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

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

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 (OECD 421).

Link to relevant study records

Reference

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

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

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.

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.

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

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.

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.

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: 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

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

Key result

Critical effects observed:

no

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.

Behaviour (functional findings):

not examined

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.

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

Key result

Critical effects observed:

no

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

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Additional information

The key study 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.

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.

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 groupF0 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 F0 males 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 consequentlyfrom 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.

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.

Effects on developmental toxicity

Description of key information

Based on the absence of maternal effects at any dosage level, 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 maternal toxicity when test material was administered via oral gavage to time-mated Crl:CD(SD) rats. Based on lower mean fetal weights noted at 700 mg/kg/day, a dosage level of 350 mg/kg/day was considered to be the NOAEL for embryo/fetal development (OECD 414).

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 August 2018 to 11 October 2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

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

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)

Details on test animals or test system and environmental conditions:

TEST SYSTEM
- Receipt: On 24 Aug 2018, time-mated female Crl:CD(SD) rats were received from Charles River Laboratories Inc., Raleigh, NC on Gestation Day 1, 2, 3, or 4. The animals were 10–12 weeks old at receipt and weighed between 200 and 248 g on Gestation Day 0.
- Justification for test system and number of animals: The Crl:CD(SD) rat is recognized as appropriate for developmental toxicity studies. Charles River Ashland has historical data on the background incidence of fetal malformations and developmental variations in the Crl:CD(SD) rat. This animal model has been proven to be susceptible to the effects of developmental toxicants. The number of animals selected for this study was based on United States EPA Health Effects Test Guidelines OPPTS 870.3700, Prenatal Developmental Toxicity Study, Aug 1998 and OECD Guidelines for the Testing of Chemicals, Guideline 414, Prenatal Developmental Toxicity Study, Jan 2001, which recommend evaluation of approximately 20 females with implantation sites at necropsy. Given the possibility of nongravid animals, unexpected deaths, or test substance-related moribundity and/or mortality, this was an appropriate number to obtain a sample size of 20 females/group at termination.
- Animal identification: Upon receipt, each animal was identified using a subcutaneously implanted electronic identification chip (BMDS system).
- Quarantine: After receipt at the Testing Facility, the Crl:CD(SD) rats were acclimated prior to the initiation of dosing.
- Selection, assignment, replacement and disposition of animals: Animals were assigned to groups by a stratified randomization scheme designed to achieve similar group mean body weights. Animals at extremes of body weight range were not assigned to groups. Before the initiation of dosing, any assigned animals considered unsuitable for use in the study were replaced by alternate animals obtained from the same shipment and maintained under the same environmental conditions. After initiation of dosing, study animals were replaced with alternate animals in the event of accidental injury, non-test substance-related health issues, or similar circumstances. The disposition of all animals was documented in the Study Records.

HUSBANDRY
- Housing: Animals were individually housed in solid-bottom cages containing appropriate bedding equipped with an automatic watering valve. Each cage was clearly labeled with a color-coded cage card indicating study, group, animal number, dosage level, and sex. Cages were arranged on the racks in group order. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals. The animal facilities at Charles River Ashland are accredited by AAALAC International.
- Environmental conditions: Target temperatures of 68 °F to 78 °F (20 °C to 26 °C) with a relative target humidity of 30 % to 70 % were maintained. A 12-hour light/12-hour dark cycle was maintained. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.
- Food: PMI Nutrition International, LLC Certified Rodent LabDiet 5002 was provided ad libitum throughout the study. The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis are provided by the supplier and are on file at the Testing Facility. It is considered that there are no known contaminants in the feed that would interfere with the objectives of the study.
- Water: Municipal tap water after treatment by reverse osmosis was freely available to each animal via an automatic watering system. Periodic analysis of the water is performed, and results of these analyses are on file at the Testing Facility. It is considered that there are no known contaminants in the water that could interfere with the outcome of the study.
- Animal enrichment: Enrichment devices were provided to all animals as appropriate throughout the study for environmental enrichment or to aid in maintaining the animals’ oral or gastrointestinal health.
- Veterinary care: Veterinary care was available throughout the course of the study, and animals were examined by the veterinary staff as warranted by clinical signs or other changes. All veterinary examinations and recommended therapeutic treatments, if any, were documented in the Study Records and reviewed by the Study Director.

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

PEG400

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

DOSE FORMULATION AND ANALYSIS
- Preparation of vehicle: The vehicle, PEG400, was dispensed weekly for administration to Group 1 control animals and adequate amounts were divided into daily aliquots, which were stored at room temperature (18 °C to 24 °C), protected from light, until use. The vehicle was stirred continuously during dosing. Details of the dispensing of the vehicle were retained in the Study Records.
- Preparation of test substance: Test substance dosing formulations were prepared at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared approximately weekly and an adequate amount of each formulation was dispensed into daily aliquots, which were stored at room temperature (18 °C to 24 °C), protected from light, until use. The dosing formulations were stirred continuously during dosing. Details of the preparation and dispensing of the test substance were retained in the Study Records.
- Sample collection and analysis: Dose formulation samples were collected for analysis on 24 August 2018 (concentration: all groups; homogeneity: groups 2 and 4) and 06 September 2018 (concentration: all groups; homogeneity: not applicable). Samples to be analyzed were transferred to the Charles River Ashland Analytical Chemistry
Department.
- Analytical method: Analyses were performed by high liquid chromatography using charged aerosol detection using a validated analytical procedure.
- Concentration analysis: Duplicate sets of samples (1.0 mL) for each sampling time point were transferred to the analytical laboratory; the remaining samples were retained at the Testing Facility as backup samples. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15 % of theoretical concentration. After acceptance of the analytical results, backup samples were discarded.
- Homogeneity analysis: Duplicate sets of samples (1.0 mL) for each sampling time point were transferred to the analytical laboratory; the remaining samples were retained at the Testing Facility as backup samples. Homogeneity results were considered acceptable if the relative standard deviation of the mean value at each sampling location was ≤ 10% and if mean sample concentration results were within or equal to ± 15 % of theoretical concentration. After acceptance of the analytical results, backup samples were discarded.
- Stability analysis: Test substance formulations have previously shown the test substance to be stable at concentrations ranging from 1 to 200 mg/mL for up to 10 days at room temperature (18 °C to 24 °C). Therefore, stability of test substance formulations was not assessed on the current study.

Details on mating procedure:

- Time-mated female Crl:CD(SD) rats were received from Charles River Laboratories Inc., Raleigh, NC on Gestation Day 1, 2, 3, or 4.

Duration of treatment / exposure:

Gestation Days 6 to 20

Frequency of treatment:

Once daily

Duration of test:

15 days

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

25 females

Control animals:

yes, concurrent vehicle

Details on study design:

EXPERIMENTAL DESIGN
- Experimental design is summarised in Table 3 (below).
- Female No. 1660 in the 175 mg/kg/day group was removed from study on Gestation Day 7 due to body weight loss, reduced food consumption, and adverse clinical observations (dermal atonia) beginning prior to the initiation of test substance administration; this female was replaced with Female No. 1734.
- Administration of test materials: The test substance and vehicle were administered as a single daily oral gavage dose during Gestation Days 6 through 20. All animals were dosed at approximately the same time each day (see Appendix 1 – Study Protocol and Deviations, attached).
- Justification of route and dose levels: The route of administration 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. Dosage levels were selected based on the results of a previous range-finding prenatal toxicity study. In the previous study, dosage levels of 35, 175, 350, and 700 mg/kg/day were administered by oral gavage from Gestation Days 6–20 in time-mated rats. Mean body weights and food consumption were generally unaffected by test substance administration. No remarkable findings were noted at the gross examinations. Mean gravid uterine weights were slightly lower at 700 mg/kg/day, and correlating lower mean fetal weights were also noted. However, the changes were slight (< 7%), and no external malformations or variations were noted at any dosage level. Based on these results, dosage levels of 175, 350, and 700 mg/kg/day were selected for the current study.

Maternal examinations:

IN-LIFE PROCEDURES, OBSERVATIONS AND MEASUREMENTS
- Viability: Throughout the study, animals were observed for general health/mortality and moribundity twice daily, once in the morning and once in the afternoon (see Appendix 1 – Study Protocol and Deviations, attached). Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.
- Observations: The animals were removed from the cage, and a detailed clinical observation was performed once daily, beginning on the day of receipt and lasting through euthanasia. During the dosing period, these observations were performed prior to dosing. On dosing days, clinical observations were also recorded 1-hour postdose.
- Body weights: Animals were weighed individually on Gestation Days 0 (by supplier) and 5–21 (daily). Gravid uterine weight was collected and net body weight (the Gestation Day 21 body weight exclusive of the weight of the uterus and contents) and net body weight change (the Gestation Day 0–21 body weight change exclusive of the weight of the uterus and contents) were calculated and presented for each gravid female at the scheduled laparohysterectomy.
- Food consumption: Food consumption was quantitatively measured on Gestation Days 5–21 (daily).
- Terminal procedures: Terminal procedures are summarized in Table 4 (below).
- Unscheduled deaths: No animals died during the course of the study.
- Scheduled euthanasia: Animals surviving until scheduled euthanasia were weighed and euthanized by carbon dioxide inhalation (including any animals that delivered).
- Necropsy: Animals were subjected to a complete necropsy examination, which included evaluation of the thoracic, abdominal, and pelvic cavities with their associated organs and tissues.
- Tissue collection and preservation: Gross lesions were collected and preserved in 10% neutral buffered formalin for possible future histopathologic examination. Representative sections of corresponding organs from a sufficient number of controls were retained for comparison, if possible.

Ovaries and uterine content:

- Ovarian and uterine examination: The uterus was weighed, and the ovaries and uterus were examined for number and distribution of corpora lutea, implantation sites, live and dead fetuses, and early and late resorptions. The placentae were also examined.

Fetal examinations:

- Fetal examinations: Fetal examinations were conducted without knowledge of treatment group. External, internal, and skeletal fetal findings were recorded as developmental variations or malformations. Representative photographs of all malformations, as appropriate, were included in the Study Records. Corresponding low magnification photographs, depicting both the malformed fetus and a comparison control fetus, or normal littermate, were also included in the Study Records as needed and as appropriate for comparison, when possible.
- External: Each viable fetus was examined in detail, sexed, weighed, tagged, and euthanized by a subcutaneous injection of sodium pentobarbital in the scapular region. The crown-rump length of late resorptions (advanced degree of autolysis) was measured, the degree of autolysis recorded, a gross external examination performed (if possible), and the tissue was discarded.
- Visceral (internal): The sex of all fetuses was confirmed by internal examination. Approximately one-half of the fetuses in each litter were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected using a technique described by Stuckhardt and Poppe. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development. The heads from these fetuses were removed and placed in Harrison’s fixative for subsequent processing and soft-tissue examination using the Wilson sectioning technique (see Appendix 1 – Study Protocol and Deviations, attached). Following examination, the carcasses and cephalic slices were discarded (see Appendix 1 – Study Protocol and Deviations, attached).
- Skeletal: The remaining fetuses (approximately one-half from each litter, excluding any carcasses without heads) were eviscerated and fixed in 100% ethyl alcohol. Following fixation in alcohol, foetuses were stained with Alizarin Red S7 and Alcian Blue.8 The skeletal examination was made following this procedure.

Statistics:

See below

Indices:

- Intrauterine data were summarized using 2 methods of calculation as indicated below:
(1) Group mean litter basis: Postimplantation Loss/Litter = (Number dead foetuses, resorptions (Early/Late)/Group) / (Number gravid females/Group)
(2) Proportional litter loss basis: Summation per group (%) = (Sum of postimplantation loss/Litter) / (Number of litters/Group) where postimplantation loss/Litter (%) = [(Number dead foetuses, resorptions (Early/Late)/Litter) / (Number implantation sites/Litter) * 100]
- The fetal developmental findings were summarized by:
(1) Presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the Group.
(2) Considering the litter as the basic unit for comparison and calculating the number of affected fetuses in a litter on a proportional basis as Summation per Group (%) = (Sum of viable fetuses affected/Litter (%)) / (Number of Litters/Group) where viable fetuses affected /Litter (%) = [(Number of viable fetuses affected/Litter) / (Number of viable foetuses/Litter) * 100]

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

- No test substance-related clinical observations were noted at the daily examinations or approximately 1-hour following dose administration at any dosage level.
- Findings noted in the test substance-treated groups, including rales, red and/or clear material, and hair loss on various body surfaces, occurred infrequently, at similar frequencies in the control group and/or in a manner that was not dose-related.

Mortality:

no mortality observed

Description (incidence):

- All females in the control, 175, 350, and 700 mg/kg/day groups survived to the scheduled necropsy on Gestation Day 21, including Female No. 1655 in the control group that delivered on the day of scheduled necropsy (Gestation Day 21).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

- Mean maternal body weights, body weight gains, net body weights, net body weight gains, and gravid uterine weights in the 175, 350, and 700 mg/kg/day groups were unaffected by test substance administration.
- Differences from the control group were slight and not statistically significant.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

- Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 175, 350, and 700 mg/kg/day groups was unaffected by test substance administration. Differences from the control group were slight and not statistically significant, with the following exceptions.
- Statistically significantly higher mean food consumption was noted in the 175 mg/kg/day group on Gestation Day 18–19 and in the 350 mg/kg/day group on Gestation Day 8–9 (g/animal/day only), 18–19, and 6–21 (g/animal/day only); because these changes were transient, did not occur in a dose related manner, and/or were not of sufficient magnitude to affect mean body weights at these dosage levels, these findings were not considered to be test substance-related.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- At the scheduled necropsy on Gestation Day 21, no test substance-related internal findings were observed at dosage levels of 175, 350, or 700 mg/kg/day.
- Macroscopic findings observed in the test substance-treated groups occurred infrequently and/or in a manner that was not dose-related.
- All females were determined to be gravid.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

not examined

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

- Intrauterine survival was unaffected by test substance administration at dosage levels of 175, 350, and 700 mg/kg/day. Parameters evaluated included mean litter proportions of postimplantation loss, mean number and percentage of viable fetuses, and fetal sex ratios.
- Differences from the control group were slight and not statistically significant. Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups.

Total litter losses by resorption:

effects observed, non-treatment-related

Description (incidence and severity):

- Intrauterine survival was unaffected by test substance administration at dosage levels of 175, 350, and 700 mg/kg/day. Parameters evaluated included mean litter proportions of postimplantation loss, mean number and percentage of viable fetuses, and fetal sex ratios.
- Differences from the control group were slight and not statistically significant. Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups.

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

- Intrauterine survival was unaffected by test substance administration at dosage levels of 175, 350, and 700 mg/kg/day. Parameters evaluated included mean litter proportions of postimplantation loss, mean number and percentage of viable fetuses, and fetal sex ratios.
- Differences from the control group were slight and not statistically significant. Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups.

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

- Intrauterine survival was unaffected by test substance administration at dosage levels of 175, 350, and 700 mg/kg/day. Parameters evaluated included mean litter proportions of postimplantation loss, mean number and percentage of viable fetuses, and fetal sex ratios.
- Differences from the control group were slight and not statistically significant. Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups.

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

not examined

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Remarks on result:

other: no effects at the highest dose level

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Mean male, female, and combined fetal weights (5.7 g, 5.5 g, and 5.6 g, respectively) were statistically significantly lower (8.1%, 5.2%, and 6.7%, respectively) in the 700 mg/kg/day group compared to the concurrent control group; in addition, the values were below the minimum mean values in the Charles River Ashland historical control data range (version 2018.01; 6.036 g, 5.762 g, and 5.908 g, respectively), and therefore were considered test substance-related and adverse. Mean fetal body weights in the 175 and 350 mg/kg/day groups were similar to the control group.

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

- Intrauterine survival was unaffected by test substance administration at dosage levels of 175, 350, and 700 mg/kg/day. Parameters evaluated included mean litter proportions of postimplantation loss, mean number and percentage of viable fetuses, and fetal sex ratios.
- Differences from the control group were slight and not statistically significant. Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups.

Changes in sex ratio:

effects observed, non-treatment-related

Description (incidence and severity):

- Intrauterine survival was unaffected by test substance administration at dosage levels of 175, 350, and 700 mg/kg/day. Parameters evaluated included mean litter proportions of postimplantation loss, mean number and percentage of viable fetuses, and fetal sex ratios.
- Differences from the control group were slight and not statistically significant. Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

- The numbers of fetuses (litters) available for morphological evaluation were 309(25), 307(25), 306(25), and 307(25) in the control, 175, 350, and 700 mg/kg/day groups, respectively.
- Malformations were observed in 0(0), 2(2), 3(3), and 1(1) fetuses (litters) in these same respective dose groups and were considered spontaneous in origin.

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

- No test substance-related external malformations were noted at any dosage level. Fetus No. 1713-10 in the 350 mg/kg/day group was noted with microstomia and mandibular micrognathia. Skeletally, mandibular micrognathia consisted of mandibles that were small and fused along mandibular symphysis; both lower incisor sockets absent; tympanic rings that were fused medially. Fetus No.1701-13 in the 175 mg/kg/day group was noted with vertebral agenesis (with anury); skeletally, this finding consisted of absent vertebrae posterior to sacral vertebra No. 2, fused arches, and an absent centrum. Because the aforementioned malformations were noted in single fetuses, did not occur in a dose-related manner, and the mean litter proportions were not statistically significantly different from the control group, they were not considered test substance-related.
- No test substance-related external developmental variations were noted. Fetus Nos. 1654-05 and 1654-06 in the 700 mg/kg/day group were noted with twinning; because this finding was a single occurrence and the mean litter proportion was not statistically significantly different from the control group, it was not considered test substance-related.

Skeletal malformations:

no effects observed

Description (incidence and severity):

- No test substance-related skeletal malformations were noted for fetuses at any dosage level. Fetus No. 1750-07 in the 350 mg/kg/day group was noted with vertebral agenesis (sacral and lumbar vertebrae and lumbar centra absent, sacral and lumbar arches unossified, and small bone precursors). The aforementioned finding was not considered test substance-related because it was noted in a single fetus and was not observed in a dose-related manner.
- No test substance-related skeletal developmental variations were noted. Findings observed in the test substance-treated groups were noted infrequently, similarly in the control group, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the Charles River Ashland historical control data.

Visceral malformations:

no effects observed

Description (incidence and severity):

- No test substance-related visceral malformations were noted in fetuses at any dosage level. Visceral malformations were noted in 1 fetus each in the 175, 350, and 700 mg/kg/day groups. In the 700 mg/kg/day group, Fetus No. 1654-05 was noted with hydrocephaly (increased cavitation of both lateral and third ventricles). Situs inversus (trachea, esophagus, heart, great and major vessels, lungs, liver, stomach, pancreas, spleen, kidneys, adrenals and/or intestines were laterally transposed) was noted in Fetus Nos. 1681-10 and 1692-08 in the 175 and 350 mg/kg/day groups, respectively; Fetus No. 1692-08 was also noted with lobular agenesis of the lungs (1 lobe present, bilateral). Because the aforementioned findings were noted in single fetuses, not observed in a dose-related manner, and/or the mean litter proportions were not significantly different from the control group, they were not considered test substance-related.
- No test substance-related visceral developmental variations were noted. Findings observed in the test substance-treated groups were noted infrequently, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the Charles River Ashland historical control data.
- Renal papillae not fully developed (Woo and Hoar Grade 1) was noted for 3, 1, 4 and 2 fetuses in the control, 175, 350, and 700 mg/kg/day groups, respectively. Fetus No. 1721-02 in the 700 mg/kg/day group had a yellow area on the liver. These findings were not classified as either a malformation or developmental variation, were not included on the summary tables, and were not considered to be test substance-related because they occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.

Other effects:

not examined

Details on embryotoxic / teratogenic effects:

SUMMARY OF EXTERNAL, VISCERAL AND SKELETAL EXAMINATIONS
- The numbers of fetuses (litters) available for morphological evaluation were 309(25), 307(25), 306(25), and 307(25) in the control, 175, 350, and 700 mg/kg/day groups, respectively.
- Malformations were observed in 0(0), 2(2), 3(3), and 1(1) fetuses (litters) in the same respective groups, and were considered to be spontaneous in origin. When the total malformations and developmental variations were evaluated on a proportional basis, no statistically significant differences from the control group were noted. Fetal malformations and developmental variations, when observed in the test substance-treated groups, occurred infrequently or at a frequency similar to that in the control group, did not occur in a dose-related manner, and/or were within the Charles River Ashland historical control data ranges. Based on these data, no fetal malformations or developmental variations were attributed to the test substance.

Key result

Dose descriptor:

NOAEL

Remarks:

maternal dose

Effect level:

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

Based on:

test mat.

Remarks:

maternal dose

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to maternal toxicity:

developmental effects in the absence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

yes

ANALYSES OF DOSING FORMULATIONS

- The analyzed dosing formulations contained 85.4% to 107% of the test substance which was within the protocol-specified range of target concentrations for suspensions (85 % to 115 %) and were homogeneous. The test substance was not detected in the analyzed vehicle formulation that was administered to the control group (Group 1).

- Results of the analyses of dosing formulations are summarized in Tables 6 and 7 (attached).

Conclusions:

Based on the absence of maternal effects at any dosage level, 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 maternal toxicity when test material was administered via oral gavage to time-mated Crl:CD(SD) rats. Based on lower mean fetal weights noted at 700 mg/kg/day, a dosage level of 350 mg/kg/day was considered to be the NOAEL for embryo/fetal development.

Executive summary:

GUIDELINE

The design of the study was based on United States EPA Guideline OPPTS 870.3700 and OECD Test Guideline 414. The objectives of the study were to determine the potential of the test substance to induce developmental toxicity after maternal exposure from implantation to 1 day prior to expected parturition, to characterize maternal toxicity at the exposure levels tested, and to determine a no-observed-adverse-effect level (NOAEL) for maternal and developmental toxicity.

 

METHODS

Female Crl:CD(SD) rats (25 per group) were dosed via oral gavage at 0, 175, 350 or 700 mg/kg bw/day once daily during Gestation Days 6 to 20. The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, gravid uterine weights, food consumption, gross necropsy findings, intrauterine growth and survival, and fetal morphology.

 

All females in the control, 175, 350, and 700 mg/kg/day groups survived to the scheduled necropsy on Gestation Day 21, including 1 female in the control group that delivered on the day of scheduled necropsy (Gestation Day 21). No test substance-related clinical observations were noted at the daily examinations or approximately 1 hour following dose administration at any dosage level.

 

Mean maternal body weights, body weight gains, net body weights, net body weight gains, gravid uterine weights, and food consumption in the 175, 350, and 700 mg/kg/day groups were unaffected by test substance administration.

 

RESULTS

There were no test substance-related macroscopic findings noted at any dosage level at the scheduled necropsy on Gestation Day 21. Mean male, female, and combined fetal weights in the 700 mg/kg/day group were lower (8.1%, 5.2%, and 6.7%, respectively) compared to the control group. Intrauterine growth at 175 and 350 mg/kg/day and survival at all dosage levels were unaffected by test substance administration. There were no test substance-related fetal malformations or developmental variations noted at any dosage level.

 

CONCLUSION

Based on the absence of maternal effects at any dosage level, 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 maternal toxicity when test material was administered via oral gavage to time-mated Crl:CD(SD) rats. Based on lower mean fetal weights noted at 700 mg/kg/day, a dosage level of 350 mg/kg/day was considered to be the NOAEL for embryo/fetal development.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

350 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

The design of the key study was based on United States EPA Guideline OPPTS 870.3700 and OECD Test Guideline 414. The objectives of the study were to determine the potential of the test substance to induce developmental toxicity after maternal exposure from implantation to 1 day prior to expected parturition, to characterize maternal toxicity at the exposure levels tested, and to determine a no-observed-adverse-effect level (NOAEL) for maternal and developmental toxicity.

Female Crl:CD(SD) rats (25 per group) were dosed via oral gavage at 0, 175, 350 or 700 mg/kg bw/day once daily during Gestation Days 6 to 20. The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, gravid uterine weights, food consumption, gross necropsy findings, intrauterine growth and survival, and fetal morphology.

 

All females in the control, 175, 350, and 700 mg/kg/day groups survived to the scheduled necropsy on Gestation Day 21, including 1 female in the control group that delivered on the day of scheduled necropsy (Gestation Day 21). No test substance-related clinical observations were noted at the daily examinations or approximately 1 hour following dose administration at any dosage level.

Mean maternal body weights, body weight gains, net body weights, net body weight gains, gravid uterine weights, and food consumption in the 175, 350, and 700 mg/kg/day groups were unaffected by test substance administration. There were no test substance-related macroscopic findings noted at any dosage level at the scheduled necropsy on Gestation Day 21.

Mean male, female, and combined fetal weights in the 700 mg/kg/day group were lower (8.1%, 5.2%, and 6.7%, respectively) compared to the control group. Intrauterine growth at 175 and 350 mg/kg/day and survival at all dosage levels were unaffected by test substance administration. There were no test substance-related fetal malformations or developmental variations noted at any dosage level.

Based on the absence of maternal effects at any dosage level, 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 maternal toxicity when test material was administered via oral gavage to time-mated Crl:CD(SD) rats. Based on lower mean fetal weights noted at 700 mg/kg/day, a dosage level of 350 mg/kg/day was considered to be the NOAEL for embryo/fetal development.

Justification for classification or non-classification

No toxicity was reported with respect to adult animals in repeated dose toxicity studies (OECD 407, OECD 421 and OECD 414) and malformations observed in foetuses (litters) were considered to be spontaneous in origin (OECD 414).

Review of the study pre-natal study suggests that the doses in the third trimester may have been in excess. Future studies with this material should be designed with the appropriate protocol adjustments to prevent this. Lower fetal weights were reported at the highest maternal dose in a prenatal development toxicity study (OECD 414) and a NOAEL of 350 mg/kg bw/day was declared for embryo/fetal development. In the absence of data to the contrary it was therefore considered to take a conservative approach to classify the material based on the effect. The substance was classified as H361d: Repr. 2 under the terms of Regulation (EC) No 1272/2008 and subsequent amendments.