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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:
15 Jan - 02 Aug 1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
purity is not provided

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report Date:
1991

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted in 1981
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted in 2001
Deviations:
no
Qualifier:
according to
Guideline:
EPA OTS 798.4900 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted in 1987
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Batch No.: 791

Test animals

Species:
rat
Strain:
other: Wistar (Bor:WISW(SPF Cpb))
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: F.Winkelmann, Borchen, Germany
- Age at study initiation: mature animals
- Weight at study initiation: 186 - 148 g (females), > 300 g (males at the time of mating)
- Housing: several females together in Makrolon Type III cages during acclimation period, individually in Makrolon Type II cages on low-dust wood granules (Ssniff GmbH, Soest, Germany) from Day 0 post-coital (females), individually in Makrolon Type III cages (males)
- Diet: Altromin 1324 standard diet (Altromin, Lage, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 ± 2
- Humidity (%): 30 - 45
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% aqueous tylose
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Weighed portions of the test compound volumes corresponding to the intended dosages were prepared and stirred smooth in the mortar using a small amount of tylose suspension. Subsequently, additional tylose suspension was added until final concentration was reached. The application formulations were sealed lighttight and used immediately (on the day of preparation) or stored in the refrigerator at approx. 4° C until use on the following days. The formulations were routinely stirred at room temperature on a magnetic stirrer prior to and during applications.

VEHICLE
- Concentration in vehicle: 3, 10 and 30 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Investigations into homogeneity and stability of the active ingredient in the application vehicle were performed prior to the start of study using GC-FID analysis. Samples were collected at all dose formulations (30, 100 and 300 mg/kg bw/day) from top, the middle and the bottom of the formulation. The investigations showed the active ingredient to be homogeneously distributed in the application vehicle for the formulations of the dose groups 30 and 100 mg/kg bw. The active ingredient content in the formulation for the 300 mg/kg bw group displayed clearly inhomogeneous distribution. The stability of the active ingredient in 0.5% aqueous tylose was demonstrated after 8-day storage. A second check of homogeneity was carried out in the 8th week after termination of treatment. Visible particles were observed in the samples of the 300 mg/kg bw formulation, which, however, did not significantly affect the distribution in the application vehicle. The active ingredient content was within the permitted tolerance range (± 20 deviation from the nominal value) in all samples of all formulations; thus, the active ingredient was homogeneously distributed in the application vehicle.
A content check of the formulations of all concentrations was carried out in the 2nd and 5th week of treatment. The results revealed no significant deviations of the active ingredient content from the nominal value in the formulations for the low and high dose group. The control analysis of the application formulations for the 100 mg/kg bw group showed a slightly reduced content (63 - 74 %) in the 5th week of treatment. As only 1/4 investigations for this group (2 homogeneity analyses, 2 content control analyses) displayed a slightly reduced content, no adverse effect on the study results is to be assumed for the 100 mg/kg bw formulations. Since the second check of homogeneity as well as the control analyses for the highest dose formulations revealed active ingredient contents within the permitted tolerance range. Thus, no adverse effect on the study results because of inhomogeneity of the formulations is to be assumed for the 300 mg/kg bw group as well.
Details on mating procedure:
- Impregnation procedure: 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 of gestation
Frequency of treatment:
daily, 7 days/week
Duration of test:
day 20 of gestation
Doses / concentrationsopen allclose all
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The range of doses has been selected based on results obtained in a range-finding study where pregnant rats (5 per group) received the test substance at 300, 500 or 1000 mg/kg bw/day. Due to severe maternal toxicity the investigations of the 500 and the 1000 mg/kg bw/day group were discontinued on day 12 p.c. and day 8 p.c., respectively. Impaired body weight development as well as clinical signs including abdominal position after application, rough coat, staggering gait and stertorous breathing were observed at 300 mg/kg bw/day from day 6 p.c. No indications of external malformations were observed.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (once daily on weekends and bank holidays)
- Cage side observations included: mortality, disturbances in the rats' general condition, appearance, behavior, alterations in the excretory products

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily (once daily on weekends and bank holidays)

BODY WEIGHT: Yes
- Time schedule for examinations: on day 0 p.c., daily from day 6 to day 15 p.c. as well as on day 20 p.c .

FOOD CONSUMPTION: Yes
Feed intake was determined for the following days of gestation: day 0 - 6, day 6 - 11, day 11 - 16 and day 16 - 20 p.c .

WATER CONSUMPTION: Yes
- during inspections by visual estimation of the quantities left over

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: uterus, placenta, visceral organs, abdominal and thoracic organs
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 and dead fetuses, sex of live fetuses, weights of live fetuses
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No data
Statistics:
A non-parametric rank sum test (Wilcoxon-Mann-Whitney-U-Test) was used for average body weight gain as well as for the number of fetuses per dam with minor skeletal deviations or malformations. The exact Fisher test (two-sided; p ≤ 0.05 and p ≤ 0.01) was applied for fertility and gestation rate as well as for the number of fetuses or litters with retardations/with 14th rib or with malformations. The F-test and t-test or t-test according to Welch was used for feed consumption and corrected body weight gain, for the number of corpora lutea per dam with implantations or with live fetuses, for the number of implantations per dam with implantations or with live fetuses, for the average number of live fetuses per dam, for the percentage of male or female fetuses per dam, for the average weight of fetuses (male, female or both; also per dam) as well as for the average placental weight. The CHI²-test (correction according to Yates) was applied for pre-implantative losses per dam with implantations and live fetuses, for the sex ratio of male to female fetuses per group, for the number of resorptions per dam with implantations/ with live fetuses as well as for the number of early resorptions per dam with implantations/ with live fetuses.
Historical control data:
Historical control data from studies conducted in-house were referred to in order to allow comparison with concurrent controls. The data were generated between 1983 and 1990 and are presented in the report.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
100 mg/kg bw/day: labored breathing in 2/25 animals on days 8 and 16 p.c.
300 mg/kg bw/day: rough coat, sunken flanks, bloody muzzle, labored breathing, reduced motility, high-stepping gait from day 8 p.c.; lying on side, somnolence, abdominal position, spastic convulasions in several animals from day 6 p.c. for a period of approx. 1 h and/or starting approx. 10 min after test compound application; gasping breathing in 1/25 animal
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
300 mg/kg bw/day: 5/25 animals died; 1/26 animal was sacrificed in moribund conditions
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
100 mg/kg bw/day: statistically significant reduced mean body weight gain during days 6 - 15 p.c.; statistically significant reduced corrected body weight gain during days 0 - 20 p.c.; reduced mean body weight gain during days 0 - 20 p.c. (not statistically significant)
300 mg/kg bw/day: statistically significant reduced mean body weight gain during days 6 - 15 p.c. and 0 - 20 p.c.; statistically significant reduced corrected body weight gain during days 0 - 20 p.c.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
100 mg/kg bw/day: statistically significant reduced food intake during the application period (statistical significance from day 11 - 16 p.c. only)
300 mg/kg bw/day: statistically significant reduced food intake during the application and gestation period
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
100 mg/kg bw/day: reduced in one animal on day 9 p.c. (assessed qualitatively only)
300 mg/kg bw/day: reduced for several animals from day 9 p.c., in some cases lasting for 1 - 6 days (assessed qualitatively only)
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:
effects observed, treatment-related
Description (incidence and severity):
300 mg/kg bw/day: 3/6 animals that dies or were sacrificed moribund had inflated intestines and bloody vaginas, respectively; individual findings included a thorax filled with serous fluid and suppurative foci in the lung tissue, reddened esophagus, an
apparently small stomach, and spleen reduced in size; no effects were observed in surviving animals
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Urine/ faeces excretion:
100 mg/kg bw/day: increased urine excretion in some isolated cases from day 9 p.c.; reduced amount of faeces over a period of 1 - 5 days in several animals from day 9 p.c.
300 mg/kg bw/day: increased urine excretion in several animals from day 8 p.c. for a period of 1 - 5 days; reduced amount of faeces in several animals from day 8 p.c. (lasting for 1 - 5 days)

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
effects observed, treatment-related
Description (incidence and severity):
300 mg/kg bw/day: statistically significant increased mean resorptions/dam due to complete resorption by 2/25 dams
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
effects observed, treatment-related
Description (incidence and severity):
300 mg/kg bw/day: reduced gestation rate due to complete resorptions in 2/18 animals (without 6 animals that died or were sacrificed moribund)
Other effects:
no effects observed

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
300 mg/kg bw/day: statistically significant decreased fetal weight
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
300 mg/kg bw/day: slightly increased rate of malformations including cryptorchism, microphthalmia or anophthalmia, multiple malformation, dysplasia of humerus, hernia of diaphragm with lung hypoplasia, heart and lung dystopia
Details on embryotoxic / teratogenic effects:
VISCERAL MALFORMATIONS
The kind of malformations observed in the high dose group had been observed in the control group of this study (cryptorchism) or had occurred as spontaneous malformations in this strain in the same laboratory. Thus, they are not to be regarded as indications of a specific teratogenic effect of the test material

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

Table 1: Maternal effects

Parameter

Controldata

30 mg/kg

100 mg/kg

300 mg/kg

Dose-response
+ / –

historical

study

0 mg/kg

Number of dams examined

-

25

25

25

25

 

Clinical findings

 

 

 

 

 

 

audible breathing sounds

-

0

0

1

4

+

gasping breathing

-

0

0

1

1

+

bloody lip

-

0

1

0

0

-

rough coat

3

0

0

0

7

+

bloody muzzle

1 (nose)

0

0

0

4

+

sunken flanks

1

0

0

0

3

+

reduced motility

-

0

0

0

2

+

abdominal knots

-

0

1

0

0

bloody forelimbs

-

0

0

0

1

high-stepping gait

-

0

0

0

1

+

reduced water intake

7#

0

0

1

12

+

light-brown, hard faeces

4

1

0

0

0

 

small amount of faeces

4

0

0

4

14

+

increased urine excretion

5#

0

0

3

9

+

after application

 

 

 

 

 

 

        gasping breathing

-

0

0

0

1

+

        lying on side

-

0

0

0

7

+

        somnolence

-

0

0

0

13

+

        abdominal position

-

0

0

0

9

+

        spastic convulsion

-

0

0

0

5

+

Mortality of dams

-

0

0

0

6

+

Body weight gain [g]

Mean day 0 – 20

73.0-101.9

98.5

95.8

90.9

66.8*

+

Body weight gain [g]

corrected, day 0 – 20

-

37.6

35.3

31.0

13.7***

+

Mean food consumption
(Day 0-20) [g/rat/day]

-

18.5

18.4

18.0

15.5***

+

Pregnancies

7-24

22

24

22

24

-

Necropsy findings in dams dead before end of test

 

 

 

 

 

 

      reddened oesophagus

-

 

 

 

1

+

     suppurative foci in lung tissue

-

 

 

 

1

+

      fluid in thorax

-

 

 

 

1

+

     thorax filled with serous fluid

-

 

 

 

1

+

      stomach appears smaller

-

 

 

 

1

+

     stomach + intestines extremely distended

-

 

 

 

1

+

      reduced spleen size

-

 

 

 

1

+

      gas-inflated intestines

-

 

 

 

2

+

     bloody vagina

1

 

 

 

3

+

     organs autolytic

-

 

 

 

1

+

Necropsy findings in dams at termination

 

 

 

 

 

 

      Ovariary cysts

1

2

0

0

0

      intestinal worms

56

3

8

7

6

-

Statistically significant difference from controls: *p < 0.05; ** p < 0.005; *** p < 0.001

 

Table 2: Litter response (Caesarean section data)

Parameter

Controldata

30 mg/kg

100 mg/kg

300 mg/kg

Dose-response
+ / –

Historical
(1984-1990)

Study
0 mg/kg

Corpora lutea[mean no./dam]

-

13.0

13.2

12.4

12.6

 

Implantations[mean no./dam]

8.3-12.5

11.4

11.3

11.2

10.6

Resorptions[mean no./dam]a

0.3-2.3

0.6

0.8

0.6

1.8***

+

Resorptions[mean no./dam]b

0.6

0.8

0.6

0.7

-

Foetuses[mean no./dam]

7.6-11.7

10.7

10.5

10.6

9.9

Foetus weight(mean) [g]b

3.17-3.68

3.71

3.69

3.65

3.42**

+

Placenta weight[mean/dam] [g]b

0.55-0.68

0.62

0.65

0.62

0.60

Skeletal changes
[mean foetus no/dam]b

1.44-3.18#

2.09

1.46

2.18

1.38

Malformations
[mean foetus no/dam]b

0.00-0.39

0.14

0.08

0.05

0.38

Sex ratio (m:f)b

-

1:0.8

1:0.9

1:1.22

1:1.03

-

awith implantations
bwith live foetuses
# Skeletal retardations
Statistically significant difference from controls: *p < 0.05; ** p < 0.005; *** p < 0.001

 

Table 3: Examination of the foetuses

Parameter

Controldata

30 mg/kg

100 mg/kg

300 mg/kg

Dose-response
+ / –

historical

Study
0 mg/kg

External Examinations

 

No. of foetuses examined

-

236

253

233

158

 

No. of foetuses malformed

 

        hydronephrosis, hydro-ureter

9

1

0

0

0

        cryptorchism

13

2

1

1

1

        microphthalmia or anophthalmia

23

0

1

0

2

        multiple malformation

1

0

0

0

1

        dysplasia of humerus

4

0

0

0

1

               hernia of diaphragm, with lung hypoplasia, heart and lung dystopia

1

0

0

0

1

Total malformed foetuses

51

3

2

1

6

Applicant's summary and conclusion

Conclusions:
The test substance had no effect on intrauterine development.
Executive summary:

The teratogenic potential of the test substance was assessed in a developmental toxicity study in Bor:WISW(SPF Cpb) Wistar rats performed according to OECD Guideline 414 and in compliance with GLP. Female rats were mated overnight with males. Three groups of 25 sperm-positive female rats received the test substance by oral gavage from day 6 to 20 postcoital (p.c.) at doses of 30, 100 or 300 mg/kg bw/day in aqueous solution of 0.5% tylose. A control group of 25 females received the vehicle. On day 20 of gestation animals delivered by caesarean section. Investigations were performed on general tolerance of the test substance by the dams as well as its effect on intra-uterine development.At doses of 30 mg/kg bw/day the test substance was tolerated without any effects. At 100 mg/kg bw/day some animals showed laboured breathing. At 300 mg/kg bw/day all dams exhibited marked clinical signs (rough coat, sunken flanks, bloody muzzle, laboured breathing, reduced mobility, high-stepping gait). Within 1 h after application, several animals of this group showed additional clinical signs of more frequently lying on side, somnolence, abdominal position, spastic convulsions and gasping breathing. Five animals of the high dose group died and one was sacrificed in moribund conditions. After treatment with ≥ 100 mg/kg bw/day body weight gain, feed and water intake as well as excretion of faeces was diminished. Urine excretion was increased in isolated cases at 100 and in several animals at 300 mg/kg bw/day.

Weight and external appearance of placentas, sex ratio of foetuses and development of the skeletal system was not affected up to and including 300 mg/kg bw/day. Gestation and resorption rates, number and weight of foetuses as well as number and kind of malformations were not affected at doses of ≤ 100 mg/kg bw/day. At 300 mg/kg bw/day foetal weight, gestation rate and the number of foetuses were diminished due to an increased resorption rate. The slightly increased number of malformations observed in this group, were considered as spontaneous malformations which were not dose-dependent. The embryotoxic effects observed correlated with the marked maternal toxicity. Thus, under the conditions of this study, the NOAEL of the test substance in terms of maternal toxicity following administration of the test substance to pregnant rats via gavage is 30 mg/kg bw/day. The NOAEL in terms of fetal toxicity is 100 mg/kg bw/day.