Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-11-08 to 2013-01-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: CD® [Crl:CD®(SD)]
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, North Carolina
- Age at study initiation: (P) x 8 wks
- Weight at study initiation: (P) Males: 285 to 316 g; Females: 191 to 222 g
- Fasting period before study: no
- Housing: individually in suspended, stainless steel, wire-mesh type cages except during pairing, near parturition, and during lactation in an environmentally controlled room. During pairing, the animals were randomly cohabitated (one male and one female from the same group) in the cage of the male. On approximately GD 20, P females were individually housed in plastic solid bottom cages containing wood chip bedding. Females were housed in these solid bottom cages with pups during the 4-day lactation period.

- Diet (e.g. ad libitum): Meal Lab Diet (Certified Rodent Diet #5002, PMI Nutrition International, Inc.), ad libitum.
- Water (e.g. ad libitum): Tap water (ad libitum)
- Acclimation period: 8 days. On Days -3 and -2, all animals were administered a sham dose of tap water in the same manner and at the same volume intended for use during the study period.

ENVIRONMENTAL CONDITIONS
- Temperature: 68 to 79 °F
- Humidity (%): 30 to 70
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
peanut oil
Remarks:
arachis oil NF
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test article was used as received from the Sponsor. No adjustment was made for purity when preparing the test article formulations. Formulations of the test article were prepared as needed at nominal concentrations of 50, 75, 150, and 250 mg/mL, and were stored at room temperature.

VEHICLE
Peanut oil (arachis oil NF)
- Justification for use and choice of vehicle (if other than water): test article is well soluble in peanut oil
- Concentration in vehicle: 50, 75, 150, and 250 mg/mL
- Amount of vehicle (if gavage): 1 mL/kg/dose for the 50 mg/kg/day group, or 2 mL/kg/dose for all other groups.
- Lot/batch no. (if required): 2BH0270
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 14 days.
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy.
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility: no.
The maximum pairing period was 14 days, at the end of which any females with no confirmed evidence of mating were returned to individual cages until scheduled euthanasia. 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 as described in Section Postmortem Examination (Parental Animals).
- After successful mating each pregnant female was caged (how): in an individual cage.
- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dosing formulations prepared for the study were evaluated for homogeneity and concentration by GC (on week 1, 5 and 8). Homogeneity analyses have been performed on sampled dose level of 50 and 500 mg/kg bw. Concentration analyses have been performed on formulations of all dose levels.
Stability has been established under Test facility for the concentration range of 20 to 300 mg/mL for at least 5 days under room temperature storage. A total of 102 samples were analyzed for the test material in dosing formulations using a validated method. The analytical data demonstrated acceptable performance of the method for all reported results.
Duration of treatment / exposure:
Dosing began 14 days prior to pairing. Dosing of the males continued through the mating and postmating period to euthanasia (43 days total), while dosing of the females continued through LD 3.
Frequency of treatment:
once daily
Details on study schedule:
None.
Remarks:
Doses / Concentrations:
0, 50, 150, 300 and 500 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were selected by the Sponsor on the basis of available data from previous studies (OECD 407; 28-day oral gavage toxicity study in the Sprague-Dawley Crl:CD BR rat) with the compound. In this study, dose levels of 15, 150, 300, and 500 mg/kg/day were previously tested at a dose volume of 2 mL/kg in arachis BP oil. Treatment-related changes were observed at 500 mg/kg/day (clinical observations, body weight, food consumption, and organ weight changes) and at 150 mg/kg/day (only organ weight changes), and no such effects were demonstrated in animals treated with 15 mg/kg/day. A maximum dose level of 500 mg/kg/day was selected as the high dose to show some morbidity and for comparison to the previous study, and a low dose level of 50 mg/kg/day was selected as the potential no effect level. The 150 and 300 mg/kg/day dose levels were selected as intermediate doses to provide additional information between the low and high-dose groups.

- Rationale for animal assignment (if not random): randomized.
Positive control:
None.
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: All animals were given a detailed clinical examination and body weights were recorded on Days -8 and -1 (the results are not reported but maintained in the study file). Daily during the study (1-2 hours postdose), each P animal was removed from the cage and given a detailed clinical examination. On occasion, clinical observations were recorded at unscheduled intervals. 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 P animals weekly. Mated females were also weighed on GD 0, 7, 14, and 20, and LD 0 and 4. Body weight change was calculated weekly for the males, and for the females over the following intervals: weekly (premating),
GD 0-7, 7-14, 14-20, and 0-20, and LD 0-4.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): no
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 not recorded for any animals. Food consumption was recorded on the corresponding gestation and lactation body weight days for mated females.
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OTHER:
Dams were housed with their litters for four days after birth (LD 0 to 4). The dams and litters were observed daily for survival and behavioral alterations in nesting and nursing, and the presence of dead pups was recorded.
Oestrous cyclicity (parental animals):
Not examined
Sperm parameters (parental animals):
Not examined
Litter observations:
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. The day on which all pups were delivered was designated as LD 0.

PARAMETERS EXAMINED
The litters were examined as soon as possible after delivery.
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.
- Dams were housed with their litters for four days after birth (LD 0 to 4). The dams and litters were observed daily for survival and behavioral alterations in nesting and nursing, and the presence of dead pups was recorded. 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 sank in water, respectively). Any dead pups found during the lactation period were examined externally and discarded.
- Pups were individually weighed and examined externally on LD 0 and 4.

GROSS EXAMINATION OF DEAD PUPS: yes, for external abnormalities;
All pups still born, found dead, and surviving to the scheduled necropsy were externally sexed and examined for external malformations and variations. Following completion of the external examination of all pups in the litter, the carcasses were discarded without further evaluation.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: At the termination of the study (Day 44) all P males were observed externally, euthanized, and subjected to a necropsy.
- Maternal animals: On LD 4, all surviving P females were euthanized and subjected to a necropsy, and the number of uterine implantation scars and corpora lutea were recorded.


GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, cranial and abdominal viscera.

The animals were examined carefully for external abnormalities including palpable masses. The skin was reflected from a ventral midline incision and any subcutaneous masses were identified and correlated with antemortem findings. The abdominal, thoracic, and cranial cavities were examined for abnormalities. The organs were removed, examined, and, where required, placed in fixative. All designated tissues were fixed in neutral buffered
formalin, except for the testes, which were fixed using a modified Davidson’s fixative. The epididymis, ovary, oviducts, prostate, seminal vesicles, testis, uterus with cervix, vagina, and gross lesions were collected from all animals.

HISTOPATHOLOGY / ORGAN WEIGHTS
Body weights and protocol-designated organ weights were recorded for all parental animals at the scheduled necropsy and appropriate organ weight ratios were calculated (relative to body weights). Paired organs were weighed together.

Microscopic examination of fixed hematoxylin and eosin-stained paraffin sections was performed on protocol-designated sections of tissues. The slides were examined by a board-certified veterinary pathologist. A four-step grading system was utilized to define gradable lesions for comparison between dose groups.
Postmortem examinations (offspring):
SACRIFICE
On LD 4, all surviving F1 pups were euthanized via intraperitoneal injection of euthanasia solution. All pups still born, found dead, and surviving to the scheduled necropsy were externally sexed and examined for external malformations and variations. Following completion of the external examination of all pups in the litter, the carcasses were discarded without further evaluation.

GROSS NECROPSY
Gross necropsy consisted of external examinations

HISTOPATHOLOGY / ORGAN WEIGTHS
Not examined
Statistics:
Please refer to "Any other information on materials and methods incl. tables".
Reproductive indices:
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.
Mating, fertility and fecundity indices were calculated:

Females Delivering Litters (%) = (No. females delivering litters/No. females pregnant) x 100;

Females with All Stillborn (%) = (No. litters with all stillborn pups / No. females delivering litters) x 100

Females with Stillborn Pups (%) = (No. litters with at least 1 stillborn pup / No. females delivering litters) x 100

Fertility Index - Males = (No. males impregnating a female / No. males paired) x 100

Fertility Index - Females = (No. females pregnant/No. females paired) x 100

Offspring viability indices:
Live Birth Index = (No. live pups at birth/ No. of pups born) x 100;
Stillborn Index = No. stillborn pups/ No. pups born;
Viability Index – Day 4 = No. pups surviving 4 days (preculling)
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
post-dose salivation
Body weight and weight changes:
no effects observed
Description (incidence and severity):
no toxicologically meaningful changes
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
no toxicologically meaningful changes
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
minimal to moderate tubular degeneration/regeneration
Other effects:
not examined
Description (incidence and severity):
Test substance intake: not applicable. Test substance was administered via gavage.
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)

The most remarkable clinical finding included post-dose salivation that was seen in both male and female animals treated with the test article, which is most likely due to the taste of the test article in the vehicle at the concentrations affected. In males, this was noted in 42, 92, and 83 % of the animals at 150, 300, and 500 mg/kg/day, respectively. In females this was noted in 25 and 67 % of the animals at 300 and 500 mg/kg/day, respectively, during the premating period. During gestation, 36 % and 44 % of the females were affected with this finding at 300 and 500 mg/kg/day, respectively. However, during lactation this finding was no longer seen in the treated females at any dose level tested. This finding was considered to be test article related. Some other clinical findings were noted, but no clear dose responsive pattern was identified to indicate a relationship to treatment.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)

No test article-related changes in mean body weight were noted in male or female animals exposed to the test article at the dose levels tested (50, 150, 300, and 500 mg/kg/day) during the study period. Statistically significant decreases in mean body weight gain were limited to animals at 500 mg/kg/day. This was noted in males during Weeks 6-7 and in females during GD 0-7. Based on the lack of statistically significant changes in mean body weight or food consumption during these periods for each gender, these decreases in body weight gain were not considered toxicologically meaningful.

Mean food consumption was unaffected by treatment with the test material at the dose levels tested. A statistically significant decrease in mean food consumption was noted in males at 150 mg/kg/day during Weeks 5-6. However, as there were no statistically significant changes at the higher dose levels, this finding was considered to be spurious in nature and not toxicologically meaningful.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)

Male and female mating, fertility and fecundity indices were unaffected in the treated animals at 50, 150, 300, and 500 mg/kg/day. No statistically significant changes in females with confirmed mating day or copulatory interval were identified in the treated animals as compared to controls.

ORGAN WEIGHTS (PARENTAL ANIMALS)

There were no apparent test article-related organ weight changes in males or females. Any organ weight differences were considered to be incidental and unrelated to treatment due to a low magnitude, the lack of a dose response, and/or the absence of correlative microscopic effects.

GROSS PATHOLOGY (PARENTAL ANIMALS)

500 mg/kg/day, which had mild tan discoloration in four males at 500 mg/kg/day, and of these, one male (animal number 250) also had moderate enlargement of the kidneys. These findings correlated microscopically with tubular degeneration/regeneration. 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.

HISTOPATHOLOGY (PARENTAL ANIMALS)

Test article-related microscopic findings were present in the kidneys of males at 500 mg/kg/day, which had minimal to moderate tubular degeneration/regeneration. 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. These findings are consistent with a well characterized condition described as α2μ-globulin nephropathy, which is limited to the male rat and not relevant for human risk assessment. Tubular degeneration/regeneration was distinct from chronic progressive nephropathy, which is a common background finding in the kidneys of rats at this age, and was present in one female at 500 mg/kg/day (animal number 313). The kidneys of this female also had mild bilateral hydronephrosis and minimal unilateral lymphocytic infiltration which are common background findings in rats and were considered incidental and unrelated to treatment. It should be noted that the kidneys were only examined microscopically in animals which had gross lesions. Because the kidneys were not collected at necropsy in other animals in this study (per the protocol), the kidneys were not examined in all animals. All other microscopic observations were either common background findings in rats or were considered incidental and unrelated to treatment.
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no significant changes in fertility, mating, and fecundity indices.
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: microscopic changes noted in the males at 500 mg/kg/day (α2μ-globulin nephropathy) was limited to the male rat and is not relevant for human risk assessment.
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
decreased at parturition (LD 0) at 150, 300, and 500 mg/kg/day and on LD4 at 300 mg/kg/day (females only), and at 500 mg/kg/day (males, females, and both genders combined).
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Of the females found to be copulation positive, all of them delivered litters and none of them had total litter loss. There were no statistically significant changes in the delivery data evaluated for the treated animals as compared to controls. This included gestation length, number of pups born per litter on LD 0, corpora lutea, liveborn pups per litter on LD 0, stillborn pups per litter on LD 0, gestation index, stillborn index, total implantations/scars per litter, and number of live pups per litter on LD 4.

VIABILITY (OFFSPRING)

F1 pup survival was unaffected by treatment with the test article. This was supported by the lack of statistically significant changes in the pup survival index on LD 4 in the treated groups as compared to controls.

CLINICAL SIGNS (OFFSPRING)

F1 pup clinical observations did not show any test article-rated changes in the treated litters as compared to controls. A few different clinical findings were noted, but they were few in number and did not follow a dose responsive pattern to indicate a relationship to treatment.

BODY WEIGHT (OFFSPRING)

Mean F1 pup body weights (males, females, and both combined) were statistically decreased at parturition (LD 0) at 150, 300, and 500 mg/kg/day. On LD 4, mean body weights continued to be reduced at 300 mg/kg/day (females only), and at 500 mg/kg/day (males, females, and both genders combined). At 50 mg/kg/day, pup weights on LD 0 and 4 were not statistically different from controls.
Although mean pup weights were lower than controls at 150, 300, and 500 mg/kg/day, they did not follow a clear dose response pattern indicative of a relationship to treatment. On LD 0, gender combined pup weights at 150, 300, and 500 mg/kg/day were 12.4, 9.7, and 11.2 % lower than controls, respectively, and on LD 4 the mean body weight was the same at these dose levels and was 10.1 % lower than controls. All pups at these dose levels showed good growth from birth to LD 4, based on body weight gain.
In comparing the mean pup body weights (gender combined) at 150, 300, and 500 mg/kg/day to the historical control range (6.45-7.06 g on LD 0 and 9.95-11.62 g on LD 4), it was found that the mean pup weights at these dose levels were lower and outside the historical control range. On LD 0 mean pup weights were 5.93, 6.11, and 6.01 g, respectively and on LD 4 mean pup weights were 9.58, 9.58, and 9.58 g, respectively. It is important to note that the live pup litter size at 150, 300, and 500 mg/kg/day was considerably higher (14.7. 14.4, and 12.9 live pups per litter, respectively) and outside the historical control range of 10.2-12.5 live pups per litter. This pattern of lower pup weight with increased litter size in rats has previously been described in the literature and this would support the premise that the decreased pup weight may be related to the increased litter size and therefore not toxicologically meaningful. Furthermore, the lack of a dose response effect on pup weight at these dose levels also confirms that this is not treatment related.

SEXUAL MATURATION (OFFSPRING)

F1 pup sex ratios (percent males per litter) did not show any statistically significant changes in the treated litters as compared to controls.

GROSS PATHOLOGY (OFFSPRING)

There were no apparent test article-related changes in the F1 pup macroscopic observations for animals that were stillborn, died on study, or survived to scheduled necropsy.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: based on a lack of toxicologically meaningful changes in the treated pups at the dose levels tested.
Reproductive effects observed:
not specified
Conclusions:
Oral administration of test article to male and female rats at 50, 150, 300, and 500 mg/kg/day during the study period produced test article-related findings that included postdose salivation at 300 and 500 mg/kg/day (males and females), and microscopic changes in the parental males at 500 mg/kg/day (renal tubular degeneration/regeneration).
Based on the results obtained from this oral reproductive/developmental toxicity screening study in rats, a No-Observed-Adverse-Effect-Level (NOAEL) for developmental toxicity was considered to be 500 mg/kg/day, based on a lack of toxicologically meaningful changes in the treated pups at the dose levels tested. As there were no significant changes in fertility, mating, and fecundity indices, a NOAEL for reproductive performance was considered to be 500 mg/kg/day. With the exception of the microscopic changes noted in the males at 500 mg/kg/day (α2μ-globulin nephropathy, which was limited to the male rat and not relevant for human risk assessment), a NOAEL for general toxicity was considered to be 500 mg/kg/day.
Executive summary:

The objective of this study was to generate limited information concerning the effects of the test article on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus, and parturition. This study met or exceeded Guideline 421, Reproduction/Developmental Toxicity Screening Test, the OECD Guideline for Testing of Chemicals, adopted July 1995. Four treatment groups of twelve CD® [Crl:CD®(SD)] rats/sex/group were administered the test article at dose levels of 50, 150, 300, or 500 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 1 or 2 mL/kg/dose. 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.

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. On LD 4, surviving F1 pups were examined externally, euthanized, and discarded.

The formulation analysis verified a lack of test article in the control samples. The results from the analytical evaluation from the Week 1 test article preparations showed lower than expected values at all concentrations, particularly at the lower dose levels, which triggered an investigation and resulted in a change in the mixing procedures. The two higher concentrations (150 and 250 mg/mL) were acceptable for Weeks 2 and forward. By Week 4, all the values were within the acceptance criteria, except for the 50 mg/mL concentration that was just barely outside. Homogeneity evaluation and concentration verification were repeated on Week 5 and all values passed the acceptance criteria established. Therefore, animals at the two higher dose levels received the targeted concentrations of the test article starting on Week 2 for the remainder of the intervals evaluated. The animals receiving the two lower concentrations (50 and 150 mg/mL) had slightly lower exposures during the first three weeks, but by Week 4 the concentrations for all treatment groups approached the nominal concentrations. By Week 5, all treated animals received the targeted concentrations of the test article.

Oral administration of the test article to male and female rats at 50, 150, 300, and 500 mg/kg/day during the study period produced test article-related findings that included postdose salivation at 300 and 500 mg/kg/day (males and females) and microscopic changes in the parental males at 500 mg/kg/day (renal tubular degeneration/regeneration). All paternal animals survived to scheduled necropsy without significant test article-related effects on body weight, food consumption, or remaining uterine or reproductive parameters evaluated, including fertility, mating and fecundity indices. F1 survival was unaffected by treatment.

Based on the results obtained from this oral reproductive/developmental toxicity screening study in rats, a No-Observed-Adverse-Effect-Level (NOAEL) for developmental toxicity was considered to be 500 mg/kg/day, based on a lack of toxicologically meaningful changes in the treated pups at the dose levels tested. As there were no significant changes in fertility, mating, and fecundity indices, a NOAEL for reproductive performance was considered to be 500 mg/kg/day. With the exception of the microscopic changes noted in the males at 500 mg/kg/day (α2μ-globulin nephropathy, which is limited to the male rat and not relevant for human risk assessment), a NOAEL for general toxicity was considered to be 500 mg/kg/day.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The key study is GLP compliant and is of high quality (Klimisch score = 1).
Additional information

A Reproduction/Developmental Toxicity Screening Test (OECD 421) was conducted with the target substance(Reaction products of diphosphorus pentaoxide and alcohol C7-9-iso, C8 rich, salted with 2-ethylhexylamine), using male and female rats. Reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus, and parturition were assessed (Thorsrud, 2013, Report No. 1928-010). Four treatment groups of twelve CD® [Crl: CD®(SD) ] rats/sex/group were administered the test article at dose levels of 50, 150, 300 or 500 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 1 or 2 mL/kg/dose. 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.

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. On LD 4, surviving F1 pups were examined externally, euthanized, and discarded.

Oral administration of the test article to male and female rats at 50, 150, 300, and 500 mg/kg/day during the study period produced test article-related findings that included post dose salivation at 300 and 500 mg/kg/day (males and females) and microscopic changes in the parental males at 500 mg/kg/day (renal tubular degeneration/regeneration). All paternal animals survived to scheduled necropsy without significant test article-related effects on body weight, food consumption, or remaining uterine or reproductive parameters evaluated, including fertility, mating and fecundity indices. F1 survival was unaffected by treatment.

Based on the results obtained from this oral reproductive/developmental toxicity screening study in rats, a No-Observed-Adverse-Effect-Level (NOAEL) for developmental toxicity was considered to be 500 mg/kg/day, based on a lack of toxicologically meaningful changes in the treated pups at the dose levels tested. As there were no significant changes in fertility, mating, and fecundity indices, a NOAEL for reproductive performance was considered to be 500 mg/kg/day. With the exception of the microscopic changes noted in the males at 500 mg/kg/day (α2μ-globulin nephropathy, which is limited to the male rat and not relevant for human risk assessment), a NOAEL for general toxicity was considered to be 500 mg/kg/day.


Short description of key information:
There were no significant changes in fertility, mating and fecundity indices. Therefore, NOAEL for reproductive performance was considered to be 500 mg/kg/day, the highest dose level tested.

Justification for selection of Effect on fertility via oral route:
Only one study available (conducted with the target substance).

Effects on developmental toxicity

Description of key information
There were no toxicologically meaningful changes in the treated pups at the dose levels tested. Therefore, NOAEL for developmental toxicity was
considered to be 500 mg/kg/day, the highest dose level tested.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-11-08 to 2013-01-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD Guideline 421, Reproduction/Developmental Toxicity Screening Test
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: CD® [Crl:CD®(SD)]
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, North Carolina
- Age at study initiation: (P) x 8 wks
- Weight at study initiation: (P) Males: 285 to 316 g; Females: 191 to 222 g
- Fasting period before study: no
- Housing: individually in suspended, stainless steel, wire-mesh type cages except during pairing, near parturition, and during lactation in an environmentally controlled room. During pairing, the animals were randomly cohabitated (one male and one female from the same group) in the cage of the male. On approximately GD 20, P females were individually housed in plastic solid bottom cages containing wood chip bedding. Females were housed in these solid bottom cages with pups during the 4-day lactation period.

- Diet (e.g. ad libitum): Meal Lab Diet (Certified Rodent Diet #5002, PMI Nutrition International, Inc.), ad libitum.
- Water (e.g. ad libitum): Tap water (ad libitum)
- Acclimation period: 8 days. On Days -3 and -2, all animals were administered a sham dose of tap water in the same manner and at the same volume intended for use during the study period.

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 68 to 79
- Humidity (%): 30 to 70
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
peanut oil
Remarks:
FN
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test article was used as received from the Sponsor. No adjustment was made for purity when preparing the test article formulations. Formulations of the test article were prepared as needed at nominal concentrations of 50, 75, 150, and 250 mg/mL, and were stored at room temperature.

VEHICLE
Peanut oil (arachis oil NF)
- Justification for use and choice of vehicle (if other than water): test article is well soluble in peanut oil
- Concentration in vehicle: 50, 75, 150, and 250 mg/mL
- Amount of vehicle (if gavage): 1 mL/kg/dose for the 50 mg/kg/day group, or 2 mL/kg/dose for all other groups.
- Lot/batch no. (if required): 2BH0270
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dosing formulations prepared for the study were evaluated for homogeneity and concentration by GC (on week 1, 5 and 8). Homogeneity analyses have been performed on sampled dose level of 50 and 500 mg/kg bw. Concentration analyses have been performed on formulations of all dose levels.
Stability has been established under Test facility for the concentration range of 20 to 300 mg/mL for at least 5 days under room temperature storage. A total of 102 samples were analyzed for the test material in dosing formulations using a validated method. The analytical data demonstrated acceptable performance of the method for all reported results.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 14 days.
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy.
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility: no.
The maximum pairing period was 14 days, at the end of which any females with no confirmed evidence of mating were returned to individual cages until scheduled euthanasia. 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 as described in Section Postmortem Examination (Parental Animals).
- After successful mating each pregnant female was caged (how): in an individual cage.
- No other deviations from standard protocol.
Duration of treatment / exposure:
Dosing began 14 days prior to pairing. Dosing of the males continued through the mating and postmating period to euthanasia (43 days total), while dosing of the females continued through LD 3.
Frequency of treatment:
once daily.
Remarks:
Doses / Concentrations:
0, 50, 150, 300 and 500 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were selected by the Sponsor on the basis of available data from previous studies (OECD 407 28-day oral gavage toxicity study in the Sprague-Dawley Crl:CD BR rat) with the compound. In this study, dose levels of 15, 150, 300, and 500 mg/kg/day were previously tested at a dose volume of 2 mL/kg in arachis BP oil. Treatment-related changes were observed at 500 mg/kg/day (clinical observations, body weight, food consumption, and organ weight changes) and at 150 mg/kg/day (only organ weight changes), and no such effects were demonstrated in animals treated with 15 mg/kg/day. A maximum dose level of 500 mg/kg/day was selected as the high dose to show some morbidity and for comparison to the previous study, and a low dose level of 50 mg/kg/day was selected as the potential no effect level. The 150 and 300 mg/kg/day dose levels were selected as intermediate doses to provide additional information between the low and high-dose groups.

- Rationale for animal assignment (if not random): randomized.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed for morbidity, mortality, injury, and the availability of food and water twice daily.
Dams were housed with their litters for four days after birth (LD 0 to 4). The dams and litters were observed daily for survival and behavioral alterations in nesting and nursing, and the presence of dead pups was recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were given a detailed clinical examination and body weights were recorded on Days -8 and -1 (the results are not reported but maintained in the study file). Daily during the study (1-2 hours postdose), each P animal was removed from the cage and given a detailed clinical examination. On occasion, clinical observations were recorded at unscheduled intervals. 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 P animals weekly. Mated females were also weighed on GD 0, 7, 14, and 20, and LD 0 and 4. Body weight change was calculated weekly for the males, and for the females over the following intervals: weekly (premating),
GD 0-7, 7-14, 14-20, and 0-20, and LD 0-4.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): no
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 not recorded for any animals. Food consumption was recorded on the corresponding gestation and lactation body weight days for mated females.
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EVALUATION:
SACRIFICE
- Male animals: At the termination of the study (Day 44) all P males were observed externally, euthanized, and subjected to a necropsy.
- Maternal animals: On LD 4, all surviving P females were euthanized and subjected to a necropsy, and the number of uterine implantation scars and corpora lutea were recorded.


GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, cranial and abdominal viscera.

The animals were examined carefully for external abnormalities including palpable masses. The skin was reflected from a ventral midline incision and any subcutaneous masses were identified and correlated with antemortem findings. The abdominal, thoracic, and cranial cavities were examined for abnormalities. The organs were removed, examined, and, where required, placed in fixative. All designated tissues were fixed in neutral buffered
formalin, except for the testes, which were fixed using a modified Davidson’s fixative. The epididymis, ovary, oviducts, prostate, seminal vesicles, testis, uterus with cervix, vagina, and gross lesions were collected from all animals.

HISTOPATHOLOGY / ORGAN WEIGHTS
Body weights and protocol-designated organ weights were recorded for all parental animals at the scheduled necropsy and appropriate organ weight ratios were calculated (relative to body weights). Paired organs were weighed together.

Microscopic examination of fixed hematoxylin and eosin-stained paraffin sections was performed on protocol-designated sections of tissues. The slides were examined by a board-certified veterinary pathologist. A four-step grading system was utilized to define gradable lesions for comparison between dose groups.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
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.

Examinations included:
- Gravid uterus weight: No (this is OECD 421 study)
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No data
- Number of late resorptions: No data
Fetal examinations:
- External examinations: Yes: [all per litter ]
The litters were examined as soon as possible after delivery.
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.
- Dams were housed with their litters for four days after birth (LD 0 to 4). The dams and litters were observed daily for survival and behavioral alterations in nesting and nursing, and the presence of dead pups was recorded. 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 sank in water, respectively). Any dead pups found during the lactation period were examined externally and discarded.
- Pups were individually weighed and examined externally on LD 0 and 4.
- All pups still born, found dead, and surviving to the scheduled necropsy were externally sexed and examined for external malformations and variations. Following completion of the external examination of all pups in the litter, the carcasses were discarded without further evaluation.
Statistics:
Please refer to "Any other information on materials and methods incl. tables".
Indices:
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.
Mating, fertility and fecundity indices were calculated:

Females Delivering Litters (%) = (No. females delivering litters/No. females pregnant) x 100;

Females with All Stillborn (%) = (No. litters with all stillborn pups / No. females delivering litters) x 100

Females with Stillborn Pups (%) = (No. litters with at least 1 stillborn pup / No. females delivering litters) x 100

Fertility Index - Males = (No. males impregnating a female / No. males paired) x 100

Fertility Index - Females = (No. females pregnant/No. females paired) x 100.

Offspring viability indices:
Live Birth Index = (No. live pups at birth/ No. of pups born) x 100;
Stillborn Index = No. stillborn pups/ No. pups born;
Viability Index – Day 4 = No. pups surviving 4 days (preculling)


Historical control data:
The historical control data on Fertility and Early Embryonic Development to Implantation Data of Sprague Dawley Rat are included in the study report.
Details on maternal toxic effects:
Maternal toxic effects:no effects. Remark: post-dose salivation; male specific findings in the kidneys

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)

The most remarkable clinical finding included post-dose salivation that was seen in both male and female animals treated with the test article, which is most likely due to the taste of the test article in the vehicle at the concentrations affected. In males, this was noted in 42, 92, and 83% of the animals at 150, 300, and 500 mg/kg/day, respectively. In females this was noted in 25 and 67% of the animals at 300 and 500 mg/kg/day, respectively, during the premating period. During gestation, 36% and 44% of the females were affected with this finding at 300 and 500 mg/kg/day, respectively. However, during lactation this finding was no longer seen in the treated females at any dose level tested. This finding was considered to be test article related. Some other clinical findings were noted, but no clear dose responsive pattern was identified to indicate a relationship to treatment.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)

No test article-related changes in mean body weight were noted in male or female animals exposed to the test article at the dose levels tested (50, 150, 300, and 500 mg/kg/day) during the study period. Statistically significant decreases in mean body weight gain were limited to animals at 500 mg/kg/day. This was noted in males during Weeks 6-7 and in females during GD 0-7. Based on the lack of statistically significant changes in mean body weight or food consumption during these periods for each gender, these decreases in body weight gain were not considered toxicologically meaningful.

Mean food consumption was unaffected by treatment with the test article at the dose levels tested. A statistically significant decrease in mean food consumption was noted in males at 150 mg/kg/day during Weeks 5-6. However, as there were no statistically significant changes at the higher dose levels, this finding was considered to be spurious in nature and not toxicologically meaningful.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)

Male and female mating, fertility and fecundity indices were unaffected in the treated animals at 50, 150, 300, and 500 mg/kg/day. No statistically significant changes in females with confirmed mating day or copulatory interval were identified in the treated animals as compared to controls.

ORGAN WEIGHTS (PARENTAL ANIMALS)

There were no apparent test article-related organ weight changes in males or females. Any organ weight differences were considered to be incidental and unrelated to treatment due to a low magnitude, the lack of a dose response, and/or the absence of correlative microscopic effects.

GROSS PATHOLOGY (PARENTAL ANIMALS)

500 mg/kg/day, which had mild tan discoloration in four males at 500 mg/kg/day, and of these, one male (animal number 250) also had moderate enlargement of the kidneys. These findings correlated microscopically with tubular degeneration/regeneration. 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.

HISTOPATHOLOGY (PARENTAL ANIMALS)

Test article-related microscopic findings were present in the kidneys of males at 500 mg/kg/day, which had minimal to moderate tubular degeneration/regeneration. 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. These findings are consistent with a well characterized condition described as α2μ-globulin nephropathy, which is limited to the male rat and not relevant for human risk assessment. Tubular degeneration/regeneration was distinct from chronic progressive nephropathy, which is a common background finding in the kidneys of rats at this age, and was present in one female at 500 mg/kg/day (animal number 313). The kidneys of this female also had mild bilateral hydronephrosis and minimal unilateral lymphocytic infiltration which are common background findings in rats and were considered incidental and unrelated to treatment. It should be noted that the kidneys were only examined microscopically in animals which had gross lesions. Because the kidneys were not collected at necropsy in other animals in this study (per the protocol), the kidneys were not examined in all animals. All other microscopic observations were either common background findings in rats or were considered incidental and unrelated to treatment.
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Of the females found to be copulation positive, all of them delivered litters and none of them had total litter loss. There were no statistically significant changes in the delivery data evaluated for the treated animals as compared to controls. This included gestation length, number of pups born per litter on LD 0, corpora lutea, liveborn pups per litter on LD 0, stillborn pups per litter on LD 0, gestation index, stillborn index, total implantations/scars per litter, and number of live pups per litter on LD 4.

VIABILITY (OFFSPRING)

F1 pup survival was unaffected by treatment with the test article. This was supported by the lack of statistically significant changes in the pup survival index on LD 4 in the treated groups as compared to controls.

CLINICAL SIGNS (OFFSPRING)

F1 pup clinical observations did not show any test article-rated changes in the treated litters as compared to controls. A few different clinical findings were noted, but they were few in number and did not follow a dose responsive pattern to indicate a relationship to treatment.

BODY WEIGHT (OFFSPRING)

Mean F1 pup body weights (males, females, and both combined) were statistically decreased at parturition (LD 0) at 150, 300, and 500 mg/kg/day. On LD 4, mean body weights continued to be reduced at 300 mg/kg/day (females only), and at 500 mg/kg/day (males, females, and both genders combined). At 50 mg/kg/day, pup weights on LD 0 and 4 were not statistically different from controls.
Although mean pup weights were lower than controls at 150, 300, and 500 mg/kg/day, they did not follow a clear dose response pattern indicative of a relationship to treatment. On LD 0, gender combined pup weights at 150, 300, and 500 mg/kg/day were 12.4, 9.7, and 11.2 % lower than controls, respectively, and on LD 4 the mean body weight was the same at these dose levels and was 10.1 % lower than controls. All pups at these dose levels showed good growth from birth to LD 4, based on body weight gain.
In comparing the mean pup body weights (gender combined) at 150, 300, and 500 mg/kg/day to the historical control range (6.45-7.06 g on LD 0 and 9.95-11.62 g on LD 4), it was found that the mean pup weights at these dose levels were lower and outside the historical control range. On LD 0 mean pup weights were 5.93, 6.11, and 6.01 g, respectively and on LD 4 mean pup weights were 9.58, 9.58, and 9.58 g, respectively. It is important to note that the live pup litter size at 150, 300, and 500 mg/kg/day was considerably higher (14.7. 14.4, and 12.9 live pups per litter, respectively) and outside the historical control range of 10.2-12.5 live pups per litter. This pattern of lower pup weight with increased litter size in rats has previously been described in the literature and this would support the premise that the decreased pup weight may be related to the increased litter size and therefore not toxicologically meaningful. Furthermore, the lack of a dose response effect on pup weight at these dose levels also confirms that this is not treatment related.

SEXUAL MATURATION (OFFSPRING)

F1 pup sex ratios (percent males per litter) did not show any statistically significant changes in the treated litters as compared to controls.

GROSS PATHOLOGY (OFFSPRING)

There were no apparent test article-related changes in the F1 pup macroscopic observations for animals that were stillborn, died on study, or survived to scheduled necropsy.
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
Oral administration of test article to male and female rats at 50, 150, 300, and 500 mg/kg/day during the study period produced test article-related findings that included postdose salivation at 300 and 500 mg/kg/day (males and females), and microscopic changes in the parental males at 500 mg/kg/day (renal tubular degeneration/regeneration).
Based on the results obtained from this oral reproductive/developmental toxicity screening study in rats, a No-Observed-Adverse-Effect-Level (NOAEL) for developmental toxicity was considered to be 500 mg/kg/day, based on a lack of toxicologically meaningful changes in the treated pups at the dose levels tested. As there were no significant changes in fertility, mating, and fecundity indices, a NOAEL for reproductive performance was considered to be 500 mg/kg/day. With the exception of the microscopic changes noted in the males at 500 mg/kg/day (α2μ-globulin nephropathy, which was limited to the male rat and not relevant for human risk assessment), a NOAEL for general toxicity was considered to be 500 mg/kg/day.
Executive summary:

The objective of this study was to generate limited information concerning the effects of the test article on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus, and parturition. This study met or exceeded Guideline 421, Reproduction/Developmental Toxicity Screening Test, the OECD Guideline for Testing of Chemicals, adopted July 1995. Four treatment groups of twelve CD® [Crl:CD®(SD)] rats/sex/group were administered the test article at dose levels of 50, 150, 300, or 500 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 1 or 2 mL/kg/dose. 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.

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. On LD 4, surviving F1 pups were examined externally, euthanized, and discarded.

The formulation analysis verified a lack of test article in the control samples. The results from the analytical evaluation from the Week 1 test article preparations showed lower than expected values at all concentrations, particularly at the lower dose levels, which triggered an investigation and resulted in a change in the mixing procedures. The two higher concentrations (150 and 250 mg/mL) were acceptable for Weeks 2 and forward. By Week 4, all the values were within the acceptance criteria, except for the 50 mg/mL concentration that was just barely outside. Homogeneity evaluation and concentration verification were repeated on Week 5 and all values passed the acceptance criteria established. Therefore, animals at the two higher dose levels received the targeted concentrations of the test article starting on Week 2 for the remainder of the intervals evaluated. The animals receiving the two lower concentrations (50 and 150 mg/mL) had slightly lower exposures during the first three weeks, but by Week 4 the concentrations for all treatment groups approached the nominal concentrations. By Week 5, all treated animals received the targeted concentrations of the test article.

Oral administration of the test article to male and female rats at 50, 150, 300 and 500 mg/kg/day during the study period produced test article-related findings that included postdose salivation at 300 and 500 mg/kg/day (males and females) and microscopic changes in the parental males at 500 mg/kg/day (renal tubular degeneration/regeneration). All paternal animals survived to scheduled necropsy without significant test article-related effects on body weight, food consumption, or remaining uterine or reproductive parameters evaluated, including fertility, mating and fecundity indices. F1 survival was unaffected by treatment. Of the females found to be copulation positive, all of them delivered litters and none of them had total litter loss. There were no statistically significant changes in the delivery data evaluated for the treated animals as compared to controls. This included gestation length, number of pups born per litter on LD 0, corpora lutea, liveborn pups per litter on LD 0, stillborn pups per litter on LD 0, gestation index, stillborn index, total implantations/scars per litter, and number of live pups per litter on LD 4. F1 pup survival was unaffected by treatment with the test article. This was supported by the lack of statistically significant changes in the pup survival index on LD 4 in the treated groups as compared to controls. F1 pup sex ratios (percent males per litter) did not show any statistically significant changes in the treated litters as compared to controls. F1 pup clinical observations did not show any test article-rated changes in the treated litters as compared to controls. A few different clinical findings were noted, but they were few in number and did not follow a dose responsive pattern to indicate a relationship to treatment. Mean F1 pup body weights (males, females, and both combined) were statistically decreased at parturition (LD 0) at 150, 300, and 500 mg/kg/day. On LD 4, mean body weights continued to be reduced at 300 mg/kg/day (females only), and at 500 mg/kg/day (males, females, and both genders combined). At 50 mg/kg/day, pup weights on LD 0 and 4 were not statistically different from controls. However, the decreased mean pup weights did not follow a clear dose response pattern indicative of a relationship to treatment. Furthermore, all pups showed good growth from birth to LD 4, based on body weight gain. Additionally, the live pup litter size at these dose levels was considerably higher and outside the historical control range pointing to the premise that the decreased pup weight may be related to the increased litter size and therefore not toxicologically meaningful. Furthermore, the lack of a dose response effect on pup weight at these dose levels also confirms that this is not treatment related. There were no apparent test article-related changes in the F1 pup macroscopic observations for animals that were stillborn, died on study, or survived to scheduled necropsy.

Based on the results obtained from this oral reproductive/developmental toxicity screening study in rats, a No-Observed-Adverse-Effect-Level (NOAEL) for developmental toxicity was considered to be 500 mg/kg/day, based on a lack of toxicologically meaningful changes in the treated pups at the dose levels tested. As there were no significant changes in fertility, mating, and fecundity indices, a NOAEL for reproductive performance was considered to be 500 mg/kg/day. With the exception of the microscopic changes noted in the males at 500 mg/kg/day (α2μ-globulin nephropathy, which is limited to the male rat and not relevant for human risk assessment), a NOAEL for general toxicity was considered to be 500 mg/kg/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The key study is GLP compliant and of high quality (Klimisch score = 1).
Additional information

The potential of developmental toxicity of the target substance (Reaction products of diphosphorus pentaoxide and alcohol C7-9-iso, C8 rich, salted with 2-ethylhexylamine) can be evaluated based on the results of the Reproduction/Developmental Toxicity Screening Test (OECD 421, Thorsrud, 2013, Report No. 1928 -010), which evaluated reproductive parameters such as gonadal function, mating behavior, conception, development of the conceptus, and parturition. Four treatment groups of twelve CD® [Crl: CD®(SD) ] rats/sex/group were administered the test article at dose levels of 50, 150, 300, or 500 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 1 or 2 mL/kg/dose. 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.

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. On LD 4, surviving F1 pups were examined externally, euthanized, and discarded.

Oral administration of the test article to male and female rats at 50, 150, 300 and 500 mg/kg/day during the study period produced test article-related findings that included post dose salivation at 300 and 500 mg/kg/day (males and females) and microscopic changes in the parental males at 500 mg/kg/day (renal tubular degeneration/regeneration). All paternal animals survived to scheduled necropsy without significant test article-related effects on body weight, food consumption, or remaining uterine or reproductive parameters evaluated, including fertility, mating and fecundity indices.

F1 survival was unaffected by treatment.Of the females found to be copulation positive, all of them delivered litters and none of them had total litter loss. There were no statistically significant changes in the delivery data evaluated for the treated animals as compared to controls. This included gestation length, number of pups born per litter on LD 0, corpora lutea, live born pups per litter on LD 0, stillborn pups per litter on LD 0, gestation index, stillborn index, total implantations/scars per litter, and number of live pups per litter on LD 4. F1 pup survival was unaffected by treatment with the test article. This was supported by the lack of statistically significant changes in the pup survival index on LD 4 in the treated groups as compared to controls. F1 pup sex ratios (percent males per litter) did not show any statistically significant changes in the treated litters as compared to controls. F1 pup clinical observations did not show any test article-rated changes in the treated litters as compared to controls. A few different clinical findings were noted, but they were few in number and did not follow a dose responsive pattern to indicate a relationship to treatment.Mean F1 pup body weights (males, females, and both combined) were statistically decreased at parturition (LD 0) at 150, 300, and 500 mg/kg/day. On LD 4, mean body weights continued to be reduced at 300 mg/kg/day (females only), and at 500 mg/kg/day (males, females, and both genders combined). At 50 mg/kg/day, pup weights on LD 0 and 4 were not statistically different from controls. However, the decreased mean pup weights did not follow a clear dose response pattern indicative of a relationship to treatment. Furthermore, all pups showed good growth from birth to LD 4, based on body weight gain. Additionally, the live pup litter size at these dose levels was considerably higher and outside the historical control range pointing to the premise that the decreased pup weight may be related to the increased litter size and therefore not toxicologically meaningful. Furthermore, the lack of a dose response effect on pup weight at these dose levels also confirms that this is not treatment related. There were no apparent test article-related changes in the F1 pup macroscopic observations for animals that were stillborn, died on study, or survived to scheduled necropsy.

Based on the results obtained from this oral reproductive/developmental toxicity screening study in rats, a No-Observed-Adverse-Effect-Level (NOAEL) for developmental toxicity was considered to be 500 mg/kg/day, based on a lack of toxicologically meaningful changes in the treated pups at the dose levels tested. As there were no significant changes in fertility, mating, and fecundity indices, a NOAEL for reproductive performance was considered to be 500 mg/kg/day. With the exception of the microscopic changes noted in the males at 500 mg/kg/day (α2μ-globulin nephropathy, which is limited to the male rat and not relevant for human risk assessment), a NOAEL for general toxicity was considered to be 500 mg/kg/day.


Justification for selection of Effect on developmental toxicity: via oral route:
Only one study available (conducted with the target substance).

Justification for classification or non-classification

Toxicity to reproduction

According to the classification criteria outlined in section 3.7.2.2 (Guidance on the Application of CLP criteria, 2012), if there is evidence from humans or experimental animals, possibly supplemented with other information, of an adverse effect on sexual function and fertility, or on development, the classification as Reproductive Toxicant is assigned. Mating, fertility, and fecundity indices were unaffected by treatment with the test item in the reproductive/development toxicity study. The NOAEL for reproductive performance was established to be 500 mg/kg bw, the highest dose level tested.

Therefore, the target substance (Reaction products of diphosphorus pentaoxide and alcohol C7-9-iso, C8 rich, salted with 2-ethylhexylamine) does not meet the requirement under EU CLP (Regulation (EC) No. 1272/2008) for classification as a reproductive toxicant.

Developmental toxicity

Based on a lack of toxicologically meaningful changes in the treated pups at the dose levels tested, the NOAEL for developmental toxicity was considered to be 500 mg/kg/day, the highest dose level tested. Therefore, the target substance (Reaction products of diphosphorus pentaoxide and alcohol C7-9-iso, C8 rich, salted with 2-ethylhexylamine) does not meet the requirement under EU CLP (Regulation (EC) No. 1272/2008) for classification as a developmental toxicant.