Desmedipham

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1991-05-22 to 1991-10-30

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

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:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Desmedipham

EC Number:

237-198-5

EC Name:

Desmedipham

Cas Number:

13684-56-5

Molecular formula:

C16 H16 N2 O4

IUPAC Name:

ethyl 3´-phenylcarbamoyloxycarbanilate

Test material form:

solid: particulate/powder

Details on test material:

The test material is Desmedipham but the purity has not been specified.

Specific details on test material used for the study:

white powder stored in the dark at ambient temperature

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: ca 4-6 months
- Weight at study initiation: ca 3.7 kg
- Housing: The animals were housed singly in aluminum grid bottomed cages measuring 48 x 61 x 46 cm. Excreta were collected on a peat covered tray, suspended beneath each cage. A separate stainless steel food hopper was provided for each cage, together with a drinking nozzle connected to an automatic watering system. The cages were suspended in racks, each containing 3 rows of 3 cages.
Excreta collection trays were cleaned twice weekly. Cage grids were changed as required. Accessible external surfaces and cage racks were cleaned once weekly.
- Diet: Standard Rabbit Diet, ad libitum until Day 3 of gestation; thereafter a weighed quantity of 250 g was available to each animal on each day.
- Water: domestic mains water, ad libitum.
- Acclimation period: 3-5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 18°C ± 3°C
- Humidity: 55% ± 10%
- Air changes (per hr): 15-20
- Photoperiod: There was automatic control of light cycle, Light hours were 0700-1900 h.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

DIET PREPARATION
Standard Rabbit Diet was available to the animals ad libitum until Day 3 of gestation; thereafter a weighed quantity of 250 g was available to each animal on each day. The food was supplied with batch analyses for nutritive constituents and a range of significant contaminants.

FORMULATION OF DOSING SUSPENSIONS
The test material was formulated as a suspension, the vehicle being carboxymethyl cellulose. Fresh dosing suspensions were prepared daily, each concentration being prepared separately. The test material was weighed into a labelled dose container and the requisite quantity of vehicle was added and mixed using a homogeniser. During dosing procedures, suspensions were maintained using a magnetic stirrer.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses for concentration, homogeneity and stability of dosing formulations were performed prior to commencement of this study. Results of these analyses indicated that formulations could be prepared to a satisfactory standard of accuracy and that they were stable for 24 hours. On the first and tenth days of treatment during the study, 3 x 1 mL aliquots were taken from each of the 3 concentrations of test material and from the Control suspension. These samples were analysed for concentration and homogeneity. Deviations from nominal concentration did not exceed 4%, indicating a satisfactory standard of accuracy in formulation.

Details on mating procedure:

Day 0 was the day of mating. No further info was provided

Duration of treatment / exposure:

6-18 days inclusive of gestation

Frequency of treatment:

Once daily.

Duration of test:

The animals were killed on Day 29 of gestation and the pregnancies were evaluated.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

Vehicle control

Doses / concentrations 2

Dose / conc.:

30 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

90 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

270 mg/kg bw/day

No. of animals per sex per dose:

16 females/group

Control animals:

yes

Details on study design:

- Dose selection rationale: Dose levels were agreed with the Sponsor after evaluation of the results of a separate dose range finding study.

Examinations

Maternal examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
All the animals were examined for reaction to treatment on each day. The onset, duration and intensity of any signs were recorded, with particular attention being paid to the period 1- 2 h after dosing.

All the animals were checked for viability at the beginning of each day, and again as late as possible on each day. Any animal showing signs of abortion or in poor condition was killed.
These observations included inspection of excreta trays.

BODY WEIGHT: Yes
- Time schedule for examinations:
The animals were weighed on the day of arrival and then on Days 6, 9, 12, 15, 19, 22, 26 and 29 of gestation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes / No / No data
- The weight of food consumed by each animal, from the 250 g offered each day, was recorded daily, commencing on Day 4 of gestation (weighed quantity first offered on Day 3).

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #29: Adult animals were killed by intravenous injection of sodium pentobarbitone, via the marginal ear vein. Foetuses were killed by intraperitoneal injection of sodium pentobarbitone. The single animal that aborted its pregnancy prior to Day 29 of gestation was killed on the day on which the abortion was discovered. This animal and the animal which died prematurely during the study were examined at necropsy with a view to diagnosis of each animal's condition. The examination proceeded, where practicable, as described below for necropsies conducted on Day 29 of gestation.

- Organs examined: The contents of the thoracic and abdominal cavities were inspected. Any lesions were described and, where appropriate, representative samples of abnormal tissue were preserved in neutral buffered 10% formalin. During the course of the necropsies, an apparent enlargement of the spleen was noted for the first High dose group animal. Spleen weights of all animals were therefore recorded. The reproductive tract was removed and weighed intact, then opened and the contents were examined. The number of corpora lutea graviditatis in each ovary and the number and position of all implant sites in the uterus were recorded. Each implant was classified as being live, a foetal death (death judged to have occurred after ca Day 18 of gestation), a late embryonic death (embryonic remains visible), or an early embryonic death (only placental remains visible). The borderline between early and late embryonic deaths in this classification lies at ca Day 12 of gestation.

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

Blood sampling:

None

Fetal examinations:

Foetuses (including foetal deaths) were examined for externally visible abnormalities. Each viable foetus was individually identified within the litter and weighed. Prior to fixation, the viable foetuses were examined macroscopically for abnormalities of the palate, and of the eyes and cranial bones following removal of the skin over these regions. Two thirds of the viable foetuses from each uterus were fixed in methylated ethyl alcohol and the remaining third in Bouin's fluid; the foetuses were sexed during subsequent dissection procedures. The foetuses fixed in alcohol were subsequently examined for visceral abnormalities by open dissection and the cranium was sectioned once through the coronal suture to allow inspection of the brain in that region. These foetuses were then eviscerated, and the carcasses cleared in potassium hydroxide and glycerol, and the skeletons stained with Alizarin Red S. Skeletal structures in these foetuses were examined for abnormalities and variants, including state of ossification. The foetuses fixed in Bouin's fluid were examined for soft tissue abnormalities by means of whole-body dissection, the internal head structures being examined by means of a free-hand razor blade sectioning technique.

Statistics:

At the discretion of the Study Director, statistical analyses were performed to determine the statistical significance of observed differences between treatment groups. Spleen weights were analysed by analysis of variance. Treatment means were compared by the F-protected Least Significant Difference procedure. Because there was no clear relationship between Day 29 of gestation body weight and spleen weight, analysis of covariance using this body weight as covariate was not considered appropriate.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 270 mg/kg bw/d, 12 out of the 14 pregnant animals had reduced faecal output during the treatment period. This finding was concomitant with periods of reduced food consumption. The incidences of reduced faecal output at 30 and 90 mg/kg bw/d were too small to be attributable to treatment. At 30 mg/kg bw/d, one animal aborted its pregnancy on Day 22 of gestation. This animal had shown a marked reduction in food consumption and a reduction in body weight. Immediately prior to necropsy on Day 29 of gestation, one animal in this group, aborted a foetus. The reason for this was not established, but it was not considered to have been related to treatment. Other occasional clinical observations and necropsy findings were considered to have been incidental.

Description (incidence):

There was no mortality

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 270 mg/kg bw/d, there was a reduction in mean body weight gain over the treatment period compared to Control, with a decrease in mean body weight over Days 6 to 12 of gestation.
Body weight performance at 90 and 30 mg/kg bw/d, was similar to Controls.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 270 mg/kg bw/d, mean food consumption was 49% lower than Control over the treatment period. The effect was most pronounced over Days 6 to 15 of gestation. Food consumption at 90 and 30 mg/kg bw/d was essentially similar to the Control value.

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

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:

effects observed, non-treatment-related

Description (incidence and severity):

At necropsy, an apparent increase in spleen size was observed in the first High dose animal. Spleen weights were therefore recorded for all animals. There was a slight increase in group mean spleen weight at 270 and 90 mg/kg bw/d. These increases did not achieve statistical significance and were considered to be of equivocal biological significance. The lower mean body weight at 270 mg/kg bw/d was not considered to have influenced the interpretation of the spleen weight data.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

An animal at 30 mg/kg bw/day dose level was killed prematurely. During the dosing procedure, red liquid was found on the catheter and the animal had dyspnoea. At necropsy, dark red lung lobes were seen. The findings were consistent with accidental damage during the dosing procedure.

Other occasional clinical observations and necropsy findings were considered to have been incidental.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

At 30 mg/kg bw/day, one animal aborted its pregnancy on Day 22 of gestation. This animal had shown a marked reduction in food consumption and a reduction in body weight. Histopathological examination revealed findings in the lung consistent with a mild reaction to an inhaled foreign body, and liver changes consistent with a period of inappetence. The aborted pregnancy was considered to have been incidental and not related to treatment with Desmedipham.

Maternal developmental toxicity

Number of abortions:

effects observed, non-treatment-related

Description (incidence and severity):

At 30 mg/kg bw/d, one animal aborted its pregnancy on Day 22 of gestation. This animal had shown a marked reduction in food consumption and a reduction in body weight. Histopathological examination revealed findings in the lung consistent with a mild reaction to an inhaled foreign body, and liver changes consistent with a period of inappetence. The aborted pregnancy was considered to have been incidental and not related to treatment with Desmedipham.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Group values for live and dead implants, after exclusion of the above animal, were within the normal background range reported in this laboratory.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

At 270 mg/kg bw/d, one animal resorbed its litter during early gestation. This animal had a low body weight and slightly low food consumption, prior to commencement of treatment. Resorption of the litter was considered to have been incidental and not related to treatment with Desmedipham.

Dead fetuses:

no effects observed

Details on maternal toxic effects:

Maternal toxicity was demonstrated at 270 mg /kg bw/d by body weight loss and markedly reduced food consumption, and by reduced faecal output. At both 270 and 90 mg kg bw/d there was a slight increase in mean spleen weight. The biological importance of this finding was uncertain.
At 90 mg/kg bw/d, maternal effects were confined to a slightly increased mean spleen weight.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

organ weights and organ / body weight ratios

Remarks on result:

other: Maternal NOAEL based on rediced spleen weight at 90 mg/kg bw/d; reduced weight gain, reduced food consumption and clinical signs at 270 mg/kg bw/d

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 270 mg/kg bw/d, there was some evidence of slightly retarded foetal development indicated by slightly reduced mean foetal weight, with the lowest weight litter demonstrating retardation of skeletal ossification. This effect was probably related to treatment and may have been secondary to the maternal toxicity.

Reduction in number of live offspring:

not examined

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

not examined

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

The incidences of the various abnormalities and variants did not indicate an adverse effect of treatment with Desmedipham.

Skeletal malformations:

no effects observed

Description (incidence and severity):

At 270 mg/kg bw/d, group incidences of the various skeletal ossification parameters indicated slight retardation of skeletal ossification.

Visceral malformations:

no effects observed

Details on embryotoxic / teratogenic effects:

At 270 mg/kg bw/d, group incidences of the various skeletal ossification parameters indicated slight retardation of skeletal ossification. This effect however was essentially due to one litter, which showed marked retardation of skeletal ossification and reduced foetal weight. When this litter was excluded from consideration, the group incidences at this level were essentially similar to the control values. The incidences of the ossification parameters at 90 and 30 mg/kg bw/d did not indicate any effect of treatment.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

fetal/pup body weight changes

Remarks on result:

other: Fetal NOAEL based on increased early embryonic death

Overall developmental toxicity

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

30 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

not specified

Relevant for humans:

not specified

Any other information on results incl. tables

See "Overall remarks, attachments" for the result's tables

Applicant's summary and conclusion

Conclusions:

There was no evidence of teratogenicity in this study. Maternal toxicity was clearly observed at the dose level of 270 mg/kg bw/d as a significant reduction in mean body weight gain and markedly reduced food consumption and reduced fecal excretion. The mean spleen weights were increased at 90 and 270 mg/kg bw/d, indicating hemolytical effects of desmedipham. Therefore, as a conservative approach, the maternal NOAEL can be settled at 30 mg/kg bw/d. Desmedipham was embryotoxic at 90 and 270 mg/kg bw/d. The observed effects included increased percentages of early embryonic deaths. Fetotoxicity was manifested as reduced fetal body weight at 270 mg/kg bw/d. The fetuses were considered not to be affected at treatment levels of 30 or 90 mg/kg bw/day. Due to the embryotoxic effect of desmedipham at 90 mg/kg bw/d, the NOAEL for developmental toxicity was 30 mg/kg bw/d.

Executive summary:

Mated female New Zealand White rabbits were randomised into 4 treatment groups, each containing 16 animals. These animals were dosed by gavage once daily over Days 6-18 inclusive of gestation, where Day 0 was the day of mating. Dose levels of test material were as follows:

Group	mg Desmedipham/kg bw/day
Control	0
Low dose	30
Intermediate dose	90
High dose	270

The test material was administered as a suspension, the vehicle being carboxymethyl cellulose.
The animals were monitored during gestation for clinical signs of toxicity, including body weight performance and food consumption.

The animals were killed on Day 29 of gestation and the pregnancies were evaluated. The foetuses were examined for visceral and skeletal abnormalities and variants.

Maternal toxicity was demonstrated at 270 mg Desmedipham/kg bw/day by body weight loss and markedly reduced food consumption, and by reduced faecal output.

At both 270 and 90 mg Desmedipham/kg bw/day, there was a slight increase in mean spleen weight. The biological importance of this finding was uncertain.

In conclusion, the developmental NOAEL is 30 mg/kg bw/d based on slight caudal pelvic shift.