Alkyl phosphites

Toxicity to reproduction

Administrative data

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:

August 2011-January 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OCSPP 870.3550

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

other: liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC.
- Age at study initiation: (P) 10 wks
- Weight at study initiation: (P) Males: 323-392 g; Females: 220-277 g
- Fasting period before study: no
- Housing: until pairing: individually in stainless steel wire-mesh cage; paired for mating in male cage; after mating males were housed in suspended wire-mesh cages; females were transferred to plastic maternity cages with nesting material; ground corncob bedding.
- Diet: ad libitum PMI Nutritional International, LLC Certified Rodent LabDiet 5002
- Water: ad libitum (reverse-osmosis-purified on-site drinking water)
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.0-21.8
- Humidity (%): 39.8-57.1
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 14 October 2011 To: 5 December 2011

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: mineral oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Test substance formulations were prepared daily as a single formulation for each dosage level and maintained at room temperature. The test substance formulations were stirred continuously throughout preparation, sampling, and dose administration procedures. Test substance concentrations were 0, 2, 10 and 30 mg/mL.
Dosage volume for all groups was 5 mL/kg.

VEHICLE
- Lot/batch no.: 2AF0729 and ZH1000

Details on mating procedure:

- M/F ratio per cage: 1/1
- Proof of pregnancy: vaginal plug or the presence of sperm following vaginal lavage referred to as day 0 of pregnancy.
- After successful mating each pregnant female was caged: individually into plastic maternity cages with nesting material, ground corncob bedding.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples for concentration determination were collected from the top, middle, and bottom strata of each test substance formulation and the middle stratum of the control group formulation and stored frozen (-70°C). Samples from the 1st, 3rd, 5th and last collection were analyzed for concentration. In addition, samples were taken for determination of homogeneity and stability.
Results of concentration analyses showed that formulations were homogenous and within the protocol-specified range for suspensions (80-120%), except for the 2 mg/mL dosing formulations and the last analyzed 10 mg/mL dosing formulation. The out-of-specification analytical results from the low and mid dose formulations did not negatively impact the outcome of the study since the the analytical results from the high dose formulation, which is the NOAEL for this study, were within specification.

Duration of treatment / exposure:

males: 14 daily doses prior to mating; throughout mating period for a total of 31-32 doses
females: 14 daily doses prior to pairing, dosed through lactation day 3 (females that delivered, for a total of 39-52 doses) or through the day prior to euthanasia (females that failed to deliver, for a total of 52 doses)

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 10, 50, 150 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on the results of previous studies, including a 28-day repeated-dose oral toxicity study in rats (dose levels of 10, 50 and 150 mg/kg bw/day). Changes were limited to local effects in the high dose males and females. Microscopic examination showed submucosal edema and/or inflammation of the forestomach, which effects were reversible (after 14-day recovery period).

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
-for moribundity and mortality, and for signs of toxicity 1-2 hours following dose administration. Females expected to deliver were observed for dystocia or other difficulties.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly for males and weekly for females until evidence of copulation. Female body weights were recorded on GD 0, 4, 7, 11, 14, 17 and 20 and on lactation days 0, 1 and 4.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No (not relevant)

Sperm parameters (parental animals):

Parameters examined in P male parental generation:
testis weight, epididymis weight; microscopic examination of testes, epididymides, seminal vesicles, coagulation gland and prostate gland from control and high dose males.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no
- all pups were euthanized on PND 4

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
litter size, 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.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals following completion of the mating period.
- Maternal animals: All surviving animals on lactation day 4 (females that delivered) or on post-cohabitation day 25 (females that failed to deliver)

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including external surface, all orifices and the cranial, thoracic, abdominal, and pelvic cavities, including viscera. Moreover the number and location of corpora lutea and implantation sites were examined, and uteri were investigated for early implantation loss.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues according to guidelines were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring was sacrificed at 4 days of age and discarded.

Statistics:

Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean. All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group by sex.

Parental mating, fertility, conception, and copulation indices were analyzed using the Chi-square test with Yates’ correction factor (Hollander and Wolfe, 1999). Mean parental body weights (weekly, gestation, and lactation), body weight changes, and food consumption, offspring body weights and body weight changes, gestation length, numbers of former implantation sites, number of pups born, live litter size on PND 0, corpora lutea, unaccounted-for sites, absolute and relative organ weights, and pre-coital intervals were subjected to a parametric one-way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test substance-treated groups to the control group. Mean litter proportions (percent per litter) of males at birth and postnatal survival were subjected to the Kruskal-Wallis nonparametric ANOVA (Kruskal and Wallis, 1952) to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunn’s test (Dunn, 1964) was used to compare the test substance-treated groups to the control group. Histopathological findings of each treated group were compared to those of the control group by Fisher’s Exact test (Steel and Torrie, 1980).

Reproductive indices:

Mating, fertility, and copulation/conception indices were calculated as follows:

Male (Female)
Mating Index (%) = (No. of Males (Females) with Evidence of Mating (or Confirmed Pregnant) / Total No. of Males (Females) Used for Mating) x 100


Male Fertility
Index (%) = (No. of Males Siring a Litter / Total No. of Males Used for Mating) x 100


Male Copulation Index (%) = (No. of Males Siring a Litter /No. of Males with Evidence of Mating (or Females with Confirmed Pregnancy)) x 100


Female Fertility
Index (%) = (No. of Females with Confirmed Pregnancy / Total No. of Females Used for Mating) x 100


Female Conception Index (%) = (No. of Females with Confirmed Pregnancy / No. of Females with Evidence of Mating (or Confirmed Pregnancy) x 100

Offspring viability indices:

Litter parameters were defined as follows:

Mean Live Litter Size = Total No. of Viable Pups on PND 0 / No. of Litters with Viable Pups PND 0

Postnatal Survival Between Birth and PND 0 or PND 4 (Pre-selection) (% Per Litter) = (Sum of (Viable Pups Per Litter on PND 0 or PND 4 [Pre-selection]/No. of Pups Born Per Litter)* / No. of Litters Per Group) x 100

Postnatal Survival for All
Other Intervals (% Per Litter)= (Sum of (Viable Pups Per Litter at End of Interval N/Viable Pups Per Litter at Start of Interval N)* / No. of Litters Per Group ) x 100

Where N = PND 01 and 14

* = Pup that was found dead due to mechanical injury was excluded from pup viability calculations.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related clinical findings were observed for males and females in the
150 mg/kg/day group and included rales, clear material around the mouth/salivation, and
red material around the mouth. These findings were observed at a low incidence at the
weekly detailed physical examinations, at the time of dose administration, and/or at
approximately 1-2 hours following dose administration beginning as early as study day 6
and continuing sporadically throughout the study. Although the incidence of these
findings was low, these findings were not observed at lower dosage levels or in the
control group. These findings are likely related to taste aversion and/or the irritant
properties of the test substance, instead of the result of systemic toxicity of the test
substance.

There were no test substance-related clinical findings observed for the 10 and
50 mg/kg/day groups at the weekly detailed physical examinations or at approximately
1-2 hours following dose administration. Other clinical findings noted in the test
substance-treated groups, including yellow material on various body surfaces, occurred
infrequently or similarly in the control group.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Female no. 29165 in the 10 mg/kg/day group was found dead on lactation day 0. Clinical
findings for this female were limited to yellow material on the hindlimbs and at the base
of the tail at the daily examinations and at approximately 1-2 hours post-dosing during
the pre-mating period and early part of gestation. During the parturition check on
gestation day 22, this female had vaginal discharge in the morning and a pale body in the
afternoon; the female was found dead the following morning. The macroscopic and
microscopic examinations revealed liver necrosis and thrombosis, which suggest
endstage disseminated intravascular coagulation. Because there were no animals in the
50 or 150 mg/kg/day groups that were found dead or moribund (i.e., absence of a dose
response), the single mortality was not considered test substance-related. However,
disseminated intravascular coagulation can be associated with dystocia. Based on the
findings observed during parturition and the macroscopic and microscopic findings for
this female, dystocia was a likely factor in this death. All males and all other females
survived to the scheduled euthanasia.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean body weights and body weight gains in the 10, 50, and 150 mg/kg/day groups were unaffected by test substance administration.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Mean food consumption, evaluated as g/animal/day and g/kg/day, was unaffected by test substance administration at 10, 50, and 150 mg/kg/day for males and females.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related microscopic findings were noted in the nonglandular stomach of the 150 mg/kg/day group males and females. Those localized reversible changes were attributed to the irritant nature of the test substance which was administered by gavage.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

No test substance-related effects on reproductive performance were observed at any dosage level. No statistically significant differences were noted between the control and test substance-treated groups. One male each in the control and 50 mg/kg/day groups did not sire a litter.

The mean numbers of days between pairing and coitus in the test substance-treated groups were not remarkably different than the control group value; none of the differences from the control group were statistically significant.

Mean gestation lengths in the 10, 50, and 150 mg/kg/day groups were similar to those in the control group.

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
at time of dosing or 1-2 hours following dose administration high dose animals showed rales, clear/red material around the mouth, and salivation prior to dosing. These effects are considered to result from taste aversion and/or the irritant properties of the substance instead of a direct toxic effect.

HISTOPATHOLOGY (PARENTAL ANIMALS):
the nonglandular stomach of high dose animals showed minimal to moderate submucosal edema and/or inflammation and/or epithelial hyperplasia and/or ulceration of the mucosa. These observations are related to the irritant effects of the test substance at the portal-of-entry (local effects).

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

sytemic

Effect level:

>= 150 mg/kg bw (total dose)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No systemic toxic effects were observed up to the highest dose tested (150 mg/kg bw/day).

2

Dose descriptor:

NOAEL

Remarks:

local

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

3

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

>= 150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

The general physical condition of all F1 pups in this study were unaffected by test substance administration.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Pups (litters) that were found dead numbered 8(5), 8(2), 4(2), and 8(5) in the control, 10, 50, and 150 mg/kg/day groups, respectively. One, 5, 1, and 2 pups in the respective groups were missing and presumed to have been cannibalized.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean male and female pup body weights and body weight changes during PND 1-4 in the 10, 50, and 150 mg/kg/day groups were unaffected by parental test substance administration.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Dose descriptor:

NOAEL

Remarks:

developmental

Generation:

F1

Effect level:

>= 150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No developmental toxicity was observed up to the highest dose level tested (150 mg/kg bw/day).

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

Treatment with EC 424-820-7 by oral gavage in male and female Sprague-Dawley rats at dose levels of 0, 10, 50 and 150 mg/kg bw/day according to OECD 421 test guideline resulted in local irritating effects in high dose rats as shown by rales and clear/red material around the mouth following dose administration and minimal to moderate submucosal edema and/or inflammation and/or epthelial hyperplasia and/or ulceration of the nonglandular stomach mucosa. Based on these observations, The NOAEL for local effects is 50 mg/kg bw/day. In the absence of systemic toxicity, the NOAEL for parental systemic toxicity is at least 150 mg/kg bw/day, the highest dose tested. No reproduction and developmental toxicity was observed for treatment up to 150 mg/kg bw/day. A reproduction and developmental NOAEL of at least 150 mg/kg bw/day was derived.

Executive summary:

EC 424-820-7 was administrated by daily oral gavage to male and female Sprague-Dawley rats at dose levels of 0, 10, 50 and 150 mg/kg bw/day according to OECD 421 guideline. Males were exposed for 2 weeks prior to mating, during mating and up to termination (total of 29 doses). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and during 3 days of lactation (39 -53 doses).

Parental toxicity was restricted to local irritating effects of the substance. In high dose animals rales and clear/red material around the mouth following dose administration and minimal to moderate submucosal edema and/or inflammation and/or epithelial hyperplasia and/or ulceration of the nonglandular stomach mucosa. Based on this observation, the NOAEL for local effects is 50 mg/kg bw/day. In the absence of systemic toxicity, the NOAEL for parental toxicity is at least 150 mg/kg bw/day, the highest dose tested. No reproduction and developmental toxicity was observed for treatment up to 150 mg/kg bw/day. A reproduction and developmental NOAEL of at least 150 mg/kg bw/day was derived.