Tetrakis(hydroxymethyl)phosphonium chloride

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 12 April 1990 to 14 august 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Comparable to guidelines.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

5 females per dose instead of 20

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Tetrakis(hydroxymethyl)phosphonium chloride

EC Number:

204-707-7

EC Name:

Tetrakis(hydroxymethyl)phosphonium chloride

Cas Number:

124-64-1

Molecular formula:

C4H12O4P.Cl

IUPAC Name:

tetrakis(hydroxymethyl)phosphonium chloride

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hazleton Research Products Inc., Pennsylvannia 17517, USA
- Age at study initiation: no data
- Weight at study initiation: 3.19 to 4.27 kg for females at mating
- Fasting period before study: no data
- Housing: individually in anodised stainless stell cages
- Diet: ad libitum, SQC standard rabbit diet, special diets services Ltd, Witham
- Water: ad libitum, filtered mains water via non-return automatic drinking valves
- Acclimation period: at least 12 days before mating


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16 to 22
- Humidity (%): 40 to 70
- Air changes (per hr): 15
- Photoperiod : 10 hrs dark / 14 hrs light (6AM to 8PM°


IN-LIFE DATES: FROM 19 APRIL 1990 TO 18 MAY 1990

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

distilled water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Solutions of the test article in distilled water were prepared daily. Nitrogen gas was bubbled through all test and control article preparations to reduce the dissolved oxygen content. Formulations were stored at ambient temperature, in containers purged with nitrogen, before use.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of formulations:
- stability: samples (100ml) of test article solutions formulated at 0.2 and 12 mg/ml were taken and analysed to determine the stability over 24 hours
- analysis for achieved concentration: samples (100 ml) of the formulations prepared on the first and last day of dosing were analysed for achieved concentration.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: no data
- Proof of pregnancy: no data, the day of mating was referred to as day 0 of gestation
- Any other deviations from standard protocol: Females that successfully completed coitus were injected intravenously via the marginal ear vein 10 I.U. of chorionic gonadotrophin to ensure ovulation.

Duration of treatment / exposure:

from day 7 to day 19 of gestation.

Frequency of treatment:

daily

Duration of test:

29 days

No. of animals per sex per dose:

5 females per dose

Control animals:

yes, concurrent vehicle

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes, twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily during gestation

BODY WEIGHT: Yes
- Time schedule for examinations: on days 0, 7, 8, 9, 12, 19, 24 29 of gestation

FOOD INTAKE ): Yes
The amount of food consumed by each female was recorded on days 3, 7, 8, 9, 12, 15, 17, 19, 21, 24, 27 and 29 of gestation and reported on the body weight interval

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: The ovaries and uteri were examined.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- 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
- Foetuses were weighed individually, examined externally and sexed by internal inspection of the gonads. Foetuses showing malformations and one control litter were fixed in industrial methylated spirit and retained, the remaining foetuses were discarded

Statistics:

No statistics were performed

Indices:

- percentage pre-implantation loss was calculated as: (number of corpora lutea - number of implantations)/ number of corpora lutea x 100
- percentage post-implantation loss was calculated as: (number of implantations- number of live foetuses)/ number of implantations x 100
- percentage male foetuses was calculated as: number of male foetuses/ number of foetuses of determined sex x 100

Historical control data:

No historical control data were provided to allow comparison with concurrent controls.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
One animal in the high dose group aborted and died on day 19 of gestation, having shown marked weight loss (15 %) and reduced food intake from the start of treatment. Necropsy revealed ulceration of the stomach and abnormal appearance of the duodenum, jejunum, liver and uterus. A small quantity of clear fluid was observed in the thoracic cavity.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

18 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Sex ratio (percentage male foetuses) was within expected limits in all groups, given the small sample size.
Mean foetal weight was lower than controls in all treated groups. Values were comparable to the expected range (39.9 to 43.5 g) for the low and intermediate dose groups and significantly reduced in the high dose group.
Pre-implantation loss was lower than the control group in all treated groups. The control value was unusually high owing to one unilateral pregnancy, which is not uncommon in this strain of rabbit.
Post-implantation loss was lower than controls in the low and intermediate dose groups and significantly increased in the high dose group. Three of the 4 high dose litters showed early or late intrauterine deaths.
Characteristic eye and/or limb malformations were observed in foetuses from all high dose litters. There was no effect of treatment at the lower doselevels investigated. No malformations were observed at 18 mg/kg/day and only one foetus from the group treated at 6 mg/kg/day showed a malformation. No malformations were observed in the control group.
Litter size was consequently lower than controls in the high dose group and comparable in the low and intermediate dose groups.

Effect levels (fetuses)

1

Dose descriptor:

NOAEL

Effect level:

18 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: teratogenicity

2

Dose descriptor:

NOAEL

Effect level:

18 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: embryotoxicity

3

Dose descriptor:

NOAEL

Effect level:

18 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: fetotoxicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Maternal toxicity

- Morbidity:

One female in the high dose group aborted and died on day 19 of gestation. This animal having shown marked weight loss (15%) and reduced food intake from the start of the treatment. At necropsy, ulceration of the stomach and abnormal appearance of the duodenum, jejunum, liver and uterus were revealed.

All other animals survived.

 

- Clinical signs:

No clinical signs related to treatment were observed.

 

- Bodyweight/food intake:

During all the study, mean bodyweight gain was comparable in the low dose group to controls. Weight losses were observed in the medium and high dose groups, with a dose relationship, -1.5% and -3.5% respectively. For the high dose group, bodyweight did not increase even if the dosing ceased.

 

Dosage-related reductions in food intake were observed in the medium and high dose groups during the treatment period. After dosing ceased, food intake was comparable to controls in the high dose group but remained lower in the medium dose group. Slight food consumption was observed for the low dose group during the days 24-29. No relation with clinical signs or necropsy findings was considered.

 

- Necropsy:

Necropsy revealed gastro-intestinal changes in 3 out of 5 animals from each treated groups. Findings in the digestive tracts were considered indicative of irritation due to the acidic test article (reddening of mucosa) rather than toxicityper se.

Foetal toxicity

- Uterine implantation (Table THPC RF Rabbit-1):

All animals in the treated groups were pregnant, and one control female was not.

 

Table THPC RF Rabbit-1: Implantation data of female rabbit daily administered with THPSat 3dose levels (low: 4.8 mg/kg bw, medium: 14.4 mg/kg bw, high: 48 mg/kg bw, main ingredient).

 

	Control	Low	Medium	High
N females	5	5	5	5*
N pregnant	4	5	5	5
Mean corpora lutea per female	10.3	9.0	9.4	11.3
Mean pre-implantation loss (%)	26.8	15.6	8.5	6.7
Mean post-implantation loss (%)	6.7	0.0	4.7	38.1
N females with live foetuses on day 29	4	5	5	4
N foetus	28	38	41	26
N foetus per female	7	7.6	8.2	6.5
% male foetuses	53.6	52.6	48.8	46.2

 

Pre-implantation loss was lower than the control group in all treated groups. The control value was unusually high owing to one unilateral pregnancy.

Post-implantation loss was lower than controls in the low and medium dose groups and significantly increased in the high dose group. Three of the 4 high dose litters showed early or late intrauterine deaths.

 

- Foetal data:

Sex ratio was within expected limits in all groups.

Mean foetal weight was lower than controls in all treated groups, but comparable to the expected range for the low and medium dose groups. It was significantly reduced in the high dose group.

Foetal malformations were observed in foetuses from all high dose litters. All the foetuses showed eye malformations (microphthalmia, retinal dysplasia) and a half exhibited limb malformations (malrotated limbs, (oligo)syndactyly). Concerning the other groups, only one foetus from the low dose group (4.8 mg/kg bw main ingredient) was malformed (arthrogryposis).

Applicant's summary and conclusion

Conclusions:

Under the conditions of this test, treatment with THPC at 60 mg/kg/day elicited severe maternal toxicity and associated embryo/foetal toxicity, demonstrated by an increased incidence of intrauterine deaths, reduced foetal weight and a high incidence of foetuses showing characteristic eye and limb malformations.

Executive summary:

In an oral (gavage) range-finding study (Hazleton UK, 1991), three groups of 5 mated New Zealand White rabbits were given THPC orally at dose levels of 6, 18 or 60 mg/kg daily from day 7 to day 19 of gestation. A similar group of rabbits, given the vehicle (distilled water) by the same route and over the same period served as controls. All animals were maintained until day 29 of gestation, killed and their uterine contents examined. At the highest dose, one animal aborted and died on day 19, having shown excessive weight loss and low food intake. Ulceration of the stomach was observed at necropsy. No other deaths were observed.

There were no treatment-related clinical changes. Signs indicative of irritation of the gastro-intestinal mucosa were observed in all treated groups but were considered to be a result of the acidic test article rather than toxicity per se. Treatment with THPC at 60 mg/kg/day elicited severe maternal toxicity and associated embryo/foetal toxicity, demonstrated by an increased incidence of intrauterine deaths, reduced foetal weight and a high incidence of foetuses showing characteristic eye and limb malformations. At 18 mg/kg/day, maternal toxicity to a lesser extent was also observed but there was no indication of an adverse effect on the developong embryo/foetus. There was no indication of toxicity, maternal or foetal, at 6 mg/kg/day.

Under the conditions of this test, THPC induced severe maternal toxicity, embryo/foetal toxicity and teratogenicity only at the highest dose level.

A maternal NOAEL of 18 mg/kg/day and a teratotoxicity and embryo/foetal toxicity NOAEL of 18 mg/kg/day were identified.