Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information
No data available
Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

no data


Short description of key information:
no data available

Justification for selection of Effect on fertility via oral route:

The repeated dose toxicity study did not show any effect on the fertility. No effects were observed on the female estrous cycles nor on the thyroid hormones levels, no effect on the male epididymal sperm (motility, morphology and count) nor on the testicular sperm (daily sperm production). The histological examination did not revealed any effect of the test substance in ovaries, uterus, vagina, mammary gland area, epididymis, prostate, seminal vesicle and testis.

Effects on developmental toxicity

Description of key information
There were no direct effects of Triethyl Phosphonoacetate on external, visceral and/or skeletal development of fetuses during the OECD 414 study.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2016-07-14 to
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
2015-09-22
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Address: Sandhofer Weg 7, D-97633, Sulzfeld, Germany) from SPF colony
- Age at study initiation: Young adult female rats, nulliparous and non-pregnant, at least approximately 11 weeks old
- Weight at study initiation: Will not exceed ± 20% of the mean weight at onset of treatment
- Housing: Standard laboratory conditions; individual housing
- Cage type: Type II (size: 18 [height] x 38 [length] x 23 [width] cm) and III (size: 18 [height] x 38 [length] x 38 [width] cm) polycarbonate
- Bedding: Lignocel® Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany); Batch number: 03018160506 (Expiry date: 06 May 2019) was used in the study.
- Enrichment: Rodents will be housed individually. Arbocel crinklets natural nesting material produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany); Batch number: 05072160121 (Expiry date: 21 January 2019) was used in the study.
- Diet (e.g. ad libitum): The animals will be provided with ssniff® SM R/M-Z+H “Autoclavable Complete Feed for Rats and Mice – Breeding and Maintenance” (ssniff Spezialdiäten GmbH, D-59494 Soest, Germany) (Batch number 278 5652, Expiry date: 30 November 2016)
- Water (e.g. ad libitum): tap water as for human consumption
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5 - 24.7 °C
- Humidity (%): 37-80 %
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES: From 2016-07-26 to 2016-08-24
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item will be formulated in the vehicle (drinking water treated by reverse osmosis using Direct-Q®-8 UV, Millipore) at the appropriate concentrations according to the dose level and volume selected, in the Pharmacy of CiToxLAB Hungary Ltd.
Formulations may be prepared fresh 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. 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.

VEHICLE
- Concentration in vehicle: 100, 300 and 1000 mg/kg
- Amount of vehicle (if gavage): 10 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
SUMMARY
The objective of this analytical study phase was to determine the concentration and homogeneity of triethyl phosphonoacetate in drinking water for dose formulations prepared at CiToxLAB Hungary Ltd. (Test Facility Study Number 15/515-105P). Control formulations were also determined for triethyl phosphonoacetate concentration.

Material and method
CiToxLAB Hungary Ltd. prepared control dose formulation at 0 mg/mL and dose formulations at 10, 30 and 100 mg/mL. Two sets of 1 mL duplicate samples were sampled from the middle of control dose formulation and from three different levels (top, middle and bottom) of each dose formulation. The two samples sets were stored at room temperature and protected from light and sent to CiToxLAB France for analysis.
CiToxLAB France performed control dose formulation and test item dose formulations analysis. The method based on Gas Chromatography with Flame Ionization Detection (GC/FID) used to determine triethyl phosphonoacetate content in control and dose formulations samples is fully documented in the validation report CiToxLAB France/Study No. 38534 VAA.
Samples from set one were diluted appropriately according to the validated method, then analysed by Gas Chromatography with Flame Ionization Detection (GC/FID). The concentration of the test item was determined by bracketing, using the mean response factor of triethyl phosphonoacetate in standard solutions (external standard).
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 (LOQ) of triethyl phosphonoacetate.
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%.

Conclusion
The results of dose formulation analysis showed a satisfactory agreement between the nominal and the measured concentrations of triethyl phosphonoacetate for each investigated dose formulation. No peak
attributed to test item was detected in the control dose formulation Dose formulation were shown to be homogeneous.

Laboratory equipment and apparatus
Gas Chromatography system with FID detector model (CP-3800) Varian
Gas Chromatography system with FID detector (430-GC) Brüker
Micro-balance, Balance Mettler-Toledo
Automatic pipette Biohit
Equipments for agitating which could be used (magnetic stirrers, vortex),
Class A volumetric flasks.


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, lenght = 30m, diameter = 0.53 mm
Mobile phase: hydrogen
Detector: FID
Sensitivity: 12
Column temperature: from 80°C (stay 1 min) to 250 °C (stay 2 min) at 10 °C/min
Constant flow rate: 5 mL/min
Split rate: 10
Software: Empower 2 (Waters)
Detector temperature: 250 °C
Injetcor temperature: 220 °C
Injected volume: 2µL
Detector gas flows: 30mL/min for makeup, 30 mL/min for hydrogen and 300 mL/min for air
Retention time: TEPA: 11.6 min
Analysis time: 20 min

Quantification:
The concentrations of the test item will be detremined from the mean response factor of TEPA in standard solution.
The sample concentrations will be determined using the following equation:
[concentration] = (Area sample/ Standard mean response factor) x dilution factor
Where:
Area sample = area of sample
Standard mean response factors = mean reposnse factor of standard solutions 1 and 2 (n= 10)
Dilution factor (Also including conversion between units if required)
response factor = area Std / concentration Std

Acceptance criteria
Precision of response factor for standard solution STD 1 : Coefficient of variation CV% < 5%
Precision of response factor for standard solution STD 2 : Coefficient of variation CV% < 5%
Precision of response factor for standard solution STD 1 and SDT 2 : Coefficient of variation CV% < 5%
Accuracy of the response actor of the standards (ration of mean response of standard solution 1 with mean resposne factor of standard solution 2) should be between 90.0% and 110.0%.

Details on mating procedure:
The oestrus cycle of female animals will be examined shortly before start of pairing. After acclimatisation the females, according to their oestrus cycle, will be paired with males for approximately 2 hours (1 male: 1females) in the morning, until at least 24 sperm-positive females/group are obtained. After the daily mating period, a vaginal smear will be prepared and stained with 1% aqueous methylene blue solution. The smear will be examined with a light microscope; the presence of a vaginal plug or sperm in the vaginal smear is considered as evidence of copulation (GD0). Sperm-positive females will be separated and caged individually.
Duration of treatment / exposure:
From gestation day 6 to gestation day 19
Frequency of treatment:
Daily (oral gavage)
Duration of test:
From Gestation day 6 to gestation day 20
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
24 mated female per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were set by the Sponsor in consultation with the Study Director based on available data, including a 4-week repeated dose toxicity study by oral gavage in rats and a Dose Range Finding (DRF) study by oral gavage in pregnant rats, with the aim of inducing toxic effects but no death or suffering at the highest dose and a NOAEL at the lowest dose.
In this study the 4-week repeated dose toxicity study, the No Observed Adverse Effect Level (NOAEL) was considered to be at 1000 mg/kg/day. In the DRF study, treatment with the test item (up to and including 1000 mg/kg bw/day) was not associated with any signs of maternal toxicity. No signs of embryotoxicity or foetotoxicity were observed in any test item treated dose groups (up to and including 1000 mg/kg bw/day).
Based on this information, the doses of 100, 300 and 1000 mg/kg bw/day are deemed suitable for the purpose of the study.
The oral route is a potential route of human exposure to the test item and is considered suitable to provide the exposure required for this developmental toxicology study.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: usually twice daily (at the beginning and end of each working day, or before treatment and when the peak of the clinical observations, if any, is observed after treatment). Only one clinical observation will be made in the afternoon on those days when detailed clinical observation is made in the morning. Furthermore, clinical observation will be made only once on necropsy days. When signs of toxicity are noted, animals may be observed more frequently.

BODY WEIGHT: Yes
- Time schedule for examinations: Gestation Day 0, 3, 6, 8, 10, 12, 14, 16, 18 and 20.

FOOD CONSUMPTION : Yes
Food will be measured with precision of ±1 g at least on Gestation Day 0, 3, 6, 8, 10, 12, 14, 16, 18 and 20. Food consumption will be calculated for each interval, including Gestation Day 0-6, Gestation Day6-20 and Gestation day D0-20.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined:
The dams’ viscera will be examined macroscopically for any structural abnormalities or pathological changes; all the gross findings will be retained in 10% buffered formalin solution (or modified Davidson fixative, in case of the eyes, if any abnormalities noted) for possible future evaluation.
The placentas will be examined macroscopically.


OTHER:
The corrected body weight will be calculated (body weight on GD20 minus weight of the gravid uterus).
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions:
- Number of late resorptions: Yes
- Other:
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: [half per litter]
Statistics:
The statistical evaluation of data will be performed with the program package SPSS PC+4.0 (SPSS Hungary Kft, Budapest) or SAS v9.2 (when using Provantis).
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related adverse effects or systemic clinical signs were noted in the treated animals. Fur thin / alopecia was recorded in some control and test item treated animals and scar on the neck was recorded for a Low dose animal, but those findings were not considered as a test item related effect.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No toxicologically significant changes were observed in the mean body weights or body weight gain values of the Low (100 mk/kg bw/day), Mid (300 mg/kg bw/day) or High (1000 mg/kg bw/day) dose groups when compared to control. Furthermore, no toxicologically or statistically significant changes were recorded in the corrected body weight, corrected body weight gain (GD 0-20) and net body weight gain (GD 6-20) values of any test item treated groups.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No significant changes were observed in the mean daily food consumption of dams in the Low (100 mg/kg bw/day), Mid (300 mg/kg bw/day) and High (1000 mg/kg bw/day) dose groups compared to the control value.
Spillage was recorded for one control animal (#1510) in the periods of GD0-3, GD3-6 and GD8-10, and for two Low dose animals (#2505 and #2519) in the periods of
GD0-3, GD3-6 and GD6-8, in those cases the measured food consumption values were not used for calculation.
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Hernia on the diaphragm was observed for a control female (#1508), fur thin was recorded for one control (#1521) and one Low dose (#2503) females. However, these facts were considered as biologically not relevant findings and not related to the test item administration.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The mean number of corpora lutea and the mean number of implantation sites were comparable with the controls in all treated groups. No adverse effect was observed in preimplantation loss of the test item treated groups when compared to the control.
The early and the late embryonic loss did not differ significantly from the control in the test item treated groups. There was no statistically significant difference in the postimplantation loss between the test item treated and control groups.
The total intrauterine mortality was comparable with the control; no statistically significant differences were observed.
There was no statistically significant difference in foetal death in any test item treated group compared to the control.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Changes in number of pregnant:
no effects observed
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: See "remarks"
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The mean weight of foetuses per litter in the Low, Mid and High dose groups (100, 300 and 1000 mg/kg bw/day, respectively) did not differ significantly from the control mean value as shown in Table 5. In the case of the parameter “mean body weight/foetus” significantly higher value (p<0.01) was observed in the Mid dose group when compared to the control, but the slightly higher foetal body weight with a lack of a dose response, was not considered as a test item related effect.
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
Although in the absolute number of foetuses, statistically significant differences were noted in the Low (100 mg/kg bw/day) and Mid (300 mg/kg bw/day) dose groups (p<0.05 and p<0.01, respectively), there was no dose response (decrease was seen in the Low dose group while increased value was seen in the Mid dose group), thus these facts were considered as not related to the test item treatment.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There was no toxicologically significant difference in the sex distribution of foetuses between the control and treatment groups
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
No external malformations and no external variations were recorded in the study.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
All of the skeletal findings correspond with the current historical control or the concurrent study control data, or were considered to be incidental findings without dose response
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
All of the visceral findings are consistent in general nature and incidence with the study concurrent control data or the existing historical control data, therefore considered as incidental findings.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat. (total fraction)
Sex:
male/female
Basis for effect level:
other: See remarks
Abnormalities:
no effects observed
Developmental effects observed:
no

Dose formulation analysis

Group

02 August 2016

16 August 2016

Mean measured concentration (mg/mL)

Relative
to the nominal
concentration (%)

Measured concentration (mg/mL)

Relative
to the nominal
concentration (%)

Control

not detectable

-

not detectable

-

10 mg/mL

10.5

105

10.4

104

30 mg/mL

31.8

106

30.3

101

100 mg/mL

105

105

101

101

Samples collected freshly and sent to the Test Site where they arrived next day and were stored at room temperature and protected from light for a maximum of 4 days before analysis.

 

Body weight parameters

Parameters

Dose (mg/kg bw/day)

 

0

100

300

1000

 

Number of evaluated dams

24

22

24

24

 

Body weight on GD20 (g)

317.1

318.5

322.4

317.7

NS

Body weight gain GD6-20 (g)

103.6

106.1

107.0

104.3

NS

Corrected body weight gain GD0-20 (g)

47.17

44.32

46.71

45.63

NS

Net body weight gain GD6-20 (g)

24.96

23.50

24.63

23.71

NS

NS: Statistically not significant when compared to the negative (vehicle) control.

Corrected and Net weight gains refer to body weight differences minus the weight of the gravid uterus.

 

Summary of pregnancy data

Parameters

Dose (mg/kg bw/day)

0

100

300

1000

Number of mated females

24

24

28

24

Pre-terminal death or euthanasia#

0

0

4

0

Number of non-pregnant females

0

2

0

0

Number of females with ≤ 5 implantation sites

0

0

0

0

Number of evaluated females
on GD20 (Caesarean section)

24

22

24

24

Pre-terminal death or euthanasia seen in the Mid dose group arrived from a technical issue and not from a treatment related effect.

 

Summary of the intrauterine evaluation

Parameters

Dose (mg/kg bw/day)

 

0

100

300

1000

 

Number of evaluated dams

24

22

24

24

 

Mean number of corpora lutea

12.83

12.36

12.46

12.29

NS

Mean number of implantations

10.54

10.86

10.46

10.42

NS

Preimplantation loss, mean

2.29

1.50

2.00

1.88

NS

Preimplantation loss (%), mean

17.13

11.86

15.25

14.63

NS

Early embryonic loss, mean

0.50

0.09

0.08

0.08

NS

Early embryonic loss (%), mean

4.88

0.77

0.88

0.79

NS

Late embryonic loss, mean

0.29

0.23

0.04

0.29

NS

Late embryonic loss (%), mean

2.88

2.91

0.46

2.83

NS

Dead foetuses, mean

0.00

0.00

0.00

0.08

NS

Dead foetuses (%), mean

0.00

0.00

0.00

0.62

NS

Postimplantation loss, mean

0.79

0.32

0.13

0.46

NS

Postimplantation loss (%), mean

7.75

3.73

1.33

4.25

NS

Total intrauterine mortality, mean

3.08

1.82

2.13

2.33

NS

Total intrauterine mortality (%), mean

23.00

14.77

16.25

18.29

NS

Viable foetuses, mean

9.75

10.55

10.33

9.96

NS

NS: Statistically not significant when compared to the negative (vehicle) control.

 

Examination of viable fetuses

Parameters

Dose (mg/kg bw/day)

 

0

100

300

1000

 

Number of examined litters

24

22

24

24

 

Viable foetuses, mean

9.75

10.55

10.33

9.96

NS

Male foetuses, mean

4.88

5.50

5.29

5.00

NS

Female foetuses, mean

4.88

5.05

5.04

4.96

NS

Total number of foetuses

234

232*

248**

239

CH

Total number of male foetuses

117

121

127

120

NS

Total number of female foetuses

117

111

121

119

NS

Mean foetal weight / litter (g)

3.456

3.501

3.535

3.483

NS

Mean body weight / foetus (g)

3.474

3.507

3.537*

3.479

DN

Number of foetuses with retarded body weight

10

4

6

9

NS

Number of affected litters (Runts)

8

4

5

7

NS

NS: Statistically not significant when compared to the negative (vehicle) cont

CH: Chi2test, DN: Duncan’s multiple range test

*: p<0.05, **: p< 0.01

 

External abnormalities

Parameter

Dose (mg/kg bw/day)

HC data

Control

100

300

1000

Total number of examined litters

24

22

24

24

485

Total number of examined foetuses

234

232

248

239

4955

Total number of intact (normal) foetuses

234

232

248

239

--

Total number of foetuses / litters
with malformation

0 / 0

0 / 0

0 / 0

0 / 0

--

Total number of foetuses / litters
with variation

0 / 0

0 / 0

0 / 0

0 / 0

--

External malformations

No external malformations were observed

External variations

No external variations were observed

HC: historical control

 

Summary table of visceral abnormalities

Parameter

Dose (mg/kg bw/day)

HC data

Control

100

300

1000

Total number of examined litters

24

22

24

24

485

Total number of examined foetuses

118

116

125

118

2484

Total number of intact (normal) foetuses

115

110

121

110

--

Total number of foetuses / litters
with malformation

2 / 2

0 / 0

0 / 0

0 / 0

--

Total number of foetuses / litters
with variation

1 / 1

6 / 6

4 / 3

8*CH/ 7

--

CH: Chi2 test, *: p<0.05

 

Details of the visceral abnormalities

Parameter

Dose (mg/kg bw/day)

HC data

Control

100

300

1000

Visceral malformations

Great vessels, transposition, malpositioned

Litter
incidence

n

2

0

0

0

0

%

8.3

0.0

0.0

0.0

0.0

Foetal
incidence

n

2

0

0

0

0

%

1.695

0.000

0.000

0.000

0.000

Visceral variations

Brachiocephalic trunk, short

Litter
incidence

n

1

0

0

0

35

%

4.5

0.0

0.0

0.0

7.2

Foetal
incidence

n

1

0

0

0

43

%

0.862

0.000

0.000

0.000

1.731

Thymic cord

Litter
incidence

n

0

4

0

4

55

%

0.0

18.2

0.0

16.7

11.3

Foetal
incidence

n

0

4*CH

0

4*CH

66

%

0.000

3.448

0.000

3.389

2.657

Kidney, misshapen, malpositioned

Litter
incidence

n

0

1

0

0

1

%

0.0

4.5

0.0

0.0

0.2

Foetal
incidence

n

0

1

0

0

1

%

0.000

0.862

0.000

0.000

0.040

Ureter, convoluted

Litter
incidence

n

0

1

1

2

3

%

0.0

4.5

4.2

8.3

0.6

Foetal
incidence

n

0

1

1

2

3

%

0.000

0.862

0.800

1.695

0.121

Ureter, dilated

Litter
incidence

n

0

0

1

1

4

%

0.0

0.0

4.2

4.2

0.8

Foetal
incidence

n

0

0

1

1

4

%

0.000

0.000

0.800

0.847

0.161

Renal papilla, small

Litter
incidence

n

0

0

2

3

16

%

0.0

0.0

8.3

12.5

3.3

Foetal
incidence

n

0

0

2

3

18

%

0.000

0.000

1.600

2.542

0.725

Numbers represent the number (n) or ratio (%) of abnormalities.

HC: historical control

CH: Chi2test, *: p<0.05, **: p<0.05

 

Summary table of the skeletal abnormalities

Parameter

Dose (mg/kg bw/day)

HC data

Control

100

300

1000

Total number of examined litters

24

22

24

24

484

Total number of examined foetuses

116

116

123

121

2467

Total number of intact (normal) foetuses

110

109

110

107

--

Total number of foetuses / litters
with malformation

0 / 0

1 / 1

0 / 0

0 / 0

--

Total number of foetuses / litters
with variation

6 / 5

6 / 6

13 / 9

14 / 10

--

 

Details of the skeletal abnormalities

Parameter

Dose (mg/kg bw/day)

HC data

Control

100

300

1000

Skeletal malformations

Carpal bone, supernumerary

Litter
incidence

n

0

1

0

0

0

%

0.0

4.5

0.0

0.0

0.0

Foetal
incidence

n

0

1

0

0

0

%

0.000

0.862

0.000

0.000

0.000

Skeletal variations

Skull, 3 or more bones incomplete ossification

Litter
incidence

n

1

2

5

4

114

%

4.2

9.1

20.8

16.7

23.5

Foetal
incidence

n

1

2

6

4

144

%

0.862

1.724

4.878

3.306

5.837

Hyoid body, incomplete ossification

Litter
incidence

n

0

1

0

0

0

%

0.0

4.5

0.0

0.0

0.0

Foetal
incidence

n

0

1

0

0

0

%

0.000

0.862

0.000

0.000

0.000

Ossified sternebra (3 or less)

Litter
incidence

n

2

2

4

5

37

%

8.3

9.1

16.6

20.8

7.6

Foetal
incidence

n

2

2

5

6

46

%

1.724

1.724

4.065

4.959

1.865

Rib, wavy, marked

Litter
incidence

n

0

0

2

0

149

%

0.0

0.0

8.3

0.0

30.8

Foetal
incidence

n

0

0

3

0

243

%

0.000

0.000

2.439

0.000

9.850

Vertebra, dumbbell or asymmetric ossification (2 or more)

Litter
incidence

n

0

0

1

1

67

%

0.0

0.0

4.2

4.2

13.8

Foetal
incidence

n

0

0

2

2

73

%

0.000

0.000

1.626

1.652

2.959

Vertebra, dumbbell shaped

Litter
incidence

n

1

1

0

1

6

%

4.2

4.5

0.0

4.2

1.2

Foetal
incidence

n

1

1

0

1

6

%

0.862

0.862

0.000

0.826

0.243

Vertebra, bipartite ossification

Litter
incidence

n

1

2

1

0

24

%

4.2

9.1

4.2

0.0

4.9

Foetal
incidence

n

1

2

1

0

24

%

0.862

1.724

0.813

0.000

0.973

Tarsal ossified ≤ 3

Litter
incidence

n

1

1

1

1

29

%

4.2

4.5

4.2

4.2

6.0

Foetal
incidence

n

2

1

1

1

43

%

1.724

0.862

0.813

0.826

1.743

Carpal ossified ≤ 2.5

Litter
incidence

n

0

0

1

0

4

%

0.0

0.0

4.2

0.0

0.8

Foetal
incidence

n

0

0

1

0

4

%

0.000

0.000

0.813

0.000

0.162

Numbers represent the number (n) or ratio (%) of abnormalities.

HC: historical control

CH: Chi2 test, *: p<0.05, **: p<0.05

Conclusions:
Triethyl phosphonoacetate, when administered daily by oral gavage to pregnant Hannover Wistar rats from gestation days GD6 to GD19, was associated with the following findings:

There was no test item effect on maternal body weight, body weight gain, maternal corrected body weight and body weight gain, and on food intake up to and including 1000 mg/kg bw/day.

There were no toxicologically significant differences, or test item related-changes in the reproductive parameters examined up to and including 1000 mg/kg bw/day.

The mean number of viable foetuses in all test item treated groups as well as their sex distribution were comparable with the control mean. The mean foetal body weight per litter was comparable with the control value in all test item treated groups.

There were no direct effects of the test item on external, visceral and/or skeletal development of foetuses in the study.

In this study, from the observations made in the dams and their foetuses, there were no changes on embryos or foetuses. The following no-observed-adverse-effect (NOAEL) levels were derived:

NOAELmaternal toxicity: 1000 mg/kg bw/day
NOAELembryotoxicity: 1000 mg/kg bw/day
NOAELfoetotoxicity: 1000 mg/kg bw/day
NOAELteratogenecity: 1000 mg/kg bw/day
Executive summary:

This developmental toxicity study was performed to assess the effects of the test itemTriethyl phosphonoacetate on the embryonic and foetal development (including the organogenesis period) of Hannover Wistar rats in their first pregnancy. The dams (one control and three test item treated groups) were treated daily by oral (gavage) administration, from gestation day GD6 up to and including GD19 (sperm positive day = 0 day of pregnancy, GD0). Caesarean sections, necropsy of dams and examination of uterine contents were performed on GD20.

The dose levels of 100, 300 and 1000 mg/kg bw/day were set by the Sponsor based on available data including the results of a 4-week repeated dose toxicity study by oral gavagein rats and a Dose Range Finding (DRF) study in the pregnant rat (CiToxLAB Hungary study code 15/515 -105PE), with the aim of inducing toxic effects but no death or suffering at the highest dose and a NOAEL at the lowest dose. Control dams were treated with the vehicle (drinking water treated by reverse osmosis) only.

Dose formulations were analysed for test item concentration and homogeneity two times during the treatment period using a validated GC-FID method.

Parameters monitored during the study included mortality and clinical observations, body weight, body weight gain and individual food consumption. Maternal reproductive parameters associated with uterine examination were evaluated, and the foetuses were weighed and examined for external, visceral and skeletal abnormalities. Placentas were examined macroscopically.

The number of confirmed pregnant, evaluated dams was 24 in the Control, 22 in the Low (100 mg/kg bw/day), 24 in the Mid (300 mg/kg bw/day) and 24 in the High (1000 mg/kg bw/day) dose groups, respectively.

 

Results

All formulations were within the range of 101 to 106% of nominal concentration and were found to be homogenous. No test item was detected in the control samples. Based on these results, test item formulations were considered suitable for the study purposes. 

There were no unscheduled mortalities or clinical signs in the study related to the selected treatment dose levels.

There was no test item effect on maternal body weight, body weight gain, maternal corrected body weight and body weight gain, and on food intakeup to and including 1000 mg/kg bw/day.

There were no toxicologically significant differences, or test item related-changes in the reproductive parameters examined up to and including 1000 mg/kg bw/day.

There were no effects on the preimplantation loss, and on the early or late embryonic loss in the test item-treated dams evaluated. 

The mean number of viable foetuses in all test item treated groups as well as their sex distribution were comparable with the control mean.

The mean foetal body weight per litter was comparable with the control value in all test item treated groups.

No remarkable internal or external observations were recorded for any pregnant animals during ecropsy.

No remarkable abnormalities were observed on the placentas in any examined groups.

There were no direct effects of the test item on external, visceral and/or skeletal development of foetuses in the study.

In conclusion,Triethyl phosphonoacetate,when administered daily by oral gavage to pregnant Hannover Wistar rats from gestation days GD6 to GD19, was associated with the following findings: 

In this study, from the observations made in the dams and their foetuses, there were no changes on embryos or foetuses. The following no-observed-adverse-effect (NOAEL) levels were derived:

NOAELmaternal toxicity: 1000 mg/kg bw/day

NOAELembryotoxicity: 1000 mg/kg bw/day

NOAELfoetotoxicity: 1000 mg/kg bw/day

NOAELteratogenecity: 1000 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
Additional information

In a GLP OECD 414 compliant study, the substance was daily administered by gavage at doses of 100, 300 or 1000 mg/kg/day, from Day 6 up to and including Day 19, by oral route (gavage), to mated female SpragueDawley rats.

There was no test item effect on maternal body weight, body weight gain, maternal corrected body weight and body weight gain, and on food intake up to and including 1000 mg/kg bw/day.

 

Therefore, on the basis of the results obtained in this study, the No Observed Adverse Effect Level (NOAEL) was considered to be 1000 mg/kg/day for both maternal parameters and for embryo-fetal development and no classification is retained for the test item.

Justification for classification or non-classification

self-classification:

Triethyl phosphonacetate showed no adverse effect on the reproductive organs (male and female) in the subacute toxicity study.

No adverse effect was noted during the Prenatal Developmental Toxicity (OECD 414).

The Triethyl Phosphonoacetate was therefore not classified for reproduction and developmental parameters.