Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From August 09, 2016 to November 11, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
in the study plan but they were considered not to have affected the outcome or the achievement of the study objectives
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650
Version / remarks:
Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000
Deviations:
yes
Remarks:
in the study plan but they were considered not to have affected the outcome or the achievement of the study objectives
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3550
Version / remarks:
Reproduction/Developmental Toxicity Screening Test, July 2000
Deviations:
yes
Remarks:
in the study plan but they were considered not to have affected the outcome or the achievement of the study objectives
Principles of method if other than guideline:
In this section, the focus was made on male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development.
GLP compliance:
yes
Limit test:
no
Justification for study design:
To evaluate the potential toxic effects of the test substance when exposed for a minimum of 28 days and the potential to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development. Parental, reproduction (up to and including implantation) and developmental (from implantation onwards) NOAELs were evaluated.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
- Name of test material (as cited in study report): Phosphoric acid, mono- and bis (C16-20 branched and linear alkyl) esters
- Batch: CH 200529/001
- Composition: UVCB
- Appearance: Lightly yellow liquid


Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl: WI (Han)
Details on species / strain selection:
One of the rodent species acceptable to the regulatory agencies. Historical control data for the strain are available at the Test Facility.
Sex:
male/female
Details on test animals and environmental conditions:
Supplier: Charles River Laboratories, Domaine des Oncins, 69210 Saint-Germain-Nuelles, France.
Number of animals in the study: 80 (40 males and 40 females).
Acclimatisation period: 7 days between animal arrival and the start of treatment.
Age at initiation of treatment: Virgin males: 9 weeks and Virgin females: 8 weeks.
Body weight range at initiation of treatment: Males: 267.0 to 307.0 g and Females: 139.8 to 172.9 g.
Housing: One air-conditioned room in a barrier protected unit (building K1).
Temperature: 22 +/- 3 °C (target range).
Relative humidity: > 35 % (target).
Air changes: At least 10 air changes per hour.
Lighting cycle: 12 hours light (artificial)/12 hours dark (except when required for technical acts).
Diet: Rat pelleted complete diet ad libitum (Diet reference A04C-10) sterilised by irradiation and analysed for a predefined list of chemical and bacteriological contaminants. (The animals were fasted for approximately 16 hours before clinical laboratory blood sampling and before scheduled necropsy.)
Water: Softened and filtered (0.2 µm) mains drinking water was available ad libitum (via an automatic watering system or via bottles). Water is analysed twice a year for chemical and bacterial contaminants by Laboratoire Santé Environnement Hygiène de Lyon, France.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Gavage
Once daily
0, 100, 300 and 1000 mg/kg bw/day in a volume of 5mL/kg bw/day
Details on mating procedure:
Animals were paired 2 weeks after the start of the treatment period, i.e. at 11 (males) or 10 (females) weeks of age on the basis of one male and one female from the same group for a maximum of 14 days.
Mating was confirmed by the presence of sperm in the vaginal smear or a vaginal plug and was recorded and taken as Day 0 of gestation (G 0). Mated females were separated from the males once mating has been confirmed and smearing was ceased.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Quadruplicate 0.5 g samples (accurately measured) were taken from the middle from group 1 and 3 formulations (0 and 300 mg/kg bw/day) for accuracy analysis and from three levels (top, middle and bottom) from group 2 and 4 formulations (100 and 1000 mg/kg bw/day) for accuracy and homogeneity analyses. The samples were stored frozen (between -15 and -25 °C), protected from light.
- No test substance was detected in the group 1 formulation (0 mg/kg bw/day). The concentrations analysed in the formulations of group 3 and group 4 (300 and 1000 mg/kg bw/day) were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). For the formulation of group 2 (100 mg/kg bw/day) prepared for use on Day 1, the mean accuracy was below the target concentration (i.e. 79% of target). Additional low dose samples were taken from formulations prepared on 10 October 2016. The concentration analysed of the second set of low dose samples was in agreement with target concentration (i.e. mean accuracies between 90% and 110%). The formulations of group 2 and group 4 (100 and 1000 mg/kg bw/day) were homogeneous (i.e. coefficient of variation ≤ 10%).
Duration of treatment / exposure:
Three groups of 10 male and 10 female Wistar Han rats were given the test substance, by oral (gavage) administration at dose levels of 100, 300 and 1000 mg/kg bw/day from 14 days before mating (Day 1) until Day 31 for males or until Day 4 of lactation for females. A control group of 10 male and 10 female Wistar Han rats was given 5 mL/kg bw/day of the vehicle (corn oil) over the same periods.
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
Gavage using a plastic cannula.5 mL/kg/day (individual dose volumes were adjusted once weekly using the latest body weight).
Rationale for choice of route of administration: oral route was selected as this is a potential route of human exposure during manufacture, handling or use of the test substance as specified in the applicable guidelines.
Positive control:
No

Examinations

Parental animals: Observations and examinations:
- Morbidity/mortality: All animals were observed at least twice daily (i.e. at the beginning and at the end of the working day, including weekends and public holidays).
- Clinical signs: The animals were observed daily during the study. To detect any clinical signs or reactions to treatment, the animals were observed before and at least once after dosing. A full clinical examination was performed on each weighing day. A particular attention was paid to hypersalivation. Toward the end of gestation, females were examined daily for signs of parturition.
- Body weight: Males: once weekly and Females: Once weekly during pre-mating and mating periods (only pre-mating data are reported), on days 0, 6, 9, 12, 15, 18 and 20 of gestation and on days 1 and 4 of lactation.
- Food consumption: Males: once weekly during the pre-mating period and Females: weekly during the pre-mating period, on days 0 to 6, 6 to 9, 9 to 12, 12 to 15, 15 to 18, 18 to 20 of gestation and on days 1 to 4 of lactation.
- Functional tests: Males: first 5 males/group, on the last day of treatment (i.e. Day 31) and Females: first 5 females/group with live pups at the time of test (i.e. lactation day 4). Tests performed: Auditory reflex, Pupillary reflex, Righting reflex, Fore- and hind-limb grip strength and Locomotor activity in an open field test.
- Clinical laboratory determinations
1. Blood sampling: First 5 animals/group/sex, at the end of the pre mating period (i.e. Day 14). Method of blood sampling: Blood was withdrawn from the retro-orbital sinus.
2. Haematology: Haemoglobin, Haemoglobin distribution width, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Packed cell volume, Red blood cell count, Red blood cell distribution width, Mean corpuscular volume, Reticulocyte count, Platelet count, Total white blood cell count and Differential white blood cell count.
3. Coagulation: Prothrombin time, Activated partial thromboplastin time and Fibrinogen.
4. Serum clinical chemistry: Sodium, Potassium, Chloride, Calcium, Inorganic phosphorus, Glucose, Urea, Total cholesterol, Total bilirubin, Total protein, Albumin, Creatinine, Alkaline phosphatase, Aspartate aminotransferase, Alanine aminotransferase and Bile acids.
Oestrous cyclicity (parental animals):
- For each female, the following data were recorded: Date of mating, Date of parturition (Day 0 of lactation or L 0), Duration of gestation and Abnormalities of delivery, nesting or nursing behaviour.
Sperm parameters (parental animals):
- Analysis of testes slides
Litter observations:
- Number of pups born (live and dead), External abnormalities/clinical observation of the pups and number, Weight and sex of pups on postnatal Days (PND) 1 and 4.

Postmortem examinations (parental animals):
- Pathology. Necropsy schedule: Adult animals were killed by carbon dioxide inhalation followed by exsanguination then necropsied according to the following schedule: Males: after completion of the mating period, Females: on Day 4 of lactation and Any apparently unmated female: 24 days after the last day of the mating period (Day 53 of study).
(Any moribund adult or female with total litter death was killed by carbon dioxide inhalation and exsanguination then necropsied.)
- All animals were submitted to a macroscopic examination. For half of the animals/group, selected organs were weighed, and organ/tissue samples were fixed and preserved at necropsy. A limited list of organ/tissue samples were fixed and preserved at necropsy for the remaining animals. Selected organs/tissues, from half of the group 1 (0 mg/kg bw/day) and 4 (1000 mg/kg bw/day) animals killed at the end of the treatment period were examined histopathologically. The reproductive organs (including additional PAS/haematoxylin-stained slides for testes) were examined histopathologically for the 5 randomly selected adult males of groups 1 and 4, all males suspected to be infertile and all females that failed to deliver healthy pups.
Postmortem examinations (offspring):
- Pathology: Surviving pups were killed by intraperitoneal injection of sodium pentobarbitone (CEVA Santé Animale) on postnatal day 4. (Pups that died were necropsied; their stomach was examined for the presence of milk, if possible. Defects or cause of death were evaluated, if possible.)
Statistics:
Statistical analyses were performed by the Provantis data acquisition system.
Reproductive indices:
Pre-coital interval (in days), Copulation index (in %), Male and female fertility index (in %), Gestation index (in %) and Pre-birth loss (in %)
Offspring viability indices:
Live birth index (in %) and F0 Viability index (in %)
(+Sex ratio (proportion of male) (in %))

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Compared with the concurrent controls, there was a slightly increased incidence of animals showing hypersalivation associated or not with abnormal foraging and/or pedalling in all treated female groups during the premating, gestation and lactation periods. The incidence of hypersalivation tended to decrease with time during lactation where only treated females showed these signs in a dose-related manner. All males (except one given 1000 mg/kg bw/day), including controls, had hypersalivation associated or not with abnormal foraging and/or pedalling. In addition, straub tail was noted for 3 and 2 females given 100 and 1000 mg/kg bw/day, respectively, during the lactation period and in 3 males given 1000 mg/kg/day. Other clinical signs noted such as hairloss or scabs were occasionally noted in all groups, including controls, are were considered to be incidental.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There was no test substance-related mortality in any group. One female given the vehicle was euthanized after the death of its entire litter.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
- Males:
There was an overall slightly lower mean body weight gain from Day 1 to Day 29 for male groups given 300 and 1000 mg/kg bw/day (-11 and -10 %, respectively) when compared with the control. However, in the absence of any dose-relationship and since there was no effect on terminal mean body weight, these minor differences were considered incidental. There was no evidence of an effect of treatment on mean body weight gain for males at 100 mg/kg bw/day.
- Females:
There was no adverse test substance-related effect on mean body weight or body weight gain during the pre-mating, gestation and lactation periods at any dose level. Lower mean body weight gain was noted for test substance-treated females when compared with controls from days 1 to 4 of lactation (-12.4, -62.6 and -68.2 % in the 100, 300 and 1000 mg/kg bw/day groups, respectively). However, the values remained within the historical control data range (3.3 -12.2 g) and terminal mean body weights were not overtly affected so the differences were considered to be of no toxicological significance.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
- Males:
Mean food consumption for males between days 9 and 15 of the premating period was comparable between all groups.
- Females:
There was a slightly lower mean food consumption in all treated female groups between days 9 and 15 of the premating period (-8, -6 and -5 % in the 100, 300 and 1000 mg/kg bw/day groups, p<0.05, respectively) when compared with the control group. Consistent with minor body weight differences, mean food consumption was slightly lower between day 1 and 4 of lactation for females given 1000 mg/kg bw/day (-8 %) when compared with the concurrent control. There was no evidence of a test substance-related effect on mean food consumption for females during the gestation period at any dose level and during the lactation period at 100 and 300 mg/kg bw/day.
In addition, when compared with the historical control data, there was a lower mean food consumption for both sexes during the pre-mating, gestation and/or lactation periods in all groups receiving the vehicle with or without the test substance. This finding was therefore considered to be associated with the vehicle, i.e. corn oil.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There was no obvious effect of treatment on pupillary reflexes for the males or females at any dose level.
Haematological findings:
no effects observed
Description (incidence and severity):
There was no obvious effect of treatment on haematology and coagulation parameters for males or females at any dose level.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There was no obvious effect of treatment on other serum clinical chemistry for males or females at any dose level.
There was a dose-related decrease in bile acids in test substance-treated males at all dose levels and females at 300 and 1000 mg/kg bw/day. This effect was considered non-adverse since no correlation was made with any histological findings or any organ weight differences.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There was no obvious effect of treatment on the auditory, pupillary and righting reflexes or gripping and openfield tests for the males or females at any dose level.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
- There was no test substance-related effect on mating performance of females in any group. All animals mated with the exception of one pair in the 1000 mg/kg bw/day. This isolated finding was considered incidental since all other females in this and the lower dose groups showed evidence of insemination within the first 4 days of pairing (approximate duration of a normal oestrous cycle). The mean pre-coital interval was consequently comparable in all groups.
- There was no effect of treatment on fertility. There was one female in the control group that did not become pregnant.
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
There was no obvious test substance-related effect on mating performance of the males and females, on fertility or duration of gestation in any group. There were 9, 10, 10 and 9 females that completed delivery in the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. All animals mated with the exception of one pair in the 1000 mg/kg bw/day group. This isolated finding was considered incidental since all other females in this and the lower dose groups showed evidence of insemination within the first 4 days of pairing (approximate duration of a normal oestrous cycle). The mean pre-coital interval was consequently comparable in all groups. There was no effect of treatment on fertility of either sex in any group. There was one female in the control group that did not become pregnant.

Details on results (P0)

The mean number of corpora lutea and implantation sites was higher for all treated groups when compared with the control group and were within the historical control data range. There was no test substance-related effect on pre-birth survival. The mean percentage pre-birth loss was slightly higher in the 100 and 300 mg/kg bw/day groups (5.6 and 7.1 %, respectively) compared with the control and 1000 mg/kg/day groups (4.0 and 3.7 %, respectively) but remained lower than the mean historical control value (8.4 %).

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Remarks:
systemic and reproductive
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
There was no pattern in the incidences of the pup observations, such as thin, cyanotic or weak appearances, haematomas or tail constriction, that suggested any association with maternal treatment.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
There was no test substance-related effect on the live birth index (number of pups born alive/number of pups born) or viability index (number of pups alive on PND 4/number of pups born alive). The mean values for all treated groups were above that of the control group and within the historical control data range.


Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no obvious test substance-related effect on mean pup body weight on PND 1 or PND 4 in any group.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
Similarly, there was no obvious test substance-related effect on pup sex ratio, weight or viability in any group.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
There was no evidence of pathological changes in either sex due to 28-day oral administration of the test substance up to 1000 mg/kg bw/day in the Wistar rat. The occasional findings encountered were consistent with the spectrum of spontaneous lesions commonly seen in the rodents.
Histopathological findings:
not examined
Other effects:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects observed
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
Under the study conditions, the NOAEL for reproductive and developmental toxicity of the test substance was determined to be 1000 mg/kg bw/day (highest dose tested).
Executive summary:

A study was conducted to determine the toxicity to reproduction of the test substance according to OECD Guideline 422 EPA OPPTS 870.3650 and EPA OPPTS 870.3550, in compliance with GLP. In this 'Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test', only the results regarding the reproductive performance and the gonadal observations are discussed. The systemic toxicity of the test substance is detailed in the section 'Repeated dose toxicity: oral'. Male and female Wistar Han rats were exposed (by gavage) to the test substance at dose levels of 100, 300 and 1000 mg/kg bw/day from 14 days before mating (Day 1) until Day 31 for males or until Day 4 of lactation for females. A control group of 10 male and 10 female rats was given 5 mL/kg bw/day of the vehicle (corn oil) over the same periods. Clinical condition, body weight and food consumption of the animals were monitored throughout the study. Clinical laboratory determinations were performed at the end of the pre‑mating period (Day 14). After two weeks of treatment, one male and one female of the same group were paired for a maximum of 14 days. The females were allowed to give birth and litter parameters, including the number of pups born, pup survival, sex and pup weights were recorded up to postnatal day 4. Functional tests were performed at the end of the treatment period for 5 selected males in each group and on Day 4 of lactation for 5 selected females in each group. The males were necropsied after the pairing period. The dams and pups were necropsied on day 4 of lactation, where applicable. All animals were submitted to a macroscopic examination. For half of the animals/group, selected organs were weighed, and organ/tissue samples were fixed and preserved at necropsy . A limited list of organ/tissue samples were fixed and preserved at necropsy for the remaining animals. Selected organs/tissues, from half of the group 1 and 4 animals killed at the end of the treatment period were examined histopathologically. The reproductive organs (including additional PAS/haematoxylin-stained slides for testes) were examined histopathologically for the 5 randomly selected adult males of groups 1 (control) and 4 (high dose), all males suspected to be infertile and all females that failed to deliver healthy pups. Oral administration of the test substance at doses of 100, 300 and 1000 mg/kg bw/day was associated with a non-adverse minor decrease in mean body weight gain for males and females during lactation at 300 and 1000 mg/kg bw/day and a slight reduction of food consumption for all treated females (from Day 9 and Day 15 of pre-mating) and from Day 1 to Day 4 of lactation at 1000 mg/kg bw/day. There was no effect of treatment on mating performance or fertility (including gonadal function, mating behaviour, conception and parturition). In addition, there were no test substance-related pathological changes in the male or female genital system. The treatment did not affect pup viability or pup weight at any dose on PND 1 and through to termination. Under the study conditions, the NOAEL for reproductive and developmental toxicity of the test substance was determined to be 1000 mg/kg bw/day (highest dose tested) (Mounier, 2017).