Ziram

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 Jun 1989 - 11 Jun 1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted in 2018

Deviations:

yes

Remarks:

the treatment was restricted to the period of organogenesis and did not cover the complete gestation period, only 4 days of acclimation period, continued under "Principles of method if other than guideline"

Principles of method if other than guideline:

Further deviations to the current guideline (adopted in 2018): anogenital distance not measured, gravid uterus weights not shown, no historical control data provided and parameters related to the detection of endocrine disrupting potential (thyroid weights, thyroid histopathology and thyroid hormones) not examined as they were not required at the time the study was conducted, no special attention paid to reproductive tract of foetusses.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Ziram

EC Number:

205-288-3

EC Name:

Ziram

Cas Number:

137-30-4

Molecular formula:

C6H12N2S4Zn

IUPAC Name:

ziram

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Portage, Michigan, USA
- Age at study initiation: 8 - 10 weeks
- Weight at study initiation: 161 - 218 g
- Housing: 5 per cage in suspended galvanised metal cages
- Diet: Labsure Laboratory Animal Diet No. 1, ad libitum
- Water: tap water, ad libitum
- Acclimation period: Pregnant females arrived at the laboratory 4 days before beginning of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23
- Humidity (%): 52 - 60
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 19 Jun 1989 To: 04 Jul 1989

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 1% Methylcellulose (MC)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Suspensions of the test article were prepared on a daily basis. The highest required concentration was prepared by suspending a weighed quantity of the test article in the vehicle. The low concentrations were prepared by serial dilution. The dose volume was 0.1 mL/100 g.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples were taken for analysis of achieved concentration on the second day of dosing. Results showed that achieved concentration was within 9% of nominal concentration.
At nominal concentrations of 1.0 and 80 mg/mL, the results indicate that the test substance produces a homogeneous suspension in 1% MC formulations. Analysis of formulation showed that homogeneity was maintained by magnetic stirring for 1 h and that the formulation could be successfully resupended 4 h after preparation during storage at ambient temperature and at 24 h after storage at 4 °C.
Further, chemical stability of the test substance in 1% MC formulations was confirmed during storage at ambient temperature in the dark for 4 h and at 4 °C for 24 h.

Details on mating procedure:

No data.- Impregnation procedure: purchased timed pregnant
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as Day 0 of pregnancy

Duration of treatment / exposure:

Day 6 - 15 post mating

Frequency of treatment:

Daily

Duration of test:

Day 20 of gestation

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

4 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

16 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

64 mg/kg bw/day (nominal)

No. of animals per sex per dose:

25 females

Control animals:

yes, concurrent vehicle

Details on study design:

Time-mated females were purchased, the first batch consisting of 78 animals followed by the second batch of 52 animals one day later. The first batch is referred to as batch A and the second batch as batch B.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked: signs of reaction to treatment

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed initially (Day 1 of pregnancy for group A and Day 2 for group A and B) and on Day 3, 6, 8, 10, 12 14, 16, 18 and 20.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No
Food consumption was measured from weigh day to weigh day.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water consumption was measured daily from Day 2 to Day 20 of pregnancy inclusive.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: Uterus and ovaries
On Day 20 of pregnancy the animals were sacrificed, dissected and examined for congenital abnormalities and macroscopic pathological changes in maternal organs. Abnormal tissues were preserved. The ovaries and uteri were examined immediately.

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

Blood sampling:

- Plasma: No

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
- Anogenital distance of all live rodent pups: no

Statistics:

Analyses were performed on litter data. Tests were two-tailed.
- Litter data: Basic sample unit was the litter, and due to the preponderance of non-normal distributions, non-parametric analyses proved most consistent.
Mean values of litter size, pre- and post implantation loss, litter weight, mean pup weight, sex ratios and the incidence of anomalous offspring were analysed by the Kruskal-Wallis test. Intergroup comparisons were made by the non-parametric equivalent of the ‘t’ test together with the Jonckheere test for an ordered series of treatments.
Where 75% of the values for a given variable consisted of one value, a Fisher’s exact test was used.
- Water, food and bodyweight data: Analysis of variance, followed by Williams’ test were used for assessing Intergroup differences in absolute values for mean water and food consumption and bodyweight during gestation.

Indices:

In assessing litter parameters, pre-implantation loss was calculated as a percentage as:
((No. of corpora lutea - no. of implantations) x 100)/ No. of corpora lutea

Post implantation loss was calculated as:
((No. of implantations - no. of live young) x 100)/ No. of implantations

Historical control data:

Historical control data were not provided.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Treatment at 64 mg/kg bw/day was associated with slight post-dosing salivation and hair loss. At 16 mg/kg bw/day, two animals showed slight post-dosing salivation. There were no obvious treatment-related signs at 4 or 1 mg/kg bw/day.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There was an initial slight body weight loss from Day 6 to Day 8 in 13/23 and 20/24 dams at 64 and 16 mg/kg bw/day, respectively, against a combined incidence of 0/69 in the control, 1 and 4 mg/kg bw/day dose group. Body weight values of dams adjusted for body weight at Day 6 were statistically significantly lower than the controls through the study period at 16 and 64 mg/kg bw/day.
Body weight gain at 64 mg/kg bw/day was slightly retarded to Day 16 and parity with controls was not regained by termination. At 16 mg/kg bw/day body weight gain to Day 16 paralleled controls. Body weight gain at 1 and 4 mg/kg bw/day was similar to controls.
For details, please refer to attachment 1 under "Overall remarks, attachments" and to table 1 under "Any other information on results incl. tables".

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Treatment at 16 and 64 mg/kg bw/day was associated with a dosage-related decrease in mean food consumption throughout the dosing period; differences from controls attained statistical significance. At the end of treatment appetite returned and was similar to controls. Treatment at 1 and 4 mg/kg bw/day did not noticeably affect mean food consumption.
For details, please refer to attachment 2 under "Overall remarks, attachments" and to table 1 under "Any other information on results incl. tables".

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

Treatment at 16 and 64 mg/kg bw/day was associated with a dose-related increased water consumption throughout the dosing period. Differences from controls attained statistical significance; during the post-dosing period (Days 16 to 19) mean consumption was similar to controls. At 4 mg/kg bw/day mean water consumption during the dosing period was marginally higher than the control, but differences were not statistically significant.
For details, please refer to attachment 2 under "Overall remarks, attachments" and to table 1 under "Any other information on results incl. tables".

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 obvious treatment-related macroscopic changes were detected at terminal autopsy.

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:

not specified

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

Although slight intergroup variation in mean pre- and post-implantation losses were observed when compared to the control group, no statistically significance was obtained and no clear dose-dependency was observed. The implantation rate was coparable among all groups.
For details, please refer to attachment 3 under "Overall remarks, attachments"

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

There was no instance of total resorption during the study.

Early or late resorptions:

no effects observed

Description (incidence and severity):

No significant difference in early or late resorption was observed in any of the treatment groups compared to the control.
For details, please refer to attachment 3 under "Overall remarks, attachments"

Dead fetuses:

no effects observed

Description (incidence and severity):

No statistically significant difference in the number of death foetuses was observed in any of the treatment groups compared to the control.
For details, please refer to attachment 3 under "Overall remarks, attachments"

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

No statistically significant changes in the number of pregnant rats were observed in any of the treatment groups compared to the control. In the control, 4, 16 and 64 mg/kg bw/day dose groups were 2, 1, 3, 2 and 1 rats non-pregnant out of 25 females, respectively.
For details, please refer to attachment 3 under "Overall remarks, attachments"

Other effects:

not examined

Effect levels (maternal animals)

1

Key result

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

4 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: no adverse effects observed at this dose level

Remarks on result:

other: main study (ZIR 15/24/89137)

2

Key result

Dose descriptor:

LOAEL

Remarks:

maternal general toxicity

Effect level:

16 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

water consumption and compound intake

Remarks on result:

other: main study (ZIR 15/24/89137)

3

Key result

Dose descriptor:

NOAEL

Remarks:

maternal developmental toxicity

Effect level:

64 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: no adverse effect observed at the highest dose level tested

Remarks on result:

other: main study (ZIR 15/24/89137)

4

Key result

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

8 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: no adverse effects observed at this dose level

Remarks on result:

other: supplementary study (PFX 002/033719)

5

Key result

Dose descriptor:

LOAEL

Remarks:

general toxicity

Effect level:

16 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

Remarks on result:

other: supplementary study (PFX 002/033719)

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean fetal body weight showed a dose-dependent reduction and reached statistical significance at 64 mg/kg bw/day when compared to the control group, which was reflected in the low mean litter weight. Therefore, the effect was considered to be related to the treatment with the test substance, but represents a secondary effect due to maternal toxicity observed at the highest dose tested.
For details, please refer to attachment 3 under "Overall remarks, attachments" and to table 2 under "Any other information on results incl. tables".

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

No difference in the number of live offspring were observed in any of the treatment groups when compared to the control group.
For details, please refer to attachment 3 under "Overall remarks, attachments".

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Sex ratio was similar among both control and treated animals.
For details, please refer to attachment 3 under "Overall remarks, attachments".

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

The slight intergroup variation in litter size was not considered treatment-related. Mean litter size at 64 mg/kg bw/day was identical to the control value. However, the litter weight was statistically significantly decreased in the high-dose group when compared to the control group. In the other treatment groups, no statistically significant difference compared to the control was observed. The reduction in litter weight in the high-dose group was considered to be related to the treatment with the test substance, but represents a secondary effect due to maternal toxicity observed at the highest dose tested.
For details, please refer to attachment 3 under "Overall remarks, attachments" and to table 2 under "Any other information on results incl. tables".

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):

Malformations:
Observed external malformations included microphthalmia observed in 1/255 foetusses in the low-dose group and 1/272 foetusses in the high-dose group, both which are considered incidental.
For details, please refer to attachment 4 under "Overall remarks, attachments".

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Malformations and anomalies:
No treatment-related skeletal malformations were observed during the study. The incidence of malformed foetuses was 4, 2, 1, 1 and 4 in control, 1, 4, 16 and 64 mg/kg bw/day dose groups.
The incidence of foetuses with skeletal anomalies was similar in both control and treated groups.

Variants:
The marginally higher incidence of foetuses at 64 mg/kg bw/day with unossified sternebrae was probably related to the lower foetal weight. Differences in percentage incidence were not statistically significant. Treatment at 1, 4 and 16 mg/kg bw/day was not considered to affect the occurrence of skeletal variants. The incidences of skeletal variants were not statistically significantly different from the control.

For details, please refer to attachment 4 under "Overall remarks, attachments".

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

Malformations and anomalies:
Visceral malformations observed included cardiac, situs inversus and anals atresia, diaphragmatic hernia and umbilicial hernia. These malformations only occured in one fetus and were considered incidental.
The incidence of foetuses with visceral anomalies was marginally higher at 16 and 64 mg/kg bw/day compared with controls. This increase was not dose-related but 5/133 and 5/134 foetuses at 16 and 64 mg/kg bw/d respectively showed thinning of the diaphragm/protrusion of the liver. Although a high proportion of foetuses at the two top doses showed the same visceral anomaly the relationship to treatment is not clear as no foetuses at these doses showed diaphragmatic hernia.
For details on visceral malformations, please refer to attachment 4 under "Overall remarks, attachments" and to table 2 under "Any other information on results incl. tables".

Effects of the test substance on the development of diaphragm in foetuses were further investigated in an supplementary study, which can be found under "Any other information on results incl. tables".

Other effects:

not examined

Effect levels (fetuses)

1

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

4 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at this dose level

Remarks on result:

other: main study (ZIR 15/24/891371 Part I)

2

Key result

Dose descriptor:

LOAEL

Remarks:

developmental toxicity

Effect level:

16 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

visceral malformations

Remarks on result:

other: main study (ZIR 15/24/891371 Part I)

3

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

16 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects at this dose level

Remarks on result:

other: supplementary study (PFX 002/033719)

4

Key result

Dose descriptor:

LOAEL

Remarks:

developmental toxicity

Effect level:

64 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

visceral malformations

Remarks on result:

other: supplementary study (PFX 002/033719)

Fetal abnormalities

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

other: visceral/soft tissue: thinning of the diaphragm

Description (incidence and severity):

the incidence of diaphragmatic thinning was lower in the offspring than in foetus; no incidence of development into diaphragmatic hernia was observed

Overall developmental toxicity

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

16 mg/kg bw/day (nominal)

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

Table 1         Maternal toxic effects
Parameter	0 mg/kg	1 mg/kg	4 mg/kg	16 mg/kg	64 mg/kg	Dose-response +/–
Number of dams examined	25	25	25	25	25
Mean water consumption (Day 6-15)			(↑)	↑**	↑**	–
Mean food consumption (Day 6-15)				↓**	↓**	+
Bodyweight loss with retarded weight gain	No	No	No	Yes**	Yes**	+
**p 0.01 Williams' test
Table 2            Embryo toxic effects
Parameter	0 mg/kg	1 mg/kg	4 mg/kg	16 mg/kg	64 mg/kg	Dose-response +/–
Number of foetuses examined	126	125	123	133	134
Litter weight					↓*	–
Mean foetal weight					↓***	+
Incidence of foetuses with visceral anomalies	8.3%	4.9%	6.9%	14.9%	12.2%	–
Thinning of diaphragm (no. of foetus affected)			1	5	5	–
*    p 0.05 ***p 0.001 Jonckheere statistic

 

Supplementary study to report ZIR 15/24/891371: Investigation of the incidence of diaphragmatic thinning and diaphragmagic hernia in the fetuses and offspring of CD rats treated by oral gavage during organogenesis (supplementary study to report)

An additional study was conducted with the objective to investigate the influence of the test substance on the development of the diaphragm in the fetus, with specific reference to diaphragmatic thinning and diaphragmatic hernia recorded in fetuses in an earlier study (1990). In order to monitor the subsequent progression of this particular abnormality postnatally, similar observations were made on culled offspring at Day 4 of age and on weanling offspring from females allowed to litter. Treatment of the parental females was restricted to the period of organogenesis (Days 6 - 15 post coitum) to match the period of treatment in the original study. The test substance was administered during the organogenesis phase (Days 6 - 15) of pregnancy to rats. Three groups of 44 mated female rats received the test item by oral gavage at dosages 0, 8, 16 or 64 mg/kg bw/day from Days 6 - 15 after mating. Twenty two females in each group were sacrificed on Day 20 after mating for fetal examination and the remaining females were allowed to litter to permit examination of offspring on Day 4 and 21 of age.
Clinical signs, body weight and food consumption were monitored. Adult females were examined macroscopically at necropsy on Day 20 after mating or after weaning. Fetuses were examined macroscopically at necropsy and subsequently by detailed examination of the diaphragm after fixation. Offspring received detailed examinations of the diaphragm at either Day 4 or Day 21 of age.
Females receiving 64 mg/kg bw/day of the test substance showed reduced activity, piloerection, reduced body temperature and partially closed eyelids approximately 6 h after dose administration on the first day of treatment only and sporadic incidences of increased salivation and rales were recorded at later stages through the dosing period. There were no deaths. There was marked body weight loss in the early treatment period and a marked reduction in food intake. Food intake and body weight gain remained low throughout treatment and animals were much lighter than controls at necropsy on Day 20 of gestation. Females allowed to litter continued to recover body weight relative to controls during the lactation period. Live litter size was slightly low at Day 20 post coitum, but this was not confirmed in animals allowed to litter. Litter weight and fetal weight were low at Day 20 post coitum, but the females that were allowed to litter showed an increase in gestation length by approximately 24 h so that offspring weights at Day 1 post partum were similar to control values. Survival and growth of the offspring was unaffected by treatment administered to the dams during the organogenesis phase of pregnancy. The incidence of diaphragmatic thinning with protrusion of the liver into the thorax in the region of the central ligament (30%) was approximately twice the incidence seen in controls (17%). The incidence of this anomaly in offspring at Day 4 or 21 of age was about 9%, whereas the observed control incidence was 1 or 3% at Day 4 and Day 21, respectively. There were no cases of diaphragmatic hernia but the liver and diaphragm tended to become fused together where there was protrusion of the liver through a thin portion of the diaphragm.
At 16 mg/kg bw/day, approximately 25% of females were cold 6 h after dosing on the first day of treatment, but there were minimal other clinical signs of reaction to dosing. One female was killed for humane reasons on Day 11 of gestation because of marked respiratory distress and reduced activity.
Animals lost weight during the first few days of treatment, overall weight gain and food consumption during treatment was low but the animals recovered between the end of treatment at Day 15 and necropsy at Day 20 of gestation. Body weight of females at the start of lactation remained low, but there was a degree of recovery towards the end of lactation. Live litter size was slightly low at Day 20 post-coitum, but this was not confirmed in animals allowed to litter. The incidence of diaphragmatic thinning with protrusion of the liver was similar to control at all examination stages and declined with increasing age of the offspring.
At 8 mg/kg bw/day there were no significant clinical signs, although food intake during early treatment and body weight gain throughout treatment were slightly low compared to control. Litter and fetal parameters at Day 20 of gestation were unaffected by treatment. There was a minor extension of gestation length but post partum litter parameters for females allowed to give birth were unaffected by maternal treatment. The incidence of diaphragmatic thinning with protrusion of the liver was similar to control at all examination stages and declined with increasing age of the offspring. There was a single incidence of diaphragmatic hernia at Day 21 of age but in the absence of cases at higher levels this was considered to be unrelated to treatment. For detailed litter data with respect to diaphragmatic anomalies, please refer to attachment 5 under "Overall remarks, attachments".
In the subsequent oral teratogenicity study for the investigation of the incidence of diaphragmatic thinning and diaphragmatic hernia in the foetuses and offspring of rats, the NOAEL for maternal toxicity was 8 mg/kg bw/day, based on decreased body weight and food consumption and clinical signs. The NOAEL for developmental toxicity was 16 mg/kg bw/day based on increased incidence of the thinning on the diaphragm at 64 mg/kg bw/day. The incidence was lower in the offspring than in foetus and there was no incidence of development into diaphragmatic hernia.

Applicant's summary and conclusion

Conclusions:

The current study was not performed under GLP conditions, but similar to guideline 414 (adopted in 2018). Deviations to the current OECD guideline included that the treatment was restricted to the period of organogenesis and did not cover the complete gestation, only 4 days of acclimation period, anogenital distance was not measured, gravid uterus weights were not noted, no historical control data were provided and parameters related to the detection of endocrine disrupting potential (thyroid weights, thyroid histopathology and thyroid hormons) were not examined as there were not required at the time the study was conducted, no special attention paid to reproductive tract of foetusses.
In conclusion, treatment with the test substance at 16 and 64 mg/kg bw/day exhibited effects on foetal and litter weight, but there was no indication of any teratogenic effect. There was no effect of treatment, maternal or foetal, at 4 mg/kg bw/day and below.
In the present oral teratogenicity study in rats, the NOAEL for maternal toxicity was 4 mg/kg bw/day based on decreased body weight and food and water consumption at 16 mg/kg bw/day and above. The NOAEL for maternal developmental toxicity was 64 mg/kg bw/day as no adverse effect were observed up to the highest dose level tested. Foetuses showed diaphragmatic thinning in the mid- and high-dose groups. Further, reduced body weight and litter weights at the highest dose tested was observed, which is considered secondary due to maternal toxicity. However, the NOAEL for developmental effects (foetuses) was set to 4 mg/kg bw/day. No teratogenic effect was observed.

An additional study (GLP-compliant) was conducted with the objective to investigate the influence of the test substance on the development of the diaphragm in the fetus, with specific reference to diaphragmatic thinning and diaphragmatic hernia recorded in fetuses in the previous study. In order to monitor the subsequent progression of this particular abnormality postnatally, similar observations were made on culled offspring at Day 4 of age and on weanling offspring from females allowed to litter. Treatment of the parental females was restricted to the period of organogenesis (Days 6 - 15 post coitum) to match the period of treatment in the original study. Female rats received the test item by oral gavage at dosages 0, 8, 16 or 64 mg/kg bw/day from Days 6 - 15 after mating. Twenty two females in each group were sacrificed on Day 20 after mating for fetal examination and the remaining females were allowed to litter to permit examination of offspring on Day 4 and 21 of age.
The majority of the changes in the diaphragm were small, but with the fixation process these shape changes were apparent when the foetus was sectioned. There was a clear indication in the current study that the incidence of diaphragmatic thinning with protrusion of the liver was increased in foetuses of females, which had been treated with 64 mg/kg bw/day during organogenesis. This would be consistent with the findings from the original study. There was no evidence, however, of any increase in the incidence of diaphragmatic thinning at 8 and 16 mg/kg bw/day, despite the detailed examinations and the fact that the numbers of fetuses examined was almost three times greater than in the original study and post partum offspring were also examined. The incidence of diaphragmatic changes was lower in the offspring than in the fetus and although this was still apparent at weaning there was no evidence that diaphragmatic thinning developed into diaphragmatic hernia after birth or that growth or survival of the offspring “at risk” was compromised. However, the observerd increasing incidences of diaphragmatic changes were related to systemic maternal toxicity, as treatment of pregnant rats with the test substance at levels of 16 or 64 mg/kg bw/day has marked effects on the mothers (dose-related reductions in food intake and body weight loss/reduced gain during treatment). Fetal weight was also reduced at 64 mg/kg bw/day.
In conclusion, the results of the detailed examinations of the fetuses/offspring in the current study clearly show that it is possible to detect high frequencies of cases where the diaphragm appears thin and the liver protrudes slightly through the diaphragm causing a bulge to appear in the cranial surface of the liver. There was an incidence of approximately 30% in fetuses from dams treated at 64 mg/kg bw/day, with about 14 - 17% incidence in controls and groups treated with 8 and 16 mg/kg bw/day. The NOAEL for maternal toxicity was 8 mg/kg bw/day, based on decreased body weight and food consumption and clinical signs. The NOAEL for developmental toxicity was 16 mg/kg bw/day, based on increased incidence of the thinning on the diaphragm at 64 mg/kg bw/day.