Triethyl phosphate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

• Study Ongoing, Draft reporting expected April 2023: OECD 443 (Oral, Cohorts 1A 1B + 2A 2B)

 

Link to relevant study records

Reference

Reference 1

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

Reason / purpose for cross-reference:

reference to other study

Remarks:

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

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]

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

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.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1: Vehicle control

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

Group 2: Low

Dose / conc.:

450 mg/kg bw/day (nominal)

Remarks:

Group 3: Intermediate

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

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.

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: 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.

Remarks on result:

not measured/tested

Remarks:

Pilot study for EOGRTS only

Critical effects observed:

no

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.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Remarks on result:

not measured/tested

Remarks:

Pilot study for EOGRTS only

Critical effects observed:

no

Reproductive effects observed:

no

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

 

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.

Reference 2

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

Reference 3

Endpoint:

fertility, other

Remarks:

other: repeated dose toxicity studies

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Principles of method if other than guideline:

OECD Guideline 407 and 408 Study

GLP compliance:

yes

Limit test:

no

Justification for study design:

repeated dose toxicity studies including reproductive parameters can be used in a weight of evidence approach for the assessment of reproductive toxicity

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: CH1/3003
- Lab specific test material number: 207699/A
- Expiration date of the lot/batch: 2018-03-09
- Purity: 99.6%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: stability was analytically confirmed for at least 5 hours over the concentration range of 1 to 200 mg/mL and in a refrigerator for at least 10 days. Test facility No. 514832
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: none

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

see endpoint study records listed under 'cross-reference'

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

see endpoint study records listed under 'cross-reference'

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

see endpoint study records listed under 'cross-reference'

Duration of treatment / exposure:

28 day and 90 day study

Frequency of treatment:

once daily

Details on study schedule:

see endpoint study records listed under 'cross-reference'

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

doses of 10, 100, and 1000 mg/kg bw/day were tested in the 28d oral toxicity study

Dose / conc.:

700 mg/kg bw/day

Remarks:

doses of 60, 200, and 700 mg/kg bw/day were tested in the 90d oral toxicity study

No. of animals per sex per dose:

5 per sex/dose in the 28 day study and 10 per sex/dose in the 90 day study

Control animals:

yes, concurrent vehicle

Details on study design:

see endpoint study records listed under 'cross-reference'

Parental animals: Observations and examinations:

Weight determinations in the 90d oral toxicity study: ovaries, testes, uterus (including cervix), prostate, seminal vesicles including coagulating glands
Macroscopic and microscopic in the 90d oral toxicity study: cervix, epididymides, female mammary gland area, ovaries, pituitary gland prostate gland, seminal vesicles incuding coagulation gland, testes, uterus vagina

Oestrous cyclicity (parental animals):

All females in the 90d oral toxicity study had a daily lavage from Day 72 up to and including Day 92 to determine the stage of estrous.

Sperm parameters (parental animals):

From all control and high dosed males of the 90d oral toxicity study additional slides of the testes were prepared to examine staging of spermatogenesis. The testes were processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No test item related changes were noted in estrous cycle determination in the 90d oral toxicity study. One control female (no. 47) showed an irregular estrous cycle length and one 200 mg/kg female (no. 65) showed extended estrus. All other females showed a normal (regular) estrous cycle of 4/5 days. The incidence of irregular estrous cycle length showed no relationship to the dose, and was therefore considered unrelated to treatment.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Spermatogenic staging profiles were normal for all males examined in the control and high dose group in the 90d oral toxicity study.

Weight determinations in the 90d oral toxicity study showed no treatment-related effects on the reproductive organs of male and female rats (ovaries, testes, uterus (including cervix), prostate, seminal vesicles including coagulating glands)
Macroscopic and microscopic investigtions in the 90d oral toxicity study showed no treatment related alterations that differ in prevalence, severity, or histologic character to incidental tissue alterations (cervix, epididymides, female mammary gland area, ovaries, pituitary gland prostate gland, seminal vesicles incuding coagulation gland, testes, uterus vagina)

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: 90 day repeated dose toxicity study

Dose descriptor:

NOAEL

Remarks:

reproductive organ toxicity

Effect level:

>= 700 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: 90 day repeated dose toxicity study

Reproductive effects observed:

no

Repeated dose toxicity of triethyl phosphate was investigated in two comprehensive guideline studies performed according to OECD TG 407 and 408 and conducted in compliance with GLP. In the 28 day study (performed 1992) the test item was given via oral gavage in doses of 0, 10, 100, and 1000 mg/kg bw/day to 5 male and 5 female Wistar rats per dose group. No treatment-related effects on reproductive organs were noted.

In the 90 day study (performed 2017) the test item was given via oral gavage in doses of 0, 60, 200 or 700 mg/kg bw/day to 10 male and 10 female Wistar rats per dose group.

The overall NOAEL for repeated dose toxicity is established at 200 mg/kg bw/day based on effects on liver weight in the 90 day study. The NOAEL for effects to reproductive organs is established with >= 700 mg/kg bw/day, the highest dose in 90 day study. Reproductive parameters assessed in this study (estrous cycle regularity, spermatogenesis staging) and morphology of gonads and accessory reproductive organs revealed no treatment-related changes.There were no effects on the weight of the gonads (males: testes and epididymes, seminal vesicles including coagulating glands; females ovaries, uterus including cervix) or the microscopic examined integrity of the gonads (males: epididymides, prostate, seminal vesicles and testes; females: uterus and ovaries, vagina) at any dose tested.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on fertility: via dermal route

Quality of whole database:

The endpoint will be concluded based on a guideline study undertaken according to GLP and an accepted OECD TG for this endpoint. A GLP OECD 443 (EOGRTS, Cohorts 1A 1B and 2A 2B) is ongoing following ECHA Decision on Compliance Check CCH-D-2114488748-25-01/F. Final reporting is expected October 2023.

Additional information

Summary of studies

 

Preliminary Extended One Generation Study in the Sprague Dawley Rat by Oral Gavage Administration (2022)

In 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), Triethyl phosphate (the test item) was administered by daily oral gavage to male and female Sprague Dawley rats at dose levels of 0, 250, 450 and 800 mg/kg bw/day. F0 animals were exposed from two weeks before pairing until Day 20 of lactation/scheduled termination. F1 animals From weaning on Day 21 to Day 34 of age. Parameters investigated covered general systemic toxic potential, including reproductive/developmental effects.

 

Parental results:

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. 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.

 

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.

 

No toxicologically significant changes were noted in any of the remaining parental parameters investigated i.e. body weight gain, food consumption, estrous cycle regularity and reproductive performance.

 

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. 

 

Reproductive/Developmental results:

No reproductive/developmental toxicity was observed up to the highest dose tested (450 mg/kg bw/day).

 

OECD 443 - EOGRTS Cohorts 1A 1B + 2A 2B (expected 2023)

Following an ECHA decision (ECHA communication/decision number CCH-D-2114513186-55-01/F.), an Extended One Generation Reproductive toxicity study (Annex X, Section 8.7.3.; test method: OECD TG 443) in rats, oral route with the registered substance was commenced. The study design includes: Ten weeks premating exposure duration for the parental (P0) generation; Dose level setting with the aim to induce systemic toxicity at the highest dose level; Cohort 1A (Reproductive toxicity); Cohort 1B (Reproductive toxicity) without extension to mate the Cohort 1B animals to produce the F2 generation); and Cohort 2 (Developmental neurotoxicity). 

 

Other relevant studies

In an early investigation (Gumbmann, 1968) not following GLP or Guideline with only 5 rats/sex and dose the effects of triethyl phosphate in food were investigated. The animals were treated for 92 days prior to mating and during mating and weaning. This study is of low significance due to the low number of animals per dose group, the absence of analytics and definition of doses, insufficient parameters investigated and inadequate description of results.

 

Repeated dose toxicity of triethyl phosphate was investigated in two comprehensive guideline studies performed according to OECD TG 407 and 408 and conducted in compliance with GLP. In the 28 day study (performed 1992) the test item was given via oral gavage in doses of 0, 10, 100, and 1000 mg/kg bw/day to 5 male and 5 female Wistar rats per dose group. No treatment-related effects on reproductive organs were noted.

 

In the 90 day study (performed 2017) the test item was given via oral gavage in doses of 0, 60, 200 or 700 mg/kg bw/day to 10 male and 10 female Wistar rats per dose group.

 

The overall NOAEL for repeated dose toxicity is established at 200 mg/kg bw/day based on effects on liver weight in the 90 day study. The NOAEL for effects to reproductive organs is established with >= 700 mg/kg bw/day, the highest dose in 90 day study. Reproductive parameters assessed in this study (estrous cycle regularity, spermatogenesis staging) and morphology of gonads and accessory reproductive organs revealed no treatment-related changes. There were no effects on the weight of the gonads (males: testes and epididymes, seminal vesicles including coagulating glands; females ovaries, uterus including cervix) or the microscopic examined integrity of the gonads (males: epididymides, prostate, seminal vesicles and testes; females: uterus and ovaries, vagina) at any dose tested.

 

Guideline- and GLP compliant developmental toxicity studies on rats and rabbits do also not point to reproductive toxicity. In rats, gestation rate, intrauterine development, resorption rate and accordingly the number of fetuses, fetal sex and weight and appearance of placentas were unaffected by treatment up to and including the highest dose. The NOAEL for developmental/reproductive toxicity was 625 mg/kg bw/day, the highest dose tested in this study. Also in rabbits no treatment related adverse effects became obvious, with a NOAEL of 250 mg/kg bw/day, representing the highest dose tested.  

 

In summary, based on the available repeated dose and developmental toxicity studies there is no indication of potential interference of the substance with reproduction.

Effects on developmental toxicity

Description of key information

• NOAEL (developmental toxicity) = at least 625 mg/kg bw/day | NOAEL (maternal) = 125 mg/kg bw/day) - OECD 414, Rat, Oral (1995)


• NOAEL (developmental toxicity) = at least 250 mg/kg bw/day | NOAEL (maternal) = 80 mg/kg bw/day) - OECD 414, Rabbit, Oral (2017)

 

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Qualifier:

according to guideline

Guideline:

other: EPA TSCA Test Guidelines (401 CFR Parts 796, 797, 798: 798.4900 Developmental Toxicity Study, September 27, 1985, and Revised Edition May 20, 1987)

Qualifier:

according to guideline

Guideline:

other: EC guidelines (Commision Directive 88/302/EEC, Official Journal of the European Communities L 133, May 30, 1988).

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Route of administration:

oral: gavage

Vehicle:

water

Analytical verification of doses or concentrations:

yes

Details on mating procedure:

The animals were mated by placing two females in a Type III cage with one male rat overnight. If sperm was found in a vaginal smear taken on the morning following mating, this day was regarded as day 0 of gestation.

Duration of treatment / exposure:

from day 6 to day 15 p.c.

Frequency of treatment:

daily

Duration of test:

The fetuses were delivered by cesarian section on the 20th day of gestation.

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

25/dose

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels were selected according to a preceding rang-finding study in 4-5 rats with doses of 300, 500, and 625 mg/kg bw/d.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes, twice daily

DETAILED CLINICAL OBSERVATIONS: Yes, twice daily

BODY WEIGHT: Yes, on days 0, 6-15, and 20 pc

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20

Ovaries and uterine content:

according to Guideline

Fetal examinations:

External, visceral and skeletal examination of the fetuses

Statistics:

Fisher's exact significance test for fertility rate and gestation rate
F-test and t-test or Welch t-test for feed intakes, body weight gains, number of corpora lutea per dam, implantations per dam, live fetuses per dam etc.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The appearance and behavior of the dams was unaffected up to and including the dosage of 125 mg/kg.The dams of the 625 mg/kg group revealed clinical signs like bloddy mouth, staggering gait and ventral posture. The staggering gait and the ventral posture are considered to be a sign of the narcotic efficacy of TEP.

Mortality:

no mortality observed

Description (incidence):

one dam of the control group died on day 18 pc

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

body weight gains of the dams in the 625 mg/kg bw group statistically significant reduced between day 6-15 pc (15.1 versus 23.9 g in control) and day 0-20 corrected (24.5 versus 31.8 g); food consumption was lower in this dose group on day 6-11 pc.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment related findings were evident at necropsy up to and including the dose of 625 mg/kg bw.

Description (incidence and severity):

The number of corpora lutea, preimplantation losses and implantations were comparable in all experimental groups, with exception of the significantly lower preimplantation losses in the 25 mg/kg bw group.

Description (incidence and severity):

none

Description (incidence and severity):

the number of resorptions

Description (incidence and severity):

no changes

Description (incidence and severity):

No. of pregnants: 22, 20, 22, 20

Dose descriptor:

NOAEL

Effect level:

125 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

none: fetal body weight. 3.48, 3.55, 3.59, 3.44 g in control, low, mid, high dose group

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

none: No. of fetuses per dam: 10.5, 11.6, 11.6, 10.6 in control, low, mid, high dose group

Changes in sex ratio:

no effects observed

External malformations:

no effects observed

Description (incidence and severity):

see Table

Skeletal malformations:

no effects observed

Description (incidence and severity):

see Table

Visceral malformations:

no effects observed

Description (incidence and severity):

see Table

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects
External, visceral and skeletal examination of the fetuses revealed no effects on intrauterine development

Dose descriptor:

NOAEL

Effect level:

625 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no treatment related effects on development at any dose

Abnormalities:

no effects observed

Developmental effects observed:

no

External, skeletal and visceral malformations in viable fetuses:

Malformation	Dose (mg/kg b.w./day)
	0	25	125	625
fetuses/ (Litters Affected) (n)	-	-	-	-
Microphtalmia	1	1	1	2 (2)
Hydrocephalus internus	-	-	-	1
Pelvis dislocated	1	-	-	-
Malformation of vertebra	4 (3)	-	1	1
Fusion of ribs	1	-	-	-
Thickened cervical rib	1	-	-	-
Malformation of forelimb bones	1	-	-	-
Exoccipitale dysplastic	1	-	-	-
Dislocation of atlanto-occipital joint (probably artifact +)	-	1	-	1
Fetuses per group (n)	231	232	242	213
malformed fetuses (n)	7	2	2	5
(%)	3,0	0,9	0,8	2,4
Litters per Group (n)	22	20	22	20
Litters with malformations (n)	5	2	1	3
(%)	22,7	10,0	5,0	15,0

+ = nevertheless included in the calculation

some fetuses revealed more than one malformation, therefore the number of malformations does not necessarily reflect the number of fetuses affected.

Executive summary:

In a developmental toxicity study following OECD 414 groups of 25 inseminated female Wistar rats per dose group were treated daily by gavage with TEP formulated in demineralised water from days 6 to day 15 p.c. in doses of 0, 25, 125 or 625 mg/kg body weight, respectively. The fetuses were delivered by cesarian section on day 20 of gestation.

The dams of the 625 mg/kg group revealed clinical signs like bloddy mouth, staggering gait and ventral posture. The staggering gait and the ventral posture are considered to be a sign of the narcotic efficacy of TEP.

Body weight gain (day 6 -15 p.c.), feed intake (day 6 -11p.c.) and accordingly the feces excretion were reduced in the 625 mg/kg group.

The intrauterine development was not affected by the treatment including external, visceral and skeletal examination of the fetuses. A teratogenic potential was not evident.

The NOAEL were:

Maternal toxicity: 125 mg/kg bw./day

Developmental toxicity: 625 mg/kg bw./day.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016/2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: stability was analytically confirmed for at least 5 hours over the concentration range of 1 to 200 mg/mL and in a refrigerator for at least 10 days, Test Facility Study No. 514832
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: none

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Chatillon sur Chalaronne, France
- Untreated females from a non-inbred laboratory colony were mated at the suppoier and were at day 0 or 1 post-coitum on arrival at the Test Facility (day 0 post-coitum is the day of successful mating)
- Age at delivery: 17-19 weeks;
- Fasting period before study: none
- Housing: individually
- Diet: ad libitum
- Water: ad libitum

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the test item. No correction was made for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle (if other than water): water was chosen based on trial formulations performed at Charles River and information provided by the Sponsor.
- Amount of vehicle (if gavage): 5 ml/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of dose preparations were taken on a single occasion during the treatment phase (20 February 2017), and were stored and dispatched on dry ice to the test site for formulation analysis. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.
Stability in the vehicle was confirmed in a separate study (Test Facility Study No. 514832).

Details on mating procedure:

Untreated females were mated at the Supplier and were at Day 0 or 1 post-coitum on arrival at the Test Facility (Day 0 post-coitum is the day of successful mating).

Duration of treatment / exposure:

Day 6 - 28 post coitum inclusive

Frequency of treatment:

once daily

Duration of test:

All animals surviving to the end of the observation period (Day 29 post-coitum) were euthanised by intravenous injection of pentobarbital (approx. 1 mL/kg Euthasol®20%) and subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs and the fetuses.

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

Each group consisted of 22 mated female rabbits.

Control animals:

yes, concurrent vehicle

Details on study design:

Rationale for dose levels
Dose levels were selected based on the results of a dose range finding study (Project 514830). In this latter study dose levels of 75, 150 and 300 mg/kg bw/day were tested in 6 pregnant rabbits per dose and control from gestational day 6 to 29 by oral gavage. One female in the mid and high dose each was euthanized in extremis on day 18 pc. Both females had shown body weight loss and (severely) reduced food consumption. Calm and/or lethargic behavior was noted in all females of the treatment groups after dosing, showing a dose related incidence and severity relationship. In all females treated at 300 mg/kg, except one, additional flat posture, ventro-lateral and/or lateral recumbency was observed on many days during treatment. Reduced food consumption between Days 6-16 post-coitum with concurrent body weight loss in this period was observed in females treated at 300 mg/kg. Both subsequently recovered. In the 150 mg/kg bw group transient periods of lower food consumption and body weight gain was observed. No effects were seen at 75 mg/kg bw. A slight, not statistically significant increase in liver weight was recorded for the high dose group. No effects on pregnancy or fetuses was seen.
Based on the results of this dose range finding study, selected dose levels for the main study were 30, 80 and 250 mg/kg bw/day.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Days 2, 6, 9, 13, 16, 20, 23, 26, 29 post-coitum

FOOD CONSUMPTION: Yes
- Days 2-6, 6-9, 9-13, 13-16, 16-20, 20-23, 23-26 and 26-29 post-coitum

WATER CONSUMPTION: Yes
- Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: external, thoracic and abdominal examination, with special attention being paid to the reproductive organs;
- Terminal body weight, liver weight and gravid uterus weight were recorded

Ovaries and uterine content:

The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated.

Fetal examinations:

External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or represent slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).

External:
Each viable fetus was examined in detail and weighed. All live fetuses were euthanized by administration of approximately 0.3 mL (= 60 mg) of sodium pentobarbital into the oral cavity using a small flexible plastic or metal feeding tube. Recognizable fetuses of females that were killed in extremis were examined externally. Nonviable fetuses (the degree of autolysis was minimal or absent) were examined and weighed. For late resorptions a gross external examination was performed (if possible). Late resorptions with malformations were fixed in 10% buffered formalin.

Visceral (Internal):
All fetuses were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected using a technique described by Stuckhardt and Poppe. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development as described by Woo and Hoar. The sex of all fetuses was determined by internal examination.

The heads were removed from approximately one-half of the fetuses in each litter and placed in Bouin's solution. Tissues were then transferred to a 70% aqueous ethanol (Klinipath, Duiven, The Netherlands) for subsequent processing and soft-tissue examination of all groups using the Wilson sectioning technique. After examination, the tissues were stored in 10% formalin. The heads from the remaining one-half of the fetuses in each litter of all groups were examined by a mid-coronal slice.

All carcasses, including the carcasses without heads, were eviscerated, skinned and fixed in identified containers containing 96% aqueous ethanol for subsequent examination of skeletons.

Skeletal:
The eviscerated fetuses from all groups, following fixation in 96% aqueous ethanol, were macerated in potassium hydroxide and stained with Alizarin Red S by a method similar to that described by Dawson. Subsequently, the skeletal examination was done on all fetuses from Groups 1 and 4. Based on a possible treatment related effect in the high dose group, skeletal examination was extended to all fetuses from the low and mid dose group.

The specimens of all groups were archived in glycerin with bronopol as preservative.

A few bones were not available for skeletal examination because they were accidentally damaged or lost during processing. The missing bones were listed in the raw data; evaluation by the fetal pathologist and study director determined there was no influence on the outcome of the individual or overall skeletal examinations, or on the integrity of the study as a whole.

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control group.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Mann Whitney test was used to compare mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and sex distribution.
- Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations might be rounded off before printing. Therefore, two groups might display the same printed means for a given parameter, yet display different test statistics values.

No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.

Indices:

For each litter the following calculations were performed:

Pre-implantation loss (%) = (number of corpora lutea - number of implantation sites) divided by the number of corpora lutea x 100
Post-implantation loss (%) = (number of implantation sites - number of live fetuses) divided by the number of implantation sites x 100
The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison, calculating the number of affected fetuses as a mean litter proportion on a total group basis, where:

Viable fetuses affected/litter (%) = number of viable fetuses affected/litter divided by the number of viable fetuses/litter x 100

Historical control data:

available

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No maternal toxicity was observed up to and including 80 mg/kg.
The majority of the high dose females treated at 250 mg/kg bw/day showed treatment related calm behavior during several days up to the entire treatment period, ranging from slight to moderate severity. Moderate effects were seen on 1-2 days only in 3 animals.
Reduced faeces production, varying in duration and severity, was noted in all groups, including the vehicle control group. The incidence was increased in the 250 mg/kg bw/day treated females compared to the concurrent controls (i.e. 13 out of 22 control females compared with 19 out of 22 high dose females).
Female no. 21 (control group) showed moderately labored respiration directly after dosing on Day 7 post-coitum. Based on the slightly labored respiration observed on Day 8 post-coitum, dosing was omitted for this female on this day and continued from Day 9 post-coitum onwards.
For female nos. 05 (control), 29 (30 mg/kg bw/day), 65, 661 (80 mg/kg bw/day) and 70 (250 mg/kg bw/day) incidental red fluid was observed on the manure tray during the treatment period. All these females were pregnant with viable fetuses. Red fluid on the manure tray was also noted for female no. 87 between Days 13-25 post-coitum. This female had resorptions only. At the incidence observed and without a dose related incidence trend, this was considered not treatment related.
The incidence of alopecia, wounds, scabs, scars, slight swelling, piloerection, dark eyes, broken teeth, lean appearance, diarrhea remained within the range of background findings to be expected for rabbits this strain and age.

Mortality:

no mortality observed

Description (incidence):

No treatment related mortality occurred up to 250 mg/kg bw/day.
Female no. 38 (30 mg/kg bw/day) and no. 69 (250 mg/kg bw/day) were euthanized in extremis on Day 16 and 18 post-coitum, respectively. Both females had limited to no food consumption for a prolonged period (up to 16 days) and showed a body weight loss ranging between 4 to 11%. For female no. 69 piloerection and pale and lean appearance were noted up to 4 days prior to euthanasia. These effects are not uncommon for rabbits of this strain and age. At the incidence observed and without a dose relationship, this was considered not treatment-related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights and body weight gain were unaffected by treatment up to and including 80 mg/kg bw/day.
At 250 mg/kg bw/day, absolute and relative food consumption was reduced from start treatment onwards up to Day 23 post-coitum. Concurrent body weight loss on Day 9 post-coitum (3 days after start treatment) was observed in the majority of the 250 mg/kg bw/day treated females, with continued body weight loss in individual animals throughout the treatment period. Between Days 13 and 23 post-coitum, mean body weight gain was reduced in these high dose females compared with the concurrent controls; not reaching statistical significance. From Day 26 post-coitum onwards, body weight gain was within the range of the control group. The absolute mean body weight was in the range of the control group from day 23 post-coitum onwards.
Body weight gain corrected for weight of the gravid uterus was unaffected by treatment up to 250 mg/kg bw/day at sacrifice.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption:
No treatment-related effects were observed up to 80 mg/kg bw/day.
In the first 3 days of treatment absolute and relative food consumption was strongly and significantly reduced in 250 mg/kg bw/day treated females compared with the vehicle control group. Up to Day 23 post-coitum, absolute and relative food consumption were still reduced in the 250 mg/kg bw dose group (only reaching statistical significance at Days 16-20 postcoitum). From Day 23 post-coitum onwards, food consumption was within the range of the control group again.
Reduced faeces production, varying in duration and severity, was noted in all groups, including the vehicle control group. However, as the incidence was increased in females treated at 250 mg/kg compared with the vehicle controls, low and mid dose groups, which could be correlated with the reduced food consumption in this group, it was therefore considered treatment related.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Absolute and relative liver weights and (gravid) uterus weight remained within the same range as the vehicle control group up to 250 mg/kg.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment-related macroscopic findings were noted up to 250 mg/kg bw/day.
A reduced size of the gallbladder was observed for one female of the 30 mg/kg bw/day group (no. 38) which was euthanized in extremis on Day 16 post-coitum; no macroscopic findings were noted for female no. 69 (250 mg/kg bw/day), which was also euthanized in extremis.
Incidental findings at macroscopic examination were within the normal range for rabbits of this strain and age.

Number of abortions:

no effects observed

Description (incidence and severity):

see Table 1

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

see Table 2: The number of corpora lutea, implantation sites and pre-implantation loss in the control and treatment groups were similar and within the range of normal biological variation.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

see Table 1: Two individual females with resorptions only i.e. female nos. 55 (80 mg/kg) and 87 (250 mg/kg) were observed as incidental finding.

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

see Tables 2 and 3: The number of early resorptions and consequently the post-implantation loss were increased in the 80 and 250 mg/kg groups compared with the concurrent controls (not statistically significant). The percentage of early resorptions per litter was 7.4 and 8.1 in the mid and high dose groups, respectively, compared with 4.0 in the concurrent control group. It should be noted that individual females with resorptions only contributed to the increased percentages of early resorptions in these groups; i.e. female nos. 55 (80 mg/kg bw/day) and 87 (250 mg/kg bw/day). When excluding these females, the total number of early resorptions was 7, 5, 4 and 6 for the control, 30, 80 and 250 mg/kg bw/day groups, respectively.

This increase was contributed to two individual females in each of these dose groups with resorptions only, which were considered incidental findings. In the remaining females the number of early resorptions and post implantation loss remained within the same range compared with the concurrent controls. Taken together, the observed effects were considered not to be treatment related.

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

see Table 1: A total of eight females were found not pregnant at necropsy; four, zero, three and one in the respective control, 30 mg/kg bw, 80 mg/kg and 250 mg/kg groups. As this occurred mainly in the control group and treatment started on Day 6 post-coitum after implantation, this was incidental and not related to treatment.

Details on maternal toxic effects:

The combination of calm behavior (slight severity), reduced food consumption and concurrent body weight effects at 250 mg/kg was considered treatment related and adverse.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: combination of calm behavior (slight severity), reduced food consumption and concurrent body weight effects at 250 mg/kg bw

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

see Table 2: No treatment related effect on fetal body weight was observed up to 250 mg/kg bw/day.
Mean combined (male and female) fetal body weights were 40.4, 38.5, 40.7 and 38.8 grams for the vehicle control, 30, 80 and 250 mg/kg bw/day groups, respectively.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

see Table 2: The male:female ratio was unaffected by treatment up to 250 mg/kg bw/day.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

see Table 2: There were no treatment related effects on litter size for any group.
Mean litter sizes were 8.4, 10.1, 9.2 and 9.2 fetuses per litter for the control, 30, 80 and 250 mg/kg bw/day groups, respectively.

External malformations:

no effects observed

Description (incidence and severity):

see Tables 4 and 5: The numbers of fetuses (litters) available for fetal morphological examination were 152 (18), 213 (21), 175 (18) and 183 (19) in the control, 30, 80 and 250 mg/kg bw/day groups, respectively. External and visceral examinations were done for all fetuses, soft tissue cephalic examination was done for approximately half of the fetuses for all groups, and skeletal examination was done for all fetuses of the control and high dose groups, initially. However, as the findings of 13th full ribs and caudal shift of pelvic girdle were noted at higher incidences at the high dose than in the control group, it was decided to extend the skeletal examinations to the low and mid dose groups.
There were no treatment related effects on external morphology following treatment up to 250 mg/kg.

Beside one fetus at the high dose (A078-06) with exencephaly, two late resorptions had a malformation. One fetus at 80 mg/kg (A047-12) had omphalocele and one at 250 mg/kg (A085-11) had anasarca. The single occurrence of these malformations do not indicate a relation to treatment and therefore were considered to be chance findings.
There were no external variations seen for any fetus in any group.

Description (incidence and severity):

see Tables 4 and 5:
The incidence of 13th full ribs in the control group was 27.8%, which is nearby the minimum historical control value of 26.8% (range 26.8% up to 71.4%). Therefore, this incidence was statistically significantly increased in all treatment groups. The increased incidences at 30 and 80 mg/kg (48.7% and 58.4% per litter, respectively) were considered not related to treatment, as the incidences remained within the historical control data and were not accompanied by an increased incidence of caudal shift of pelvic girdle (10.5% and 8.9% per litter, respectively compared with 9.0% per litter in the controls). The incidence at 250 mg/kg (74.3%), however, was higher than the historical control maximum value (71.4% per litter) and was accompanied by a statistically significantly increased incidence of caudal shift of pelvic girdle (33.0% versus control value 9.0% per litter). Therefore, the combination of these findings was considered treatment related. However, as these developmental variation findings were only seen at the highest dose leading to maternal toxicity and do not affect further development and viability of the fetuses, these findings were judged as not adverse.
Historical control 13th full ribs (% per litter in 26 studies in New Zealand White rabbits with in total 3934 control fetuses in 434 litters): mean = 48.7; min = 26.8; max = 71.4

The number of fetuses with fused sternebrae at 250 mg/kg/day was higher than in the other groups. This skeletal malformation was observed in 2 (2), 1 (1), 1 (1) and 5 (3) fetuses (litters) at an incidence of 1.8 %, 0.4%, 0.5% and 2.7% per litter in the control, 30, 80 and 250 mg/kg groups, respectively. The incidence at 250 mg/kg was just above the historical control maximum value (2.4% per litter), but given the absence of a dose response and statistical significance and because it is the most common skeletal malformation in the historical control database, it was not considered to be treatment related.

Remaining skeletal malformations occurred singly and as these occurred in different groups, they were not considered to be treatment related.

Visceral malformations:

no effects observed

Description (incidence and severity):

see Tables 4 and 5: There were no treatment related effects on visceral morphology following treatment up to 250 mg/kg.
Visceral malformations occurred in 2 (2), 2 (2), 2 (1) and 2 (2) fetuses (litters) in the control, 30, 80 and 250 mg/kg groups, respectively. Absence of a lung lobe was most frequently noted and it occurred in 0, 1, 2 and 2 fetuses in these respective groups. As a dose response for this finding could not be established and as it is also the most common visceral malformation in historical controls (highest incidence in historical control groups is three ), it was not considered to be treatment related.
The other visceral malformations occurred singly and therefore were considered to be chance findings.
All the variations noted, were not considered treatment related as they occurred infrequently, occurred at frequencies that were within the range of available historical control data or were observed in control fetuses only.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No toxicologically relevant adverse changes were noted in any of the developmental parameters investigated in this study.

Abnormalities:

no effects observed

Description (incidence and severity):

no adverse effects observed

Developmental effects observed:

no

Accuracy, homogeneity and stability of formulations were demonstrated by analyses. Stability of the test item was confirmed.

Table 1: SUMMARY OF MATERNAL SURVIVAL AND PREGNANCY STATUS

Dose group	control		30		80		250
	No.	%	No.	%	No.	%	No.	%
Females on study	22		22		22		22
Females that aborted or delivered	0	0.0	0	0.0	0	0.0	0	0.0
Females that died	0	0.0	0	0.0	0	0.0	0	0.0
Females that aborted	0	0.0	0	0.0	0	0.0	0	0.0
Nongravid	0	0.0	0	0.0	0	0.0	0	0.0
Gravid	0	0.0	0	0.0	0	0.0		0.0
Females that were euthanized	0	0.0	1	4.5	0	0.0	1	4.5
Nongravid	0	0.0	0	0.0	0	0.0	0	0.0
Gravid	0	0.0	1	100.0	0	0.0	1	100.0
Females examined at scheduled necropsy	22	100.0	21	95.5	22	100.0	21	95 .5
Nongravid	4	18.2	0	0.0	3	13.6	1	4.8
Gravid	18	100.0	21	100.0	19	86.4	20	95.2
with resorptions only	0	0	0	0.0	1	5.3	1	5.0
with viable fetuses	18	100.0	21	100.0	18	94.7	19	95.0
Total females gravid	18	81.8	22	100.0	19	86.4	21	95.0

Table 2: SUMMARY OF FETAL DATA AT SCHEDULED NECROPSY

Group		Sex  M	Sex  F	Viable fetuses	Dead fetuses	Resorptions  Early	Resorptions Late	Post implantation loss	Implantation sites	Corpora lutea	Pre implantation loss	Fetal weights in grams	No. of gravid females
control	Total	83	60	152	0	7	5	12	164	175	12	NA	18
	Mean	4.6	3.8	8.4	0.0	0.4	0.3	0.7	9.1	9.7	0.7	40.4
30 mg/kg	Total	107	106	213	0	5	0	5	218	224	6	NA	21
	Mean	5.1	5.0	10.1	0.0	0.2	0.0	0.2	10.4	10.7	0.3	38.5
80 mg/kg	Total	88	87	175	0	10 (4)#	5	15	190	197	7	NA	19
	Mean	4.6	4.6	9.2	0.0	0.5	0.3	0.8	10.0	10.4	0.4	40.7
250 mg/kg	Total	88	95	183	0	17 (6)#	4	21	204	221	17	NA	20
	Mean	4.4	4.8	9.2	0.0	0.9	0.2	1.1	10.2	11.1	0.8	38.8

None significantly different from control group

NA = NOT APPLICABLE

MEAN NUMBER OF VIABLE FETUSES, MEAN NUMBER OF IMPLANTATION SITES, MEAN NUMBER OF CORPORA LUTEA,

FETAL WEIGHTS COMPARED USING DUNNETT'S TEST

# substracting females No. 55 (80 mg/kg) and No. 87 (250 mg/kg) with early resorptions only the percentages shown in parenthesis result

Table 3: SUMMARY OF FETAL DATA AT SCHEDULED NECROPSY [% PER LITTER]

Group	0 MG/KG	30 MG/KG	80 MG/KG	250 MG/KG
Number of litters	18	21	19	20
Corpora lutea
Mean	9.7	10.7	10.4	11.1
Implantation sites
Mean	9.1	10.4	10.0	10.2
Viable fetuses (%)
Mean	93.9	97.9	90.6	90.2
Dead fetuses (%)
Mean	0.0	0.0	0.0	0.0
Early resorptions (%)
Mean	4.0	2.1	7.4#	8.1#
Late resorptions (%)
Mean	2.2	0.0	2.0	1.7
Total resorptions (%)
Mean	6.2	2.1	9.4#	9.8#
Pre-implantation loss (%)
Mean	6.6	2.6	3.8	6.8
Post-implantation loss (%)
Mean	6.2	2.1	9.4	9.8
Males (%)
Mean	58.7	49.8	49.5	46.0
Females (%)
Mean	41.3	50.2	50.5	54.0
Male fetal weights (g)
Mean	40.2	38.5	40.5	54.0
Female fetal weights (g)
Mean	39.4	38.5	40.8	38.5
Combined fetal weights (g)
Mean	40.4	38.5	40.7	38.8

PROPORTIONAL (%) DATA COMPARED USING THE MANN-WHITNEY TEST

FETAL WEIGHTS COMPARED USING DUNNETT'S TEST

None significantly different from control group

# please take into consideration the explanation given in Table 2

Table 4: SUMMARY OF FETUSES AND LITTERS WITH MALFORMATIONS [ABSOLUTE NO. (% per litter)]

	Fetuses				Litters
Dose group (mg/kg bw per day:	0	30	80	250	0	30	80	250
Number examined	152	213	175	183	18	21	18	19
EXTERNALLY: exencephaly	0	0	0	1	0	0	0	1 (0.5%)
VISCERALLY: teratology of Fallot	0	1	0	0	0	1 (0.4%)	0	0
lung- absent lobe (s)	0	1	2	2	0	1 (0.4%)	1 (0.9%)	2 (1.0%)
right subclavian - originating from the pulmonary trunk	1	0	0	0	1 (0.7%)	0	0	0
vena cava - malpositioned	0	0	1	0	0	0	1 (0.4%)	0
lung - abnormal lobation	1	0	0	0	1 (0.7%)	0	0	0
SKELETALLY: vertebral anomaly with or without associated rib anomaly	1	0	0	0	1 (0.7%)	0	0	0
sternebrae fused	2	1	1	5	2 (1.8%)	1 (0.4%)	1 (0.4%)	3 (2.7%)
Vertebral centra anomaly	0	0	1	0	0	0	1 (0.5%)	0
Total number with malformations
External:	0	0	0	1	0	0	0	1 (0.5%)
Soft tissue:	2	2	2	2	2 (1.4%)	2 (0.8%)	1 (0.9%)	2 (1.0%)
Skeletal:	2	1	2	6	2 (1.8%)	1 (0.4%)	1 (1.0%)	3 (3.1%)
Combined:	3	3	4	9	3 (2.5%)	3 (1.3%)	2 (1.9%)	5 (4.7%)

Table 5: SUMMARY OF FETUSES AND LITTERS WITH VARIATIONS [ABSOLUTE NO. (% per litter)]

	Fetuses				Litters
Dose group (mg/kg bw per day:	0	30	80	250	0	30	80	250
Number examined	152	213	175	183	18	21	18	19
SKELETALLY: 13th full rib(s)	39	98	100	137	14 (27.8%)	20 (48.7%*)	17 (58.4%**)	19 (74.3%**)
sternebra (e) malaligned (slight or moderate)	11	23	19	14	8 (7.6%)	10 (10.2%)	11 (10.1%)	9 (8.6%)
metacarpal (s) and/or metatarsal (s) unossified	10	6	7	6	4 (5.0%)	3 (2.6%)	5 (3.6%)	4 (2.8%)
sternebra (e) #5 and/or #6 unossified	29	53	51	28	11 (19.0%)	18 24.0%)	14 (27.9%)	14 (15.7%)
pelvic girdle - caudal shift	10	22	18	61	4 (9.0%)	8 (10.5%)	8 (8.9%)	16 (33.0%**)
13th rudimentary rib(s)	17	20	22	16	9 (11.7%)	12 (9.3%)	11 (11.4%)	12 (8.7%)
sternebra (e) - branched	1	0	0	0	1 (0.7%)	0	0	0
hyoid body and/or arches unossified	0	2	1	2	0	2 (0.9%)	1 (0.5%)	2 (1.0%)

* = Significantly different from the control group at 0.05

** = Significantly different from the control group at 0.01

Executive summary:

Triethyl phosphate was tested in a prenatal developmental toxicity study on 22 pregnant New Zealand rabbits per dose group following OECD TG 414. The test item was administered once daily by oral gavage from Days 6 to 28 post-coitum at doses of 0, 30, 80 and 250 mg/kg bw/day in water. The doses were chosen on the basis of a dose range finding study with doses of up to 500 mg/kg bw/day.

No maternal toxicity was observed up to and including 80 mg/kg bw/day. At 250 mg/kg bw/day, the majority of the high dose females showed treatment related calm behavior during several days up to the entire treatment period, ranging from slight to moderate severity. Reduced faeces production, varying in duration and severity, was noted in all groups, including the vehicle control group. However, as the incidence was increased in females treated at 250 mg/kg compared with the vehicle controls, low and mid dose groups, which could be correlated with the reduced food consumption in this group, it was therefore considered treatment related.

At 250 mg/kg, absolute and relative food consumption was reduced from start of treatment onwards up to Day 23 post-coitum. Concurrent body weight loss on Day 9 post-coitum (after 3 days of treatment) and reduced body weight gain between Day 13 and 23 post-coitum were also noted. From Day 23 and 26 post-coitum onwards absolute body weight and body weight gain were within the range of the control group, respectively. Body weight gain corrected for weight of the gravid uterus was unaffected by treatment up to 250 mg/kg bw/day.

The combination of calm behavior, ranging from slight to moderate severity, and reduced food consumption with concurrent body weight effects at 250 mg/kg was considered treatment related and adverse.

No adverse effects on development were observed in the 30, 80 and 250 mg/kg groups.

An increased incidence of non-adverse treatment related variations (i.e. 13th full ribs and caudal shift of the pelvic girdle) was noted at 250 mg/kg. The incidence of 13th full ribs was statistically significantly increased in all treatment groups. It should be noted that the control value was at the lower end of the historical control data. The increased incidences at 30 and 80 mg/kg bw/day (48.7% and 58.4% per litter, respectively) were considered not related to treatment, as the incidences remained within the historical control data and were not accompanied by an increased incidence of caudal shift of pelvic girdle (10.5% and 8.9% per litter, respectively compared with 9.0% per litter in the controls). The incidence at 250 mg/kg bw/day (74.3%), however, was higher than the historical control maximum value (71.4% per litter) and was accompanied by a statistically significantly increased incidence of caudal shift of pelvic girdle (33.0% versus control value 9.0% per litter). Therefore, the combination of these findings was considered treatment related. However, as these developmental variation findings do not affect further development and viability of the fetuses, these findings were not adverse. Furthermore, this finding was observed at a dose level resulting in maternal toxicity.

The number of early resorptions and consequently the post-implantation loss were increased in the 80 and 250 mg/kg bw/day groups compared with the concurrent controls. This increase was contributed to two individual females in each of these dose groups with resorptions only, which were considered incidental findings. In the remaining females the number of early resorptions and post implantation loss remained within the same range compared with the

concurrent controls. Taken together, the observed effects were considered not to be treatment related.

Based on the results in this prenatal developmental toxicity study the maternal No Observed Adverse Effect Level (NOAEL) for Triethyl phosphate was established as being 80 mg/kg bw/day based on calm behavior, reduced food consumption and transient body weight effects at 250 mg/kg bw/day. The developmental NOAEL was determined to be at least 250 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Quality of whole database:

The endpoint is concluded based on two guideline studies undertaken according to GLP and an accepted OECD TG for this endpoint (OECD 414). Both are assigned a Klimisch rating of 1: reliable without restrictions.

Additional information

Summary of studies 

 

Prenatal developmental toxicity in first species (OECD 414, rat, 1995)

In a developmental toxicity study following OECD 414 groups of 25 inseminated female Wistar rats per dose group were treated daily by gavage with TEP formulated in demineralised water from days 6 to day 15 p.c. in doses of 0, 25, 125 or 625 mg/kg body weight, respectively. The fetuses were delivered by cesarian section on day 20 of gestation.

 

The dams of the 625 mg/kg group revealed clinical signs like bloddy mouth, staggering gait and ventral posture. The staggering gait and the ventral posture are considered to be a sign of the narcotic efficacy of TEP.

 

Body weight gain (day 6 -15 p.c.), feed intake (day 6 -11p.c.) and accordingly the feces excretion were reduced in the 625 mg/kg group.

 

The intrauterine development was not affected by the treatment including external, visceral and skeletal examination of the fetuses. A teratogenic potential was not evident.

 

The NOAEL were:

Maternal toxicity: 125 mg/kg bw/day

Developmental toxicity: 625 mg/kg bw/day.

 

Prenatal developmental toxicity in second species (OECD 414, rabbit, 2017)

Triethyl phosphate was tested in a prenatal developmental toxicity study on 22 pregnant New Zealand rabbits per dose group following OECD TG 414. The test item was administered once daily by oral gavage from Days 6 to 28 post-coitum at doses of 0, 30, 80 and 250 mg/kg bw/day in water. The doses were chosen on the basis of a dose range finding study with doses of up to 500 mg/kg bw/day.

 

No maternal toxicity was observed up to and including 80 mg/kg bw/day. At 250 mg/kg bw/day, the majority of the high dose females showed treatment related calm behavior during several days up to the entire treatment period, ranging from slight to moderate severity. Reduced faeces production, varying in duration and severity, was noted in all groups, including the vehicle control group. However, as the incidence was increased in females treated at 250 mg/kg compared with the vehicle controls, low and mid dose groups, which could be correlated with the reduced food consumption in this group, it was therefore considered treatment related.

 

At 250 mg/kg, absolute and relative food consumption was reduced from start of treatment onwards up to Day 23 post-coitum. Concurrent body weight loss on Day 9 post-coitum (after 3 days of treatment) and reduced body weight gain between Day 13 and 23 post-coitum were also noted. From Day 23 and 26 post-coitum onwards absolute body weight and body weight gain were within the range of the control group, respectively. Body weight gain corrected for weight of the gravid uterus was unaffected by treatment up to 250 mg/kg bw/day.

 

The combination of calm behavior, ranging from slight to moderate severity, and reduced food consumption with concurrent body weight effects at 250 mg/kg was considered treatment related and adverse.

 

No adverse effects on development were observed in the 30, 80 and 250 mg/kg groups. An increased incidence of non-adverse treatment related variations (i.e. 13th full ribs and caudal shift of the pelvic girdle) was noted at 250 mg/kg.

 

Based on the results in this prenatal developmental toxicity study the maternal No Observed Adverse Effect Level (NOAEL) for Triethyl phosphate was established as being: 

Maternal NOAEL = 80 mg/kg bw/day (based on calm behavior, reduced food consumption and transient body weight effects at 250 mg/kg bw/day).

Developmental NOAEL = at least 250 mg/kg bw/day.

Justification for classification or non-classification

Based on a weight of evidence evaluation of the available data for reproduction toxicity and Guideline-compliant developmental toxicity studies on rats and rabbits a classification for fertility and/or developmental toxicity according to EU Regulation 1272/2008 is not warranted.