Phosphoric acid, mono- and di-isotridecyl esters, compd. with 2,2'-iminobis[ethanol]

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From September 07, 2016 to December 19, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

but were considered not to have affected the outcome or the achievement of the study objectives

Qualifier:

according to guideline

Guideline:

other: US EPA OPPTS 870.3650

Version / remarks:

Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000.

Deviations:

yes

Remarks:

but were considered not to have affected the outcome or the achievement of the study objectives

Qualifier:

according to guideline

Guideline:

other: US EPA OPPTS 870.3550

Version / remarks:

Reproduction/Developmental Toxicity Screening Test, July 2000.

Deviations:

yes

Remarks:

but were considered not to have affected the outcome or the achievement of the study objectives

GLP compliance:

yes

Limit test:

no

Justification for study design:

Exposure 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 reproductive (up to and including implantation) and developmental (from implantation onwards) NOAELs were evaluated.

Specific details on test material used for the study:

Batch no.: RE 10-9
Purity (dry matter): 10.0 % (taken into account for dose calculations)
Appearance: whitish liquid

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI (Han)

Details on species / strain selection:

One of the rodent species acceptable to the regulatory authorities. Historical control data for the strain are available at the Test Facility.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species/strain: Wistar rats: Crl: WI (Han).
Supplier: Charles River Laboratories France, Domaine des Oncins, 69210 Saint Germain sur l'Arbresle, France.
Acclimatisation period: 9 days between animal arrival and the start of treatment.
Number of animals in the study: 40 males and 40 females.
Age at initiation of treatment: Virgin males: approximately 9 weeks; Virgin females: approximately 8 weeks.
Body weight range at initiation of treatment: Virgin males: 307 to 341 g; Virgin females: 167 to 201 g.
Housing: One air-conditioned room in a barrier protected unit.
Temperature: 22 +/- 3°C.
Relative humidity: > 35%.
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)
Water: Softened and filtered (0.2 µm) mains drinking water was available ad libitum
Group housed males and females and individual housed females, including females during mating and with litters, were housed in plastic cages meeting European directive 2010/63/EU requirements.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Method of administration: Oral gavage using a plastic cannula.
Volume of administration: 10 mL/kg/day.
Individual dose volumes were calculated 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.)

Details on mating procedure:

After 2 weeks of treatment, animals were paired on the basis of one male and one female from the same group for a maximum of 10 days. The day of mating was confirmed by the presence of sperm in a 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:

- Accurately measured 0.5 g samples were taken from each formulation, used on the first day of treatment immediately after preparation (Time 0) and after 6 hours (Time T+6) stored at ambient temperature. The samples were then stored frozen (between -25 and -15 °C).
- Analysis of the samples (Charles River Laboratories Den Bosch BV. project 515269) was performed at the Test Site according to a validated method (Charles River Laboratories Den Bosch BV. project 514913).
- Formulations at the entire range were stable when stored in a freezer (≤ -15°C) for at least 7 days. Therefore, the stability under sample shipment conditions was demonstrated. The accuracy of preparation and homogeneity of the test substance in formulations was determined.
The concentrations analysed in the formulations of 30, 100 and 300 mg/kg bw/day were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). No test substance was detected in the control group formulations. The formulations of 30 and 300 mg/kg bw/day were homogeneous (i.e. coefficient of variation ≤ 10%).

Duration of treatment / exposure:

For males: 14 days before mating, throughout the mating period and up to the day before necropsy (i.e. 30 days).
For females: 14 days before mating to Day 3 of lactation. During gestation (the first day of gestation was designated as G 0) and during lactation (the first day of birth is designated as L 0). Apparently one unmated female was treated 29 days after the last day of the mating period (i.e. 54 days). Females that fail to produce a viable litter were treated up to Day 25 of gestation.

Frequency of treatment:

Daily

Details on study schedule:

- Three groups of 10 male and 10 female Wistar Han rats were given the test substance, by daily oral (gavage) administration at dose levels of 30, 100 and 300 mg/kg bw/day from 14 days before mating (Day 1) until Day 30 for males or until Day 3 of lactation for females. A control group of 10 male and 10 female Wistar Han rats was given 10 mL/kg/day of the vehicle (water for injection).
- 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 10 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 (Day 30) for 5 selected males in each group and on Day 3 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. All animals were submitted to a macroscopic examination and selected organs were weighed. Selected organ/tissue samples were fixed and preserved at necropsy for half of the animals/group and reproductive organs samples were fixed and preserved at necropsy from all animals. Histopathological examinations were performed on selected organs/tissues from any dead animal, from half of the group 30 and 300 mg/kg bw/day animals killed at the end of the treatment period, from all males suspected to be infertile or that failed to sire, and from all females that failed to deliver healthy pups.

Dose / conc.:

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

Remarks:

Dose volume: 10 mL/kg/day
Dose concentration: 0 mg/g

Dose / conc.:

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

Remarks:

Dose volume: 10 mL/kg/day
Dose concentration: 3 mg/g

Dose / conc.:

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

Remarks:

Dose volume: 10 mL/kg/day
Dose concentration: 10 mg/g

Dose / conc.:

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

Remarks:

Dose volume: 10 mL/kg/day
Dose concentration: 30 mg/g

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Three groups of 10 male and 10 female Wistar Han rats were given the test substance, by daily oral (gavage) administration at dose levels of 30, 100 and 300 mg/kg bw/day from 14 days before mating (Day 1) until Day 30 for males or until Day 3 of lactation for females. A control group of 10 male and 10 female Wistar Han rats was given 10 mL/kg/day of the vehicle (water for injection).
- 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 10 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 (Day 30) for 5 selected males in each group and on Day 3 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. All animals were submitted to a macroscopic examination and selected organs were weighed. Selected organ/tissue samples were fixed and preserved at necropsy for half of the animals/group and reproductive organs samples were fixed and preserved at necropsy from all animals. Histopathological examinations were performed on selected organs/tissues from any dead animal, from half of the group 30 and 300 mg/kg bw/day animals killed at the end of the treatment period, from all males suspected to be infertile or that failed to sire, and from all females that failed to deliver healthy pups.

Positive control:

-

Parental animals: Observations and examinations:

- Clinical signs: Towards the end of the gestation, females were examined daily for signs of parturition.
- Pregnancy and parturition:
For each female, the following data were recorded: date of mating, date of parturition (day 0 of lactation or L 0), duration of gestation, abnormalities of nesting or nursing behaviour.

Oestrous cyclicity (parental animals):

- Evaluated

Sperm parameters (parental animals):

- Testes analysis

Litter observations:

- Offspring: once daily. (Animals and pups found dead were submitted to necropsy).
- Litter data:
For each litter, the following data were recorded: number of pups born (live and dead), external abnormalities of the pups, number, weight and sex of pups alive on PND’s 1 and 4.

Postmortem examinations (parental animals):

All adult males and females were killed by carbon dioxide inhalation, exsanguinated and then submitted to a macroscopic examination with special attention being paid to the reproductive organs:
1. males: after completion of the mating period, corresponding to a minimum of 28 days of oral administration
2. females: on day 4 of lactation. Females that failed to produce a viable litter by day 26 post coitum were killed and necropsied
- The organs were weighed at scheduled necropsy for the 5 randomly selected adult males and females of each group (for females, only those with live pups). For the other 5 males of each group, only epididymides and testes were weighed. Paired organs were weighed together. The organs were weighed after dissection of fat and other contiguous tissues. Organ weights were expressed as absolute values (g) and relative values (g per 100 g of body weight).
- The organs/tissues were sampled for the 5 randomly selected adult males and females of each group (only females with live pups) and any animal found dead. Only a limited list of organs/tissues was sampled for the remaining males and females, including females that failed to produce a viable litter by day 26 post-coitum (unmated females or mated females that did not deliver after 26 days post-coitum) or females with total litter death. All organs/tissues sampled were fixed and preserved in 10% neutral formalin except for Harderian glands, testes, epididymides, eyes and optic nerves which were fixed in modified Davidson's fluid. The same sampling and trimming procedures was used for all applicable tissues in all applicable dose groups. Paraffin-embedded blocks were prepared only for tissues which were evaluated histopathologically.
Histopathology:
Histological evaluation was performed as follows: for all organs/tissues from all animals found dead during the study, for all macroscopic lesions from all dose group animals , for all organs/tissues from the 5 randomly selected adult males and females of groups control and 300 mg/kg bw/day (for females, only those with live pups), for the testes from the 5 randomly selected adult males of groups control and 300 mg/kg bw/day and all males suspected to be infertile.
A detailed qualitative evaluation of the testes was made, taking into account the tubular stages of the spermatogenic cycle. The cell- or stage-specificity of any testicular findings were recorded, where appropriate: for the reproductive organs of all males that fail to sire and all females that fail to deliver healthy pups and the liver, thyroid glands from group 2 and 3 males and females, and the uterus from group 2 and 3 females.
- Histological slides were prepared and stained with haematoxylin and eosin (HE). In addition, Periodic Acid Schiff-(PAS)-stained sections were prepared and examined for the testes.
- Severity grades were assigned to non-neoplastic histologic diagnoses, using a theoretical maximum range of grades.

Postmortem examinations (offspring):

Pups were also necropsied. Their stomach was examined for the presence of milk, where possible. Defects or cause of death were evaluated, where possible.

Statistics:

Statistical analyses were performed by the Provantis data acquisition system and analysed using a SAS software package

Reproductive indices:

Pre-coital interval (in days), Male and female copulation index (in %), Male and female fertility index (in %), Pre-birth loss (%).

Offspring viability indices:

Live birth index (in %), Viability index (in %), Sex ratio (proportion of male pups in %).

Clinical signs:

effects observed, treatment-related

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Clinical biochemistry findings:

effects observed, non-treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the uterus, the lower group mean uterus weight in females given 300 mg/kg bw/day was considered to correlate with the lower incidence of implantation traces noted microscopically, which may reflect the treatment-related lower litter size and number of implantation sites observed at this dose.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

All mated females 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.

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

- There was no test substance-related effect on mating performance or fertility of the males and females in any group.
- The mean duration of gestation was higher at 300 mg/kg bw/day than in the control and lower dose groups.
- There were 7, 9, 9 and 7 females that completed delivery in the control, 30, 100 and 300 mg/kg bw/day groups, respectively.
- There was a lower number of corpora lutea and an increased percentage pre-implantation and pre-birth loss in the 300 mg/kg bw/day group, compared with control and lower dose groups, inducing a lower litter size at birth.

At 300 mg/kg bw/day, there was a decreased number of corpora lutea, increased pre-implantation and pre-birth loss (inducing lower litter size at birth), longer gestation duration and decreased male ratio. Thereafter, developmental changes included decreased viability index and pup weight at 300 mg/kg bw/day.

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Reproductive toxicity - decreased number of corpora lutea, increased pre-implantation and pre-birth loss, longer gestation duration, decreased male ratio at 300 mg/kg bw/day

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

other: reproductive toxicity

Organ:

uterus

Treatment related:

yes

Dose response relationship:

yes

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

All pups from two litters in the 300 mg/kg bw/day group were cold to touch from L1 to L4. There were no other pup observations that suggested any association with maternal treatment.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

- There was a lower number of corpora lutea and an increased percentage pre-implantation and pre-birth loss in the 300 mg/kg bw/day group, compared with control and lower dose groups, inducing a lower litter size at birth.
- Thereafter, there was a decreased viability index (up to PND 4) and pup body weight in the 300 mg/kg bw/day group, compared with control and lower dose groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean pup weight at birth and through to PND 4 was approximately 10 (L1) to 30 (L4) % lower than control pups in the 300 mg/kg bw/day group in both sexes and was below the historical control. range.

Food consumption and compound intake (if feeding study):

no effects observed

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:

effects observed, treatment-related

Description (incidence and severity):

There was a decreased male pup ratio in the 300 mg/kg bw/day group, compared with control and lower dose groups.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Developmental toxicity - decreased viability index and pup weight at 300 mg/kg bw/day

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

other: fetal developmental toxicity

Organ:

other: decreased viability index and pup weight

Treatment related:

yes

Dose response relationship:

yes

Key result

Reproductive effects observed:

no

Conclusions:

Under the study conditions, the NOAEL of the substance for reproductive and developmental toxicity in rats was determined to be 100 mg/kg bw/day.

Executive summary:

A study was conducted to determine the repeated dose toxicity of the substance according to OECD Guideline 422, US EPA OPPTS 870.3650 and 870.3550, in compliance with GLP. The test substance was administered by daily oral (gavage) to male and female Wistar Han rats (10 males and10 females per dose group) from 14 d before mating (males and females), during organogenesis and until Day 3 of lactation (females) at doses of 0, 30, 100 and 300 mg/kg bw/day. Mortality, 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 10 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 (Day 30) for 5 selected males in each group and on Day 3 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. All animals were submitted to a macroscopic examination and selected organs were weighed. Selected organ/tissue samples were fixed and preserved at necropsy for half of the animals/group and reproductive organs samples were fixed and preserved at necropsy from all animals. Histopathological examinations were performed on selected organs/tissues from any dead animal, from half of the group 30 and 300 mg/kg bw/day animals killed at the end of the treatment period, from all males suspected to be infertile or that failed to sire, and from all females that failed to deliver healthy pups. For details on systemic toxicity, please refer to section 7.5.1. 'Repeated dose toxicity: oral'. There was no evidence of test substance-related changes on mating performance, fertility and histopathological examination of reproductive organs at any dose level. At 300 mg/kg bw/day, there was a decreased number of corpora lutea, increased pre-implantation and pre-birth loss (inducing lower litter size at birth), longer gestation duration and decreased male ratio. The developmental changes included decreased viability index and pup weight at 300 mg/kg bw/day. The NOAEL for reproductive and developmental toxicity in rats was determined to be 100 mg/kg bw/day (Pique, 2017).

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A study was conducted to determine the reproductive and developmental toxicity of the substance according to OECD Guideline 422, US EPA OPPTS 870.3650 and 870.3550, in compliance with GLP. The test substance was administered by daily oral (gavage) to male and female Wistar Han rats (10 males and10 females per dose group) from 14 d before mating (males and females), during organogenesis and until Day 3 of lactation (females) at doses of 0, 30, 100 and 300 mg/kg bw/day. Mortality, 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 10 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 (Day 30) for 5 selected males in each group and on Day 3 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. All animals were submitted to a macroscopic examination and selected organs were weighed. Selected organ/tissue samples were fixed and preserved at necropsy for half of the animals/group and reproductive organs samples were fixed and preserved at necropsy from all animals. Histopathological examinations were performed on selected organs/tissues from any dead animal, from half of the group 30 and 300 mg/kg bw/day animals killed at the end of the treatment period, from all males suspected to be infertile or that failed to sire, and from all females that failed to deliver healthy pups. For details on systemic toxicity, please refer to section 7.5.1. 'Repeated dose toxicity: oral'. There was no evidence of test substance-related changes on mating performance, fertility and histopathological examination of reproductive organs at any dose level. At 300 mg/kg bw/day, there was a decreased number of corpora lutea, increased pre-implantation and pre-birth loss (inducing lower litter size at birth), longer gestation duration and decreased male ratio. The developmental changes included decreased viability index and pup weight at 300 mg/kg bw/day. The NOAEL for reproductive and developmental toxicity in rats was determined to be 100 mg/kg bw/day (Pique, 2017).

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the combined repeated dose and reproductive-developmental toxicity study with the substance, no classification for reproductive toxicity is warranted according to EU CLP (EC 1272/2008) criteria.