 5 females in the control and high-dose groups were not used for mating and were treated beginning on study day 0; following 28 doses for the males and 40 doses for the females, these animals were assigned to the post treatment period and remained on study for a 14-day non-dosing period. These animals were not evaluated for reproductive parameters.
Frequency of treatment:
Once daily at approximately the same time each day.

No. of animals per sex per dose:
The low- and mid-dose groups each consisted of 10 rats/sex and the high-dose group consisted of 15 rats/sex. Concurrent control group of 15 rats/sex



Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dosage levels were determined from the results of previous studies conducted
- Rationale for animal assignment: body weight stratification in a block design using a computer randomization procedure

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All rats were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality. Mated females expected to deliver were also observed twice daily during the period of expected parturition and at parturition for dystocia (prolonged labor, delayed labor) or other difficulties.

DETAILED CLINICAL OBSERVATIONS: Yes, A detailed physical examination was conducted weekly on each animal beginning approximately 3 days prior to the initiation of dose administration. Each male and female was also observed for signs of toxicity approximately 1 hour following dose administration. The absence or presence of findings was recorded for individual animals. In addition, the presence of findings at the time of dose administration was recorded for individual animals. Mated females expected to deliver were also observed twice daily during the period of expected parturition and at parturition for dystocia (prolonged labor, delayed labor) or other difficulties.

FUNCTIONAL OBSERVATIONAL BATTERY (FOB) AND LOCOMOTOR ACTIVITY: Yes, FOB assessments were recorded for 5 animals/sex/group prior to dose administration on study day 27 (males selected for pairing) and on lactation day 4 (females). FOB testing was performed without knowledge of the animal’s group assignment. The FOB was performed in a sound attenuated room equipped with a white noise generator set to operate at 70 ± 10 dB. Home cage, handling, open field, sensory, neuromuscular, and physiological parameters were observed. Forelimb and hindlimb grip strength were measured.
Locomotor activity counts were recorded for 5 animals/sex/group prior to dose administration on study day 27 (males selected for pairing) and on lactation day 4 (females); the same animals evaluated for FOB were selected for locomotor activity assessment. Locomotor activity, recorded after the completion of the FOB, was measured automatically using a personal computer controlled system utilizes a series of infrared photobeams surrounding a clear plastic, rectangular cage to quantify each animal’s motor activity. Four-sided black plastic enclosures were used to surround the clear plastic boxes to decrease the potential for distraction from extraneous environmental stimuli or stimuli from biologists or adjacent animals. The black enclosures rested on top of the photobeam frame and did not interfere with the path of the beams. The testing of treatment groups was done according to replicate sequence. Each animal was tested separately. Data were collected in 5 minute epochs and the test session duration was 60 minutes. These data were compiled as six, 10-minute subintervals for tabulation. Data for ambulatory and total motor activity were tabulated. Total motor activity was defined as a combination of fine motor skills (i.e., grooming, interruption of 1 photobeam) and ambulatory motor activity (interruption of 2 or more consecutive photobeams).

CLINICAL PATHOLOGY: Yes, Blood samples for clinical pathology evaluations (hematology and serum chemistry) were collected from 5 animals/sex/group at the scheduled necropsies (study day 28 for males selected for breeding and lactation day 4 for females) and from 5 animals/sex in the control and high-dose groups following a 14-day non-dosing post-treatment period (study day 42 for males and study 53 for females). All males (including those not scheduled for clinical pathology assessments) and the post-treatment phase females were fasted overnight prior to blood collection with water available. Blood for serum chemistry and hematology was collected from the retro orbital sinus following isoflurane anesthesia. Blood for coagulation parameters was collected from the vena cava at the time of necropsy. Blood was collected into tubes containing EDTA (hematology), sodium citrate (clotting determinations), or no anticoagulant (serum chemistry).

BODY WEIGHT: Yes
- Time schedule for examinations: Individual male body weights were recorded 1 week prior to the initiation of dose administration, on the first day of dose administration, and weekly throughout the study and prior to the scheduled euthanasia. Individual female body weights were recorded beginning approximately 1 week prior to the initiation of dose administration, on the first day of dose administration, and weekly thereafter until evidence of copulation was observed for females selected for pairing. Once evidence of mating was observed, female body weights were recorded on gestation days 0, 4, 7, 11, 14, 17, and 20 and on lactation days 0 (when possible), 1, and 4.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Individual food consumption was recorded for both males and females on the corresponding weekly body weight days until pairing. Food consumption continued to be recorded for males and females not selected for pairing until euthanasia. For animals selected for paiting, once evidence of mating was observed, food consumption was recorded for females on gestation days 0, 4, 7, 11, 14, 17, and 20 and on lactation days 1 and 4.

SACRIFICE
- All animals were euthanized by carbon dioxide inhalation. Males selected for pairing were euthanized following completion of the mating period. Males not selected for pairing were euthanized following the 14-day non-dosing post-treatment period. Females that delivered were euthanized on lactation day 4. Females (with evidence of mating) that failed to deliver were euthanized on post mating day 25.

GROSS NECROPSY
- A complete necropsy was conducted on animals euthanized in extremis or at the scheduled necropsies. Necropsy included examination of the external surface, all orifices, the cranial cavity, the external surface of the brain, and the thoracic, abdominal, and pelvic cavities, including viscera.

HISTOPATHOLOGY
- The following tissues were examined microscopically from all treatment phase animals in the control and high dose groups and from the high dose male that was euthanized in extremis. Adrenal glands (2), Aorta, Bone with marrow (sternebrae), Bone marrow smeara, Brain, Cerebrum level 1, Cerebrum level 2, Cerebellum with medul/pons, Coagulating gland, Eyes with optic nerve (2)b, Gastrointestinal tract, Esophagus, Stomach, Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum, Heart, Kidneys (2), Liver (sections of 2 lobes), Lungs (including bronchi, fixed by inflation with fixative), Lymph node (Axillary, Mesenteric, Mandibular), Ovaries and oviducts (2), Pancreas, Peripheral nerve (sciatic), Pituitary gland, Prostate gland, Mandibular salivary glands (2), Seminal vesicles (2), Skeletal muscle (rectus femoris), Skin with mammary glandc, Spinal cord (cervical), Spleen, Testes with epididymidesd (2), Thymus, Thyroids [with parathyroids, if present (2)], Trachea, Urinary bladder, Uteruse with cervix and vagina, All gross lesions (all groups)
In addition, the non-glandular portion of the stomach, thymus, and all gross lesions (all animals) and the testes at all dosage levels were examined microscopically at the scheduled necropsies for both treatment and post treatment phase animals.

ORGAN WEIGHTS
- The following organs were weighed from all F0 animals at the scheduled necropsies: Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries with oviducts, Spleen, Testes, Thymus gland, Thyroids with parathyroids.
Statistics:
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. Due to the use of significant figures and the different rounding conventions inherent in the types of software used, the means and standard deviations on the summary and individual tables may differ slightly. Data obtained from nongravid females were excluded from statistical analyses following the mating period. Where applicable, the litter was used as the experimental unit.
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 by sex.
Indices:
Litter parameters were calculated for: Mean Live Litter Size, Postnatal Survival Between Birth and PND 0 or PND 4 (% Per Litter), Postnatal Survival for All Other Intervals (% Per Litter).

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: portal-of-entry

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
One male in the 160 mg/kg/day group was euthanized in extremis on study day 17; a gross observation of a thickened stomach was noted at necropsy. Clinical findings noted for this male approximately 1 hour following dose administration on the day of euthanasia consisted of yellow material on various body surfaces, clear material around the mouth, unkempt appearance, decreased defecation, and labored respiration. Microscopically, this male was noted with inflammation, edema, and ulceration in the non-glandular stomach, erosion and inflammation in the trachea, and lymphoid depletion in the thymus, spleen, and lymph nodes (mesenteric, mandibular, and axillary). The lesions in the non glandular stomach were considered test item-related and may have contributed to the moribund state of this male. All other animals in all dosage groups survived to the scheduled necropsies. Test item related clinical findings were noted in the 160 mg/kg/day group males and females and included rales, decreased, shallow, and/or labored respiration and salivation related findings. These findings were noted at the daily examinations, at the time of dosing, and/or approximately 1 hour following dose administration primarily during the treatment period. However, because of their sporadic occurrence, these cardio pulmonary findings were considered to be incidental and secondary to the nature of the test item and the route of administration.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Mean body weights, body weight changes, and food consumption were unaffected by test item administration in the 10, 40, and 160 mg/kg/day groups throughout the treatment and post-treatment periods.

FUNCTIONAL OBSERVATIONAL BATTERY (FOB) AND LOCOMOTOR ACTIVITY (PARENTAL ANIMALS)
No test item-related effects were noted during the FOB or locomotor activity evaluations at any dosage level.

CLINICAL PATHOLOGY (PARENTAL ANIMALS)
There were no test substance-related effects on serum chemistry, hematology, or coagulation parameters in the 10, 40, and 160 mg/kg/day groups.

ORGAN WEIGHTS (PARENTAL ANIMALS)
There were no test item-related alterations in final body weight or organ weights at any dosage level. Significant (p<0.05) differences were observed when the control and high dose group males were compared at the recovery (post-treatment) necropsy and consisted of lower mean kidney weight relative to body weight, higher mean spleen weight relative to brain weight, higher mean left testis weight relative to brain weight, and higher mean right testis weights (absolute and relative to brain weight). There was no case where all 3 measures (absolute, relative to body weight, and relative to brain weight) were statistically significant. Thus, since the absolute weights and weights relative to body or brain weight were discordant, these organ weight changes were considered to be spurious.

GROSS PATHOLOGY (PARENTAL ANIMALS)
One male (no. 61739) in the 160 mg/kg/day group was euthanized in extremis on study day 17. The thickened stomach noted macroscopically for this male was related to test item administration. There were no other test item related internal findings observed for either sex at any dosage level at the scheduled necropsies. Macroscopic findings observed in the test item groups occurred infrequently and/or in a manner that was not dose related.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Test item-related histologic observations of epithelial hyperplasia, hyperkeratosis, and inflammation of the non-glandular stomach, typically at the limiting ridge, but sometimes more widespread, were noted in the 160 mg/kg/day group males and females.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOEL
Remarks:
portal-of-entry
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Remarks on result:
other:
Remarks:
localised injury to nonglandular portion of the stomach
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
160 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Remarks on result:
other:
Remarks:
no effects observed at any dose level

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Effect levels (fetuses)

Dose descriptor:
NOEL
Effect level:
160 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
maternal dose
Sex:
male/female
Basis for effect level:
other: development toxicity
Remarks on result:
other:
Remarks:
no effects on general physical condition of F1 pups

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

VIABILITY (OFFSPRING): Mean numbers of corpora lutea and unaccounted-for sites, mean number of pups born, live litter size, the percentage of males at birth, and postnatal survival in the 10, 40, and 160 mg/kg/day groups were similar to the control group values.

CLINICAL SIGNS (OFFSPRING): The general physical condition of all F1 pups in this study were unaffected by test item administration. No test item-related clinical findings were noted for the F1 pups.

BODY WEIGHT (OFFSPRING): Mean male and female pup body weights and body weight gains in the 10, 40, and 160 mg/kg/day groups were unaffected by test item administration during PND 1-4. No statistically significant differences from the control group were noted.

GROSS PATHOLOGY (OFFSPRING): There were no remarkable macroscopic findings in the F1 pups at the scheduled necropsy at any dosage level.

Read-Across Justification for EC 218-679-9

EC 218-679-9 has been tested for reproduction – developmental toxicity, however experimental data from a key study with the structurally related substance EC 270-608-0 was available and suitable for read-across.

Consistent with ECHA and OECD Guidance, read-across can be performed to fill data gaps for a substance when one or more analogues have similarity from multiple lines of evidence including structural, physical-chemical, mechanistic, toxicological and/or ecotoxicological bases (REFERENCES: 1. ECHA Chapter R.7a: Endpoint specific guidance. Guidance on Information Requirements and Chemical Safety Assessment, http://wko.at/up/enet/chemie/TL_ChapterR7a.pdf; 2. ECHA Practical Guide 6: How to Report Read-Across and Categories, http://echa.europa.eu/doc/publications/practical_guides/pg_report_readacross_categ.pdf; 3. OECD 2007. Guidance on grouping of chemicals. ENV/JM/MONO(2007)28).

The registered substance EC 218-679-9, zinc O,O,O',O'-tetrakis(1,3-dimethylbutyl) bis(phosphorodithioate), is a member of the group of inter-related ZDDP substances of similar structure and chemical properties that have previously been assessed as a category under the HPV program. For the purposes of read-across to fill data gaps for this substance the analogue ZDDP EC 270-608-0, phosphorodithioic acid, mixed O,O-bis(iso-Bu and pentyl) esters, zinc salts, is justified for use based on its similar structure, physical chemical properties, and fate and effects profile. For some endpoints where multiple reliable analogs exist, “worst case” data is selected based on the most precautionary test result, or based on reading across from lower molecular weight to higher or from higher water solubility to lower.

The following discussion provides multiple lines of evidence justifying this read across approach:

I. Category: EC 218-679-9 substance and EC 270-608-0 analog have been demonstrated to show sufficient structural and physicochemical similarity to be included in the High Production Volume (HPV) Chemical Challenge Program under the Zinc Dialkyldithiophosphate (ZDDP) category.

II. Manufacture/Usage:  EC 218-679-9 substance and EC 270-608-0 analog are substances that are generically referred to as zinc dialkylthiophosphate (ZDDP) that are produced under similar manufacturing procedures and are intended for multifunctional use as oil additives for antioxidancy and antiwear.

III. Chemical Similarity:EC 218-679-9 substance and EC 270-608-0 analog have the general empirical formula of C#H#O4P2S4Zn and are coordination complexes of zinc metal bonded to alkyldithiophosphate ligands. ZDDP complexes exist in reversible monomeric or dimeric forms (equilibrium dependent on temperature) and a basic form. The stereochemistry of the basic form can be described as four Zn atoms arranged around a tetrahedral oxide with six alkyldithiophosphate ligands. As a group, these ZDDPs share similar alcohol ester of dithiophosphate core structures, and variations that relate to alkyl chain length and the degree of branching of the alcohol. Using Tanimoto Fingerprint (ToxMatch Version 1.06 software) to model the chemical structures of the substances and its analog showed comparable values for relevant molecular descriptors (e.g., number of H bond acceptor atoms), and gave a similarity index greater than 0.8 (values range from 0, no similarity to 1, identical). Peer reviewed literature indicates that values greater than 0.6 are significantly similar and read-across is supported.

IV. Physicochemical Properties:EC 218-679-9 substance and EC 270-608-0 analog have similar values for average molecular weight (based on the monomer structure), log Kow, water solubility, and vapor pressure; or in some instances for read-across purposes “worst case” values are selected by going from a lower to a higher molecular weight, or from a higher to a lower water solubility.

V. Biologically Active Functional Groups: The ester group is a common functional group present in each of the analogue members, and is expected to exhibit similar biological activities with little influence from the length of carbon chain. Any potential breakdown products, via physical or biological processes, are also expected to result in structurally similar chemicals. In addition, non-random patterns have been observed for the toxicological effects (e.g., available data showed low levels of acute toxicity, lack of mutagenic potential, and a trend of change in ecotoxicity potential based on molecular weight). These common behaviors and consistent trends suggest a common mechanism and mode of action thereby providing further supporting evidence for the read-across among the ZDDP members.

 

Applicant's summary and conclusion

Executive summary:

In a guideline repeated dose and reproduction / developmental screening study (OECD 422) conducted according to Good Laboratory Practices, WIL Research Labs (2010) evaluated the potential toxic effects of phosphorodithioic acid, mixed O,O-bis(iso-Bu and pentyl) esters, zinc saltswhen administered to rats. This study was designed to evaluate the toxic effects, including neurobehavioral effects, of the test material to parental animals and to evaluate the potential to effect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition, and early postnatal development. The test material was administered orally by gavage once daily to 3 groups of Crl:CD(SD) rats at levels of 10, 40 or 160 mg/kg/day. The low- and mid-dose groups each consisted of 10 rats/sex and the high-dose group consisted of 15 rats/sex. A concurrent control group of 15 rats/sex received the vehicle, mineral oil USP, on a comparable regimen. Ten males/group selected for pairing were dosed for 14 days prior to mating through 1 day prior to euthanasia for a total of 28 doses. Ten females/group selected for pairing were dosed for 14 days prior to mating through lactation day 3 for a total of 40-52 doses; females that failed to deliver were dosed through the day prior to euthanasia (post-mating day 25) for a total of 40 doses. The extra 5 males and 5 females in the control and high-dose groups were not used for mating and were treated beginning on study day 0; following 28 doses for the males and 40 doses for the females, these animals were assigned to the post‑treatment period and remained on study for a 14-day non-dosing period.

Test item-related moribundity, clinical findings, and microscopic findings in the non glandular portion of the stomach, characterized by epithelial hyperplasia, hyperkeratosis, and inflammation, were observed in the 160 mg/kg/day group. The injury to the nonglandular portion of the stomach was localized and considered to be irritation from test item portal-of-entry effects. Based on these results, the NOEL for portal-of-entry effects was considered to be 40 mg/kg/day, and excluding the histologic injury to the nonglandular stomach, the no-observed-adverse-effect level (NOAEL) for systemic toxicity was considered to be 160 mg/kg/day. In the absence of effects on the general physical condition of the F1pups, the NOEL for neonatal toxicity was 160 mg/kg/day.