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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 1992 June 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: well documented study performed according to OECD guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report Date:
1993

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
once-only false dosing of 17 animals in low dose group
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): Marlotherm S
- Substance type: pure active substance
- Physical state: yellow liquid
- Lot/batch No.: 200 l barrel from August 21, 1991
- Stability under test conditions: unlimited
- Storage condition of test material: no data
Specific details on test material used for the study:
The purity of the material used for the test was lower than the purity of the actual material. It contained ca. 26% of isomers of dimethyl tribenzenes. However, based on comparable basic structure of the impurities and the assumption that any toxic effects would increase with higher molecular weight and lipophilicity, we regard that the studies conducted with the lower purity material being "worst case" and can be used for the evaluation of the actual product.

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Wiga GmbH, Sulzfeld, Germany
- Age at study initiation: about 8 weeks
- Weight at study initiation: 197 to 280 g
- Fasting period before study: no
- Housing: single, in Macrolon cages
- Diet : "Ssniff R" pelleted diet by Sniff Spezialdiäten GmbH, Soest, Germany; ad libitum
- Water: tap water ad libitum
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.5 °C +/- 1.5 °C
- Humidity (%): 50 - 70 %
- Air changes (per hr): 16
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: April 23, 1992 To: June 2, 1992

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5 % Sodium Carboxymethylcellulose
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Preparation of doses by homogenizing the test substance with the vehicle by means of ultraturrax, stirring constantly prior to administration in order to maintain homogeneity.

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Concentration in vehicle: according to the dose levels 2.5, 15 and 100 g/l, respectively
- Amount of vehicle (if gavage): 10 ml/kg bw
- Lot/batch no. (if required): 109 F 0361 (supplier: SIGMA, Deisenhofen, Germany)
- Purity: no data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical verification of doses and stability control by photometry (at fixed wavelenght of 262 nm) after extraction of test substance from dose suspensions by dichloromethane.

Analyses of samples of dosing emulsions from all dose groups at commencement and termination of treatment. Samples of 100 ml per group were drawn and analyses for identity and concentration were performed. The allowance limit of concentration was +/- 10%.
The stability and homogeneity of the test article in the vehicle had been proven by analysing a representative sample at different intervals after preparation during dose range finding study.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/2
- Length of cohabitation: overnight
- Further matings after two unsuccessful attempts: no data
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
from day 6 to day 15 of gestation
Frequency of treatment:
once daily
Duration of test:
until day 20 of gestation
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
25 mg/kg
Basis:
actual ingested
; 17 animals were treated erroneously once with dose of 150 mg/kg either on day 6 or 7 of gestation
Remarks:
Doses / Concentrations:
150 mg/kg
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Preliminary dose range finding was conducted with dose levels of 100, 500 and 1000 mg/kg. The test substance was administered to 8 female rats per dose once daily by gavage from thoughout gestation and lactation up to day 4 post partum. Observations: Significantly decreased weight gains during the first week of treatment (days 0 to 7 of gestation) in pregnant rats at all dose levels. Food consumption was significantly decreased in the high dose group from days 1 to 7 and 1 to 20 of gestation. Food conversion ratios were decreased in all dose groups on day 7 of gestation. The pre-birth loss index in the high dose group was increased. In the F1 generation in the high dose group the mean litter size was significantly reduced at birth and at day 4 post partum and in correspondence, the number of males was significantly decreased. The pup loss index at birth and the number of litters with dead pups was found to be increased in the high dose group. Rationale: The dose level of 1000 mg/kg was selected according to the recommendations of the OECD guideline as high dose level resulting in toxic effects. The low and mid dose group were chosen to obtain information about possible dose-related effects and in order to establish a clear NOAEL.
- Animal assignment: random

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily over the entire period of the investigation, viability twice daily
Special regard on sensory and motor behaviour, coat, urine and fecal excretion, condition of body orifices and signs of ill health. Additionally, during the treatment period, dose rsponse examinations were carried out at appropriate intervals after administration of the test article. During gestation females were observed closely for signs of abortion or premature delivery.

BODY WEIGHT: Yes
- Time schedule for examinations: daily but recorded only on days 0, 6, 10, 15 and 20 of gestation
Body weight development was evaluated over the following phases: 0-6, 6-15, 15-20 and 0-20 days

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes (on days 0, 6, 10, 15 and 20 of gestation)
- Food consumption for each animal determined: Yes
- Mean daily diet consumption was 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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on day 20 of gestation
- Organs examined: macroscopic examination of internal viscera (not specified)

OTHER:
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: location of implantation sites; number and location of live and dead fetuses, weight of fetuses and placentae, head/tail presentation of fetuses, uteri ore individual uterine horns without visible implantations were stained to reveal evidence of embryonic death at early stages of implantation
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter (in-situ dissection modified according to H. Sterz (1977) ["Routine examination of rat and rabbit fetuses for malformation of internal organs: Combination of Barrow's and Wilson's methods, in: D. Neubert, H.-J. Merker and T. E: Kwasigroch (eds) Methods in prenatal toxicology, Thieme Publishers, Stuttgart, pp. 113-122])
- Skeletal examinations: Yes: half per litter (skeletal structures were stained with alizarin red S according to Kawamura et al. [Kawamura, S. , A. Hirohashi, T. Kato and M. Yasuda (1990) Bone staining technique for fetal rat specimens without skinning and removing adipose tissue, Cong. Anom. 30, 93-95.
- Head examinations: No data
- Other: sex determination by ano-genital distance
Statistics:
Analyses of variance with a subsequent multiple range test for body weight changes, food consumption and reproductioons parameters, or, if indicated, group mean values were compared by the "Kruskal-Wallis test" and "Mann-Whitney-U-test". The maternal body weight on day 20 of gestation was corrected for gravid uterine weight and the corrected rate of body weight gain (days 0-20 of gestation) was calculated.
Indices were compared statistically by the "Mann-Whitney-U-test".
References:
- Winer, B. J. (1971) Statistical principles in experimental design, International student edition, McGraw Hill Kogakusha Ltd.
- Snedecor, G. W. and W. G. Cochran (1967) Statistical methods. 6th ed. Iowa State University Press Ames, Iowa.
Indices:
Abortion rate = 100 x No. of females with abortion/Total No. of pregnant females
Resorption rate = 100 x No. of resorptions/No. of imlantations
Pre-implantation Loss Index = 100 x No. of corpora lutea - No. of implantations/ No. of corpora lutea
Post-implantation Loss index = 100 x No. of implentations - No. of implantations/ No. of implantations
Live Birth Index = 100 x No. of live fetuses/total No. of fetuses
Runts Index = 100 x No. of runts/Total No. of runts
Variation Index = 100 x No. of live fetuses with variations/total No. of live fetuses examined
Anomaly Index = 100 x No. of live fetuses with variations/total No. of live fetuses examined
Malformation Index = 100 x No. of live malformed fetuses/total No. of live fetuses examined
Historical control data:
Recent historical data containing findings of visceral and skeletal examinations of fetuses derived from females of the control groups.

Number of fetuses/litters examined:
Visceral abnormalities:
Head: 1506 fetuses/218 litters; other soft tissue: 145 fetuses/22 litters
Skeletal examination: 1593 fetuses
Skeletal abnormalities: 1593 fetuses/218 litters
Variations and anomalies: 1446 fetuses/197 litters

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Body weight gain:
Dose related significantly decreased body weight gain from gestation days 6 to 15 at 150 and 1000 mg/kg bw . At 1000 mg/kg bw also significantly decreased body weight gain throughout the entire gestation period (days 0 to 20). At 150 mg/kg bw decreased body weight gain throughout gestation days 0 to 20 but not statistically significant (see Tables 1 and 2). Corrected body weights (body weight on gestation day 20 minus gravid uterine weigth) were slightly decreased at 150 and 1000 mg/kg bw but did not reveal significance (see Table 3).
The weight gain of the animals of the low dose group which were treated erroneously once with the mid dose on day 6 or 7 of gestation was increased from day 6 to 10 and day 6 to 15 of gestation compared to the 8 "right-treated" animals of the low dose group (see Table 4). There is no indication that the wrong dosing had any impact on the outcome of the study.

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Fetal weights:
Mean fetal body weights were significantly reduced in both males and females at 1000 mg/kg bw. Also the combined group of males and females at this dose showed a significant reduction (see Table 5).


Number of runts/runts index:
One runt each were found in the low and mid dose groups. 6 runts were found at 1000 mg/kg bw. Consequently the runts index was increased (see Table 6). The increased runts index is most likely to be in correspondence with the reduced fetal body weights found in high dose group.


Number of resorptions/resorption index:
Significantly increased total number of resorptions (both early and late resorptions) were observed at 1000 mg/kg bw (see Table 7). Consequently the resorption index was significantly increased in this group. The number of females without resorptions was decreased and the number of females with two or more resorptions was increased at 1000 mg/kg bw (see Table 8).

Post-implantation Loss index:
Since no other post-implantation losses apart from resorptions were found the post-implantation index is identical with the resorption index and significantly increased at 1000 mg/kg bw (Table 9).


Visceral examination:
Visceral examination of fetuses revealed diaphragmatic hernia in three fetuses at 1000 mg/kg bw, two of them in the same litter. A slightly increased incidence of dilated ureters was observed at 1000 mg/kg bw.


Skeletal examination:
Skeletal examination showed a slight increase of fetuses with less than 4 ossification centres and also an increase of the frequency of affected litters at 1000 mg/kg bw (Table 11). Though the incidence of affected fetuses was within the historical range the historical mean value was distinctly lower.
The incidence of cleaved sternebrae was slightly increased in the high dose group with respect to the affected litters (Table 12). This frequency was above the historical range. Also the number of fetuses with 3 ossified metacarpals was increased in the high dose group with a frequency of affected fetuses slightly above the historical range (Table 11).
Cleaved or dumpbell shaped thoracic vertebral bodies were clearly more frequently observed in fetuses at 1000 mg/kg bw. The frequency of both affected fetuses and litters were elevated. The incidence of cleaved vertebrae was distinctly above the historical range and that of the dumpbell shaped vertebrae was at the upper limit. A slightly dose-related trend of increased frequency is seen in at 150 mg/kg bw. (Table 12)
Non-ossification of coccygeal vertebrae was observed in a higher frequency in at 1000 mg/kg bw, the number of affected litters being slightly above the range of historical data. (Table 12)

These findings in the skeletal examination are considered to be signs of retarded fetal ossification and provide a further indication of developmental delay in fetuses of the high dose group.

The anomaly indices were increased with increasing doses. However, the anomaly index of the control group was low compared to those of historical studies. At 25 mg/kg bw both the anomaly index and the frequency of litters with anomalies were below the respective historical mean values. At 150 mg/kg the anomaly index was close to the mean historical value but the frequency of litters with anomalies was slightly increased. At 1000 mg/kg bw both parameters were distinctly above the respective historical mean values and at the upper limit of the historical range. (Table 13)

The visceral and skeletal malformation index in the high dose group was 1.6 % and was based on the occurrence of three different types of malformations, i. e. one skeletal and two different visceral ones. (Table 13)
Fetal weights:
Mean fetal body weights were significantly reduced in both males and females at 1000 mg/kg bw. Also the combined group of males and females at this dose showed a significant reduction (see Table 5).


Number of runts/runts index:
One runt each were found in the low and mid dose groups. 6 runts were found at 1000 mg/kg bw. Consequently the runts index was increased (see Table 6). The increased runts index is most likely to be in correspondence with the reduced fetal body weights found in high dose group.


Number of resorptions/resorption index:
Significantly increased total number of resorptions (both early and late resorptions) were observed at 1000 mg/kg bw (see Table 7). Consequently the resorption index was significantly increased in this group. The number of females without resorptions was decreased and the number of females with two or more resorptions was increased at 1000 mg/kg bw (see Table 8).

Post-implantation Loss index:
Since no other post-implantation losses apart from resorptions were found the post-implantation index is identical with the resorption index and significantly increased at 1000 mg/kg bw (Table 9).


Visceral examination:
Visceral examination of fetuses revealed diaphragmatic hernia in three fetuses at 1000 mg/kg bw, two of them in the same litter. A slightly increased incidence of dilated ureters was observed at 1000 mg/kg bw.


Skeletal examination:
Skeletal examination showed a slight increase of fetuses with less than 4 ossification centres and also an increase of the frequency of affected litters at 1000 mg/kg bw (Table 11). Though the incidence of affected fetuses was within the historical range the historical mean value was distinctly lower.
The incidence of cleaved sternebrae was slightly increased in the high dose group with respect to the affected litters (Table 12). This frequency was above the historical range. Also the number of fetuses with 3 ossified metacarpals was increased in the high dose group with a frequency of affected fetuses slightly above the historical range (Table 11).
Cleaved or dumpbell shaped thoracic vertebral bodies were clearly more frequently observed in fetuses at 1000 mg/kg bw. The frequency of both affected fetuses and litters were elevated. The incidence of cleaved vertebrae was distinctly above the historical range and that of the dumpbell shaped vertebrae was at the upper limit. A slightly dose-related trend of increased frequency is seen in at 150 mg/kg bw. (Table 12)
Non-ossification of coccygeal vertebrae was observed in a higher frequency in at 1000 mg/kg bw, the number of affected litters being slightly above the range of historical data. (Table 12)

These findings in the skeletal examination are considered to be signs of retarded fetal ossification and provide a further indication of developmental delay in fetuses of the high dose group.

The anomaly indices were increased with increasing doses. However, the anomaly index of the control group was low compared to those of historical studies. At 25 mg/kg bw both the anomaly index and the frequency of litters with anomalies were below the respective historical mean values. At 150 mg/kg the anomaly index was close to the mean historical value but the frequency of litters with anomalies was slightly increased. At 1000 mg/kg bw both parameters were distinctly above the respective historical mean values and at the upper limit of the historical range. (Table 13)

The visceral and skeletal malformation index in the high dose group was 1.6 % and was based on the occurrence of three different types of malformations, i. e. one skeletal and two different visceral ones. (Table 13)





Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes

Fetal abnormalities

Key result
Abnormalities:
not specified

Overall developmental toxicity

Key result
Developmental effects observed:
not specified

Any other information on results incl. tables

Body weight gain:

Table 1. Body weight changes [g] (Analysis of variance, mean values, standard deviations)

 

days of gestation

group

0-6

6-15

15-20

0-20

0 mg/kg

37.1± 6.7

55.8 ± 5.8

80.0 ± 11.4

173.0 ± 15.7

25 mg/kg

40.8 ± 6.1

51.5 ± 8.3

79.8 ± 11.8

172.0 ± 18.5

150 mg/kg

35.3 ± 11.7

47.8x ± 8.3

78.2 ± 15.1

161.2 ± 21.9

1000 mg/kg

38.1 ± 8.9

40.5x ± 9.0

81.9 ± 15.0

160.5x ± 20.1

MSB

129.95

1 16.03

56.75

1095.58

MSW

74.36

63.03

180.16

368.17

F

1.75

16.12***

0.32

2.98*

DF

3/   93

3/   93

3/   93

3/   93

P

0.161

0.000

0.817

0.035

x  = p 0.05 (Dunnett-test); * = p 0.05 (F-test); *** = p 0.001 (F-test)

Table 2. Mean weight development [g] (mean values and standard deviations)

 

day of gestation 

group

0

6

10

15

20

0 mg/kg

225.1 ± 9.5

262.2 ± 11.2

285.0 ± 10.6

318.0 ± 12.4

398.0 ± 17.6

25 mg/kg

225.3 ± 13.8

266.1 ± 14.6

286.8 ± 15.0

317.6 ± 18.9

397.4 ± 26.3

150 mg/kg

231.3 ± 22.5

266.5 ± 21.1

284.3 ± 21.0

314.3 ± 23.8

392.5 ± 31.5

1000 mg/kg

234.0 ± 16.8

272.1 ± 18.4

286.6 ± 19.8

312.6 ± 19.5

394.5 ± 28.6

Table 3. Corrected body weights and weight changes [g] (Analysis of variance, mean values, standard deviations)

group

Day 20 (corrected)

Day 0-20 (corrected)

0 mg/kg

311.9 ± 13.1

86.8 ± 11.4

25 mg/kg

313.0 ± 18.8

86.7 ± 13.6

150 mg/kg

311.9 ± 22.5

80.6 ±15.5

1000 mg/kg

313.6 ± 22.3

79.6 ± 16.3

MSB

16.03

362.14

MSW

381.72

204.75

F

0.04

1.77

DF

3/93

3/93

P

0.989

0.159

Table 4. Weight changes [g] and mean weight development [g] of low dose group (25 mg/kg bw)  (Mean values and standard deviations)

 

Group 25 mg/kg

Body weights

a

b

Day 0

232.4 ± 19.0

222.0 ± 9.5

Day 6

272.1 ± 21.4

263.2 ± 9.5

Day 10

288.9 ± 25.8

285.8 ± 6.6

Day 15

322.3 ± 30.5

315.4 ± 10.5

Day 20

401.1 ± 36.8

395.6 ±20.9

Weight changes

 

 

Days 6-10

16.8 ± 7.0

22.5 ± 5.4

Days 10-15

33.4 ± 8.2

29.6 ± 6.4

Days 6-15

50.1 ± 11.3

52.2 ± 6.7

Group a: treated with 25 mg/kg bw

Group b: treated with 25 mg/kg bw, but erroneously once with 150 mg/kg bw

Food consumption:

Food consumption did not differ significantly between all groups during the evaluated intervals.

Clinical signs:

No treatment-related clinical signs were noted during the course of the study. The only clinical findings were alopecia in one animal of mid dose group and conjunctivitis in one animal of high dose group both in observation period of days 6 -15 of gestation.

No females were found dead or had to be killed in extremis prior to caesarean section on day 20.

Pregnancy performance:

No significant differences in mating performance of treatment groups were found compared to control group. No females showed signs of abortion or premature delivery during the course of the study.

Necropsy findings:

No treatment-related findings were observed during necropsy of females.

Caesarean section:

The mean number of live fetuses (per dam) and the number of fetuses (per dam) in the left or right uterine horns did not differ significantly among groups.

No biological relevant differences were determined among groups concerning the number of fetuses with head or tail presentation. The sex ratio and distribution show intergroup variation, but no biologically relevant differences are evident.

Fetal weights:

Table 5. Fetal weights (mean value ± sd)

 

Fetuses

Group

Males

Females

Males + Females

0 mg/kg

3.94 ± 0.28

3.76 ± 0.25

3.84 ± 0.27

25 mg/kg

4.06 ± 0.29

3.86 ± 0.28

3.95 ± 0.25

150 mg/kg

3.88 ± 0.22

3.68 + 0.18

3.79 ± 0.20

1000 mg/kg

3.59x ± 0.33

3.37 ± 0.35

3.51x ± 0.27

MSB

0.9723

1.0801

0.8549

MSW

0.0775

0.0747

0.0632

F

12.55***

14.45***

13.52***

DF

3/93

3/92

3/93

P

0.000

0.000

0.000

x   = p 0.05   (Dunnett-test); *** = p 0.001 (F-test)

Placental weights:

Mean placental weights were comparable between control and the dose groups.

Dead fetuses and live births index:

There were no dead fetuses in any test group. Consequently the Live Births Index was 100% in all groups.

Number of runts and Runts Index:

Table 6.No. of runts, runts index

group

No. of runts

Runts Index [%]

0 mg/kg

0

0

25 mg/kg

1

0.3

150 mg/kg

1

0.3

1000 mg/kg

6

1.6

Number of Resorptions and resorption index:

Table 7.Number of resorptions, resorption index (mean value ± sd)

group

early resorptions

late resorptions

total resorptions

Resorption index [%]

0 mg/kg

0.7 ± 0.9

0.0 ± 0.0

0.7 ± 0.9

4.4 ± 5.7

25 mg/kg

0.8 ± 0.9

0.1 ± 0.3

0.9 ± 0.9

6.0 ± 5.7

150 mg/kg

0.7 ± 0.8

0.1 ± 0.3

0.8 ± 0.8

5.3 ± 5.0

1000 mg/kg

1.3 ± 1.4

0.4 ± 1.1

1.7x ± 2.0

10.2 ± 12.0

x = p 0.05 (U-test)

Table 8. Relative frequency of females with resorptions (%)

group

0 mg/kg

25 mg/kg

150 mg/kg

1000 mg/kg

Without resorptions

54

36

42

25

With 1 resorption

25

44

33

33

With 2 or more resorptions

21

20

25

42

Pre-implantation loss index:

The pre-implantation loss indices did not differ significantly between the groups.


Post-implantation Loss Index:

Table 9.Post-implantation Loss Index (mean value ± sd)

group

Post-implantation Loss Index [%]

0 mg/kg

4.4 ± 5.7

25 mg/kg

6.0 ± 5.7

150 mg/kg

5.3 ± 5.0

1000 mg/kg

10.2 ± 12.0

Implantations:

Mean numbers of implantations showed no significant differences between groups and no biologically relevant differences were found in the left/right intrauterine distribution.

Corpora lutea:

Significantly increased number of corpora lutea at 1000 mg/kg bw. Accordingly, the number of copora lutea in each ovary was also increased. This finding is considered to be incidental, since the treatment commenced only on day 6 of gestation, when corpora lutea had already developed (table 10).

Table 10. No. of Corpora Lutea (Analysis of variance, mean values, standard deviations)

group

left

right

total

0 mg/kg

8.4 ± 1.9

9.0 ± 2.2

17.4 ± 2.1

25 mg/kg

8.5 ± 1.8

9.1 ± 2.1

17.6 ± 2.3

150 mg/kg

8.7 ± 1.8

9.1 ± 2.9

17.8 ± 2.9

1000 mg/kg

9.6 ± 2.6

10.2 ± 2.4

19.8x ± 2.8

MSB

7.6149

7.8864

30.8978

MSW

4.1831

5.8540

6.4009

F

1.82

1.35

4.83**

DF

3/93

3/93

3/93

P

0.147

0.263

0.004

x = p 0.05 (Dunnet-test); ** = p 0.01 (F-test)

External examination:

One incidental finding was observed in the control group. In the high dose group the hindlimbs appeared slightly crooked in two fetuses. Since no bent bones of the hindlimbs were found and only two fetuses were affected, this finding is considered to be without toxicological importance.

Visceral examination:

Slight to moderate hydrocephalus was observed in two fetuses of the high dose group in two different litters. A clear treatment relation cannot be considered for this malformation since this finding occurred in control fetuses of the historical studies even with a slightly higher incidence.

The finding of diaphragmatic hernia found in three fetuses of two litters of the high dose group was also found in literature studies with a spontaneous occurrence of 2.7 % in albino rats [Andersen d. H. (1949) Effect of diet during pregnancy upon the incidence of congenital hereditary diaphragmatic hernia in the rat. Am. J. Pathol. 25, 163 -185] and up to 0.9 % of fetuses of vehicle treated groups and in up to 5.6 % of litters [Charles River Laboratories (1988) Embryo and fetal developmental toxicity (teratology) control data in the Charles River Crl:CD(R) BR rat. Charles River Laboratories, ]. Andersen (1949) denominated this kind of abnormality as a right incomplete diaphragmatic hernia and stated, that this lesion appeared to have no effect on the health or growth of the affected fetuses. Diaphragmatic henia with protusion of a part of the liver can be considered as a result of delayed development of pleuraoperitoneal membranes. This delay of development may be associated with the general growth retardation which was seen in fetuses of high dose group females.

A slight tendency of increased occurrence of dilated ureters may be stated with respect to affected fetuses in the dose groups. However, the relative frequency of affected litters was very similar in control, low and mid dose group. The slight increase of dilated ureters in the high dose group may be associated with the general growth retardation in fetuses of the high dose group.

Dilation of renal pelvis was noted in fetuses of all test groups with a similar incidence and no dose relationship.

All other abnormalities of soft tissues were minor findings and were noted in single fetuses only. Thus they are considered to be without toxicological relevance.

Skeletal examination:

One malformed fetus was found in the high dose group showing as major finding a kyphosis of the thoracic vertebral column. Since it occurred only once and scoliosis, which denotes a similar abnormality of the vertebral column, was found even in historical control fetuses, this finding is considered to be incidental.

The relative frequency of non-ossified sternebrae was very similar between all test groups with respect to both affected fetuses and litters. The incidence of other skeletal abnormalities provided no evidence of a treatment effect.

Table 11. Skeletal examination: Number of ossification centers (Relative frequencies [%] of affected fetuses and litters)

Group

0 mg/kg

25 mg/kg

150 mg/kg

1000 mg/kg

No. of examined fetuses/litters

Fetuses / litters

188 / 24

189 / 24

180 / 24

191 / 24

Number of ossification centers

Sternum

6

Fetuses/litters

73 / 96

77 / 100

75 / 96

68 / 96

5

22 / 75

16 / 64

23 / 58

25 / 88

4

4 / 21

6 / 32

2 / 13

5 / 29

4

1 / 4

1 / 4

1 / 4

3 / 21

Metacarpals

4

Fetuses / litters

82 / 100

88 / 100

84 / 100

66 / 100

3

18 / 58

12 / 52

16 / 67

34 / 88

3

1 / 4

0 / 0

0 / 0

0 / 0

Metatarsals

¿ 4

Fetuses /litters

100 / 100

100 / 100

99 / 100

100 / 100

3

0 / 0

0 / 0

1 / 4

0 / 0

3

0 / 0

0 / 0

0 / 0

0 / 0

Table 12: Skeletal examination: (Selected) skeletal abnormalities

 

group

0 mg/kg

25 mg/kg

150 mg/kg

1000 mg/kg

 

 

fetuses (%)

litters (%)

fetuses (%)

litters (%)

fetuses (%)

litters (%)

fetuses (%)

litters (%)

No. of examined fetuses/litters

188 (100)

24 (100)

189 (100)

25 (100)

180 (100)

24 (100)

191 (100)

24 (100)

Sternum (ossification centers)

150 (80)

24 (100)

150 (79)

25 (100)

128 (71)

24 (100)

158 (83)

24 (100)

 

irregular shape

34 (18)

20 (83)

54 (29)

20 (80)

39 (22)

17 (71)

58 (30)

22 (92)

 

cleaved

1 (1)

1 (4)

1 (1)

1 (4)

1 (1)

1 (4)

5 (3)

5 (21)

 

rudimentary

48 (26)

20 (83)

30 (16)

15 (60)

28 (16)

15 (63)

51 (27)

19 (79)

 

incomplete ossification

93 (49)

24 (100)

98 (52)

25 (100)

94 (52)

23 (96)

120 (63)

24 (100)

 

not ossified

52 (28)

20 (83)

43 (23)

18 (72)

45 (25)

15 (63)

58 (30)

20 (83)

Vetebral column

27 (14)

11 (46)

26 (14)

12 (48)

45 (25)

18 (75)

81 (42)

23 (96)

 

Thoracic Vertebrae

27 (14)

11 (46)

26 (14)

12 (48)

45 (25)

18 (75)

78 (41)

21 (88)

 

kyphosis

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

1 (1)

1 (4)

 

vertebral bodies

27 (14)

11 (46)

26 (14)

12 (48)

45 (25)

18 (75)

77 (40)

21 (88)

 

cleaved

0 (0)

0 (0)

0 (0)

0 (0)

4 (2)

3 (13)

16 (8)

9 (38)

 

dumpbell shaped

27 (14)

11 (46)

26 (14)

12 (48)

44 (24)

18 (75)

74 (39)

21 (88)

Coccygeal Vertebrae

0 (0)

0 (0)

1 (1)

1 (4)

1 (1)

1 (4)

5 (3)

4 (17)

 

not ossified

0 (0)

0 (0)

1 (1)

1 (4)

1 (1)

1 (4)

5 (3)

4 (17)

Variation indices:

Variation indices did not show any biologically significant differences.

Anomaly index and malformation index:

Table 13.Anomaly index and malformation index

group

0 mg/kg

25 mg/kg

150 mg/kg

1000 mg/kg

Number of anomalies skeletal

29

35

45

81

Anomaly Index skeletal

15.4

18.5

25.0

42.4

Litters affected skeletal

11

14

18

23

%

45.8

56

75

95.8

Number of malformed fetuses

0

0

0

6

malformaion Index

0.0

0.0

0.0

1.6

Litters affected

0

0

0

4

%

0.0

0.0

0.0

16.7

Applicant's summary and conclusion

Conclusions:
The test substance caused a clear maternal toxic effect at 1000 mg/kg bw/d shown as a dose-dependent decreased body weight gain. Body weight effects at 150 mg/kg were only transitory and slight.
Embryotoxic effects were found as distinct growth retardation of fetuses and to an increased number of resorptions at 1000 mg/kg bw/d. The growth retardation is considered to be the cause of many of the findings seen during fetal examination, e. g. diaphragmatic hernia, increase of dilated ureters, skeletal findings. At doses of 150 mg/kg bw/d only a very slight occurrence of delayed ossification was found.

Under the conditions of this study, the No Observed Adverse Effect Level (NOAEL) for maternal toxicity was considered to be 150 mg/kg bw/d. The No Observed Adverse Effect Level (NOAEL) for developmental toxicity was considered to be 150 mg/kg bw/d, since at this dose only a very slight occurrence of delayed ossification was observed, accompanied by a distinct maternal toxicity.
Executive summary:

The test material was administered to pregnant Wistar rats via oral gavage from day 6 to 15. At doses of 25 and 150 mg/kg bw/d no maternal toxicity was observed. At a dose of 1000 mg/kg/d clear maternal toxicity, such as reduced body weight gain was observed.

At 25 mg/kg bw/d no effects on offspring was observed. At a dose of 150 mg/kg bw/d only a very slight delay of ossificaiton occurred. Treatment of the females with the high dose (1000 mg/kg bw/d) led to a distinct growth retardation of fetuses and an increased resorption. The growth retardation can be seen as cause of the various findings observed during fetal examination.