Triethyl phosphonoacetate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2016-02-25 to 2017-02-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted in compliance with international standard guidelines under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

2015-09-22

Test material

Test material information

Constituent 1

Reference substance name:

Triethyl phosphonoacetate

EC Number:

212-757-6

EC Name:

Triethyl phosphonoacetate

Cas Number:

867-13-0

Molecular formula:

C8H17O5P

IUPAC Name:

ethyl 2-(diethoxyphosphoryl)acetate

Test material form:

liquid

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D- 97633 Sulzfeld
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Young adult rats (6-7 weeks) at start of treatment.
- Weight at study initiation: Not exceed ± 20% of the mean weight for each sex at onset of treatment males: 247-279 g, females: 169-195 g
- Housing: Not exceed ± 20% of the mean weight for each sex at onset of treatment males: 247-279 g, females: 169-195 g
- Diet (e.g. ad libitum): Animals received ssniff® SM R/M "Autoclavable complete diet for rats and mice –breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany
- Water (e.g. ad libitum): tap water from municipal supply, as for human consumption from 500 mL bottles
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.0 - 26.5°C
- Humidity (%): 34 - 79 %
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): artificial lighting 12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES: From: 19 April 2016 To: 19 July 2016

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The oral route was selected as it is a possible route of exposure to the test item in humans.

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle, at the appropriate concentrations according to the dose level and volume selected, in the Pharmacy of CiToxLAB Hungary Ltd. Formulations were prepared freshly prior to administration to animals or at the appropriate frequency to allow their use according to stability assessment results of the analytical method validation study of the Test Site (CiToxLAB France study codes are 38534VAA (2012) and 38535 AHS (2012)). Based on the results, formulations in the 2-200 mg/mL concentration range (covering the range of 20-2000 mg/kg bw/day) were found to be stable after 9 days of storage at room temperature and protected from light.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of test item formulations for concentration and homogeneity were performed at the Test Site according to the validated analytical method. Top, middle and bottom duplicate samples (1 mL weighted accurately) were taken from test item formulations at four times (during the first, fifth, ninth and last week of the study), one set to analyze and one set as a back-up. Similarly, one sample was taken in duplicate from the Group 1 (control) solution for concentration measurements.
Formulation samples were kept at room temperature protected from light until shipment. Samples (both sets) were shipped under the requested conditions as soon as possible after collection for concentration and homogeneity measurement.

DETERMINATION OF triethyl phosphonoacetate CONCENTRATIONS IN DOSE FORMULATION SAMPLES
Dose formulation samples preparation
The dose formulations were appropriately diluted according to the validated analytical method and analyzed by Gas Chromatography with Flame Ionization Detection within their stability period. Single analysis was performed for each sample.

Acceptance criteria
Control dose formulation: no peak should be observed at the test item retention time or any interference peak at the test item retention time should be below the Limit Of Quantification.

Dose formulations:
The mean measured concentration should be within ± 10% of the theoretical concentration. The precision (Coefficient of Variation CV%, n = 3) should be ≤ 5%.

Dose formulations stability
Stability data of test item stored at room temperature and protected from light for 9 days were obtained under GLP conditions in CiToxLAB France/Study No. 38535 AHS.
Dose formulations prepared by CiToxLAB Hungary Ltd. were analyzed within this period, which was calculated as the max delay between the preparation of the dose formulations at CiToxLAB Hungary Ltd. and their analysis at CiToxLAB France.

Computerized systems
Critical computerized systems: Description of data collected and/or analyzed
Empower 2 version Build 2154: Acquisition and management of chromatographic data.
CITPharma (CITAC) version 2: Test item receipt and inventories, reagent, matrix. Panorama E2 version 2.60.0000 Acquisition of temperature and humidity in study rooms (study and laboratory rooms, cold chambers).
CITAC - CIT master version 2: CIT Application Center: Web business portal. Master schedule sheet (including Study Note). Master schedule sheet - Study event.

Chromatographic conditions
Column: DB-17 (Agilent), Film = 1µm, length = 30m, diameter = 0.53 mm
Mobile phase: Hydrogen
Detector: FID
Sensitivity: 12
Column temperature: From 80°C (stay 1min) to 250 °C (Stay 2 min) at 10°C/min
Constant flow rate: 5 mL/min
Split rate: 10
Software: Empoxer 2 (Waters)
Detector temperature: 250 °C
Injector temperature: 220 °C
Injected volume: 2 µL
Detector gas flow: 30 mL/min for make up, 30 mL/min for hydrogen and 300 mL/min for air.
Retention time: TEPA: 11.6 min
Analysis time: 20 min.

Quantification:
The concentration of the test item will be determined from the mean response factor of TEPA in standard solutions
The sample concentrations will be determined using the following equation:
[concentration] = (Area sample/ Standard mean response factor) x dilution factor where:
Standards mean response factor = Mean response factor of standard solutions 1 and 2 (n = 10)
Dilution factor (Also including conversion between units if required)
Response factor = Area standard / concentration standard

Acceptance criteria
Precision of response factor for Standard solution 1: Coefficient of variation CV% < 5%
Precision of response factor for Standard solution 2: Coefficient of variation CV% < 5%
Precision of response factor for Standard solution 1 and 2 : Coefficient of variation CV% < 5%
Accuracy of the response factor of the standards (ratio of mean response factor of standard solution 1 with mean response factor of standard solution 2) should be between 90% and 110%

Duration of treatment / exposure:

90 days

Frequency of treatment:

Once a day

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

No. of animals per sex per dose:

10 animals per sex and dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The doses were selected by the Sponsor based on available information and results of 28-day study by oral gavage in rats performed at the Test Site [CiToxLAB France Study
code: 38533 TSR (2012)].
In this study the No Observed Adverse Effect Level (NOAEL) was considered to be at 1000 mg/kg/day.
Based on these results, the 1000 mg/kg bw/day dose was considered to be acceptable as top dose in the 90-day repeated dose study.

Positive control:

No

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: once a week
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmoscopy was conducted in all animals before treatment (Day -1/-2), and in the control group and high dose group animals, during Week 12 (Day 84/83).

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the treatment period, prior to scheduled necropsy on Day 91
- Anaesthetic used for blood collection: Yes: by heart puncture under pentobarbital anaesthesia
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked in table Haematology and blood clotting times.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the treatment period, prior to scheduled necropsy on Day 91
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table Clinical chemistry

URINALYSIS: Yes
- Time schedule for collection of urine: At the end of the treatment period, prior to scheduled necropsy on Day 91
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table Urinalysis

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During week 13
- Dose groups that were examined: All animals
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No

OTHER:
Examination of vaginal smears
Prior to necropsy, the oestrus cycle of all females was determined by taking vaginal smears, which was prepared and stained with 1% aqueous methylene blue solution. The smear was examined with a light microscope, in order to provide information regarding the stage of oestrus cycle at the time of sacrifice and assist in histological evaluation of oestrogen sensitive tissues.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table histopathology)

HISTOPATHOLOGY: (Yes see table histopathology)

Statistics:

Data were collected using the software PROVANTIS v.9 (Instem LSS Ltd. UK) or were recorded on the appropriate forms from the relevant SOPs of CiToxLAB Hungary Ltd., then tabulated using the Microsoft Office Word and/or Excel, as appropriate.


Group mean and standard deviation were calculated for numerical data. Statistical analysis was performed using SAS 9.2 (built in Provantis System).

The following decision tree was automatically applied within the validated Provantis system for statistical evaluation of numeric data:

The normality and heterogeneity of variance between groups was checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests showed no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test was carried out. If the obtained result was positive, Dunnett (Multiple Range) test was used to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01 as appropriate. This parametric analysis it the better option when the normality and heterogeneity assumptions implicit in the tests are adequate.

If either of the Shapiro-Wilk or Levene tests showed significance on the data, then the ANOVA type approach was not valid and a non-parametric analysis was required. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons was performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

There were no treatment related clinical findings during the study.

Mortality:

no mortality observed

Description (incidence):

There was no mortality during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no treatment related effects on bodyweight at any dose levels.
The mean body weights of test item treated males were comparable to the control throughout the treatment period. The overall body weight gains (Days 1-90) of males were similar to the control.
Minor difference to control was recorded in body weight gain in males, on one occasion attaining statistical significance (p<0.05): higher mean body weight gain was noticed between Day 8 and 15 in males in the low dose, which was without any biological significance.
In females mid and high doses the mean body weights were statistically significantly (p<0.05 or p<0.01) lower than the control in several periods throughout the study. In these doses the body weights were significantly lower than the control in the period of Days 1-90 too; however the difference was bigger in the mid dose than the high dose, with no dose relationship. The body weight gain was also significantly (p<0.05) lower in the mid dose when compared to the control in the period of Days 1-90. In the body weight data in females there was no dose dependence observed and the values observed were all within the normal range (treated group means were closer to the historic
control than was the concurrent control means) hence these statistical differences are not considered to reflect a treatment related effect.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

In both sexes the food consumption was comparable to the controls throughout the treatment period. The total and mean food consumption (Days 1-90) of both sexes were similar to the controls, however in males was statistically higher than control (p<0.01).
Minor statistically significances were seen in males in some periods, however they were always increases and without any biological significance. In females minor statistically
significant decreases were seen in a few periods during the study, however the values were all well within the normal range, hence this difference is not considered to reflect a treatment related effect.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Description (incidence and severity):

No test item related changes compared to pre-treatment were noted at ophthalmoscopy examination.

Haematological findings:

no effects observed

Description (incidence and severity):

Haematology parameters evaluated at the completion of the 90-day treatment period did not show signs of toxicity. Variations were noted in a few parameters in the treated animals, on few occasions attaining statistical significance.
In males, the Red Blood Cell (erythrocyte) count (RBC) in the low dose group, the Haematocrit (Hct) in the low and mid dose groups, and the Red Cell width (RDW) in the high dose group was statistically significantly (p<0.05) higher than the control. All values were well within the historical control ranges.
These minor statistical differences were considered to be unrelated to treatment and of no toxicological significance.

The Prothrombin Time (PTT) was statistically (p<0.05) higher in the female high dose, and statistically (p<0.05 or p<0.01) lower in the low and mid doses in males when compared to the control. All values were all considered to be in the normal range, the statistical differences were not considered to represent an adverse effect.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Clinical chemistry parameters evaluated at the completion of the 90-day treatment period did not show signs of toxicity. Variations were noted in a few parameters in the treated animals, on few occasions attaining statistical significance in males.
In males, statistically significant (p<0.05) increase in Total protein values in the mid dose were observed. The Albumin values were also statistically higher in the mid (p<0.05) and high (p<0.01) doses, and consequently the Albumin/Globulin ratio A/G ratio in the high dose (p<0.01). The mean values of the groups were well within the normal control range and are considered to have no toxicological significance.
A statistically significantly (p<0.05) increased Aspartate Aminotransferase activity (AST/GOT) was observed in the mid dose males without dose relationship, which was regarded as incidental and not to be treatment related, therefore considered have no toxicological significance.
The Bile Acid value of the mid and high doses in males were statistically significantly (p<0.01) higher than the control. The individual values were very variable and all were well within the normal range, there was no dose dependence and the differences are not considered to have no toxicological significance.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No effects were noted on the urinalysis parameters evaluated, which were considered to be related to the test item.
The urinalysis parameters were comparable to the controls in all test treated groups. Variations occurred and consisted of minor differences to controls, these were unrelated to treatment and within the normal range.
Urine sediment analysis in the groups showed similar results, no treatment related effect was noted.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no treatment related effects.
There were no toxicologically significant changes in the animal behaviour, general physical condition, in the reactions to different type of stimuli in the control or treated groups.
There was no effect of treatment noted during the assessment of landing foot splay, grip strength or motor activity.
Statistically significant numerical differences between groups were noted in landing foot splay tests in males in the mid and high doses. The differences were statistically significant (p<0.01), however they did not show a clear dose dependence and the values were all well within the normal range for this strain of rat in our laboratory, hence this difference is not considered to reflect a treatment related effect.
Statistically significant difference were recorded for high dose females for the total travelled distance, during a single five-minute interval (20-25 min p<0.05). This difference is considered to be without any biological significance.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Slightly enlarged kidneys were found in high dose males, 11.9% above control. The kidney weight data related to body weight as well as related to brain weight, compared to controls, showed a statistical significant increase (p<0.05 or p<0.01, respectively). In the absence of any histopathology changes, or changes in the female high dose, the weight difference is of equivocal relationship with treatment; it is plausible that an adaptive change occurred relating to excretion of the test item, but any such change is clearly not adverse.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test item-related macroscopic changes were observed in the experimental animals.
Changes such as small epididymis (1/10 in control, 1/10 in low dose), small testes (1/10 in control, 1/10 in low dose), enlarged testes (2/10 in low dose), soft testes (1/10 in low dose) in males and dilatation of the uterine body, horn (4/10 in control and 1/10 in low dose), firm single nodule in cervix (1/10 in mid dose) in females, were all considered as incidental or background findings.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No test item related microscopic findings were observed.
Focal/multifocal tubular degeneration (vacuolated or flattened tubular cells with peritubular/ interstitial mixed cellular/mononuclear cell infiltrate), proteinaceus casts and basophilia of the tubule in the kidney was observed in 7/20 control and 6/20 high dose animals. In the heart of 2/20 control and 3/20 high dose animals, focal/multifocal, myocardial degeneration and mononuclear cell infiltrate were seen. Tubular degeneration/atrophy or dilated tubules in the testes, reduced sperm content in the epididymis, congestion/haemorrhage in the thymus and signs of oestrus were also seen. All of these findings were considered as incidental or background.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment related effect

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Selected clinical parameters are summurised in the following table

 

Summary of selected clinical chemistry parameters (%)
		Males			Females
		Albumin	A/G	Bile acid	Albumin	A/G	Bile acid
Control	Mean	29.58	1.12	14.165	35.89	1.42	12.620
Low dose	Mean	30.51	1.16	17.817	34.07	1.35	12.483
(100 mg/kg bw)	Difference (%)	3.1	3.6	25.8	-5.1	-4.9	-1.1
Mid dose	Mean	31.19*	1.13	23.834**	32.39	1.29	17.295
(300 mg/kg bw)	Difference (%)	5.4	0.9	68.3	-10.0	-9.2	37.0
High dose	Mean	31.45**	1.19**	23.622**	34.74	1.39	16.619
(1000 mg/kg bw)	Difference (%)	6.3	6.3	66.8	-3.2	-2.1	31.7

Notes:

1.       * = Significant (p<0.05, Dunnett 2 Sided Test)

2.       ** = Significant (p<0.01, Dunnett 2 Sided Test orDunn 2 Sided Test)

 

Organ weight: Kidney

Slightly enlarged kidneys were found in high dose males, 11.9% above control. The kidney weight data related to body weight as well as related to brain weight, compared to controls, showed a statistical significant increase (p<0.05 or p<0.01, respectively). In the absence of any histopathology changes, or changes in the female high dose, the weight difference is of equivocal relationship with treatment; it is plausible that an adaptive change occurred relating to excretion of the test item, but any such change is clearly not adverse.

KIDNEY	Dose (mg/kg bw/day)
	Control	100	300	1000	Control	100	300	1000
	Males				Females
Terminal body weight (g)	521.3	552.2	522.5	531.0	302.4	291.0	278.1**	280.9*
Kidney weight Absolute (g)	3.257	3.239	3.296	3.644*	1.840	1.808	1.736	1.821
differences %		-0.6	1.2	11.9		-1.7	-5.7	-1.0
Kidney weight Body weight relative (%)	0.62385	0.58865	0.63234	0.68970*	0.60864	0.62131	0.62401	0.64856
differences %		-5.6	1.4	10.6		2.1	2.5%	6.6
Kidney weight Brain relative (%)	144.616	143.689	145.285	165.979**	91.616	89.195	85.856	91.732
differences %		-0.6	0.5	14.8		-2.6	-6.3%	0.1

Notes:

1.       * = Significant (p<0.05, Dunnett 2 Sided Test)

2.       ** = Significant (p<0.01, Dunnett 2 Sided Test)

Examination of vaginal smears

There were no test item related changes in the animal oestrus cycle evaluated immediately prior to necropsy, the animals showed the normal distribution of the oestrus phases.

Applicant's summary and conclusion

Conclusions:

Based on the results, Triethyl phosphonoacetate administrated to Wistar rats at 100, 300 and 1000 mg/kg bw/day, daily for 90 consecutive days, by gavage in water at a dose volume of 5 mL/kg bw, was not associated with any overt adverse effects that could be ascribed to test item administration.

In conclusion, the NOAEL of Triethyl phosphonoacetate is considered to be the high-dose level of 1000 mg/kg bw/day.

Executive summary:

Description

This 90-day study was performed to obtain information on the toxicity of Triethyl phosphonoacetate when administered by oral gavage to male and female Wistar rats at 3 dose levels + control group (100, 300 and 1000 m/kg) according to OECD 408 guideline under GLP.

Analysis of test item formulations for concentration and homogeneity was performed four times during the treatment period (during the first, fifth, ninth and last week of treatment) using a validated Gas Chromatography with Flame Ionization Detection (GC-FID) method. No test item was detected in the control solution samples. All formulations were shown to be generally homogeneous. All formulations were found to be in the range of 99.2-108.5 % of the nominal concentrations.

The examinations included clinical signs, mortality, body weights, food consumption, ophthalmoscopy, neurological assessment including functional observation battery (FOB), measurements of the landing foot splay and grip strength, clinical pathology, vaginal smears, gross pathology, organ weights and histopathology. Full histopathology was performed in Groups 1 (Control) and 4 (High dose).

Results

There was no mortality and no toxicologically significant or test-item related clinical findings during the study.

There were no toxicologically significant changes in the animal behaviour, general physical condition, in the reactions to different type of stimuli in the control or treated groups. There was no effect of treatment noted during the assessment of landing foot splay, grip strength or motor activity.

No test item related changes compared to pre-treatment were noted at ophthalmoscopy examination.

There were no effects on bodyweight at any dose levels and the food consumption was comparable to the controls throughout the treatment period.

Haematology, clinical chemistry and urinalysis parameters, evaluated at the completion of the 90-day treatment period did not show signs of toxicity. Minor statistical differences were not considered to be related to treatment.

There were no test item related changes in the animal oestrus cycle evaluated prior to necropsy and the animals showed the normal distribution of the oestrus phases.

At necropsy, no test item-related macroscopic changes were observed in the experimental animals.

In the organ weights, slightly enlarged kidneys were found in high dose males (11.9% above control). The difference, in the absence of histopathology or urinalysis effects is considered not to reflect an adverse effect.

No test item related microscopic findings were observed.

Conclusion

Based on the results, Triethyl phosphonoacetate administrated to Wistar rats at 100, 300 and 1000 mg/kg bw/day, daily for 90 consecutive days, by gavage in water at a dose volume of 5 mL/kg bw, was not associated with any overt adverse effects that could be ascribed to test item administration.

In conclusion, the NOAEL of Triethyl phosphonoacetate is considered to be the high-dose level of 1000 mg/kg bw/day.

 

This study is considered as acceptable as it satisfied the criteria of the OECD Guideline 408.