Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented Guideline study with GLP compliance.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
Minimal deviations occurred that did not affect the quality or integrity of the study.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- Stability under test conditions: Stability has been established for the concentration range of 75 to 500 mg/mL for at least 15 days under room temperature storage.
- Storage condition of test material: The samples, including backup samples, were stored at room temperature and protected from light pending analyses or final disposition.
- Other: No adjustment was made for purity when preparing the test article formulations. Formulations of the test article were prepared weekly at nominal concentrations of 75, 250, and 500 mg/mL, and were stored at room temperature.

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Portage, Michigan, United States
- Age at study initiation: (P) 8 weeks
- Weight at study initiation: males: 271 - 303 g; females: 185-213 g
- Fasting period before study: no fasting
- Housing: suspended, stainless steel, wire-mesh type cages
- Use of restrainers for preventing ingestion (if dermal): no
- Diet (e.g. ad libitum): Meal Lab Diet (Certified Rodent Diet #5002, PMI Nutrition International, Inc.) was available ad libitum.
- Water (e.g. ad libitum): Tap water was available ad libitum via an automatic watering system.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 68 to 79°F
- Humidity (%): 30 to 70%
- Photoperiod (hrs dark / hrs light): Fluorescent lighting was provided for approximately 12 hours per day.

IN-LIFE DATES: From: 2012-03-10 To: 2012-04-10

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
The vehicle and test article were administered once per day to all groups via oral gavage.

VEHICLE
Documentation of the strength/purity, composition, stability, and other pertinent information for the lot of vehicle used on study was limited to the information listed on the label of this commercially available product.
Details on mating procedure:
- M/F ratio per cage: Animals were individually housed. During pairing, the animals were randomly cohabitated (one male and one female from the same group) in the cage of the male.
- Length of cohabitation: The maximum pairing period was 14 days.
- Proof of pregnancy: Positive evidence of copulation was established by daily inspection for a copulatory plug in the vagina and/or a vaginal lavage with the presence of sperm.
- At the end of the cohabitation any females with no confirmed evidence of mating were returned to individual cages until scheduled euthanasia.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individual cage
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
All analytical work was conducted by MPI Research, Inc., using an analytical method developed and validated under MPI Research, Inc., Study Number 1928-004.
Duration of treatment / exposure:
Dosing began 14 days prior to pairing and continued to euthanasia (43 days total) for the parental (P) males. Dosing of the females began 14 days prior to pairing, through the mating period, up to and including Lactation Day (LD) 3.
Frequency of treatment:
daily
Details on study schedule:
Not applicable.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
150 mg/kg bw
Basis:
actual ingested
gavage
Remarks:
Doses / Concentrations:
500 mg/kg bw
Basis:
actual ingested
gavage
Remarks:
Doses / Concentrations:
1000 mg/kg bw
Basis:
actual ingested
gavage
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
yes, historical
Details on study design:
- Dose selection rationale: The dose levels were selected by the Sponsor, or in consultation with the Sponsor, on the basis of available data from a 2-week dose range finding toxicity study in rats (MPI Research, Inc., Study Number 1928-008).

-Dose volume: 2 mL/kg/dose
Positive control:
Not applicable.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed for morbidity, mortality, injury, and the availability of food and water twice daily.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily during the study (1-2 hours postdose), each P animal was removed from the cage and given a detailed clinical examination. The observations included, but were not limited to, evaluation of the skin, fur, eyes, ears, nose, oral cavity, thorax, abdomen, external genitalia, limbs and feet, as well as evaluation of respiration.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded for all P0 animals weekly. Mated females were also weighed on GD 0, 7, 14, and 20, and LD 0 and 4.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
Food consumption for all P animals was recorded weekly prior to pairing for mating. During the first 14 days of the pairing period, food consumption was recorded for any animals. Beginning on Day 15 of the pairing period, any males that completed mating were placed back on measured food consumption weekly until euthanasia. Food consumption was recorded on the corresponding gestation and lactation body weight days for mated females.
OTHER:
Toward the end of the gestation period, P females were examined twice daily for signs of parturition. The mated females were allowed to give birth (F1). The duration of gestation was calculated, and any difficulties occurring at parturition were recorded.

Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: [yes]
- If yes, maximum of [all] pups/litter; excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
[number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities]

GROSS EXAMINATION OF DEAD PUPS:
[yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.]. Pups found dead at birth were given an external examination, and the lungs were removed and evaluated for liveborn or stillborn status (lungs floated or sunk in water, respectively). Any dead pups found during the lactation period were examined externally and discarded.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals [on day 44, the day of study termination]
- Maternal animals: All surviving animals [on lactation day 4]. Also, all P females with no confirmed mating date that appeared to be nonpregnant on the basis of body weight and shape were euthanized 25 days after the last scheduled pairing day and examined. Females that appeared to be pregnant on the basis of body weight and shape were euthanized as identified to prevent delivery in the cage and loss of the litter. Dams with total litter loss were euthanized and subjected to a necropsy.
In surviving animals, the uterus and ovaries were exposed using a mid-abdominal incision. Beginning at the distal end of the left uterine horn, the number of total implantation scars was recorded. The number of corpora lutea on each ovary was also recorded. Uteri from females that appeared nongravid were opened and placed in 10% ammonium sulfide solution for detection of implantation sites. No foci were seen, and the females were considered nonpregnant.

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]: yes

HISTOPATHOLOGY / ORGAN WEIGHTS: yes
Postmortem examinations (offspring):
SACRIFICE
- F1 animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: All pups still born, euthanized in extremis, found dead, and surviving to the scheduled necropsy on LD 4 were externally sexed and examined for external malformations and variations. Following completion of the external examination of all pups in the litter, each pup was euthanized via intraperitoneal injection of euthanasia solution, and the carcasses were discarded without further evaluation. Up to a total of ten pups from any dams with total litter loss were saved and preserved (half of the pups in 10% neutral buffered formalin and the other half snap frozen in liquid nitrogen) for possible future examination. An approximately equal number of pups from the control group were preserved under the same conditions for comparative purposes.

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]
Statistics:
The raw data were tabulated within each time interval, and the mean and standard deviation were calculated for each endpoint by sex and group. For each endpoint, treatment groups were compared to the control group by using Fisher´s Exact Test, Group Pair-wise Comparisons and Arcsin-Square-Root Transformation.

Group Pair-wise Comparisons (Levene’s/ANOVA-Dunnett’s/Welch’s)

If sample sizes for all groups were three or greater, Levene’s test was used to assess homogeneity of group variances for each specified endpoint and for all collection intervals. If Levene’s test was not significant (p>0.01), a pooled estimate of the variance (Mean Square Error or MSE) was computed from a one-way analysis of variance (ANOVA) and utilized by a Dunnett’s comparison of each treatment group with the control group. If Levene’s test was significant (p<0.01), comparisons with the control group were made using Welch’s t-test with a Bonferroni correction.
In the case that sample size was less than three for at least one treatment group, Levene’s method could not be implemented. Groups with sample sizes less than three were excluded from the analysis and control-treatment pair-wise comparisons that satisfied the sample size assumption (n>3) were conducted using Welch’s t-test with a Bonferroni correction.
If there were only two groups involved, the above methodology applied and the Dunnett’s test reduced to a Student’s t-test.
Results of all pair-wise comparisons are reported at the 0.05 and 0.01 significance levels. All endpoints were analyzed using two-tailed tests.
Reproductive indices:
P reproductive and fertility indices are reported. The reproductive and fertility indices evaluated for the treatment groups at 150, 500, and 1000 mg/kg/day did not result in any statistically significant changes. The male and female mating, fertility, and fecundity indices for the treatment groups ranged from 83.3 to 100% and did not show any dose responsive patterns to indicate a treatment-related change. In addition, there were no statistically significant changes in the copulatory interval measured between the treatment groups, which ranged from 2.5 to 2.9 days, as compared to the controls at 3.3 days.
Offspring viability indices:
Reduced viability indices (number of pups surviving to LD 4 divided by the number of live pups at delivery times 100) are reported. F1 pup survival was statistically decreased at 500 and 1000 mg/kg/day as evidenced by significantly reduced viability indices of 66.17 and 0.00%, respectively.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
postdose salivation; discoloration of the hair
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Females (1000 mg/kg bw): decreases in body weight and food consumption
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Females (1000 mg/kg bw): decreases in body weight and food consumption
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
organ weight changes present in the ovaries, liver, kidneys, adrenal glands, and thymus
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
adrenal gland, thymus, and stomach.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
ovaries, liver, kidneys, adrenal glands, stomach, and duodenum.
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)

Postdose salivation was one of the more commonly noted clinical finding in both male and female animals particularly at 500 and 1000 mg/kg/day during the dosing period. In the males this was seen in 83% of the animals at 500 and 1000 mg/kg/day (Days 1-44). During the premating period this finding was also noted in the females in a dose responsive manner and affected 75 and 50% of the animals at 500 and 1000 mg/kg/day, respectively. During gestation this affected 18, 70, and 91% of the females at 150, 500, and 1000 mg/kg/day, respectively. This finding was less pronounced during lactation and was noted in 30 and 25% of the females at 500 and 1000 mg/kg/day, respectively.
In the males, yellow discoloration of the hair (abdominal and scrotal regions) was also noted predominantly at 1000 mg/kg/day during the dosing period (Days 1-44) and affected up to one half of the animals.
During the premating period, females were noted with yellow discoloration of the hair (anogenital and ventral regions) at both 500 and 1000 mg/kg/day at incidences up to 50 and 58%, respectively. Findings of increased activity were noted in 33 and 100% of the females at 500 and 1000 mg/kg/day, and few/absent feces was seen in 50% of the females at 1000 mg/kg/day during the same period.
During gestation, similar clinical findings were noted that included yellow discoloration of the hair (anogenital and ventral regions), as well as brown and red discharge of the vulva predominatly at1000 mg/kg/day, but with a few occurrences at 500 mg/kg/day.
During lactation, the only remarkable clinical observation included yellow discoloration of the hair in 75% of the animals at1000 mg/kg/day.
Other clinical findings were noted, but they were spurious in nature or did not follow a dose responsive pattern to indicate a relationship to treatment.

No animals were found dead during the course of this study at 150, 500, or 500 mg/kg/day. Two females at 1000 mg/kg/day (animal numbers 188 and 193) were euthanized in extremis at the expected time of parturition on GD 21 and 22, respectively. On the day of moribund euthanasia, female number 188 had clinical findings that included distended abdomen, wet hair in the anogenital region, red material around the nose, skin pale, and cold to touch (entire body). The necropsy findings indicated bilateral adrenal enlargement and tan discoloration of multiple lobes. The microscopic evaluation indicated bilateral adrenal cortical hypertrophy and panlobular hepatocyte hypertrophy and centrilobular vacuolation, as well as some renal tubular vacuolation. Female number 193 had clinical findings that included decreased activity, difficult and shallow breathing, and dystocia. The necropsy findings indicated bilateral enlarged adrenals, tan discolored liver lobes, black foci in the glandular stomach, and small thymus. The microscopic evaluation revealed bilateral adrenal cortical hypertrophy, renal tubular vacuolation, panlobular hepatocyte hypertrophy with minimal necrosis and centrilobular vacuolation, duodenal necrosis, mild erosion of the glandular stomach and thymic lymphoid depletion. Adrenal cortical hypertrophy and generalized thymic lymphoid depletion suggest that these animals may have been under stress. Another female at 1000 mg/kg/day (animal number 190) was found to be pregnant with an incomplete delivery on GD 22. This animal did not have any significant clinical findings on the day of delivery. The macroscopic evaluation revealed a small thymus and the microscopic examination revealed some minimal focal liver necrosis and thymic lymphoid depletion. These findings were considered to be test article related.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)

There were no adverse effects on mean body weight for the males during the dosing period (Weeks 1-7). Statistically significant increases in mean body weight were noted at 500 mg/kg/day during Weeks 3 and 5. However, these changes were not considered to be toxicologically meaningful as the mean body weight at 1000 mg/kg/day was comparable to controls during this period.
Test article-related decreases in mean body weight were noted in females at 1000 mg/kg/day during the premating, gestation, and lactation periods that were statistically identified. During the premating period (Week 2) mean body weight at 1000 mg/kg/day was 7% lower than controls. During gestation mean body weight at 1000 mg/kg/day was 6 and 16% lower than controls on GD 14 and 20, respectively. During lactation, mean body weight at 1000 mg/kg/day was 13% lower than controls.
Body weight change was statistically decreased in males at 1000 mg/kg/day during the postmating period (Weeks 6-7); however, as there were no statistically significant changes in mean body weight or food consumption during this period, the decrease in body weight change is not considered to be toxicologically meaningful. In the females, statistically significant decreases in body weight change were noted at 1000 mg/kg/day during the premating period (Weeks 1-2) and gestation period (GD 7-14, 14-20 and 0-20). These changes were considered to be test article related, as they corresponded to statistically significant decreases in mean body weight and food consumption for the females at 1000 mg/kg/day during the same period. A mean body weight loss was noted at 500 mg/kg/day during lactation (-0.9 g vs. 7.9 g in the controls), which, although not statistically significant, did correspond to a statistically significant decrease in food consumption at this dose level during the same period and therefore may be toxicologically relevant.

No adverse effects on food consumption were noted for the males treated with the test item at 150, 500, or 1000 mg/kg/day during the dosing period (Weeks 1-7). Statistically significant increases in food consumption were observed during Weeks 2-3 and 5-6 at both 500 and 1000 mg/kg/day. As there were no statistically significant changes in mean body weight during these intervals and at these dose levels, these changes were not considered toxicologically meaningful.
For the females, there were statistically significant decreases at 1000 mg/kg/day during the premating and gestation periods (Week 1 and GD 14-20, respectively). In addition, mean food consumption was statistically decreased at 500 mg/kg/day during lactation. These changes were considered to be test article related.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)

The reproductive and fertility indices evaluated for the treatment groups at 150, 500, and 1000 mg/kg/day did not result in any statistically significant changes. The male and female mating, fertility, and fecundity indices for the treatment groups ranged from 83.3 to 100% and did not show any dose responsive patterns to indicate a treatment-related change. In addition, there were no statistically significant changes in the copulatory interval measured between the treatment groups, which ranged from 2.5 to 2.9 days, as compared to the controls at 3.3 days.

ORGAN WEIGHTS (PARENTAL ANIMALS)

Test article-related organ weight changes were present in the ovaries, liver, kidneys, adrenal glands, and thymus. The terminal body weights of females were lower than controls at 500 and 1000 mg/kg/day, but were not statistically significantly lower at 500 mg/kg/day. Statistically significant organ weight differences in the brain, kidneys, lung, and thyroid/parathyroid glands of females compared to controls were considered to be incidental to changes in body weight.
Mean ovary weights compared to body weight were statistically significantly greater than those of controls in females at 1000 mg/kg/day. While low body weights in these animals potentially affected ovary weights, increased ovary weights correlated microscopically with increased corpora lutea.
Mean absolute and relative liver weights (to body and brain weights) in males at 500 and 1000 mg/kg/day were greater than those of controls; however, at 500 mg/kg/day, mean absolute liver weights were not statistically significantly different from controls. In females at 1000 mg/kg/day, only mean liver weights compared to body weight were statistically significantly greater than controls. Increased liver weights correlated microscopically with centrilobular hepatocellular hypertrophy in males and females at 1000 mg/kg/day.
Mean absolute and relative kidney weights (to body and brain weights) in males at ≥150 mg/kg/day were dose-dependently greater than those of controls; however, at 150 mg/kg/day, only mean kidney weights relative to brain weight were statistically significantly different from controls. Increased kidney weights correlated microscopically with tubular degeneration/regeneration in males at ≥150 mg/kg/day.
Mean absolute and relative adrenal gland weights (to body and brain weights) in females at 500 and 1000 mg/kg/day were statistically significantly greater than those of controls. Increased adrenal gland weights correlated macroscopically with enlargement in females at 500 and 1000 mg/kg/day and correlated microscopically with diffuse cortical hypertrophy in females at 1000 mg/kg/day.
Mean absolute and relative thymus weights (to body and brain weights) in females at 500 and 1000 mg/kg/day were lower than those of controls; however, at 500 mg/kg/day, only mean thymus weights relative to brain weight were statistically significantly different from those of controls. Low thymus weights correlated microscopically with generalized lymphoid depletion in females at 500 and 1000 mg/kg/day.
All other statistically significant organ weight differences were considered to be incidental due to the lack of a dose response, the absence of correlative microscopic effects, and/or the lack of similar findings in both sexes.

MACROSCOPIC FINDINGS (PARENTAL ANIMALS)
Test article-related macroscopic findings were present in the adrenal gland, thymus, and stomach.
The adrenal glands were mildly enlarged in females at 1000 mg/kg/day which correlated microscopically with diffuse cortical hypertrophy. One female at 500 mg/kg/day had macroscopically mildly enlarged adrenal glands, and while there was no microscopic correlate for this finding, adrenal gland weights were increased compared to controls at both 500 and 1000 mg/kg/day. The thymus was mildly to moderately small in females at 1000 mg/kg/day, which correlated microscopically with generalized lymphoid depletion and correlated with low thymus weights. These findings are most likely indicative of stress in these animals, which appear to be test article related.
The glandular stomach had a mild black focus in one female at 1000 mg/kg/day (animal number 193) which was euthanized in extremis. The black focus correlated microscopically with a mild erosion/ulcer located near the junction of the pylorus and duodenum.
One male at 1000 mg/kg/day (animal number 142) had a 2 mm nodule located on the lip at the corner of the mouth which correlated microscopically with a benign squamous cell papilloma. The relationship of this finding to test article administration was unclear.
Mild tan discoloration was present in the liver of the two females at 1000 mg/kg/day that were euthanized in extremis. In animal number 188, tan discoloration correlated microscopically with moderate centrilobular vacuolation; however, in animal 193, there was no microscopic correlate. Tan discoloration due to centrilobular vacuolation was most likely a (peanut oil) vehicle-related finding.
All other macroscopic findings were either common background findings in rats or were considered incidental and unrelated to treatment due to the lack of a dose response, the absence of correlative microscopic effects, and/or the lack of similar findings in both sexes.

MICROSCOPIC FINDINGS (PARENTAL ANIMALS)
Test article-related microscopic findings were present in the ovaries, liver, kidneys, adrenal glands, stomach, and duodenum.
The ovaries had minimally to mildly increased corpora lutea in females at 1000 mg/kg/day. While corpora lutea in concurrent controls exhibited heterogeneity in size and stage of regression, corpora lutea in females at 1000 mg/kg/day were uniformly enlarged (increased in size) and there was an absence of regressing corpora lutea.
The liver had minimal centrilobular hepatocellular hypertrophy in males and females at 1000 mg/kg/day and minimal panlobular hepatocellular hypertrophy in the two females at 1000 mg/kg/day that were euthanized in extremis. One female at 1000 mg/kg/day (animal number 193) that was euthanized in extremis also had minimally increased mitotic figures within hepatocytes. Centrilobular vacuolation was present within a low number of females including one control female and was most likely a (peanut oil) vehicle-related finding.
The kidneys had dose-dependent, minimal to moderate tubular degeneration/regeneration in males at ≥150 mg/kg/day. Tubular degeneration/regeneration was most prominent within the outer medulla and inner cortex characterized by tubules lined by swollen, hypereosinophilic epithelial cells (degeneration) associated with tubules lined by increased numbers of small, basophilic epithelial cells (regeneration). With increased severity (mild to moderate), tubular degeneration/regeneration was also often associated with casts of cellular debris at the junction of proximal tubules and the loop of Henle. Tubular degeneration/regeneration was distinct from chronic progressive nephropathy in that chronic progressive nephropathy was associated with thickening of the tubular basement membrane and mixed interstitial infiltrates of mononuclear inflammatory cells. Chronic progressive nephropathy is a common background finding in the kidneys of rats at this age and was present in all groups including controls; however, in males, the incidence of chronic progressive nephropathy was increased at 500 and 1000 mg/kg/day which potentially indicated a test article-related exacerbation of this finding. The two females at 1000 mg/kg/day that were euthanized in extremis had minimal to mild tubular vacuolation in the kidneys. The relationship of this finding to test article administration was unclear.
The adrenal glands had minimal diffuse cortical hypertrophy in females at 1000 mg/kg/day characterized by thickening of the zona fasciculata. The thymus had minimal to severe generalized lymphoid depletion in females at 1000 mg/kg/day and minimal to mild generalized lymphoid depletion in females at 500 mg/kg/day. While minimal generalized lymphoid depletion was present in control females, the incidence and severity were increased in females at 500 and 1000 mg/kg/day. Diffuse cortical hypertrophy of the adrenal glands and associated generalized lymphoid depletion in the thymus are most likely indicative of stress in these animals.
The glandular stomach had a mild erosion/ulcer located near the junction of the pylorus and duodenum in one female at 1000 mg/kg/day (animal number 193) which was euthanized in extremis. The adjacent duodenum had mild diffuse necrosis characterized by necrosis of the mucosal epithelial cells lining the intestinal crypts and villi. It should be noted that the stomach and adjacent duodenum were only examined in this animal due to the presence of a gross lesion (black focus) in the glandular stomach. While gastric ulcers can occur sporadically as a background finding in rats, the concurrent necrosis of the duodenum suggests that these findings are most likely test article related.
All other microscopic observations were either common background findings in rats or were considered incidental and unrelated to treatment due to the lack of a dose response and/or the lack of similar findings in both sexes.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: mating, fertility, and fecundity indices were unaffected by treatment at dose levels up to 1000 mg/kg/day.
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: significant clinical findings, decreases in body weight and food consumption (primarily females), organ weight changes and microscopic findings at 500 and 1000 mg/kg/day

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
decreased activity; skin cold
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
survival decrease
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
body weight decrease
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)

F1 pup survival was statistically decreased at 500 and 1000 mg/kg/day as evidenced by significantly reduced viability indices (number of pups surviving to LD 4 divided by the number of live pups at delivery times 100) of 66.17 and 0.00%, respectively. In addition, at 1000 mg/kg/day there was a statistically significant decrease in the total number of pups born per litter on LD 0 (9.9 pups vs. 12. 9 pups in the controls) and a significant decrease in the number of liveborn pups per litter on LD 0 (6.6 vs. 12.9 in the controls). By LD 4, the number of liveborn pups per litter was statistically reduced at 500 mg/kg/day (9.0 pups vs. 12.8 pups in the controls). The decreases F1 survival appears to be associated with considerable maternal toxicity, particularly at 1000 mg/kg/day. During the last week of gestation (GD 14-20), the dams at 1000 mg/kg/day were noted with mean body weight that was over 50% lower than controls and food consumption that was almost 20% lower than controls. Most of the F1 offspring at 1000 mg/kg/day were either stillborn or died shortly after birth, with none of them surviving to LD 4. At 500 mg/kg/day the dams did not show any significant changes in body weight or food consumption during gestation (prior to delivery), but during the lactation period (LD 0 to 4) these dams did exhibit a significant decrease in mean food consumption, which was 27% lower than controls and a net body weight loss during this period. The decreased survival of the F1 offspring at 500 mg/kg/day, in association with the maternal toxicity observed, may have been the result of either one or a combination of the following scenarios: decreased maternal care, an inability to produce enough milk, or offspring lacking the ability or interest to properly nurse. These changes in F1 pup survival at 500 and 1000 mg/kg/day were considered to be test article related.

CLINICAL SIGNS (OFFSPRING)
During the lactation period there were some remarkable clinical findings in the pups from the treated females. At 500 mg/kg/day, 10% of the pups were noted with decreased activity. Another finding that included skin cold to touch (entire body) was noted in 11 and 79% of all surviving pups at 500 and 1000 mg/kg/day, respectively. These findings at 500 and 1000 mg/kg/day were considered to be test article related.

BODY WEIGHT (OFFSPRING)
On LD 0 the F1 mean body weight was statistically decreased in the female and both sexes combined at 500 and 1000 mg/kg/day. In addition, on LD 4 the F1 mean body weight was statistically reduced at 500 mg/kg/day. No pups survived to LD 4 at 1000 mg/kg/day. These body weight changes at 500 and 1000 mg/kg/day were considered to be test article related.

SEX RATIOS (OFFSPRING)
No statistically significant changes in the F1 pup sex ratio (percent males per litter) were noted at 150, 500, or 1000 mg/kg/day as compared to controls.

MACROSCOPIC FINDINGS (OFFSPRING)
The macroscopic findings for the stillborn pups and those that died were either within normal limits or too cannibalized for evaluation. At scheduled necropsy on LD 4 there were no pups available for evaluation at 1000 mg/kg/day and those at 0, 150, and 500 mg/kg/day did not show any significant treatment-related findings.

Effect levels (F1)

Dose descriptor:
NOEL
Generation:
F1
Effect level:
150 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: significant decreases in survival and mean body weight at 500 and 1000 mg/kg/day

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

P Parturition Data

Out of the 12 females per group that were mated to treated males, all the controls were found to be pregnant and delivered litters for evaluation. At 150 mg/kg/day, 11 females were found to be pregnant and 10 of them delivered litters. At 500 mg/kg/day, 10 females were found to be pregnant and all of them delivered litters. At 1000 mg/kg/day, 11 females were found to be pregnant and 9 delivered litters. Of the females delivering litters, there were 3, 2, and 7 females with at least one stillborn pup per litter at 150, 500 and 1000 mg/kg/day, respectively. The stillborn index (number of stillborn pups divided by the number of pups born times 100) was statistically increased at 1000 mg/kg/day (32.87% vs. 0.0% in the controls). Gestation length was slightly reduced at 500 mg/kg/day (21.2 days vs. 22.1 days in the controls). This was not considered to be toxicologically meaningful, as this value falls within the expected gestation period for this species and strain of rat and it was limited to the mid-dose group, without an apparent dose response pattern to indicate a relationship to treatment. There were no other statistically significant changes in the parturition data for the treated females as compared to the controls.

Table 1. Test Article-related Organ Weight Changes ‑ Terminal

Male and Female (Percent change relative to control)

Dose level: mg/kg/day

150

500

1000

Sex

M

F

M

F

M

F

Number Examined

12

12

12

12

12

10

 

 

 

 

 

 

Adrenal glands (g)

6.45

6.58

12.90

26.32b

12.90

38.16b

Adrenal glands/BWt%

3.03

3.50

9.09

33.46b

12.88

57.59b

Adrenal glands/BrWt ratio

9.57

5.70

13.53

26.42b

10.23

44.04b

 

 

 

 

 

 

 

Kidneys (g)

7.06

0.14

18.04b

2.83

12.97b

7.96

Kidneys/BWt%

4.42

2.27

14.92b

8.99b

13.88b

3.80

Kidneys/BrWt ratio

11.41a

0.95

19.39b

2.39

11.27a

5.16

 

 

 

 

 

 

 

Liver (g)

5.09

3.75

10.93

4.36

18.32b

1.05

Liver/BWt%

2.38

5.48

8.55a

1.59

19.87b

14.32b

Liver/BrWt ratio

9.21

4.34

12.24a

4.58

16.32b

4.13

 

 

 

 

 

 

 

Ovaries (g)

NA

3.48

NA

0.87

NA

0.87

Ovaries/BWt%

NA

5.15

NA

5.15

NA

13.40a

Ovaries/BrWt ratio

NA

3.76

NA

1.20

NA

3.93

 

 

 

 

 

 

 

Thymus (g)

4.07

7.34

5.02

23.08

1.91

54.20b

Thymus/BWt%

1.25

4.55

2.51

19.01

0.68

49.48b

Thymus/BrWt ratio

8.26

5.68

6.10

23.90a

3.69

53.36b

 

 

aSignificantly different from control; (p<0.05)

bSignificantly different from control; (p<0.01)

BWt ‑ Body Weight

BrWt ‑ Brain Weight

NA - Not applicable

M - Male

F ‑ Female

Applicant's summary and conclusion

Conclusions:
Clinical observations and findings were noted predominantly at dose levels of 500 and 1000 mg/kg/day. Test article-related macroscopic findings, microscopic findings and organ weight changes were present at 1000 mg/kg/day and partly at 500 mg/kg/day. Therefore, the No-Observed-Adverse-Effect-Level (NOAEL) for general toxicity in parental male and female animals was considered to be 150 mg/kg/day while a No Observed-Effect-Level (NOEL) of 150 mg/kg bw was determined for the F1 offspring for developmental toxicity.
Executive summary:

This study was conducted to generate limited information concerning the effects of the test item (reaction product of amines, C12-14,-tert-alkyl, alcohol, C14-18, C18 unsat, and phosphorus pentoxide) on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus, and parturition. According to OECD Guideline 421, three treatment groups of twelve CD [Crl:CD(SD)] rats/sex/group were administered the test article at dose levels of 150, 500, or 1000 mg/kg/day. One additional group of twelve animals/sex served as the control and received the vehicle, peanut oil (arachis oil NF). The vehicle or test article was administered to all groups daily via oral gavage at a dose volume of 2 mL/kg/dose. Dosing began 14 days prior to pairing and continued to euthanasia (43 days total) for the parental (P) males. 250 observations of the P animals included clinical signs, body weights and body weight change, and food consumption during the premating/mating, gestation, and lactation periods, and parturition and litter data. Observations of the offspring (F1) included survival at birth and during lactation, individual clinical signs, pup body weights and sex, and gross abnormalities. At study termination, necropsy examinations were performed on all surviving P animals, and organs and tissues were collected, weighed, and examined for select groups. Dams losing their entire litter were subjected to a necropsy, and up to ten pups from the litters were saved in fixative for possible future examination. On LD 4, surviving F1 pups were examined externally, euthanized, and discarded.  

Once daily administration of "reaction product of amines, C12-14,-tert-alkyl, alcohol, C14-18, C18 unsat, and phosphorus pentoxide" by oral gavage to male rats for 7 weeks and female rats for two weeks prior to mating, through gestation and lactation at dose levels of 150, 500, and mg/kg/day produced clinical observations consisting of postdose salivation and yellow discolored hair in both male and female animals predominantly at 500 and 1000 mg/kg/day. Additional clinical findings were noted in the females predominantly at 500 and 1000 mg/kg/day that included increased activity, few/absent feces, and vulvar discharge. Two females at 1000 mg/kg/day had to be euthanized moribund close to the time of parturition. Test article-related changes in body weight and food consumption were limited to females at 500 and 1000 mg/kg/day. The F1 pup survival index and mean body weight were adversely affected by treatment with the test item at 500 and 1000 mg/kg/day.

Test article-related macroscopic findings were present in the adrenal gland, thymus, and stomach of females at 1000 mg/kg/day. Test article-related organ weight changes were present in the ovaries, liver, kidneys, adrenal glands, and thymus at 500 and 1000 mg/kg/day. Test article-related microscopic findings were present in the ovaries, liver, kidneys, adrenal glands, stomach, and duodenum. Most findings were predominantly observed at 1000 mg/kg/day, but were also noted to some extent in animals at 500 mg/kg/day.

Based on the results obtained from this study, the No-Observed-Adverse-Effect-Level (NOAEL) for general toxicity in parental male and female animals was considered to be 150 mg/kg/day, which was generally based on, but not limited to, significant clinical findings, decreases in body weight and food consumption (primarily females), as well are organ weight changes and microscopic findings at 500 and 1000 mg/kg/day. A No-Observed-Effect-Level (NOEL) for F1offspring for developmental toxicity was considered to be 150 mg/kg/day, based on the significant decreases in survival and mean body weight at 500 and 1000 mg/kg/day. However, mating, fertility, and fecundity indices were unaffected by treatment with the test item at dose levels up to 1000 mg/kg/day, the highest dose level tested.