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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 March - 08 June 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report date:
1997

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 12 May 1981
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese MAFF guidelines (Guidance on Toxicology Study Data for Application of Agricultural Chemical Registration, Requirements for Safety Evaluation of Agricultural Chemicals, Teratogenicity Study" )
Version / remarks:
January 28, 1985
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: US EPA guidelines (Pesticide Assessment Guidelines, Subdivision F, Hazard Evaluation: Human and Domestic Animals", Series 83-3, "Teratogenicity Study"
Version / remarks:
November 1984
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EEC guidelines (Comission Directive 88/302/EEC, Official Journal of the European Communities L 133)
Version / remarks:
May 30, 1988
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

1
Chemical structure
Reference substance name:
4-(2-chlorophenyl)-N-cyclohexyl-N-ethyl-5-oxo-4,5-dihydro-1H-1,2,3,4-tetrazole-1-carboxamide
EC Number:
605-140-1
Cas Number:
158237-07-1
Molecular formula:
C16H20ClN5O2
IUPAC Name:
4-(2-chlorophenyl)-N-cyclohexyl-N-ethyl-5-oxo-4,5-dihydro-1H-1,2,3,4-tetrazole-1-carboxamide

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Wistar rats Hsd/Cpb:WU, Harlan-Winkelmann GmbH, Borchen
- Weight at study initiation: > 300 g (males), 187 - 240 g (females)
- Housing: females were kept in groups in Type III Makrolon® cages during adaptation period and individually in Type II Makrolon® cages from gestation day 0 Makrolon®, low-dust wood shavings, males were kept individually in Type III Makrolon® cages
- Diet: Altromin® 1324 8 (Altromin Company, Lage, Germany), ad libitum
- Water: tap water (drinking water quality), ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 - 25
- Humidity (%): 35 - 60
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 29 March 1995 To: 08 June 1995

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% tylose suspension
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Administration formulations were prepared using a 0.5% tylose suspension in demineralized water, the administration formulations were stored for the duration of their use at room temperature

VEHICLE
- Justification for use and choice of vehicle: tylose suspension in demineralized water, the vehicle has no effect on the parameters investigated
- Concentration in vehicle: 0.5%

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Investigations on the stability of the active ingredient in samples of 1 mg/mL and 200 mg/mL (dose volume 10 mL/kg bw) covering the range of concentrations used in this study revealed no significant deviations after 8-day storage from the content determined on the day of preparation. The homogeneity of the administration formulations at the 1 mg/mL and the 200 mg/mL concentration also complied. A content check of the formulations of all concentrations was carried out during the inlife period of the study in week 1 and week 3 (April 4 and April 20, 1995 respectively) after initiation of treatment of the first animals. The results revealed no significant deviation (+ 20%) of the active ingredient content from the nominal value in the formulations in any of the three treatment groups.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/2
- Length of cohabitation: overnight
- Proof of pregnancy: 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:
20 Days
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day
Remarks:
concentration: 10 mg/mL
volume: 10 mL/kg bw
Dose / conc.:
300 mg/kg bw/day
Remarks:
concentration: 30 mg/mL
volume: 10 mL/kg bw
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
concentration: 100 mg/mL
volume: 10 mL/kg bw
No. of animals per sex per dose:
28
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels used were selected according to a preceding developmental toxicity screening study.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily from day 0 - 20 post mating
- The following cage side observations were checked: appearance and behavior, feed and water consumption, the appearance of their excretory products, the body weight development and mortality of the animals, as well as on the basis of pathological findings

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: Day 0, daily from Day 6 - 15 and on Day 20 post mating, corrected body weight gain was calculated by subtracting the weight of the uterus on day 20 p.c. from the body weight gain over the period from day 0 to day 20 p.c.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, feed consumption of the animals on gestation days 0-6, 6-11, 11-16 and 16-20 was determined based on the differences in weight of feed provided and feed which remained unconsumed.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes, water consumption was determined by estimation of the remaining quantities in the water bottles.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20


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
- Other: number of live fetuses, sex of live fetuses, individual weights of fetuses
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: [all per litter / half per litter / #? per litter ]
Statistics:
Differences between the control and test substance-treated groups were considered significant when p<0.05

Analysis of Variance (ANOVA) and in case of significant results Dunnett's t-Test as posthoc test for:
- feed intakes
- body weights and body weight gains
- uterus weights
- corrected body weight gains
- number of corpora lutea per dam
- number of implantations per dam
- number of live fetuses per dam and as percentage of implantantions per dam
- placental weights
- fetal weights

CH|2 test (correction according to Yates) for:
- fertility rate
- gestation rate
- number of fetuses or litters with malformations

2 by N CH|2 test; in case of significant differences Fisher's exact test with Bonferroni correction for:
- number of implantations per group
- number of preimplantation losses per group
- number of postimplantation losses, early resorptions, late resorptions or dead fetuses per group
- number of live fetuses per group in percent of implantations
- number of male or female fetuses or fetuses with undeterminable sex per group
- number of fetuses or litters with skeletal findings

Kruskall-Wallis test and in case of significant differences Dunn's test for:
- number of preimplantation losses per dam
- number of postimplantation losses, early resorptions, late resorptions or dead fetuses per dam
- number of male or female fetuses or fetuses with undeterminable sex per dam


Historical control data:
Please refer to attached PDF (M-011586-02-1_Developmental Toxicity study in rats historical control data) in the "attached background material" section and "Any other information on materials and methods incl. tables" section.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
yes 1000 mg/kg bw/day: red stains around the nose were observed in two animals during the treatment period (days 10 and 11) as this observations was occasionally observed in control animals in previous studies, the effect is considered as non-adverse.
yes 1000 mg/kg bw/day: 7 dams showed light discoloration of the feces which is attributed to the elimination of test material which is a white powder (non-adverse). Please refer to table 1 in the "any other information on results incl. tables " section. For historical control data please refer to attached PDF (M-011586-02-1_Developmental Toxicity study in rats historical control data) in the "attached background material" section.
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
no data
Mortality:
no mortality observed
Description (incidence):
no moratility observed. Please refer to table 1 in the "any other information on results incl. tables " section.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight and weight gain were comparable among the groups. Please refer to table 1 in the "any other information on results incl. tables " section.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was not affected. Please refer to table 1 in the "any other information on results incl. tables " section.
Food efficiency:
no effects observed
Description (incidence and severity):
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Water consumption was not affected to a toxicologically significant extent by the treatment at doses of up to and including 1000 mg/kg.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
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:
no effects observed
Description (incidence and severity):
no effects observed
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
yes 1000 mg/kg bw/day: pre-implantation loss in the high dose group was slightly elevated when compared to controls, but well within the normal range of historical control values (non adverse)
Please refer to table 1 in the "any other information on results incl. tables " section. For historical control data, please refer to table 1 and 2 in the "any other information on materials and methods incl. tables" section and the attached PDF (M-011586-02-1_Developmental Toxicity study in rats historical control data) in the "attached background material" section.
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
yes 1000 mg/kg bw/day: in 2 females total resorption of the conceptus was observed, which is known to occur spontaneously in the rat strain used as demonstrated by historical data and a preceding developmental toxicity screening study (Supporting study, M-011047-01-1, Nihon Bayer Agrochem K.K., 1994). Thus, the effect is interpreted as incidental and non-adverse. Otherwise, the number of fetuses and the incidence of late resoprtions is comparable among the groups.
Please refer to table 1 in the "any other information on results incl. tables " section. For historical control data, please refer to table 1 and 2 in the "any other information on materials and methods incl. tables" section and the attached PDF (M-011586-02-1_Developmental Toxicity study in rats historical control data) in the "attached background material" section.
Early or late resorptions:
no effects observed
Description (incidence and severity):
Please refer to table 1 in the "any other information on results incl. tables " section. For historical control data, please refer to table 1 and 2 in the "any other information on materials and methods incl. tables" section and the attached PDF (M-011586-02-1_Developmental Toxicity study in rats historical control data) in the "attached background material" section.
Dead fetuses:
no effects observed
Description (incidence and severity):
The number of fetuses per female was not affected by the treatment in any dose group. Please refer to table 1 in the "any other information on results incl. tables " section.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
The number of pregnant females was not affected by treatment. Please refer to table 1 in the "any other information on results incl. tables " section.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
yes 1000 mg/kg bw/day: placental weights were slightly but statistically significantly increased when data were calculated on an individual basis but not when evaluated on a litter basis. Since this increase in mean placenta! weight was also within the range of historical control values it is not considered to be related to treatment. Necrotic placental borders were observed in all dose groups including the control animals without apparent dose-dependency. Since this very common finding which was also observed at only marginally lower levels in untreated animals of former studies, it is not considered to be related to the treatment with the test compound. A slightly increased number of engorged placentas was observed in the 1000 mg/kg dose group. Since there was no clear dose-response for this finding when data were evaluated on a per litter basis and considering that this finding was also observed in control groups of previous studies at similar incidence rates it is not assumed to be related to treatment with the test compound. (non adverse). Please refer to table 1 in the "any other information on results incl. tables " section. For historical control data, please refer to table 1, 2 and 3 in the "any other information on materials and methods incl. tables" section and the attached PDF (M-011586-02-1_Developmental Toxicity study in rats historical control data) in the "attached background material" section.
Details on maternal toxic effects:
Total litter losses by resorption:
Total resorption of the conceptus was observed in two females at the 1000 mg/kg dose level. Historical data collected during 1991 to 1994 in this laboratory, indicate that single total resorptions occur by chance, apparently independent of treatment. Furthermore a preceding developmental toxicity screening study did not reveal any total resorptions in 20 treated animals even at dose levels of 2000 mg/kg bw/day (M-011047-01-1, Report No. RS94072,1994). It is therefore concluded that the two incidences of complete postimplantation loss observed in the high dose group of this study were incidental and that the gestation rate was unaffected by the treatment at doses of up to and including 1000 mg/kg bw/day.

Other effects:
The weight of the placentas was slightly but statistically significantly increased at 1000 mg/kg bw/day when data are calculated on an individual basis but not when evaluated on a litter basis. Since this increase in mean placental weight was also within the range of historical control values it is not considered to be related to treatment. Necrotic placental borders were observed in all dose groups including the control animals without apparent dose-dependency. Since this very common finding which was also observed at only marginally lower levels in untreated animals of former studies, it is not considered to be related to the treatment with the test compound. A slightly increased number of engorged placentas were observed in the 1000 mg/kg dose group. Since there was no clear dose-response for this finding when data were evaluated on a per litter basis and considering that this finding was also observed in control groups of previous studies at similar incidence rates it is not assumed to be related to treatment with the test compound.

General reproductive data:
Fertility rates (percentage of inseminated animals with implantations) in the dose groups did not differ to a statistically significant extent from the control dams and were also within the range of historical control data. Further, the number of corpora lutea and implantations was comparable among the groups.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Remarks:
Maternal toxicity
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: no adverse effects observed at this dose

Results (fetuses)

Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
yes 1000 mg/kg bw/day: slightly statistically significantly elevated fetal weight, when calculated on an individual basis, but not when the data are calculated on a per litter basis and are within the range of historical controls (non adverse).
Please refer to table 2 in the "any other information on results incl. tables " section. For historical control data, please refer to table 1 and 2 in the "any other information on materials and methods incl. tables" section and the attached PDF (M-011586-02-1_Developmental Toxicity study in rats historical control data) in the "attached background material" section.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
The mean number of fetuses per dam was not affected by treatment. Please refer to table 2 in the "any other information on results incl. tables " section.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The percentages of male and female fetuses did not differ from those in the control groups to any marked extent at levels up to and including 1000 mg/kg. Please refer to table 1 in the "any other information on results incl. tables " section.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Litter size and weights were not affected by treatment.
Please refer to table 2 in the "any other information on results incl. tables " section.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
yes 300 mg/kg bw/day: slightly elevated malformation rate (external, skeletal and visceral) (non-adverse) Otherwise, the type and distribution of anomalies and deviations regarding external findings in the fetuses did not reveal compound-related effects in any of the dose groups. Please refer to table 2 in the "any other information on results incl. tables " section. For historical control data, please refer to table 4 in the "any other information on materials and methods incl. tables" section.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
yes 300 mg/kg bw/day: slightly elevated malformation rate (external, skeletal and visceral). Because dose-dependency was missing and similar malformation rates and types have been reported in control groups of this and previously conducted studies with the same rat strain, the elevated malformation rate was considered incidental (non-adverse). Please refer to table 2 in the "any other information on results incl. tables " section and to attached PDF (M-011586-02-1_Developmental Toxicity study in rats selected tables) in the "attached background material" section. For historical control data, please refer to table 4 in the "any other information on materials and methods incl. tables" section.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
yes 300 mg/kg bw/day: slightly elevated malformation rate (external, skeletal and visceral), which is considered incidental because of lack of dose-dependency. Further, similar malformation rates and types of malformations have also been reported in control groups of this and previously conducted studies with the same rat strain in this laboratory (non-adverse) Please refer to table 2 in the "any other information on results incl. tables " section and to attached PDF (M-011586-02-1_Developmental Toxicity study in rats selected tables) in the "attached background material" section. For historical control data, please refer to table 4 in the "any other information on materials and methods incl. tables" section.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Skeletal System Deviations (Variations, Retardations):
yes 1000 mg/kg bw/day: when elevated on a fetal basis, there was a dose-dependent increase in the incidence of dumbbell shaped 12th thoracic vertebral body, advanced ossification of the 4th sacral and 2nd caudal vertebral arches as well as the 6th caudal vertebral body, unossified hyoid body and enlarged fontanelle. None of those findings was statistically significantly different from control values when the data were calculated on a per litter basis except for-advanced ossification of the 4th sacral vertebral arch. All of the above mentioned findings have either also been reported at similar incidence levels in control fetuses of previous studies (dumbbell shaped 12th thoracic vertebral body, advanced ossification of the 4th sacral and 2nd caudal vertebral arches as well as the 6th caudal vertebral body and enlarged fontanelle; or do not display a clear dose-response (ossification of hyoid body) and are for those reasons not considered to be related to treatment.(non adverse) Please refer to attached PDF (M-011586-02-1_Developmental Toxicity study in rats selected tables) in the "attached background material" section.
Details on embryotoxic / teratogenic effects:
Other effects:
Skeletal System Deviations (Variations, Retardations):
There was a dose-dependent increase in the incidence of dumbbell shaped 12th thoracic vertebral body, advanced ossification of the 4th sacral and 2nd caudal vertebral arches as well as the 6th caudal vertebral body, unossified hyoid body and enlarged fontanelle- which were significantly different at the 1000 mg/kg dose level when evaluated on a fetal basis. None of those findings was statistically significantly different from control values when the data were calculated on a per litter basis except for-advanced ossification of the 4th sacral vertebral arches. All of the above mentioned findings have either also been reported at similar incidence levels in control fetuses of previous studies (dumbbell shaped 12th thoracic vertebral body, advanced ossification of the 4th sacral and 2nd caudal vertebral arches as well as the 6th caudal vertebral body and enlarged fontanelle) or do not display a clear dose-response (ossification of hyoid body) and are for those reasons not considered to be related to treatment. It is also highly unlikely that the same compound would elicit advanced and/or delayed ossification, respectively in different skeletal localizations at the same time and that only a few single localizations would be affected. It was therefore concluded that a substance-related effect on ossification (variations and retardations) can be excluded.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Remarks:
Developmental toxicity
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed on intrauterine development at this dose

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Table 1: Maternal effects and caesarean section data  

Parameter

Controldata

100 mg/kg bw/day

300 mg/kg bw/day

1000 mg/kg bw/day

Number of dams examined

28

28

28

28

Clinical findings

 

 

 

 

Red strains around nose [no. animals]

0

0

0

2

Light discoloration feces [no. animals]

0

1

0

7

Mortality of dams [%]

0

0

0

0

Body weight gain [g]

Day 6-15 p.c.

32.0

34.8

32.1

30.5

Day 0-20 p.c.

105.5

104.6

101.8

101.9

Day 0-20 p.ccorrected

38.9

43.0

40.0

37.1

Food consumption, range [g/animal/day]

Day 0-6 p.c.

17.7

18.0

17.8

17.7

Day 6-11 p.c.

17.7

18.3

18.3

18.2

Day 11-16 p.c.

19.4

19.9

19.7

19.4

Day 16-20 p.c.

20.9

21.2

20.9

21.5

Inseminated animals

28

28

28

28

Animals with implantations (a)

Total

24

25

26

26

In [%] of those inseminated

85.7

89.3

92.9

92.9

Animals with viable fetuses (b)

Total

24

25

26

24

in [%] of animals with implantations

100

100

100

92.3

Mean values per dam with implantations (a) / viable fetuses (b)

Corpora Lutea (b)

14.3

14.0

13.7

14.6

Preimplantation loss (b)

1.7

2.4

2.2

2.9

Implantations (b)

12.6

11.5

11.5

11.7

Placental weight in [g] (b)

0.6

0.63

0.62

0.64

Number of fetuses (b)

11.7

10.9

10.7

11.0

Late resorptions (a)

0.9

0.6

0.8

1.1

Late resorptions (b)

0.9

0.6

0.8

0.7

Early resorptions (a)

0.0

0.0

0.0

0.1

Early resorptions (b)

0.0

0.0

0.0

0.0

Sex ratio [%] males

46.9

45.2

47.6

46.3

Fetal weight in [g] (b)

3.74

3.70

3.79

3.84

Postimplantation loss (a)

0.9

0.6

0.8

1.2

Postimplantation loss (b)

0.9

0.6

0.8

0.7

Dead fetuses (a)

0.0

0.0

0.0

0.0

Dead fetuses (b)

0.0

0.0

0.0

0.0

 Table 2: Examination of fetuses

Parameter

Controldata

100 mg/kg

300 mg/kg

1000 mg/kg

Number of fetuses per group

281

272

277

264

Total number of fetuses with malformations

3

3

11

4

[%] of fetuses with malformations

1.1

1.1

4.0

1.5

Litters per group

24

25

26

24

Litters with malformations

3

3

8

3

Litters with malformations [%]

12.5

12.0

30.8

12.5

Weight of live fetuses (litter basis) total mean [g]

3.74

3.70

3.79

3.84

Weight of live fetuses (individual basis) total mean [g]

3.73

3.67

3.76

3.84**

External, skeletal and visceral malformations - number of fetuses (litters) affected

Multiple Malformations

0

1

0

0

Microphthalmia/Anophthalmia

2(2)

1

3(2)

2(2)

Situs inversus totalis

0

1

0

0

Globular shaped heart; lung reduced

0

0

0

0

In size; slightly oedematous

0

0

1

0

Lobe of thyroid gland reduced/absent

1

0

2(2)

2(1)

Hernia, left body wall

0

0

1

0

Malformation of vertebra with or without

0

0

0

0

Supernumerary ribs

0

0

2(1)

0

Pelvis shift

0

0

1

0

Dysplasia of scapula

0

0

1

0

** p<0.01

 

Applicant's summary and conclusion

Conclusions:
CLP: not classified