Phosphonic acid, mixed C12-20-alkyl and C14-18-unsatd. alkyl derivs.

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:

Between February 2012 and June 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Valid and conclusive guideline study under GLP

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

1995

Deviations:

no

GLP compliance:

yes

Remarks:

United States EPA GLP Standards 40 CFR Part 160 and 40 CFR Part 792 (16 October 1989 and 18 September 1989, respectively), OECD Principles of GLP [C(97) 186/Final] (26 November 1997)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Phosphonic acid, mixed C12-20-alkyl and C14-18-unsatd. alkyl derivs.

EC Number:

300-326-6

EC Name:

Phosphonic acid, mixed C12-20-alkyl and C14-18-unsatd. alkyl derivs.

Cas Number:

93925-25-8

Molecular formula:

Complex

IUPAC Name:

Phosphonic acid, mixed C12-20-alkyl and C14-18-unsatd. alkyl derivs.

Test material form:

liquid: viscous

Details on test material:

- Substance type: Organic
- Physical state: White, opaque, viscous liquid
- Analytical purity: Considered to be 100 % due to the UVCB character
- Expiration date of the lot/batch: 31 May 2013
- Storage condition of test material: Stored at room temperature, protected from light

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, U.S.A.
- Age at study initiation: (P) 10 weeks
- Weight at study initiation: (P) Males: 330-410 g; Females: 221-269 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 Nutrition International, LLC Certified Rodent LabDiet 5002
- Water: Ad libitum (reverse-osmosis-purified on-site drinking water)
- Acclimation period: 14 d

ENVIRONMENTAL CONDITIONS
- Temperature: 21.2 to 22.2 °C
- Humidity: 43.1 to 51.4, % relative humidity
- Air changes: 10/h (at least)
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: From 28 February 2012 to 21 April 2012

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

peanut oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test item formulations were prepared approximately weekly for each dosage level, divided into daily aliquots and stored at room temperature, protected from light. Test item concentrations were 0, 6, 60 and 200 mg/mL.
Dosage volume for all groups was 5 mL/kg.

VEHICLE
- Lot/batch no.: 2AI0653

Details on mating procedure:

- M/F ratio per cage: 1/1
- Proof of pregnancy: Vaginal plug or the presence sperm following a 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 were collected from the middle stratum of each dosing formulation (including the control group) prepared during the last week during which all groups were present for dose administration. Analyses were conducted by means of a validated HPLC method with UV absorbance detection. Acceptance criteria for intersession variability (precision, RSD): ≤ 15 % (85 to 115 % of target). Acceptance for accuracy (RE): within ± 15 %. Mean concentrations ranged from 88.7 to 104 % of target concentration.
Homogeneity and stability were also determined. Results of homogeneity analyses showed mean % of target ranging from 88.7 to 114 %
Stability analyses (10-d room temperature storage) demonstrated mean concentrations of 104 % (6 mg/mL group) and 91.1 % (200 mg/mL group).

Duration of treatment / exposure:

Males: 14 daily doses prior to mating; throughout mating period for a total of 29 doses
Females: 14 daily doses prior to pairing, dosed through to lactation day 3 (females that delivered) or post-mating day 25 (females that failed to deliver) for a total of 39-53 doses

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 30, 300 and 1000 mg/kg bw per d
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; oral route was considered to be the potential route of human exposure, and historically this route has been used extensively for studies of this nature.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily for moribundity and mortality, and for signs of toxicity approximately 1 h following dose administration. Females expected to deliver were observed for dystocia (prolonged labor, delayed labor) or other difficulties.

DETAILED CLINICAL OBSERVATIONS: Yes, 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: Yes

WATER CONSUMPTION AND COMPOUND INTAKE: No

Sperm parameters (parental animals):

Parameters examined in all/P/F1/F2 male parental generations: Testis weight, epididymis weight; microscopic examination of testes, epididymides, seminal vesicles, coagulation glands and prostate gland

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No

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 and 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-mating 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 cavity, the external surface of the brain, and the thoracic, abdominal, and pelvic cavities, including viscera. Moreover, the number of corpora lutea and implantation sites were examined and uteri were investigated for early implantation loss.

HISTOPATHOLOGY / ORGAN WEIGHTS
Microscopically the following tissues were investigated: All gross (internal) lesions, Cervix , Coagulating glands, Epididymidesa, Kidneys (females), Liver (males), Mammary gland (females), Ovaries, Pituitary gland, Prostate gland, Seminal vesicles, Testesa, Uterus, Vagina and Vas deferens
The following organs were weighted: Brain, Epididymides, Kidneys, Liver, Ovaries with oviducts, Pituitary gland and Testes

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring was sacrificed at day 4 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 item-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. 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 to determine intergroup differences. If the ANOVA revealed significant (p < 0.05) intergroup variance, Dunnett's test was used to compare the test item-treated groups to the control group. Mean litter proportions (percent per litter) of males at birth and postnatal survival were subjected to the Kruskal-Wallis nonparametric ANOVA to determine intergroup differences. If the ANOVA revealed significant (p < 0.05) intergroup variance, Dunn’s test was used to compare the test item-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.

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) ∙ 100
- Male Fertility Index [%] = (No. of Males Siring a Litter / Total No. of Males Used for Mating) ∙ 100
- Male Copulation Index [%] = (No. of Males Siring a Litter /No. of Males with Evidence of Mating (or Females with Confirmed Pregnancy)) ∙ 100
- Female Fertility Index [%] = (No. of Females with Confirmed Pregnancy / Total No. of Females Used for Mating) ∙ 100
- Female Conception Index [%] = (No. of Females with Confirmed Pregnancy / No. of Females with Evidence of Mating (or Confirmed Pregnancy)) ∙ 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) ∙ 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) ∙ 100
Where
N = PND 0-1 and 1-4
* = 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):

Local effects at nose and mouth in higher doses

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

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): In high dose males and in mid and high dose females increased incidences of clear material around nose and mouth (1 h following dose administration, and did not persist to the daily examinations). Observations of this nature are commonly observed following the oral administration of an unpalatable or slightly irritant test item formulation and in isolation are considered not to be of toxicological importance.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS): Mean BW and BW gain were unaffected by the test item administration. Differences from the control group were slight, not statistically significant, and/or did not occur in a dose-related manner.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS): Mean food consumption in the test groups was similar to that in the control group throughout the study. The only significant (p < 0.01) difference noted from the control group was higher mean g/kg per d food consumption in the 1000 mg/kg per d group males during study days 27-29. However, this increase was not considered test item-related because the mean g/animal per d value was similar to the control group for the corresponding interval.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS): Not examined

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS): Not examined

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): No test item-related effects on reproductive performance were observed at any dosage level. The mean numbers of days between pairing and coitus in the test item-treated groups were similar to the control group value.

ORGAN WEIGHTS (PARENTAL ANIMALS): Increased mean relative liver weight of high dose males (8.2 %) and mean relative kidney weight of high dose females (7.9 %) compared to controls. No effect was observed at histopathological examination, and therefore these effects are not considered to be adverse.

GROSS PATHOLOGY (PARENTAL ANIMALS): No test item-related internal findings were observed at any dosage level in females that failed to deliver or males and females at the scheduled necropsy.

HISTOPATHOLOGY (PARENTAL ANIMALS): There were no test item-related changes in the F0 generation males or females. All histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than administration of the test item. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

OTHER FINDINGS (PARENTAL ANIMALS): None

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No parental systemic toxicity observed up to the highest dose level tested

2

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related effects noted in any of the reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, numbers of corpora lutea and implantation sites) up to the highest dose level tested

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

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): The mean number of pups born, live litter size, and the percentage of males at birth in the test groups were similar to the control group values.

CLINICAL SIGNS (OFFSPRING): The general physical condition of all F1 pups in this study was unaffected by test item administration.

BODY WEIGHTS (OFFSPRING): On PND 4 lower mean pup body weights were noted for high dose males (9.3 %) and high dose females (7.7 %), without statistical significance. Moreover, mean control values were slightly higher due to increased mean pup body weights in 3 litters on PND 4.

SEXUAL MATURATION (OFFSPRING): Not examined

ORGAN WEIGHTS (OFFSPRING): Not examined

GROSS PATHOLOGY (OFFSPRING): Compared to controls, no differences were observed in the test groups.

HISTOPATHOLOGY (OFFSPRING): Not examined

OTHER FINDINGS (OFFSPRING): None

Effect levels (F1)

Dose descriptor:

NOAEL

Remarks:

neonatal toxicity (development)

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

maternal dose

Sex:

male/female

Basis for effect level:

other: No developmental toxicity observed up to the highest dose level tested

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

NOAEL for parental systemic toxicity and for reproduction and developmental ≥ 1000 mg/kg bw per d (the highest dose tested), derived in absence of effects

Executive summary:

The toxicity of the test item to reproduction and development was investigated in GLP-compliant study by screening for effects on reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition using male and female Sprague-Dawley rats according to the OECD TG 421 (1995) protocol. The experiment is deemed valid, conclusive and thus suitable for assessment without restrictions.

The test item, in the vehicle peanut oil was administered orally by gavage once daily to 3 groups of rats, each group consisting of 12 males and 12 females. Dosage levels were 30, 300, and 1000 mg/kg bw per d administered at a dosage volume of 5 mL/kg bw. A concurrent control group of 12 rats per sex received the vehicle on a comparable regimen. Males and females were approximately 10 weeks of age at the beginning of test item administration. Males received 14 daily doses prior to mating. Males were dosed throughout the mating period through 1 d prior to euthanasia for a total of 29 doses. Females received 14 daily doses prior to pairing and were dosed through the day prior to euthanasia (lactation day 3 for females that delivered and post-mating day 25 for females that failed to deliver) for a total of 39-53 doses. All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. All F0 females were allowed to deliver and rear their pups until lactation day 4. F1 clinical observations and body weights were recorded on Post-Natal Day (PND) 1 and 4. Necropsies were performed on pups found dead. Surviving pups were euthanized on PND 4 and discarded without examination. F0 males were euthanized following completion of the mating period and F0 females were euthanized on lactation day 4. One F0 female that failed to deliver was euthanized on post-mating day 25. Complete necropsies were conducted on all F0 animals, and selected organs were weighed. Selected tissues were examined microscopically from all F0 animals in the control and high-dose groups.

All F0 male and female rats survived to the scheduled necropsy. Parental toxicity was restricted to local irritating effects of the submission item and no relevant findings were noted at the weekly detailed physical examinations. There were no test item-related effects on mean body weights, body weight gains, or food consumption for males throughout the treatment period or for females during the pre-mating, gestation, or lactation treatment periods at any dosage level. There were no test item-related effects on male and female reproductive performance (mating, fertility, copulation and conception indices and pre-coital intervals), mean gestation lengths or the process of parturition at any dosage level. Changes in relative organ weight were of small magnitude, did not have histologic correlates and were not considered to be adverse. There were no test item-related microscopic changes in the F0 males or females. All histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than administration of the test item. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations. There were no test item-related effects on the numbers of pups born, live litter size, percentage of males at birth, pup survival or on mean male and female pup body weights and body weight gains during PND 1-4. No test item-related clinical findings were noted for pups at any dosage level; no test item-related macroscopic findings were observed in pups that were found dead.

In the absence of test item-related effects on reproductive performance, gestation lengths, parturition, live litter size, and numbers of pups born, a dosage level of ≥ 1000 mg/kg per d (the highest dosage level tested) was considered to be the NOAEL for reproductive toxicity of the test item. Based on the absence of adverse effects on parental survival, clinical condition, mean body weights, body weight changes, food consumption, organ weights, or macroscopic and microscopic changes at all dosage levels, the NOAEL for F0 male and female systemic toxicity was considered to be ≥ 1000 mg/kg per d. In addition, the NOAEL for neonatal toxicity was ≥ 1000 mg/kg per d based on the absence of adverse effects on postnatal survival or pup body weights.