Phosphonic acid, monoethyl ester, sodium salt (1:1)

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

7 Sept - 8 Oct 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

draft document August 1996

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted in August 1998

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Aluminium triethyl triphosphonate

EC Number:

254-320-2

EC Name:

Aluminium triethyl triphosphonate

Cas Number:

39148-24-8

Molecular formula:

C2H7O3P.1/3Al

IUPAC Name:

aluminum tris(ethyl phosphonate)

Test animals

Species:

rabbit

Strain:

other: New Zealand White Rabbits Hra (NZW) SPF

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Covance Research Products, Denver, USA
- Age at study initiation: 5 - 6 months (days 0 - 2 of gestation)
- Weight at study initiation: 3 - 5 kg (pregnant females)
- Fasting period before study: no
- Housing: individually in suspended stainless steel wire mesh cages
- Diet: pelleted diet (Certified Rabbit Diet, No. 5322 (PMI Nutrition International, St .Louis, MO)), 50 g feed on the day of arrival, 125 g on the second day and approx. 200 g thereafter
- Water: facility water, ad libitum
- Acclimation period: 2 - 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 25
- Humidity (%): 40 - 96
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 7 Sept 1999 To: 8 Oct 1999

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: methyl cellulose (Methocel ® (0.5% w/v in water))

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of test substance was added to methyl cellulose and stirred on a magnetic stir plate for at least 30 min and continued to be stirred until completion of dosing.

VEHICLE
- Amount of vehicle (if gavage): 2 mL/kg bw day
- Lot/batch no. (if required): 97H0980
- Purity: 100%
The aqueous solution of methyl cellulose was prepared weekly and stored refrigerated at 2 - 8 °C.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of dosing solutions were tested for homogeneity and dose accuracy via HPLC-UV. Homogeneity analysis revealed a recovery rate of 96.4% for all concentrations thereby demonstrating homogeneity of the solution. Moreover, dose level confirmatory tests determined concentrations of dosing solutions of ±10% of the nominal concentration which demonstrates the presence of acceptable concentrations.

Details on mating procedure:

not applicable (pregnant rats were obtained from the supplier)

Duration of treatment / exposure:

Day 4 - 28 of gestation

Frequency of treatment:

daily, 7 days/week

Duration of test:

25 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

50 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)

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: dose selection was based on a range-finding teratology study performed with 75, 125, 250, 500 and 1000 mg Fosetyl-Al/kg bw/day administered via gavage during Days 4 - 28 of gestation. Maternal toxicity was observed at the two top doses tested identified by reduced body weight gain and food comsumption. At 250 mg/kg bw/day, some evidence of decreased maternal body weight gain was still observed. Furthermore, 250 and 500 mg/kg bw/day induced slight decreases in the number of life fetuses and a slight increase in post-implantation loss. Due to the small animal number included in the study of only 5 dams per dose group, data on alterations in uterine implantation is inconclusive. No adverse effects have been observed either on the dams or fetuses at the two lowest doses applied. Thus, doses ranging from 50 - 300 mg/kg bw/day were chosen as applicable for the prenatal developmental toxicty study including a high-dose which is expected to produce slight maternal toxicity and a low-dose, which should represent the NOEL. The mid-dose was proposed to provide dose-response information.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily (including post-dose observations approx. 1.5 to 2 h after dosing)
- Cage side observations included observations for mortality, morbidity and signs of toxicity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
- Observations included general condition, skin and fur, eyes, nose, oral cavity, abdomen and external genitalia and evaluation of respiration.

BODY WEIGHT: Yes
- Time schedule for examinations: gestation day 0 and daily thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: uterus, lung, kidney, bladder, ovary

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: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: [all per litter]

Statistics:

Mean values and standard errors were calculated from the examined parameters. Statistical analysis were performed with ANOVA followed by a Dunnett's test to identify differences between the control and treatment groups (in regard to body weights, body weight change, feed consumption, number of corpura lutea, uterine implantation data, pre- and post-implantation loss, number of male and female fetuses, fetal weight distinguished by sex and as a composite for both sexes). A Kruskal-Wallis test was performed to test equality of means. All statistical tests were conducted at the 5 and 1%, two-sided risk levels. Statistical evaluation of equality of means was made by the appropriate one way analysis of variance (ANOVA) technique, followed by a multiple comparison procedure, if needed. If ANOVA showed no difference, no additional comparisons were made. If ANOVA was significant, Dunnett's test was used to determine which data, if any, differed from the control.

The Kruskal-Wallis test (2Xn) was used to test equality of means. If no differences were seen, no additional analyses were performed. If differences were indicated, a 2X2 Kruskal-Wallis test was used to determine which treatments differed from control.

Further, a Fisher Exact Test with Bonferonni correction was performed to identify differences in mortality rate, pregnancy rates, incidence of females with only resorptions and incidence of litters containing fetuses with malformations. All statistical tests were conducted at the 5 and 1%, two-sided risk levels.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs of systemic toxicity were observed despite scabs, alopecia and decreased faecal volume. As these effects were determined only at low incidence in the control and dose groups and they represent common laboratory findings, they are interpreted as fortuitous and not substance-specific.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

50 mg/kg bw/day: 1/25 animals was euthanized on Gestation Day 27 as a result of an apparent abortion, fetus found under the cage. Since abortions where not seen in the other treatment groups and spontaneous abortions are common among rabbits when stressed, the death is not considered to be related to treatment. Moreover, postmortem examinations revelead a discoloured uterus and a fluid-filled uterine horn on the left side.

100 mg/kg bw/day: 1/25 animals found dead on Day 11 of gestation. Postmortem examination, revealed possible gavage trauma in 5 of 6 dead rabbits since the lungs were discolored and/or the lungs and trachea were fluid filled. This theory is supported as these animals did not exhibit any overt clinical observations prior to death (non-treatment-related).

300 mg/kg bw/day: 6/25 animals were found dead during the study on Days 6 (two unscheduled death), 9, 12, 18 and 27. Postmortem examination, revealed possible gavage trauma in 5 of 6 dead rabbits since the lungs were discolored and/or the lungs and trachea were fluid filled. This theory is supported as these animals did not exhibit any overt clinical observations prior to death (non-treatment-related).
The remaining animal of this dose group, died on Gestation Day 27 postmortem examination was unable to determine the cause of death, however findings included discolored lung foci and hemorrhagic kidneys (due to the single apperance, the death is rather considered unrelated to treatment).

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

100 and 300 mg/kg bw/day: Despite a significant increase in food consumption from Day 27 - 29 of gestation in mid- and high-dose females, food consumption was unremarkable in all animals. As body weight gain appeared normal in all animals, the biological significance of the altered food consumption remains questionable (non-adverse).

Food efficiency:

no effects observed

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:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No effect of treatment was evident from the macroscopic postmortem examination for the female rabbits.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Maternal developmental toxicity

Number of abortions:

effects observed, non-treatment-related

Description (incidence and severity):

50 mg/kg bw/day: 1/25 animals was euthanized on Gestation Day 27 as a result of an apparent abortion, fetus found under the cage.

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

not examined

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Gravid uterine weight: gravide uterine weight were comparable among the groups (please refer to Table 1).

Corpora Lutea: no effects observed

Details on maternal toxic effects:

Pregnancy rates and gravide uterine weight were comparable among the groups (please refer to Table 1). Uterine implantation data, number of corpora lutea and pre-and postimplantation loss and mean number of live fetuses were unremarkable.

Effect levels (maternal animals)

1

Key result

Dose descriptor:

NOEL

Remarks:

systmic toxicity

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal systemic toxicity

2

Key result

Dose descriptor:

NOEL

Remarks:

maternal developmental toxicity

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal developmental effects

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Fetal body weights were not affected by treatment when compared to the control group.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

No effect of treatment at any dose level was observed on mean number of live fetuses per female rabbit compared to control group.

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There was one dead fetus in the 50 mg/kg/day treated group which exhibited external malformations of gastroschisis, hindlimbs hyperflexion, and agenesis of the tail. The malformations in the dead fetus are not considered a teratogenic treatment-related effect since they occurred at a low incidence and were not observed in the other treatment groups.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

One commonly observed skeletal malformation (fused stemebrae) was present in all treatment groups, but at a single incidence in each group. This skeletal malformation has been observed previously in this strain of rabbit, are considered to be of spontaneous origin, and are not attributed to treatment. Skeletal variations were observed in the treatment groups, but the variations appeared at an incidence which were comparable to that of the control group, and thus were considered to be of spontaneous origin, and not attributed to treatment.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Visceral variations were observed in the treatment groups, but the variations appeared at an incidence which were comparable to that of the control group, and thus were considered to be of spontaneous origin, and not attributed to treatment.

Other effects:

not examined

Details on embryotoxic / teratogenic effects:

Details on embryotoxic / teratogenic effects:
Fetal body weights, fetal sex ratio and the number of live fetuses per female rabbit were comparable among the groups (please refer to Table 2). External, visceral and skeletal examinations did not reveal treatment-related effects and malformations were observed to a low incidence of 0.5 and 2% without being dose-related (please refer to Table 3). Fused sternebrae observed as skeletal malformation was present in all test groups in only 1 fetus. Due to the low incidence at any applied dose and the fact that fused sternebrae has been previously observed in this strain, the appearance of this skeletal malformation is considered as incidental and not treatment-related. Moreover, the death of 1 low-dose fetus which exhibited several external malformations including gastroschisis, hindlimbs hyperflexion and agenesis of the tail is not interpreted as treatment-related due to the low incidence and a missing dose-response relationship. External malformations were only present in control and low-dose fetuses. Moreover, visceral malformations were detected in 1 fetus each of control and high-dose animals (please refer to Table 3).

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no substance-related effects were observed on fetuses on the evaluated endpoints

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

Table 1. Maternal effects

 

Endpoint		Control	50 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day
Number of dams mated		25	25	25	25
Number of pregnant dams		22	24	23 (pregnancy undetermined in 1 animals)	19 (pregnancy undetermined in 5 animals)
Number of nonpregnant dams		3	1	1	1
Pregnancy rate (%)		88	96	96^#	95^#
Number of dams with viable fetuses		22 (88%)	23 (95.8%)	23 (95.8%)	18 (94.7%)
Mortality of dams		0	1 (elective sacrifice, GD 27)	1 (GD 11)	6  (GD 6 (2 animals), 9, 12, 18 and 27)
Examined at caesarean section		25	24	24	19
Clinical findings(frequency/animals)
General appearance	Red exudate from extremities	1/1	0/0	1/1	0/0
	Swollen vulva	1/1	4/1	0/0	3/2
Dermal (general)	Scabs	37/2	13/1	1/1	21/1
	Alopecia – extremities/snout	66/6	1/1	8/2	21/4
	Black/brown anogenital staining	0/0	0/0	7/3	7/1
	Yellow anogenital staining	7/2	1/1	0/0	0/0
	Ulceration	2/1	0/0	1/1	0/0
	Alopecia (general)	22/3	17/2	25/5	9/4
Miscellaneous	Decreased faecal volume	24/10	18/7	12/6	22/10
	Watery stool	0/0	1/1	2/2	1/1
	One fetus found in the cage	0/0	1/1	0/0	0/0
Post-dose	Excessive salivation (immed post dose)	0/0	0/0	0/0	1/1
	Palpable masses	0/0	0/0	2/1	20/1
Body weight gain (g) [GD 0 – 29]		501 ± 229.4	531 ± 189.9	552 ± 209.6	476±  208.6
Food consumption (g/kg bw/day) [GD 27 – 29]		21± 8.2	26± 10.6	31± 9.1**	29± 7.5*
Food consumption (g/kg bw/day) [GD 0 – 29]		39± 7.5	40± 7.6	41± 7.0	38± 6.1
Gravid uterus weight (g)		561±119.2	593±113.6	543±145.6	527±  135.8

#: The number of dams with undetermined pregnancy was excluded from calculations.

* p < 0.05

** p < 0.01

 

Table 2. Litter response

Endpoint	Control	50 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day
Corpora lutea (total number)	240	271	246	208
Corpora lutea (no. per aimal [mean])	10.9 ± 1.85	11.8 ± 2.24	10.7 ± 2.95	11.6 ± 2.20
Implantations (total)	192	219	195	157
Implantations (no. per animal [mean])	8.7 ± 2.07	9.5 ± 1.83	8.5 ± 2.87	8.7 ± 2.27
Total number of live foetuses	183	200	190	148
Number of live fetuses per animal [mean]	8.3 ± 2.01	8.7 ± 1.84	8.3 ± 2.68	8.2 ± 2.51
Total number of dead foetuses	0	1	0	0
Pre-implantation loss (% per animal)	20.2 ± 14.97	18.8 ± 8.93	22.1 ± 12.76	24.6 ± 17.75
Post-implantation loss (no per animal)	0.4 ± 0.59	0.8 ± 1.23	0.2 ± 0.42	0.5 ± 0.71
total number of litters	22	23	23	18
Foetus weight (mean) [g]	45.3 ± 3.91	44.7 ± 3.44	45.6 ± 4.33	43.8 ± 4.77
Foetal sex (ratio (% male)	52.1 ± 18.56	55.1 ± 13.39	44.6 ± 15.69	52 ± 12.46

 

Table 3. Examination of fetuses

Endpoint	Control	50 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day
Number of fetuses per group	183	201	190	148
Total number of fetuses with malformations	1	4	1	3
% of fetuses with malformations	0.5	2	0.5	2
External malformations	1 (gastroschisis, domed cranium, proboscis and open eyelid)	2 (spinal bifida, gastroschisis, agenesis of tail and hindlimb hyperflexion)	0	0
Skeletal malformations	0	1 (fused sternebarae)	1 (fused sternebarae)	2 (fused sternebarae and ribs fused and/or branched)
Visceral malformations	1 (heart and/or great vessel anomaly)	1 (heart and/or great vessel anomaly)	0	1 (hydrocephaly)

 

Applicant's summary and conclusion

Conclusions:

Based on the results obtained in this prenatal developmental toxicity study in rabbits, the NOAEL for maternal and developmental toxicity can be set at => 300 mg/kg bw/day.