Reaction products of dimethyl phosphonate with 2-alkyl-2-alkylpropanediol and alkanediol

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-07-20 to 2018-04-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

various deviations with no impact on results or integrity of the study (see Appendix 1, attached)

GLP compliance:

yes

Limit test:

no

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

liquid: viscous

Details on test material:

- Appearance/physical state: Clear viscous liquid
- Storage conditions: Room temperature in the dark

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The animal model, the Crl:CD(SD) rat, is recognized as appropriate for reproductive toxicity studies and has been proven to be susceptible to the effects of reproductive toxicants. In addition, Charles River Ashland has reproductive historical control data in the Crl:CD(SD) rat.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC on 27 Jun 2017
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: ~ 10 weeks
- Weight at study initiation: male: 295 g to 439 g
female: 208 g to 260 g
- Housing: F0 animals: 2-3 per cage by sex in clean, solid-bottom cages with bedding material
after breeding period: males were individually housed in solid-bottom cages
after mating: females were individually housed in solid-bottom cages with bedding material.
Control group: remained housed in groups of 2-3 in clean solid-bottom cages
- Diet (e.g. ad libitum): ad libitum (exception: All F0 males and females (including animals not selected for clinical pathology evaluation) were fasted overnight prior to clinical pathology blood collection when food was withheld.)
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 21 days

DETAILS OF FOOD AND WATER QUALITY: 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 Charles River. Municipal water supplying the facility was sampled for contaminants according to Charles River SOPs. No contaminants were present in animal feed or water at concentrations sufficient to interfere with the objectives of this study.

ENVIRONMENTAL CONDITIONS
The room temperature and relative humidity controls were set to maintain environmental conditions of 68°F to 78°F (20°C to 26°C) and 30% to 70%, 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 72.3°F to 72.7°F (22.4°C to 22.6°C) and mean daily relative humidity ranged from 38.6% to 58.6% during the study. Fluorescent lighting provided illumination for a 12 hour light (0600 hours to 1800 hours)/12 hour dark photoperiod. The light status (on or off) was recorded once every 15 minutes. Air handling units were set to provide a minimum of 10 fresh air changes per hour.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on exposure:

The vehicle and test substance formulations were administered orally by gavage, via appropriately sized flexible, disposable, plastic feeding tubes (Instech Laboratories, Inc., Plymouth Meeting, PA) once daily. The males selected for pairing were dosed during Study Days 0–27 (14 days prior to pairing through 1 day prior to scheduled euthanasia) for a total of 28 doses. The females selected for pairing were dosed during Study Days 0 through the day prior to euthanasia (14 days prior to pairing through Lactation Day 12) for a total of 48–58 doses. Females with no evidence of mating or that failed to deliver were dosed through the day prior to euthanasia (Postmating or Postcohabitation Day 25) for a total of 40–52 doses. The extra 5 rats/sex in the control and high-dose groups were not used for mating, but were treated on a comparable regimen beginning on Study Day 0; following 28 doses for males and 48 doses for females, these animals remained on study for a 15-day recovery period. The dose 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.

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. A breeding record containing the male and female identification numbers and the start date of cohabitation was maintained. Each female was cohabitated with 1 male in a solid-bottom cage containing bedding material. 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 and the animals were separated. If evidence of copulation was not detected after 14 days of pairing, the animals were separated without further opportunity for mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples for homogeneity and/or concentration determination were collected from the top, middle, and bottom strata of the first 7, 70, and 140 mg/mL dosing formulations and from the middle stratum of the first control and 35 mg/mL dosing formulations. In addition, samples for resuspension homogeneity determination were collected from the top and bottom strata of an aliquot taken from the first 7, 70, and 140 mg/mL dosing suspensions following room temperature storage for 10 days; aliquots were mixed for a minimum of 30 minutes prior to sample collection. Samples for concentration analysis were also collected from the middle stratum of the fourth and last dosing formulations (including the control group) prepared during the study. One set of samples from each collection was subjected to the appropriate analyses. All remaining samples were stored at room temperature as back-up.

Duration of treatment / exposure:

Males received 14 daily doses prior to mating. Males were dosed throughout the mating period through 1 day prior to euthanasia for a total of 28 doses. Females received 14 daily doses prior to pairing and were dosed through Lactation Day 12 for a total of 48–58 doses; females with no evidence of mating or that failed to deliver were dosed through the day prior to euthanasia (Postcohabitation Day 25 or Postmating Day 25, respectively) for a total of 40–52 doses. The extra 5 males and 5 females in the control and high dose groups that were not used for mating were treated beginning on Study Day 0; following 28 doses for males and 48 doses for females, these animals were assigned to a 15-day nondosing recovery period.

Frequency of treatment:

Males received 14 daily doses prior to mating. Males were dosed throughout the mating period through 1 day prior to euthanasia for a total of 28 doses. Females received 14 daily doses prior to pairing and were dosed through Lactation Day 12 for a total of 48–58 doses; females with no evidence of mating or that failed to deliver were dosed through the day prior to euthanasia (Postcohabitation Day 25 or Postmating Day 25, respectively) for a total of 40–52 doses. The extra 5 males and 5 females in the control and high dose groups that were not used for mating were treated beginning on Study Day 0; following 28 doses for males and 48 doses for females, these animals were assigned to a 15-day nondosing recovery period.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

35 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

175 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

350 mg/kg bw/day (actual dose received)

Doses / concentrations 4

Dose / conc.:

700 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

The low- and mid dose groups (Groups 2–4) each consisted of 10 rats/sex and the high-dose group (Group 5) consisted of 15 rats/sex. A concurrent control group of 15 rats/sex received the vehicle on a comparable regimen.

Control animals:

yes, concurrent vehicle

Details on study design:

The test substance, in the vehicle (polyethylene glycol 400 [PEG400]) was administered orally by gavage once daily to 4 groups of Crl:CD(SD) rats. The low- and mid dose groups (Groups 2–4) each consisted of 10 rats/sex and the high-dose group (Group 5) consisted of 15 rats/sex. Dosage levels were 35, 175, 350, and 700 mg/kg/day. A concurrent control group of 15 rats/sex received the vehicle on a comparable regimen. The dose volume was 5 mL/kg for all groups. Males and females were approximately 10 weeks of age at the beginning of test substance administration. Males received 14 daily doses prior to mating. Males were dosed throughout the mating period through 1 day prior to euthanasia for a total of 28 doses. Females received 14 daily doses prior to pairing and were dosed through Lactation Day 12 for a total of 48–58 doses; females with no evidence of mating or that failed to deliver were dosed through the day prior to euthanasia (Postcohabitation Day 25 or Postmating Day 25, respectively) for a total of 40–52 doses. The extra 5 males and 5 females in the control and high dose groups that were not used for mating were treated beginning on Study Day 0; following 28 doses for males and 48 doses for females, these animals were assigned to a 15-day nondosing recovery period.

Examinations

Parental animals: Observations and examinations:

All rats were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality. Individual clinical observations were recorded daily and detailed physical examinations were conducted weekly (prior to dose administration during the treatment period). Each male and female was also observed for signs of toxicity at the time of dosing and approximately 1 hour following dose administration. 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. In addition, the social groups were observed at the appropriate intervals for findings that could not be attributed to a single animal; only positive findings were recorded.

Individual male body weights were recorded weekly throughout the study and prior to the scheduled euthanasia.

Food consumption was recorded on the corresponding weekly body weight days until pairing (for animals paired for breeding) or euthanasia (for animals assigned to the recovery period).

Oestrous cyclicity (parental animals):

Vaginal lavages were performed daily and the slides were evaluated microscopically to determine the stage of the estrous cycle of each female for 14 days prior to randomization and continuing for females selected for the breeding phase until evidence of copulation was observed or until termination of the mating period for females with no evidence of mating. he average cycle length was calculated and reported for complete estrous cycles (i.e., the total number of returns to metestrus [M] or diestrus [D] from estrus [E] or proestrus [P] until the detection of evidence of mating), beginning with the first day of dose administration. Estrous cycle length was determined by counting the number of days from the first M or D in a cycle to the first M or D in a subsequent cycle. For breeding phase females, the cycle during which evidence of mating was observed for a given animal was not included in the mean individual estrous cycle length calculation. Vaginal lavages were also performed on the day of necropsy to determine the stage of the estrous cycle.

Sperm parameters (parental animals):

Histology and microscopic examinations: Target tissues of testes, epididymis were examined from all F0 animals in all groups.

Litter observations:

Each litter was examined daily for survival and behaviour, and all deaths were recorded. To reduce variability among the litters, 8 pups/litter, 4 pups/sex when possible, were randomly selected on PND 4. Each pup received a clinical examination on PND 1, 4, 7, 10, and 13. Any abnormalities in nesting and nursing behavior were recorded. The anogenital distance of all F1 pups was measured on PND 1. Pups were individually weighed on PND 1, 4, 7, 10, and 13. Pups were individually sexed on PND 0, 4, and 13. On PND 13, all F1 male offspring were evaluated for the presence of thoracic nipples/areolae in accordance with previous established methods.

Postmortem examinations (parental animals):

Males were euthanized following completion of the mating period or following the 15-day recovery period; blood samples were collected for thyroid hormone analysis immediately prior to euthanasia. Females that failed to deliver were euthanized on Postmating Day 25 (females with evidence of mating) or Postcohabitation Day 25 (females with no evidence of mating); 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.
Microscopic examination was performed on all tissues -
Brain
Coagulating glands
Kidneys
Liver
Mammary glands
Ovaries and oviducts (2)
Pituitary gland
Prostate gland
Seminal vesicles (2)
Testes with epididymides a (2) and vas deferens
Cowper’s glands
Glans Penis
Uterus b with cervix and vagina
All gross lesionsc

Organ weights measured on -
Adrenal glands
Brain
Epididymides a
Heart
Kidneys
Liver
Ovaries with oviducts
Pituitary gland
Prostate gland
Seminal vesicle (with coagulating gland and fluid)
Spleen
Testes a
Thymus gland
Thyroids with parathyroids

Postmortem examinations (offspring):

On PND 13, surviving F1 rats were euthanized via an intraperitoneal injection of sodium pentobarbital. Blood samples were collected for thyroid hormone analysis immediately prior to euthanasia. Gross necropsies were conducted and the thyroids (with parathyroids, if present) were placed in 10% neutral-buffered formalin for possible histopathological examination. Females that delivered were euthanized on Lactation Day 13; blood samples were collected for thyroid hormone analysis on the day prior to euthanasia.

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. Statistical analyses were not conducted if the number of animals was 2 or less.
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-treated group to the control group by sex.
Parental mating, fertility, conception, and copulation indices were analyzed using the Chi square test with Yates’ correction factor. Parental body weights (weekly, gestation, and lactation), body weight changes, and food consumption, offspring body weights and body weight changes, estrous cycle length, precoital intervals, gestation length, numbers of former implantation sites and unaccounted-for sites, number of pups born, live litter size on PND 0, absolute and relative organ weights, thyroid hormone values, anogenital distance (absolute and relative to the cube root of body weight), and the number of nipples/areolae 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 substance-treated groups to the control group. Mean litter proportions (percent per litter) of males at birth and postnatal survival were subjected to the Kruskal-Wallis nonparametric ANOVA to determine intergroup differences. If the nonparametric ANOVA revealed significant (p < 0.05) intergroup variance, Dunn’s test was used to compare the test substance-treated groups to the control group.

Reproductive indices:

Not performed on F1 litter

Offspring viability indices:

Each litter was examined daily for survival, and all deaths were recorded. All pups were individually identified by application of tattoo markings on the digits following completion of parturition. A daily record of litter size was maintained. Intact offspring that were found dead from PND 0 to 4 were necropsied using a fresh dissection technique, which included examination of the heart and major vessels. Tissues were preserved in 10% neutral buffered formalin for possible future histopathologic examination only as deemed necessary by the gross findings.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical observations of rales were noted in the 175, 350, and 700 mg/kg/day group F0 males and females at the time of dosing and/or approximately 1 hour following dose administration sporadically throughout the dosing period; rales were also noted at the weekly detailed physical examinations and the daily examinations for males in the 350 mg/kg/day group and males and females in the 700 mg/kg/day group. Increased occurrences of red material around the nose were noted at the time of dose administration for males and females in the 700 mg/kg/day group. In addition, increased occurrences of salivation at the time of dose administration and clear and/or red material around the mouth at approximately 1 hour following dose administration were noted for males and/or females in the 175, 350, and 700 mg/kg/day groups; these findings were noted throughout the dosing period. The aforementioned clinical observations were not considered adverse because they generally did not persist to the daily examinations the following day.
No remarkable clinical observations were noted for F0 males and females in the 35 mg/kg/day group during the dosing period or for males and females in the 700 mg/kg/day group during the recovery period. Other observations noted in the treated groups, including hair loss on various body surfaces, soft feces, and brown material around the anogenital area, occurred infrequently, similarly in the control group, and/or in a manner that was not dose–related.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female in the 35 mg/kg/day group was found dead on Study Day 4. This female had no noteworthy clinical observations or changes in body weight or food consumption prior to death. This female was noted with a perforated esophagus, clear fluid in the thoracic cavity, and dark red discoloration of the lungs and pituitary gland at the necropsy, with no microscopic correlates; these findings were indicative of an intubation error as the cause of death. All other males and females survived to the scheduled necropsies.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean body weight gains in the 700 mg/kg/day group F0 males were generally lower than the control group throughout the dosing period; however, in the absence of an effect on mean absolute body weights, these changes were not considered adverse. There were no effects on mean bodyweights and body weight gains in the 35, 175, and 350 mg/kg/day group F0 males during the dosing period or in the 700 mg/kg/day group F0 males during the recovery period. There were no effects on mean body weights and body weight gains for F0 females at any dosage level throughout the study (including during gestation and lactation).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No effects on F0 food consumption were noted for males throughout the study or for females during the premating period or during gestation at any dosage level. Mean food consumption in the 700 mg/kg/day group F0 females was lower than the control group throughout lactation; however, because there were no corresponding effects on mean body weights or body weight gains during lactation, the lower mean food consumption was not considered adverse. There were no effects on lactation food consumption at 35, 175, and 350 mg/kg/day.

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:

no effects observed

Description (incidence and severity):

Thyroid hormone (T4) levels in the F0 males were unaffected. There were no effects on thyroid hormone values in the F1 males and females at any dosage level on PND 13.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No effects on reproductive performance were observed at any dosage level. No statistically significant differences were noted between the control and test substance treated groups.

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

700 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects at the highest dose tested

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The general physical condition (defined as the occurrence and severity of clinical findings) of all F1 pups in this study was unaffected by test substance administration. Three (3), 1(1), 4(2), 2(2), and 22(5) pups (litters) in the control, 35, 175, 350, and 700 mg/kg/day groups, respectively, were found dead from birth to PND 13. A single pup in the 350 mg/kg/day group and 19 pups from 5 litters in the 700 mg/kg/day group were missing and presumed to have been cannibalized. The increased incidence of found dead and missing pups in the 700 mg/kg/day group was considered adverse.

Dermal irritation (if dermal study):

not specified

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

The mean number of pups born, live litter size and the percentage of males at birth in the 35, 175, 350, and 700 mg/kg/day groups were similar to the control group values.
Postnatal survival in the 700 mg/kg/day group was lower than the control group during PND 0 1 and 1–4 (pre-selection), and consequently from birth to PND 4 (69.3% versus 97.9% in the control group). One female in this group had a total litter loss (0.0% survival) on Lactation Day 1, while 4 other females in this group had ≤ 66.7% postnatal survival from birth to PND 4. All females in the control group had ≥ 92.3% postnatal survival during this period.
Postnatal survival in the 35, 175, and 350 mg/kg/day groups was unaffected by test substance administration.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean male and female birth weights (PND 1) in the 700 mg/kg/day group were 9.6% and 10.0% lower, respectively, than the control group; differences were not statistically significant. Mean pups body weight gains in this group were similar to the control group during PND 1–4, but significantly (p < 0.05 or p < 0.01) lower than the control group beginning on PND 4 and continuing through PND 13. As a result, mean male and female pup body weights in the 700 mg/kg/day group were significantly (p < 0.05 or p < 0.01) lower (11.2% to 16.5% and 13.6% to 17.9%, respectively) than the control group from PND 7–13.
Mean male and female pup body weights and body weight changes during PND 1–13 in the 35, 175, and 350 mg/kg/day groups were unaffected by parental administration of the test substance. No statistically significant differences from the control group were noted, with the exception of a significantly (p < 0.05) lower mean body weight gain in the 175 mg/kg/day group females during PND 10–13 that was not dose-related and had no effect on mean absolute body weight.

Food consumption and compound intake (if feeding study):

not examined

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:

effects observed, non-treatment-related

Description (incidence and severity):

There were no effects on thyroid hormone values in the F1 males and females at any dosage level on PND 13. Differences from the control group were considered to be the result of normal biological variation and were not considered to be of toxicological significance.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Three (3), 1(1), 4(2), 2(2), and 22(5) pups (litters) in the control, 35, 175, 350, and 700 mg/kg/day groups, respectively, were found dead. Aside from the presence or absence of milk in the stomach, internal findings were limited to a single pup (No. 3531-01) in the 350 mg/kg/day group that had a laceration on the liver (portion of the median lobe of the liver protruded outside of the abdominal cavity through a laceration in the ventral abdomen).

Histopathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No internal findings that could be attributed to parental test substance administration were noted at the necropsy of pups euthanized on PND 13. The only internal finding noted was dark red discoloration of the salivary mandibular glands for Pup No. 3536-02 in the 35 mg/kg/day group

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

The anogenital distances (absolute and relative to the cube root of pup body weight) in the 35, 175, 350, and 700 mg/kg/day groups were similar to the control group values. Differences from the control group were slight and not statistically significant.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

no effects observed

Description (incidence and severity):

There were no effects on thyroid hormone values in the F1 males and females at any dosage level on PND 13. Differences from the control group were considered to be the result of normal biological variation and were not considered to be of toxicological significance.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

maternal dose

Generation:

F1

Effect level:

> 350 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

mortality

body weight and weight gain

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

350 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects in the absence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

yes

Applicant's summary and conclusion

Conclusions:

Under the conditions of this screening study, a dosage level of 700 mg/kg/day (the highest dosage level evaluated) was considered to be the no-observed-adverse-effect level (NOAEL) for F0 systemic and reproductive toxicity of test material when administered orally by gavage to Crl:CD(SD) rats. The NOAEL for F1 neonatal toxicity was 350 mg/kg/day based lower postnatal survival and pup body weights and body weight gains in the 700 mg/kg/day group.

Executive summary:

GUIDELINE

The study protocol was designed in general accordance with the OECD Guideline for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test, 29 Jul 2016. The objectives of the study were to provide preliminary information on the potential adverse effects of the test substance on male and female reproduction within the scope of a screening study. This will encompass gonadal function, mating behavior, conception, parturition, and lactation of the parental generation and the development of offspring from conception through Day 13 of postnatal life, as well as to evaluate the recovery, persistence or progression of any effects following a minimum of a 14-day recovery period.

      

METHODS

The low- and mid‑dose groups (Groups 2–4) each consisted of 10 rats/sex and the high-dose group (Group 5) consisted of 15 rats/sex. Dosage levels were 35, 175, 350, and 700 mg/kg/day. A concurrent control group of 15 rats/sex received the vehicle on a comparable regimen. The dose volume was 5 mL/kg for all groups. Males and females were approximately 10 weeks of age at the beginning of test substance administration. Males received 14 daily doses prior to mating. Males were dosed throughout the mating period through 1 day prior to euthanasia for a total of 28 doses. Females received 14 daily doses prior to pairing and were dosed through Lactation Day 12 for a total of 48–58 doses; females with no evidence of mating or that failed to deliver were dosed through the day prior to euthanasia (Postcohabitation Day 25 or Postmating Day 25, respectively) for a total of 40–52 doses. The extra 5 males and 5 females in the control and high‑dose groups that were not used for mating were treated beginning on Study Day 0; following 28 doses for males and 48 doses for females, these animals were assigned to a 15-day nondosing recovery period.

All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. All F₀ females selected for pairing were allowed to deliver and rear their pups until Lactation Day 13. F₁ clinical observations, body weights, and sexes were recorded at appropriate intervals and anogenital distance was recorded on PND 1. To reduce variability among the litters, 8 pups/litter, 4 pups/sex when possible, were randomly selected on PND 4; blood samples for possible thyroid hormone analysis were collected from the culled pups (at least 2/litter). All F₁ male pups were evaluated for areolae/nipple anlagen on PND 13. Remaining F₁ pups were euthanized on PND 13; blood samples for thyroid hormone analysis were collected from 1 pup/sex/litter. Blood samples for thyroid hormone analysis were collected from F₀males at the primary and recovery necropsy and from F₀ females on Lactation Day 13; only male samples collected at the primary necropsy were analyzed. F₀males were euthanized following completion of the mating period or 15-day recovery period and F₀ females were euthanized on Lactation Day 13 for females that delivered, Postcohabitation or Postmating Day 25 for females that failed to deliver, or following the 15-day recovery period. Complete necropsies were conducted on all F₀ animals, and selected organs were weighed and examined microscopically.

RESULTS

There were no effects on survival. One male in the 350 mg/kg/day group was euthanized in extremis on Study Day 2 following clinical observations of rales and labored respiration on Study Days 1 and/or 2; similar moribundity was not observed at higher dosage levels. One female in the 35 mg/kg/day group was found dead on Study Day 4; a perforated esophagus was noted for this female at the necropsy, indicating intubation error as a cause of death. Nonadverse clinical observations of rales, salivation, and clear and/or red material around the mouth in the 175, 350, and 700 mg/kg/day group F₀males and/or females and red material around the nose in the 700 mg/kg/day group F₀males and females were noted throughout the dosing period. Due to their sporadic occurrence and/or because they were mostly limited to being observed at the time of dosing and/or approximately 1 hour following dose administration, these findings were not considered adverse. No other noteworthy clinical observations were noted at the daily examinations, detailed physical examinations, at the time of dosing, or approximately 1 hour after dose administration at any dosage level. 

Mean body weight gains in the 700 mg/kg/day group F₀ males were generally lower than the control group throughout the dosing period; however, in the absence of an effect on mean absolute body weights, these changes were not considered adverse. There were no effects on mean bodyweights and body weight gains in the 35, 175, and 350 mg/kg/day group F₀ males during the dosing period or in the 700 mg/kg/day group F₀ males during the recovery period. There were no effects on mean body weights and body weight gains for F₀ females at any dosage level throughout the study (including during gestation and lactation).

No effects on F₀food consumption were noted for males throughout the study or for females during the premating period or during gestation at any dosage level. Mean food consumption in the 700 mg/kg/day group F₀ females was lower than the control group throughout lactation; however, because there were no corresponding effects on mean body weights or body weight gains during lactation, the lower mean food consumption was not considered adverse. There were no effects on lactation food consumption at 35, 175, and 350 mg/kg/day.

No effects were noted for F₀ gestation length, estrous cycle length, reproductive performance (mating, fertility, and copulation/conception indices), and the process of parturition at any dosage level.

Thyroid hormone (T4) levels in the F₀ males were unaffected. There were no effects on organ weights or noteworthy macroscopic findings for F₀males and females at any dosage level at the primary and/or recovery necropsies. An increased incidence of renal tubular hyaline droplet accumulation was noted in the 175, 350, and 700 mg/kg/day group F₀males at the primary necropsy; a slightly higher severity grade was additionally noted in some of the 700 mg/kg/day group males. The incidence and severity grade of hyaline droplet accumulation in the 700 mg/kg/day and control group males were similar at the recovery necropsy, consistent with recovery. Hyaline droplet accumulation is common in young control group male rats; higher incidence and/or severity grade of the finding in the 175, 350 and 700 mg/kg/day group males was considered to be accentuation of spontaneous change and nonadverse. While the composition of hyaline droplets was not specifically identified in this study, the finding in male rats is most often associated with accumulation of alpha-2u globulin, a male rat-specific phenomenon not observed in female rats or other species including primates. There were no remarkable microscopic findings noted for F₀ females.

Postnatal survival in the 700 mg/kg/day group was lower than then control group during PND 0‑1 and 1–4 (pre-selection), and consequently from birth to PND 4 (69.3% versus 97.9% in the control group). In addition, although mean pups body weight gains in the 700 mg/kg/day group were similar to the control group during PND 1–4, lower mean pup birth weights (PND 1) and lower mean pup body weight gains during PND 4–13 resulted in mean male and female pup body weights that were 16.5% and 17.9% lower, respectively, than the control group on PND 13. There were no effects on F₁postnatal survival or pup body weights or body weight gains at 35, 175, and 350 mg/kg/day. There were no effects on mean numbers of F₁ pups born, live litter size, percentage of males at birth, and the general physical condition of the F₁pups at any dosage level. There were no effects noted on F₁ anogenital distance or areolae/nipple anlagen (males only) in the 35, 175, 350, and 700 mg/kg/day groups. No remarkable necropsy findings were noted for F₁ pups that were found dead or euthanized on PND 13. There were no remarkable changes in mean serum T4 levels in F₁ males or females on PND 13.

CONCLUSION

Under the conditions of this screening study, a dosage level of 700 mg/kg/day (the highest dosage level evaluated) was considered to be the no-observed-adverse-effect level (NOAEL) for F0 systemic and reproductive toxicity of test material when administered orally by gavage to Crl:CD(SD) rats. The NOAEL for F1 neonatal toxicity was 350 mg/kg/day based lower postnatal survival and pup body weights and body weight gains in the 700 mg/kg/day group.