[No public or meaningful name is available]

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 23 July 1990 and 24 August 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study. The study is adequately described. The test conditions are reasonably in compliance with guidelines, however the treatment period did not cover the whole period of organogenesis

Cross-reference

Reason / purpose for cross-reference:

read-across: supporting information

Reference

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

See attached justification document

Reason / purpose for cross-reference:

read-across source

GLP compliance:

yes

Species:

rabbit

Dose descriptor:

NOAEL

Effect level:

> 20 - < 30 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

clinical signs

urinalysis

Dose descriptor:

NOAEL

Effect level:

> 20 - < 30 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

external malformations

skeletal malformations

Developmental effects observed:

not specified

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

exposure was limited to the period of organogenesis (GD 7 to 19 only)

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OTS 798.4900 (Prenatal Developmental Toxicity Study)

Deviations:

not specified

Principles of method if other than guideline:

Study to investigate the effects of the test material on the embryonic and fetal development of the rabbit when administered during the period of organogenesis.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Tetrakis(hydroxymethyl)phosphonium chloride, oligomeric reaction products with urea

EC Number:

500-057-6

EC Name:

Tetrakis(hydroxymethyl)phosphonium chloride, oligomeric reaction products with urea

Cas Number:

27104-30-9

Molecular formula:

Not applicable - UVCB

IUPAC Name:

Tetrakis(hydroxymethyl)phosphonium chloride, oligomeric reaction products with urea

Test material form:

other: liquid stored at room temperature in the dark

Details on test material:

- Substance type: straw coloured liquid
- Storage condition of test material: room temperature in the dark

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.
- Age at study initiation: 5-7 months
- Weight at study initiation: Females in weight range 2.98-3.88 kg
- Fasting period before study: None
- Housing: individually
- Diet: pelleted (ad libitum)
- Water: filtered mains (ad libitum)
- Acclimation period: 6 weeks

ENVIRONMENTAL CONDITIONS
– Temperature: 16-22°C
- Humidity: 40-70%
- Air changes: 15 per hr
- Photoperiod: 10hrs dark / 14hrs light

IN-LIFE DATES:
From: 06 June 1990
To: 24 August 1990

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): no data
- Mixing appropriate amounts with (Type of food): no data
- Storage temperature of food: no data


VEHICLE
- Concentration in vehicle: 0, 2, 6, 20 mg/ml
- Amount of vehicle (if gavage): Constant dose volume of 5 ml/kg bw was used

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For each dose group the test article was dissolved in distilled water and made to volume. Formulations were made daily. The formulation was stirred and deoxygenated by bubbling nitrogen through it. Samples were removed for analysis from formulations of the highest and lowest concentrations (2 and 20mg/ml), prepared on days 1 and 16 of dosing.
The analytical method involved dilution of dosing solutions with deoxygenated water followed by titration against a 0.1N solution of iodine.

Stability analyses confirmed that the test article content of formulations decreased rapidly in the presence of oxygen, particularly in the lowest concentration. Analyses showed that the actual concentrations achieved were outside of the normal acceptance criteria. Doses are expressed as nominal but actual doses achieved were in the range 7.5 to 8.1mg/kg bw/day for the low dose group, 26.1 to 27mg/kg bw/day for the intermediate dose group and 89 to 92mg/kg bw/day for the high dose group.
Precautions were taken to minimise this loss e.g. distilled water was deoxygenated at the start of the formulation preparation instead of the end and animals were dosed within two hours of the formulation’s preparation.
The stability problem was not thought to affect the validity of the study since NOAELs could still be established.

Details on mating procedure:

Impregnation procedure: Each female was mated with one proven New Zealand white buck rabbit. Following successful mating females were injected intravenously via the marginal ear vein with chorionic gonadotrophin to ensure ovulation. The day of mating was designated as day 0.

Duration of treatment / exposure:

Days 7-19 gestation

Frequency of treatment:

Daily

Duration of test:

29 days

No. of animals per sex per dose:

16 mated female animals per dose group

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: - Results of a range-finding study guided the choice of dose levels for this study
- Rationale for animal assignment (if not random): the females were assigned to treatment groups using a randomisation procedure based on stratified body weight

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
Time schedule: twice daily examinations for morbidity and mortality. All dead or moribund animals were subjected to necropsy

DETAILED CLINICAL OBSERVATIONS: Yes, daily, examinations for abnormalities of appearance or behaviour or other signs of reaction to treatment or ill-health recorded.


BODY WEIGHT: Yes, body weight of each female recorded on days 0, 7, 8, 9, 12, 19, 24 and 29 of gestation.

FOOD CONSUMPTION: recorded for days 0-3, 3-7, 7-8, 8-9, 9-12, 12-15, 15-17, 17-19, 19-21, 21-24, 24-27 and 27-29 of gestation.

POST-MORTEM EXAMINATIONS: Yes, surviving females were sacrifice on gestation day 29 by an intravenous injection of sodium pentobarbitone (200mg.ml) and examined macroscopically.
- Organs examined: ovaries and uteri

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
- Other: Pregnancy status and number of alive and dead fetuses, intrauterine positions of fetuses.

Fetal examinations:

- External examinations: Yes: [all]
- Soft tissue examinations: Yes: [all]
- Skeletal examinations: Yes: [all ]
- Head examinations: Yes: one half in each litter

Statistics:

Data were processed where appropriate, to give litter mean values, group mean values, and standard deviations.
The following parameters were analysed as follows:
Analysis of variance, t-test:
Body weight - day 0

Analysis of variance, Dunnett’s test, regression analysis:
Body weight gain – days 0-7, 9-12, 12-19, 19-29
Food intake – days 0-7, 7-9, 9-12, 12-19, 19-29
Litter weights

Kruskal-wallis, Terpstra-Jonckheere and Wilcoxon rank-sum test:
Body weight gain – days 7-9
Number of corpora lutea
Number of implantations
Number of fetuses
% Pre-implantation loss
Fetal weight – male, female, combined
% Male fetuses
External/visceral variations

Cochran-Armitage and Fischer’s exact test:
Proportion of litters with;
Early intrauterine death
Late intrauterine death
Post-implantation loss
External/visceral variations
Skeletal malformations
Either external/visceral malformations or skeletal malformations
All fetuses showing skeletal variations.

Indices:

Percentage pre-implantation loss, percentage post-implantation loss and percentage male fetuses were calculated as follows:

Percentage pre-implantation loss:
(No. corpora lutea-No. implantations/ No. corpora lutea ) X 100

Percentage post-implantation loss:
(No. implantations – No. live fetuses/No. implantations) X 100

Percentage male fetuses:
(No. male fetuses/No. fetuses of determined sex) X 100

Historical control data:

Historical data for the six preceding teratology studies were provided. The source of the data was the test house (Hazleton UK) conducting the current study.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
1- Mortality:
One animal from the intermediate dose group was found dead on day 20 of gestation, which necropsy determined to be due to a dosing error. Abnormal respiration was noted in one animal of the high dose group, which was killed in day 13 of gestation. Dark eyes and coloured urine were detected in this animal. Necropsy revealed mottling of the liver and darkening of the lungs. All implantation were non-viable. Since none of these effects were observed in other animals they were not considered to be treatment related.

Other results were as follows:

High dose group (100 mg/kg bw/day):
Coloured urine in 11/16.
Weight loss between days 7 to 9 gestation in 13/16 animals leading to a statistically significant (p<0.05) reduction in the group mean weight.
Statistically significant (p<0.01) reduction in weight gain in latter half of treatment period (days 12-19).
Once treatment had finished body weight gain was comparable to the control group.
Food intake statistically significantly (p<0.05 days 7-9; p<0.01 days 9-12; p<0.001 days 12-19) lower than the control group throughout the treatment period, but comparable thereafter.
Lower number of implantations leading to a dose-response (p<0.05) for pre-implantation loss. This effect could have been due to the significant weight loss at the beginning of the treatment period. However, implantation is expected to occur before day 7 of gestation, i.e. before treatment began, therefore no association can be made between treatment and pre-implantation loss. In addition, individual animal data showed no direct relationship between animals who lost weight and those with losses.

Intermediate dose group (30 mg/kg bw/day):
No effect on body weight.
No effect on food consumption.

Low dose group (10 mg/kg bw/day):
No effect on body weight.
No effect on food consumption.

The number of corpora lutea was comparable amongst all groups. All values for post-implantation loss and litter size were in the expected ranges, so treatment did not appear to affect these parameters. Pregnancy rate was 93.8% or 100% in all groups. One animal in the low dose group showed a total resorption, but this was not thought to be related to treatment as one of the control animals also showed a total resorption.

Please refer to Table 7.8.2/1 for details of caesarean section observations.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

19.5 mg/kg bw/day

Based on:

act. ingr.

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
The sex ratio was comparable to controls in the high and intermediate dose groups. The high proportion (not statistically significant) of males in the low dose group was not considered to be treatment-related due to the lack of dose response in the two higher dose levels.
Decreased mean litter weight was observed at 100 mg/kg bw/day, but was considered to be due to a lower number of foetus due to increased pre-implantation loss.
No clear treatment-related changes in the number of fetuses showed external/visceral and/or skeletal malformations were noted. However:
At 100 mg/kg bw/day:
- One foetus presented: retinal dysplasia, Brachydactyly, Major fusion of sternebrae, Forelimb phalanx/ges absent. These types of malformations (eye and limb) were not reported neither in the study control data, nor in the Historical control data.
- One foetus presented: Fused ribs
At 30 mg/kg bw/day:
- One foetus presented multiple malformations including: open eye (s), Iris malformed, Eye socket (s) reduced in size, Zygomatic arch(es) malformed
parietal malformed, Maxilla (e) malformed, Nasal malformed , Frontal malformed hydroencephalocoele, plagiocephaly,cleft palate malrotated hindlimbs, Talipes, Brachydactyly, sinistrocardia, pre-ductal coarctation of aortic arch, eye socket (s) reduced in size, zygomatic arch(es) malformed, parietal (s) malformed, maxilla (e) malformed, nasal malformed, frontal malformed, Major fusion of sternebrae
- One foetus presented: stenosis of ascending aorta
- At 10 mg/kg bw/day:
- one foetus presented: hydronephrosis
- Two foetuses presented: internal hydrocephaly
- Control:
- 2 foetuses: Scoliosis, fused vertebral centra, hemivertebrae, fused ribs
- 1 foetus: Ribs confluent, proximally fused
- 1 foetus: duplicated gall bladder
The incidence of fetuses with external/visceral variations increased in the intermediate and high dose groups (the trend was statistically significant: p<0.05). The variations involved are: abnormal common carotid and agenesis of the intermediate lung lobe. In addition, a slightly increased incidence of bilateral and unilateral insertion of pelvic girdle on the second sacral vertebra was noted at 30 and 100 mg/kg bw/day. As this latter finding was also observed in the same proportions in controls, it was considered not to be treatment-related.
There was no effect of treatment on the overall incidence of skeletal variations. There were no dose-related increases in the incidence of any specific skeletal variation. The proportion of fetuses with extra thoracic ribs was increased in all dose groups compared with controls. However, the toxicological significance of this is unknown, as this variation was observed at a similar incidence in the control group (68.7%) of a subsequent study (HUK Project number 555/11)

Please refer to Table 7.8.2/2 and Table 7.8.3/3 for details of developmental results.

Effect levels (fetuses)

1

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: teratogenicity

2

Dose descriptor:

NOAEL

Effect level:

19.5 mg/kg bw/day

Based on:

act. ingr.

Basis for effect level:

other: teratogenicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Table 7.8.2/1: Maternal toxicity and pregnancy records

 

Parameter	controldata		low dose^4	medium dose^2	high dose^2, 3
	Historical^1	Study^4
Number of dams examined	120	16	16	16	16
Clinical findings during application of test substance	ND	- Fur staining, rough haircoat.	- Fur staining, rough haircoat.	- Fur staining, rough haircoat.	- Fur staining, rough haircoat, - Coloured urine, - Abnormal respiration
Mortality of dams (#/%)	ND	-	-	1/6.25^2	1/6.25^2
Abortions (N females)	ND	-	-	-	-
Body weight gain (%) day 0-29 (end of test) day 7-19 (dosing period)	ND	22.8 7.3	19.9 6.5	21.1 6.9	15.8* 0.5**
Food consumption (g/rabbit/day) day 0-29 (end of test) day 7-19 (dosing period) day 19-29 (post dosing)	ND	168 179 143	166 178 139	170 183 140	148* 123** 142
Pregnancies (# / %)	ND / 96.5 (93.8-100)	100	100	100	15 / 93.75
Necropsy findings in dams dead before end of test	ND	-	-	-	- mottling of the liver - lung darkening.

¹Historical control data were issued from the 6 rabbit teratology studies immediately preceding the current study.

²Due to early death (one female from the medium dose group died because of an intubation error, one female from the high dose group was killed on day 13 of gestation) one female were excluded from bodyweight gain and food consumption calculations.

³Due to no pregnancy, one female was excluded from bodyweight gain and food consumption calculations.

⁴Due to total embryo-foetal resorption, one female from control group and one from low dose group were excluded from bodyweight gain and food consumption calculations.

ND: No Data

Statistical analysis: *: 0.05>p>0.01; **: 0.01>p

 

 

 

Table 7.8.2/2: Cesarian section observations.

 

Parameter	controldata			low dose	medium dose	high dose
	Historical^1	study
Corpora lutea (# total / # per dam)	ND / 9.4 (8.8-10.4)		134 / 8.9	134 / 8.9	142 / 9.5	129 / 9.2
Implantations (# total / # per dam)	ND / 8.5 (8.3-9.1)		127 / 8.5	119 / 7.9	127 / 8.5	106 / 7.6
Resorptions^a(# total / # per dam) Early resorption Late resorption	ND / 0.5 (0.1-1)   ND / 0.3 (0-0.8) ND / 0.2 (0.1-0.5)		4 / 0.2   2 / 0.1 2 /0.1	9 / 0.6   3 / 0.2 6 / 0.4	6 / 0.4   4 / 0.3 2 / 0.1	4 / 0.2   2 / 0.1 2 /0.1
Number of foetuses (# total / # per litter)	916 / 7.9 (7.4-8.2)		123 / 8.2	110 / 7.3	121 / 8.1	102 / 7.3
Pre-implantation loss (%)	9.6 (6.1-14.6)		5.2	11.2	10.6	17.8*
Post-implantation loss (%)	6.4 (1.7-11.9)		3.1	7.6	4.7	3.8
Total number of litters	120		15	15	15	14
Live foetuses / litter	8.0 (7.4-8.7)		8.2	7.3	8.1	7.3
Dead foetuses / litter	0		0	0	0	0
Foetus weight (mean, g)	42.1 (39.9-43.5)		43.8	45.0	43.6	43.7
Foetal male ratio (%)	48.4 (41.5-53.5)		44.3	45.3	42.9	44.0

 

¹Historical control data were issued from the 6 rabbit terotology studies immediately preceding the current study.

^aResorption = Early resorption (intrauterine death with decidual or placental tissue only) + Late resorption (intrauterine death with embryonic or foetal tissue in addition to placental tissue),

Statistical analysis: *: 0.05>p>0.01

ND: No Data

 

 

Table 7.8.2/3: External, Visceral and Skeletal Examinations

  

Parameter	controldata		low dose	medium dose	high dose
	Historical^1	study
Number of foetuses examined	916	123	110	121	102
External/Visceral malformations (# / %)	10 / 1.1 (0-2.4) - Abdomen (gall bladder agenesis) - Heart (dilatation/interruption of aortic arch).	1 / 0.8 - Abdomen (additional gall bladder).	3 / 2.7 - Head (hydrocephaly), - Abdomen (hydronephrosis)	2 / 1.7 - Heart (stenosis of ascending aorta), - Foetus with multiple malformations	1 / 1.0 - Eye (retinal dysplasia).
External/Visceral anomalies (#/%)	340 / 37.1 (21.8-45.5)	56 / 45.5	50 / 45.5	64 / 52.9	61 / 59.8*
Skeletal malformations (#/%)	5 / 0.6 (0-0.9) - Skull (cervical vertebral centrum absent), - Vertebrae (vertebrae absent/fused), Ribs (fused).	3 / 2.4 - Ribs (confluent ribs). - Scoliosis, - Skull (fused vertebral centra), - Vertebrae (hemivertebrae).	0 / 0.0	1 / 0.8 - Foetus with multiple malformations	2 / 2.0 - Limbs (forelimbs phalanges absent, brachydactyly), - Ribs (confluent ribs).
Skeletal anomalies (# / %) Extra thoraco-lumbar ribs (# / %)	855 / 93.3 (88.6-97.4) ND/ND	118 / 95.9 56/45.5	107 / 97.3 70/63.6	117 / 96.7 84/69.4	91 / 89.2 65/63.4

¹Historical control data were issued from the 6 rabbit teratology studies immediately preceding the current study.

ND: No Data

Statistical analysis: *: 0.05>p>0.01; **: 0.01>p>0.001; ***: 0.0001>p

 

Applicant's summary and conclusion

Conclusions:

Under the test conditions, Treatment with Tetrakis (hydroxymethyl) phosphonium chloride oligomeric reaction products with urea (THPC-urea) during the period of organogenesis (GD7 to 19), even at doses causing significant maternal toxicity. The NOAELs were identified: NOAEL (maternal): 19.5 mg Active Ingredient (AI)/kg bw/day, NOAEL (teratogenicity): 19.5 mg AI/kg bw/day.

Executive summary:

In a teratogenic study (L. Barker, 1992), according to OECD 414 and in compliance with GLP, pregnant rabbits received consecutive daily oral doses of Tetrakis (hydroxymethyl) phosphonium chloride oligomeric reaction products with urea (THPC-urea) at dose levels of 6.5, 19.5 and 65 mg Active Ingredient (AI)/kg bw/day in water from the 7th to 19th day of gestation. The animals employed were a New Zealand White strain rabbit and 16 pregnant rabbits were used in each group. A similar group of 16 rabbits given the vehicle (distilled water) by the same route and over the same period served as controls. There were no treatment related mortalities at any dose tested. Maternal effects were noted at 65 mg AI/kg bw/day and included coloured urine (11/16), reduced body weight gains and reduced food consumption. No toxicological significant changes in foetal weight were noted. Decreased mean litter weight was observed at 65 mg AI/kg bw/day, but was considered to be due to a lower number of foetuses due to increased pre-implantation loss. No clear treatment related changes in the number of foetuses showing external, visceral or skeletal malformations were noted. However, at 65 mg AI/kg bw/day, one foetus showed retinal dysplasia, absent forelimb phalanges, brachydactyly and fused sternebrae. At 19.5 mg AI/kg bw/day, one foetus presented multiple malformations including open eye, Iris malformed, Eye socket reduced in size and zygomatic arches malformed. In the high dose groups, an increased incidence (statistically significant) in foetuses showing external and visceral variations was noted, and included abnormal common carotid and agenesis of the intermediate lobe, in addition, a slightly increased incidence of bilateral and unilateral insertion of pelvic girdle on the second sacral vertebra was noted at 19.5 and 65 mg AI/kg bw/day. The slightly increased incidence of extra thoraco-lumbar ribs noted at all dose levels tested was considered to be due to a low incidence in control animals, when compared to historical control data.

Under the test conditions of this study, the NOAEL (maternal): 19.5 mg AI/kg bw/day based on decreased body weight and food consumption in females and the NOAEL (teratogenicity): 19.5 mg AI/kg bw/day based mainly on eye and/or limb malformations.

The malformations (oligosyndactyly, microphtalmia and retinal displasia (1 foetus/65 mg AI/kg bw/day), and oligosyndactyly, brachymelia, microphtalmia and retinal dysplasia (4 foetuses/97.5 mg AI/kg bw/day) observed in a range finding study in rabbits (See: tox. repro V2 1991 Bark) were considered to be secondary non-specific effects (for explanation, see summary in the Head Chapter of this section).

The developmental toxicity study in the rabbit is judged as acceptable and satisfies the guideline requirement for a developmental toxicity study (OECD 414) in rabbit.