Triethyl phosphate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

Pilot study for OECD 443

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental phase: 28 January 2021 - 24 January 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Remarks:

Main study (OECD 443) for which this is the pilot

Reference

Endpoint:

extended one-generation reproductive toxicity - with developmental neurotoxicity (Cohorts 1A, 1B without extension, 2A and 2B)

Study period:

10 November 2021 - 24 April 2023 (audited draft report)

Data waiving:

other justification

Justification for data waiving:

other:

Justification for type of information:

This study is based on ECHA communication/decision number CCH-D-2114513186-55-01/F.

TESTING ON VERTEBRATE ANIMALS
Assessment of the influence of Triethyl phosphate on reproductive performance when administered by oral gavage to Sprague-Dawley rats. Cohorts of F1 animals will be used to assess the potential for systemic toxicity, and potential effects on sexual maturation and estrus cycles. In addition, cohorts of F1 animals will be used to assess developmental neurotoxicity.

NON-CONFIDENTIAL NAME OF SUBSTANCE:
Triethyl phosphate

FURTHER INFORMATION ON TESTING:
- Details on study design / methodology are provided within this study record.

FURTHER INFORMATION:
An EOGRTS according to OECD 443 is currently underway, including Cohorts 1 and 2. The Experimental start date was 10 November 2021, and full data gathering following end of in-life is expected by January 2023.
Draft reporting is expected April 24 2023.

Reason / purpose for cross-reference:

reference to same study

Remarks:

Pilot Phase

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

Adopted 25 June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:

- Premating exposure duration for parental (P0) animals: Ten weeks premating exposure duration is required to obtain results adequate for classification and labelling and /or risk assessment. There is no substance specific information in the dossier supporting shorter premating exposure duration

- Basis for dose level selection: Pilot study (see cross-linked study Stannard, 2022)

- Inclusion/exclusion of extension of Cohort 1B : The conditions to include the extension of Cohort 1B are currently not met.

- Termination time for F2: N/A - no F2 extension

- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B: previous studies report a concern for neurotoxicity, including changes in total movement levels, foregrip strength, hindgrip strength. Further, alterations in cholinesterase levels and in thyroid histopathology were identified.

- Inclusion/exclusion of developmental immunotoxicity Cohort 3: no triggers for the inclusion of Cohort 3 (developmental immunotoxicity) were identified.

- Route of administration: Oral

- Other considerations, e.g. on choice of species, strain, vehicle and number of animals: N/A

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)

Details on species / strain selection:

The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Sprague-Dawley [Crl:CD(SD)] strain was used because of the historical control data available at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) 4-5 wks; (F1) 0 wks
- Weight at study initiation: within a 20g weight range
- Fasting period before study: N/A
- Housing: polycarbonate cages, up to 4 same sex animals per cage
- Use of restrainers for preventing ingestion (if dermal): N/A
- Diet (e.g. ad libitum): ad libitum SDS VRF1 Certified, pelleted diet
- Water (e.g. ad libitum): ad libitum
- Acclimation period: ≥5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 10 Nov 2021 To: 27 May 2022 (assuming Breeding F1 cohort 1B not triggered)

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Purified water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): N/A
- Concentration in vehicle: validated 1-100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg/day
- Lot/batch no. (if required): N/A
- Purity: N/A

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: up to 2 weeks, no mating change-overs
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): single female in polycarbonate cage

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

To be added on completion of study

Duration of treatment / exposure:

P Males: 10 weeks before mating until termination after litters are weaned
P Females: 10 weeks before mating until termination after litters are weaned
F1 unselected: indirect birth - weaning, no direct treatment (sacrifice PND 22)
F1 Cohort 1A: indirect birth - weaning, direct PND 21 (weaning) until 13 weeks (day 90)
F1 Cohort 1B: indirect birth - weaning, direct PND 21 (weaning) until 14 weeks (day 97)
F1 Cohort 1C: indirect birth - weaning, direct PND 21 (weaning) until 6-8 weeks (sexual maturation)
F1 Cohort 2A: indirect birth - weaning, direct PND 21 (weaning) until day 75
F1 Cohort 2B: indirect birth - weaning, no direct treatment

Frequency of treatment:

daily, 7 days each week

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

low

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

intermediate

Dose / conc.:

450 mg/kg bw/day (nominal)

Remarks:

high

No. of animals per sex per dose:

108 male and 108 female animals (216 total). 100 F0 animals per sex allocated to study, 8 F0 animals per sex spare.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels selected according to pilot study (Labcorp study 8453576)
- Rationale for animal assignment (if not random): By sex. After exclusion of animals showing signs of ill- health. Animals at the extremes of the body weight range will not be selected if alternatives are available.
- Fasting period before blood sampling for clinical biochemistry: overnight

Positive control:

N/A

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: F0 males each week and before necropsy. F0 females each week until mating detected, Days 0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 after mating, Days 1, 4, 7, 14, 21 and 28 of lactation and before necropsy. §

Oestrous cyclicity (parental animals):

Dry smears: For 15 days before pairing, using cotton swabs. | Wet smears: Daily after pairing until evidence of mating confirmed, using pipette lavage. For four days before scheduled termination (nominally Days 25 to 28 post partum).

Sperm parameters (parental animals):

Parameters examined in all male parental generations:
testis weight, epididymis weight, daily sperm production, sperm count in testes, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility, sperm morphology

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of [10] pups/litter (5/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups, other. Particular attention should be paid to the external reproductive genitals which should be examined for signs of altered development; gross evaluation of external genitalia

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: YES (details to be added on completion of study)
Sensory Function – Quantitative:
At Day 24±1 of age, the animals will be tested in an automated system for auditory startle habituation. Startle amplitudes measured over five consecutive blocks of 10 trials (total 50 trials).

Neurobehavioral Screening:
The functional observational battery recordings will be performed at approximately the same time of day at approximately Day 63-75 of age (nominally Day 70±1). Not all animals will be tested in one day, but the time of testing will be balanced across the groups.
In performing the functional observational battery, the treatment of the individual animal will not be known by the observer. At any point during the observations, comments can be made about behaviour as free text. Full tabulated details of exams will be added on completion of study.

Motor Activity:
Between Days 63-75 of age (nominally Day 65±1) motor activity will be measured by automated infra-red equipment. High and low beams record rearing and cage floor activity, respectively.
For testing, designated animals are placed singly into observation cages. The test session for each animal will be one hour. Data will be automatically collected and reported at regular intervals throughout the session.

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: NO

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals After at least 18 weeks of treatment and after weaning of the F1 animals, after confirmation that no further mating is required.
- Maternal animals: All surviving animals Day 28 postpartum.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [TBC] were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at [22] days of age. Further details on examinations to be added on completion of study.

Statistics:

The following data types will be analyzed at each timepoint separately, in support of interpretation:
body weight, using absolute weights and gains over appropriate study periods. food consumption, over appropriate study periods.
estrous cycles, vaginal opening to first estrus and pre-coital interval.
mating performance and fertility.
gestation length.
litter size and survival indices.
pre-weaning examination (ano-genital distance, surface and righting reflexes) sexual maturation, age and body weight at completion.
clinical pathology (hematology, blood chemistry, urinalysis).
thyroid hormone analysis.
immunophenotyping.
behavioral data, rearing and activity counts.
organ weights, both absolute and relative to terminal body weight.
sperm analysis, motility, morphology and counts.
corpora lutea and ovarian primordial follicle counts.

For categorical data, the proportion of animals will be analyzed for each treated group (as appropriate) versus the control group.
For continuous data, Bartlett’s test will first be applied to test the homogeneity of variance between the groups. Using tests dependent on the outcome of Bartlett’s test, treated groups will then be compared with the control group, incorporating adjustment for multiple comparisons where necessary.
Under the advice of the Associate Director, Global Statistics, or other qualified Statistician, alternative or additional methods may be carried out if deemed appropriate following data review. Details will be included in the study report.

Reproductive indices:

Percentage mating, Conception rate, Fertility index

Offspring viability indices:

Gestation index, Post-implantation survival index, Live birth index, Viability index, Lactation index, Sex ratio

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2023

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

Deviations are detailed within 'Any other information on results incl. tables' section. The study plan deviations neither affected the overall interpretation of study findings nor compromised the integrity of the study.

Principles of method if other than guideline:

Using a study design similar to an extended OECD 421, animals were dosed from 2 weeks prior to pairing, until after successful littering by F0 females (F0 males), day 21 of lactation (F0 females), PND 21 (F1 unselected) or PND 35 (selected F1).

GLP compliance:

no

Remarks:

Facility operates in accordance with GLP but this study was not designed as GLP-compliant.

Limit test:

no

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS [please address all points below]:

- Premating exposure duration for parental (P0) animals: 2 weeks
- Basis for dose level selection: The dose levels selected for investigation in this study were chosen in conjunction with the Sponsor and based on the outcome of a 90-day gavage repeat dose toxicity study in Han Wistar rats (Charles River Report No. 514858).
- Inclusion/exclusion of extension of Cohort 1B: N/A
- Termination time for F2: N/A
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B: N/A
- Inclusion/exclusion of developmental immunotoxicity Cohort 3: N/A
- Route of administration: N/A
- Other considerations, e.g. on choice of species, strain, vehicle and number of animals [if applicable]

Test material

Test material information

Constituent 1

Reference substance name:

Triethyl phosphate

EC Number:

201-114-5

EC Name:

Triethyl phosphate

Cas Number:

78-40-0

Molecular formula:

C6H15O4P

IUPAC Name:

triethyl phosphate

Test material form:

liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD) rat.

Details on species / strain selection:

The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Sprague-Dawley [Crl:CD(SD)] strain was used because of the historical control data available at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) Males 75 to 81 days old; Females 68 to 74 days old. (F1) 21 days at allocation to treatment groups.
- Weight at study initiation: (P) Males: 350 to 436 g; Females: 227 to 268 g
- Fasting period before study: no
- Housing: Solid (polycarbonate) bottom cages except during pairing.
Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily.
- Use of restrainers for preventing ingestion (if dermal): N/A
- Diet (e.g. ad libitum): ad libitum SDS VRF1 Certified, pelleted diet (supplied by Special Diet Services, Essex, UK). The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent; phytoestrogen content is 144 ppm.
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days before treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 10 February 2021 To: 01 May 2021

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Purified water (supplied by Crystal Medic, UK).

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Prepared weekly, formulation stored Refrigerated (2 to 8°C).

VEHICLE
- Justification for use and choice of vehicle (if other than water): N/A
- Concentration in vehicle: 25/45/80 mg/mL
- Amount of vehicle (if gavage): Dose Volume 10 mL/kg
- Lot/batch no. (if required): N/A
- Purity: N/A

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: up to 2 weeks
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After unsuccessful pairing replacement of first male by another male with proven fertility: not specified
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): singly
- Any other deviations from standard protocol: No

Analytical verification of doses or concentrations:

yes

Remarks:

The homogeneity and stability of formulations during storage were confirmed as part of Labcorp Study Number 8453574.

Details on analytical verification of doses or concentrations:

Formulations in the range 1 to 100 mg/mL were determined to be stable for:
• One day at ambient temperature (15 to 25˚C)
• 15 days when stored refrigerated (2 to 8˚C)

Samples of each formulation prepared for administration in Week 1 of treatment were analyzed for achieved concentration of the test item.

Duration of treatment / exposure:

F0 animals For two weeks before pairing until Day 20 of lactation/scheduled termination.
F1 animals From weaning on Day 21 to Day 34 of age.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1: Vehicle control

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

Group 2: Low

Doses / concentrations 3

Dose / conc.:

450 mg/kg bw/day (nominal)

Remarks:

Group 3: Intermediate

Doses / concentrations 4

Dose / conc.:

800 mg/kg bw/day (nominal)

Remarks:

Group 4: High
F0: dose level was prematurely terminated on day 1. From Day 2 of treatment the study thererefore consisted of one control group and two treated groups.

No. of animals per sex per dose:

F0: 8 animals/sex/dose
F1: 10 animals /sex/dose (250/450 mg/kg bw/day only)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels selected for investigation in this study were chosen in conjunction with the Sponsor and based on the outcome of a 90-day gavage repeat dose toxicity study in Han Wistar rats (Charles River Report No. 514858).

In that study, dose levels of 60, 200 and 700 mg/kg bw/day were investigated and were not associated with any test item-related mortalities, changes in general clinical condition, or effects on body weight or food intake. Administration of Triethyl phosphate at 700 mg/kg bw/day was associated with histopathological changes in the kidneys of males (hyaline droplet accumulation, accompanied by tubular basophilia and granular casts) and increased kidney weights, considered to represent α2μ-globulin accumulation. Hyaline droplets and subsequent associated kidney pathology was considered adverse in the animals affected, since degenerate changes were also present, but are findings specific to the male rat and are generally not considered to be toxicologically significant in humans. In addition, an increase in liver weights in males and females at 700 mg/kg bw/day was associated with minimal/slight hepatocellular hypertrophy. Although these findings were considered adverse, or potentially adverse, within the context of the 90-day study, in isolation they were considered not to preclude the use of a dose level close to, or slightly above, 700 mg/kg bw/day in this current study.

Since the No Observed Adverse Effect Level in a previous 28-day repeat dose toxicity study was 1000 mg/kg bw/day, and the half maximal oral LD50 was 800 mg/kg bw, the high dose level for this current study was therefore set at 800 mg/kg bw/day. The intermediate and low dose levels of 450 and 250 mg/kg bw/day, respectively were chosen to achieve a dose response and to aid in the determination of a suitable high dose level for the subsequent main Extended One-generation Reproductive Toxicity Study (OECD TG 443).

- Rationale for animal assignment (if not random): On arrival by non-selective allocation to cages.

Positive control:

None

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
F0: Week 1: Daily. Weeks 2 to 4: twice weekly (middle and end of the week). Week 5 onwards: once each week, including Days 0, 7, 14 and 20 after mating and Days 1, 7, 14 and 20 of lactation for F0 females. Physical examination: Once each week. For F0 females after mating, conducted on Days 0, 5, 12, 18 and 20 after mating and Days 1, 7, 14 and 21 of lactation.
F1: Daily from days 21-34 of age

BODY WEIGHT: Yes
- Time schedule for examinations:
F0 males: Day that treatment commenced. Each week. Before necropsy.
F0 females: Day that treatment commenced. Each week until mating detected. Days 0, 7, 14 and 20 after mating. Days 1, 7, 14 and 21 of lactation. Before necropsy.
F1 selected animals: Days 21, 23, 25, 27, 29, 31, 33 and 35 of age.


FOOD CONSUMPTION:
F0 males/females: Weekly before pairing, from the day that treatment commenced.
For F0 females after mating food consumption was performed to match the body weight recording:
Days 0-3, 3-7, 7-11, 11-14, 14-17 and 17-20 after mating. Days 1-4, 4-7, 7-11, 11-14, 14-18 and 18-21 of lactation.
F1 selected: Days 21-23, 23-25, 25-27, 27-29, 29-31, 31-33 and 33-35 of age.
From these records the mean weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each relevant phase.

Oestrous cyclicity (parental animals):

Dry and wet smears were taken as follows:
Dry smears For 15 days before pairing, using cotton swabs.
Wet smears After pairing until evidence of mating confirmed.

Sperm parameters (parental animals):

Not analysed

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Selected F1 animals: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities. All were subject to complete macroscopic examination. Abnormal tissues were retained in an appropriate fixative.

GROSS EXAMINATION OF DEAD PUPS:
Culled offspring with clinical signs on Day 4 of age were subject to complete macroscopic examination with assessment of stomach for presence of milk, where possible.
Unselected offspring at scheduled kill on Day 21 of age and selected spares: Subject to complete macroscopic examination. Abnormal tissues were retained in an appropriate fixative.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals After successful littering by F0 females.
- Maternal animals: All surviving animals Day 21 of lactation.

GROSS NECROPSY
All F0 females, unselected F1 offspring and selected F1 generation animals were subject to a detailed necropsy.
After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative. For females, Number of implantation sites were recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
Abnormal tissues and the liver of each animal were prepared for microscopic examination. The liver was weighed.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all selected F1 offspring were sacrificed at 21 days of age.
- These animals were subjected to postmortem examinations (macroscopic examination).

GROSS NECROPSY
Premature deaths before weaning:
Missing offspring could not be examined. All other offspring dying before weaning were subject to complete macroscopic examination with assessment of stomach for presence of milk, where possible. No abnormal tissues requiring retention were observed.
Culled offspring on day 4:
Culled offspring with clinical signs on Day 4 of age were subject to complete macroscopic examination with assessment of stomach for presence of milk, where possible. No abnormal tissues requiring retention were observed. Culled offspring with no clinical signs on Day 4 of age were killed and discarded without necropsy examination.
Unselected offspring at scheduled kill on day 21 and selected spares:
Subject to complete macroscopic examination. Abnormal tissues were retained in an appropriate fixative.
Selected F1 animals:
Subject to complete macroscopic examination. Abnormal tissues were retained in an appropriate fixative.

Statistics:

Statistical analyses were performed on the majority of data presented and results of these tests, whether significant or non-significant, are presented on the relevant tables. For estrous cycles, pre-coital interval, mating performance and fertility and gestation index the similarity of the data was such that analyses were not considered to be necessary.
All statistical analyses were carried out separately for males and females. Data relating to food consumption were analyzed on a cage basis. For all other adult parameters, the analyses were carried out using the individual animal as the basic experimental unit. For litter/fetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.
The following data types were analyzed at each timepoint separately:
Body weight, using absolute weights and gains over appropriate study periods
Food consumption, over appropriate study periods (except before pairing)
Gestation length
Litter (implantations, litter size, sex ratio - percentage male, post implantation survival index, live birth index and viability index), for before cull study periods
Liver weight, both absolute and body weight relative.
The following comparisons were performed: Group 1 vs 2, and 3

Further detail of statistical methods are presented in Any other information on materials and methods.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related clinical signs observed throughout the study at 250 or 450 mg/kg bw/day. Signs in relation to dose administration were observed in animals given 450 mg/kg bw/day during the first five days of treatment, manifest as transient incidences of unsteady/swaying gait, underactive behaviour, excessive chewing behaviour, partially closed eyes, flattened/hunched posture and reddening of the ears. The severity/duration of these signs was lower than observed in animals given 800 mg/kg bw/day. From Day 6 of treatment onwards, there were no test item-related signs observed in relation to dosing at 450 mg/kg bw/day, and no signs were observed at 250 mg/kg bw/day throughout the study.
For test item related changes following dosing with 800 mg/kg bw/day see 'Mortality'.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Administration of Triethyl phosphate at 800 mg/kg bw/day was not tolerated, with all animals killed for reasons of animal welfare approximately 3 hours after dosing on Day 1. Analysis of the formulation that had been administered confirmed that the concentration given was as intended (achieved concentration 80.4 mg/mL; target concentration 80 mg/mL).
Signs in association with dose administration became apparent approximately 20-30 minutes after dosing, with all males and 7/8 females showing unsteady gait and 2/8 females showing underactive behaviour. In addition, one female was observed with laboured respiration, prostrate posture and was unresponsive; as a consequence, this female was immediately killed for reasons of animal welfare.
At approximately 2 hours after dosing, all males and 6/7 of the surviving females showed unsteady gait and underactive behaviour, all males and all surviving females had partially closed eyes and one male had wet rales. One of the females also showed prostrate posture and was unresponsive, and a further female was very cold to touch; both of these females were therefore immediately killed for reasons of animal welfare.
At approximately 3 hours post-dose, the condition of all eight males and the surviving five females had further deteriorated. The animals continued to show signs of unsteady gait, partially closed eyes and underactive behaviour, but in addition one female had laboured respiration and was unresponsive, two males had wet rales, two males and two females had prostrate posture, one female showed hunched posture and one male showed excessive chewing behaviour. In view of the continued decline in clinical condition, all surviving animals in the 800 mg/kg bw/day group were killed for reasons of animal welfare.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no effect of Triethyl phosphate administration at 250 or 450 mg/kg bw/day on group mean body weight gain throughout the study.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no effect of Triethyl phosphate administration at 250 or 450 mg/kg bw/day on group mean food consumption throughout the study.

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

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):

Changes related to treatment with Triethyl phosphate were seen in the liver in both sexes.
Minimal or slight diffuse centrilobular hepatocyte hypertrophy was seen in males and females receiving 250 or 450 mg/kg bw/day Triethyl phosphate. Centrilobular hepatocyte hypertrophy is an indication of enzyme induction and considered an adaptive change.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrous cycle regularity during the 2-week pre-pairing dosing period was unaffected by Triethyl phosphate administration.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

There was no effect of Triethyl phosphate administration at 250 or 450 mg/kg bw/day on pre- coital interval, with all mating pairs showing positive evidence of mating within four days of pairing (ie. at the first estrus opportunity). Mating performance and fertility were similarly unaffected, with conception rate and fertility index being 100% in all groups. All females successfully gave birth to live young, with the gestation length of all females being within the expected range of 22 to 23 days; gestation index was 100% in all groups.

Effect levels (P0)

Remarks on result:

not measured/tested

Remarks:

Pilot study for EOGRTS only

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs observed amongst the F1 offspring prior to weaning which were related to parental treatment with Triethyl phosphate at 250 or 450 mg/kg bw/day.
There were no test item-related clinical signs or post-dosing signs observed in the selected F1 generation animals.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

There was no effect of parental treatment with Triethyl phosphate at doses up to and including 450 mg/kg bw/day on litter size, or on in utero or post-partum survival. Offspring mortality between birth and Day 4 of age was limited to 9, 11 and 6 pups in the 0, 250 or 450 mg/kg bw/day groups, respectively.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no effect of parental treatment with Triethyl phosphate at 250 or 450 mg/kg bw/day on the mean birth weights of the offspring, or on their subsequent body weight gain up to weaning on Day 21 of age.
There was no clear effect of treatment on mean body weight gain of the selected
F1 generation animals between Day 21 and Day 35 of age at 250 or 450 mg/kg bw/day.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Mean food intake of the F1 generation animals was unaffected by Triethyl phosphate administration at 250 or 450 mg/kg bw/day.

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:

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

Description (incidence and severity):

There were no macroscopic abnormalities detected among the low number of F1 offspring which died between birth and Day 4 of age indicative of an effect of parental treatment with Triethyl phosphate. Findings were primarily limited to an absence of milk in the stomach, a common macroscopic abnormality in newborn offspring. No macroscopic abnormalities were detected among the unselected F1 offspring on Day 21 of age.
There were no test item-related macroscopic abnormalities detected at scheduled termination of the F1 generation animals on Day 35 of age.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Sex ratio, as assessed by the percentage of male offspring per litter, was in line with expectation, and no effect of treatment was inferred.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Remarks on result:

not measured/tested

Remarks:

Pilot study for EOGRTS only

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

Table 02: Deviations from Study plan 

Category	Deviations
Animal Data Collection, Missed/Extra Procedure	The study plan required a litter size/mortality assessment to be performed daily from Day 1 to Day 21 of lactation. On Day 7 of lactation, this data collection was omitted for the following litters: Group 1 No’s. 41, 43, 45, 46 and 48; Group 2 No’s. 49, 51, 52, 53, 54, 55, and 56; Group 3 No’s. 57, 58, 62, 63, and 64. For all affected litters, individual offspring body weights were required on Day 7 of age as required by the study plan, thus confirming the presence of each pup in the litter on this day and therefore the pup status data could be retrospectively back entered. Since all omitted data points could be verified and back entered from other litter observation recordings, no data were lost, and therefore this has no impact on the scientific integrity of the study.
Animal Room Environment	On one occasion (06 April 2021) the humidity recorded in the animal room was 39%, marginally below the target range of 40-70%. The condition of the animals was unaffected.

These study plan deviations neither affected the overall interpretation of study findings nor compromised the integrity of the study.

 

Table 03: Incidence and Severity of Triethyl Phosphate-Related Microscopic Findings – Terminal Sacrifice (F0)

Triethyl phosphate
Sex		Males			Females
Dose Level (mg/kg bw/day)		0	250	450	0	250	450
Liver
Number Examined		8	8	8	8	8	8
Hypertrophy, Hepatocellular, Centrilobular
Minimal		0	4	5	0	2	5
Slight		0	0	3	0	0	0

 

Applicant's summary and conclusion

Conclusions:

Based on the results of this preliminary extended one-generation study it was concluded that 450 mg/kg bw/day would be suitable for use as the high dose level for the forthcoming main extended one-generation (OECD 443 test guideline) study.

Executive summary:

The purpose of this study was to assess the general systemic toxic potential, including reproductive/developmental effects, following oral gavage administration of Triethyl phosphate to Sprague Dawley rats, in order to act as a preliminary study to assist with setting dose levels for an Extended One-generation Reproductive Toxicity Study (OECD TG 443) requested by the ECHA (decision number CCH-D-2114513186-55-01/F).

 

Initially, in the F0 generation, three groups of eight male and eight female rats received Triethyl phosphate at dose levels of 250, 450 or 800 mg/kg bw/day by gavage at a volume dose of 10 mL/kg/day. A similarly constituted Control group received the vehicle, purified water, at the same volume dose as treated groups. All males and females given 800 mg/kg bw/day were euthanized for welfare reasons approximately three hours after dosing on Day 1 due to the onset and progression of adverse signs which resulted in a marked decline in clinical condition. The remainder of the study therefore consisted of the control group and the two surviving treated groups. F0 males in those groups were treated for two weeks before pairing and throughout pairing, up to scheduled necropsy after the F1 litters were weaned.

 

F0 females were treated for two weeks before pairing, throughout pairing up to necropsy on Day 21 of lactation. In the F1 generation, ten males and ten females were treated at the same dose levels and employed the same dose volume as the F0 generation from Day 21 to Day 34 of age.

 

During the study, clinical condition, body weight, food consumption, mating performance, gestation length, parturition observations, liver weight, macropathology and liver histopathology investigations were undertaken on F0 adult animals. The clinical condition, litter size and survival, sex ratio, body weight and macropathology of all F1 offspring were assessed. After weaning, selected F1 generation animals were assessed for clinical condition, body weight, food consumption and macropathology investigations.

 

Results

The mean concentrations of Triethyl phosphate in test formulations administered in Week 1 were within 3 % of the nominal concentration, confirming the accuracy of formulation. The coefficient of variation remained within 2%.

 

Parental responses

Administration of Triethyl phosphate at 800 mg/kg bw/day exceeded the maximum tolerated dose, with all F0 animals killed for reasons of animal welfare within approximately 3 hours of dosing on Day 1 due to the onset and progression of adverse signs including, but not limited to, unsteady gait, underactive behaviour, prostrate posture, respiratory changes and partially closed eyes; three females were also unresponsive. No macroscopic abnormalities were detected among these animals. 

 

There were no premature deaths or test item-related clinical signs observed throughout the study at 250 or 450 mg/kg bw/day. Signs in relation to dose administration were observed in animals given 450 mg/kg bw/day during the first five days of treatment, manifest as transient incidences of unsteady/swaying gait, underactive behaviour, excessive chewing behaviour, partially closed eyes, flattened/hunched posture and reddening of the ears. The severity and duration of these signs was lower than observed in animals given 800 mg/kg bw/day. From Day 6 of treatment onwards, there were no test item-related signs observed in relation to dosing at 450 mg/kg bw/day, and no signs were observed at 250 mg/kg bw/day throughout the study.

 

Body weight gain, food consumption, estrous cycle regularity and reproductive performance were unaffected at 250 or 450 mg/kg bw/day, and there were no macroscopic abnormalities observed. Mean absolute and body weight-relative liver weights were statistically significantly increased in a dose-dependent manner in both sexes given 250 or 450 mg/kg bw/day, up to a maximum of 46% higher than Controls in males given 450 mg/kg bw/day. Histopathological evaluation of the livers revealed a dose-dependent increased incidence of minimal/slight centrilobular hepatocyte hypertrophy, a non-adverse adaptive response to the administration of a xenobiotic.

 

Litter responses

General clinical condition, litter size, offspring survival, sex ratio, birth weight of the F1 offspring and subsequent body weight gain of the F1 offspring were unaffected by parental treatment with Triethyl phosphate at 250 or 450 mg/kg bw/day.

 

Macroscopic examination of the F1 offspring that died prematurely and those at scheduled termination, revealed no findings that could be related to Triethyl phosphate.

 

F1 generation responses

There were no signs at routine physical examination related to administration of Triethyl phosphate at 250 or 450 mg/kg bw/day and no post-dosing signs were observed.

 

There was no effect of oral gavage administration of Triethyl phosphate on body weight gain or food consumption for the selected F1 generation males and females.

 

No test item-related macroscopic abnormalities were detected at scheduled termination on Day 35 of age.

 

Conclusion

Based on the results of this preliminary extended one-generation study it was concluded that a dose level of 800 mg/kg bw/day exceeded the maximum tolerated dose and is unsuitable for further investigation. Although some evidence of systemic toxicity was observed in the F0 animals at 250 or 450 mg/kg bw/day (post-dosing signs during the first five days of dosing at 450 mg/kg bw/day and increased liver weights associated with minimal/slight and non-adverse centrilobular hepatocyte hypertrophy in both sexes given 250 or 450 mg/kg bw/day), there was no effect on survival, clinical condition, body weight gain, food intake or reproductive performance, and no effects of parental treatment on the survival or growth of the F1 litters or the selected F1 generation animals to Day 35 of age. It was therefore concluded that 450 mg/kg bw/day would be suitable for use as the high dose level for the forthcoming main extended one-generation (OECD 443 test guideline) study.