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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted in accordance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
(adopted 1981)
Deviations:
no
GLP compliance:
yes
Remarks:
(Hoechst Marion Roussel Global Preclinical Development Drug Safety)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Trioxan
Purity: 99.77%
Physical state: colourless liquide
Storage: at approximately 20°C, protected from light
Homogeneity and stability of the test compound in the vehicle water are guaranteed

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
Designation: Hoe: WISKf(SPF71) Wistar
Sex: female
Source: Hoechst AG, Kastengrund, SPF breeding colony
Age at test initiation: approximately 8 - 10 weeks
Housing: individually, in fully air-conditioned rooms in makrolon cages (type 111) on soft wood granulate
Room temperature: 19 - 22 °C
Relative humidity: 35 -75 %
Lighting time: 12 hours daily
Ventilation rate: 16 - 20 air changes per hour
Feed: Ssniff R-Z (V1 324), ad libitum
Drinking: tap water in plastic bottles, ad libitum
Identification: ear tags and cage numbering
Acclimatisation: at least five days under study conditions

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
- Trioxane was used as a 20% aqueous solution;
- The formulation was freshly prepared each day. If crystals were present, they were dissolved by slight heating (maximum 40 °C in the dark);
- All groups received a dose volume of 5 ml/kg body weight, with adjustment of the individual volume to the most recently recorded body weight;
- The test compound was prepared daily, immediately before dosing.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For each concentration, samples were taken towards the start and end of the dosing period (day 7 to 20).
All samples were stored deep frozen prior to analysis.
Details on mating procedure:
In the laboratory's own breeding facility, virgin female animals in the pre-oestrus or oestrus phase were mated overnight with sexually mature males in the ratio 1 male :1 female and were caged individually after detection of sperm in vaginal smears. The day of sperm detection was defined as day 1 of gestation, and the day of mating was defined as day 0 of pregnancy.
Pregnancy was confirmed at necropsy by the detection of implantation sites or normally developed corpora lutea.
Duration of treatment / exposure:
From day 7 to day 20 of pregnancy
Frequency of treatment:
once daily
Duration of test:
From day 1 to day 21 of pregnancy
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 315, 1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
23 mated females/group
Control animals:
yes, concurrent no treatment
yes, historical

Examinations

Maternal examinations:
- All animals were examined before the start of the study and were shown to be in good general health condition;
- The behaviour and the general health condition of the animals were observed several times daily (on weekends and public holidays once daily);
- Body weights were determined on days 1, 4, 7, 10, 14, 17, 19 and 21 of pregnancy;
- Food consumption was recorded between days 1-4, 4-7, 7-10, 10-14, 14-17, 17-19 and 19-21 of pregnancy;
- The dams were sacrificed on day 21 of pregnancy for the purpose of Caesarean section; all animals were examined externally and internally (thoracic and abdominal contents) for macroscopically visible changes, with emphasis on the uterus.
Ovaries and uterine content:
- Following sacrifice of the dams, the foetuses were removed by Caesarean section.
- Gravid uterus weight was determined;
- The live and dead foetuses in the uterus as well as resorptions and corpora Iutea were counted and examined macroscopically;
- The implantation sites in the uterus were counted after staining with ammonium sulphide.
Fetal examinations:
- The foetuses were removed from the uterus, weighed or measured and examined for gross external abnormalities;
- the crown-rump length was recorded;
- The foetuses were then sacrificed.
- Foetuses found dead in the uterus at caesarean section were fixed in alcohol and examined for external abnormalities;
- About 50% of the foetuses of each litter were fixed in alcohol, necropsied, sexed and checked for abnormalities of the internal organs. The carcasses were placed in a solution of potassium hydroxide for clearing and were stained with Alizarin red S. The skeletons were examined and checked for stage of development and abnormalities using a stereomicroscope.
- The remaining foetuses were transferred in Bouin's solution, examined for organ anomalies referring to Wilson's slicing technique (Wilson JG, Embryological considerations in teratology. In Teratology: Principles and Techniques: 251-277, JG Wilson, J Warkany, Ed., University of Chicago Press, Chicago, IL, 1965) and sexed.
Statistics:
- The statistical evaluation was based on the assumption of a monotone dose-response relationship.
- Statistical comparisons of the low doses with the simultaneous control were carried out if significant effects were detectable in the high dose group.
- In the univariate analysis, two-sided questions (body weight of dams, relative food consumption, crown-rump length, foetal weight and placental weight) were generally tested as follows: a two-sided comparison with the high dose group was followed by a one-sided test for the low-dose group.
- For the foetuses data at Caesarean section (e.g. foetal weight), multivariate statistics were used.
- For individual parameters, sequential comparisons with the high dose group and sequential tests at the 5% level for the low dose were done.
- The t-tests and the test statistics of Wilks are based on common variance estimations for all study groups.
- For the Wilcoxon test the exact distribution of the meaned ranks was calculated.
- For the daily food consumption, the mean consumption per 100 g bw was calculated between 2 successive measurement times and evaluated by the rank sum test after Wilcoxon.
- Change in body weight was determined in comparison to the initial weight.
- The univariate evaluation was based on t-tests. T
- Foetuses data at Caesarean section were used to calculate litter mean values.
- Multivariate evaluation was carried out using the test statistics of Wilks.
- In the univariate analysis, t-tests were used.
- The number of corpora Iutea, implantation sites and live foetuses, and quotas of dead embryonic primordia undergoing resorption in the animals were likewise analysed using one-sided Wilcoxon tests.
- Findings at necropsy and at body cross-section and skeletal foetal examination were evaluated separately for foetuses and for litters by Fisher's Exact test (sign. levels 5% and 1%); these parameters also were compared with those of corresponding findings in previous control groups
Historical control data:
yes

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
MORTALITY AND CLINICAL SYMPTOMS OF TOXICITY:
Neither mortalities nor clinical signs of toxicity were observed in the dams.

BODY WEIGHT GAIN:
Mean corrected body weight gain (day 21 body weight minus body weight on day 7 minus gravid uterus weight) was decreased with statistically significance p < 0.05 for all dosed groups. In fact, the values for corrected body weight gains were 27.13 g (19.2% of control, low dose group), 28.96 g (14% compared to control, intermediate dose group) and 18.88 g (43.8% of contol, high dose group), versus 33.60 g (control).

FOOD CONSUMPTION:
- Food consumption was slightly, but statistically significantly decreased in the high dose group (6-14%) from beginning on GD10 until study termination.
- Food consumption also was statistically significantly (3-4%) reduced in the mid dose group from day 7 and 10 and from day 17 to 19 of the study.
- No difference between the control and the low dose group could be evidenced statistically.

NECROPSY:
- Necropsy of the dams revealed no compound-related effect.
- The gravid uterus weights were comparable in all groups.
- Three control females and 2 females from each of the treatment groups did not become pregnant.
- In one animal from the low and high dose each, only implantation sites were found at Caesarean section.
- One animal from the low and high dose each showed only implantation sites. Therefore a statistically significant increase in resorptions (60%) was reported for the high dose group; however, since no dose-dependency was recognizable and since similar values were known from laboratory historical controls, the finding was not considered to be treatment-related.
- Five dead fetuses were observed in 5 litters in the high-dose group and this was statistically significant compared to the control group; a compound-related effect could not be ruled out for fetal death at the high dose. Two dead foetuses were found in one litter of the mid dose group. As only one litter was affected, a treatment-related effect in this case was questionable.

Effect levels (maternal animals)

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

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
FINDINGS AT CAESAREAN SECTION:
- Litter size was comparable in all groups.
- Foetal body weight and crown-rump length were slightly and statistically significantly decreased in the high dose group.
- Placental weights were statistically significantly increased in the high dose group. These parameters were inconspicuous in the low and mid dose groups.
- Sex ratio of the foetuses was similar in all groups.

RETARDED FETUSES:
- The incidence of retarded foetuses was statistically significantly increased in the high dose group (control: 6/123, 100 mg/kg bw: 8/117, 315 mg/kg bw: 8/139, 1000 mg/kg bw: 29/131). The values were outside the historical range of the rat strain used, indicating that the finding was treatment-related.
- Two foetuses from the high dose group showed aplasia of the tail; as this finding was not observed in 6025 historical control foetuses, a treatment-related effect could not be ruled out.

SKELETAL FINDINGS:

MAJOR SKELETAL FINDINGS:
The two foetuses of the high dose group with aplasia of the tail (see above) showed aplasia of sacral vertebral arch, sacral vertebral centres and 1st and 2nd caudal vertebral centres.

MINOR SKELETAL FINDINGS (see Tab. 2):
- The incidence of longitudinally displaced, fused or fragmented sternebrae (2/123, 3/117, 1/139, 17/131), wavy and/or thickened ribs (12/123, 14/117, 56/139, 84/131), bent or shortened scapula (0/123, 0/117, 2/139, 12/131), bent, shortened or dysplastic humerus (0/123, 0/117, 1/139, 12/131) and bent or shortened radius (0/123, 0/117, 0/139, 5/131) were statistically sgnificantly increased in the foetuses of the high dose group. Two foetuses with the latter finding showed bent ulna. The incidences were above the historical range of the rat strain used. Therefore, a treatment-related effect is probable.
- The incidence of fragmented thoracic vertebral centres (0/123, 0/117, 2/139, 4/131) was above historical control range in the high dose group and therefore treatment-related;
- The incidence of wavy and or thickened ribs was also statistically significantly increased in the mid dose group, the incidence being above the historical range of the rat strain used. Therefore, a treatment-related effect could not be ruled out;

VARIATIONS:
No treatment-related effects were reported. One of the 2 high dose fetuses with aplasia of the tail also showed anlage of only 5 lumbar vertebrae.

SKELETAL REDARDATIONS (see Tab. 3):

1000 mg/kg bw group:
- Statistical evaluation revealed numerous retardations; slight or non-ossification of individual skull bones (42/123, 50/117, 74/139, 80/131), weakly or non-ossified cervical vertebral arch (0/123, 0/117, 3/139, 13/131), weakly ossified lumbar vertebral arch (0/123, 0/117, 2/139, 13/131), weakly or non-ossified sacral vertebral arch (3/123, 3/117, 2/139, 14/131), ossification of less than two caudal vertebral centres (26/123, 50/117, 85/139, 98/131), weakly ossified ribs (0/123, 1/117, 3/139, 9/131), weakly ossified metacarpale 2 (0/123, 0/117, 0/139, 5/131), non-ossified metacarpale 5 (58/123,61/117, 92/139, 86/131) and non-ossified metatarsale 5 (3/123, 2/117, 1/139, 11/131). These incidences were above the histonical range of the rat strain used and were considered to be treatment-related.
- Further statistically significant changes in the high dose group consisted of non-or weakly ossified sternebrae (18/123, 42/117, 45/139, 70/131) and non-ossified phalanx III of the 1st to 5th toe of the hindpaw (1/123, 7/117, 3/139, 18/131). In these cases, the values were within the historical range of the rat strain used. However, with respect to the findings mentioned above, a treatment-related effect is probable. The incidences of weakly or non-ossified thoracic vertebral arch (1/123, 0/117, 2/139, 3/131), weakly or non-ossified thoracic (1/123, 0/117, 0/139, 4/131) and sacral vertebral centres (3/123, 3/117, 2/139, 10/131) and weakly ossified metacarpale 4 (0/123, 0/117, 0/139, 3/131) were above the historical range of the rat strain used;

315 mg/kg bw group:
The incidences of slight or non-ossification of individual skullbones, ossification of less than two caudal vertebral centres and non-ossified metacarpale 5 were also statistically significantly increased. As the incidences were above the historical range, a treatment-related effect cannot be ruled out. The incidences of weakly or non-ossified cervical and thoracic vertebral arch, weakly ossified lumbar vertebral arch and weakly ossified ribs were slightly above the historical range.

100 mg/kg bw group:
Findings in the low dose group were partly above historical control values, but since findings showed no dose-relationship and were without statistical significance, they were not considered to be treatment-related.

All other findings were within the historical range of the rat strain used and/or were not statistically significant.

EXTERNAL AND VISCERAL RETARDATIONS AT BODY CROSS-SECTION EXAMINATION:
116, 109, 125 and 121 fetuses of the 0, 100, 315 and 1000 mg/kg bw group, respectively, were subjected to cross section examination; the incidence of retarded fetuses was 0/116, 2/109, 1/125 and 17/121. The increase observed in the high dose group was statistically significant and was outside of the historical control range. The finding therefore was treatment-related.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Tab 1. Caesarean section, main data:

Parameters

Dose group (mg/kg bw)

0

100

315

1000

Pregnancies

20/23

21/23

21/23

21/23

Females with abortion

0

1

0

1

Corpora lutea (total)

280

287

316

298

Implantations (total)

246

245

284

276

Pre-implantation loss (mean %)

12.78

14.10

10.08

7.35

Post-implantation loss (mean %)

2.62

7.65

7.22

8.36

Live fetuses (total)

239

226

264

252

Intrauterine deaths (total)

7

19

20

24

Males (%)

48.1

59.7

55.7

50.8

Mean body weight (g)

3.3

3.3

3.2

2.8*

Mean crown-rump length (mm)

35.1

35.0

34.5

32.8*

Mean placental weight (g)

0.46

0.47

0.49

0.52

Mean uterus weight (g)

60.40

57.57

62.57

59.47

*, statistically significant difference from control

Tab 2. Foetal data, skeletal defects:

Fetal minor skeletal defects

Dose group (mg/kg bw)

0

100

315

1000

Longitudinally displaced, fused or fragmented sternebrae

2/123

3/117

1/139

17/131*

Wavy and/or thickened ribs

12/123

14/117

56/139*

84/131*

Bent or shortened scapula

0/123

0/117

2/139

12/131*

Bent, shortened or dysplastic humerus

0/123

0/117

1/139

12/131*

Bent, shortened radius

0/123

0/117

0/139

5/131*

Fragmented thoracic vertebral centers

0/123

0/117

2/139

4/131

*, statistically significant difference from control

Tab 3. Foetal data, retardations:

Retardations

Dose group (mg/kg bw)

0

100

315

1000

Slight or non-ossification of skull bone

42/123

50/117

74/139*

80/131*

Weak or non-ossification of cervical vertebral arch

0/123

0/117

3/139

13/131*

Weak ossification of lumbar vertebral arch

0/123

0/117

2/139

13/131*

Weak or non-ossification of sacral vertebral arch

3/123

3/117

2/139

14/131*

Ossification of less than 2 causal vertebral centers

26/123

50/117*

85/139*

98/131*

Weak ossification of ribs

0/123

1/117

3/139

9/131*

Weak ossification of metacarpal 2

0/123

0/117

0/139

5/131*

Non ossification of metacarpal 5

58/123

61/117

92/139*

86/131*

Non ossification of metatarsal 5

3/123

2/117

1/139

11/131*

Weak or non ossification of sternebrae

18/123

42/117*

45/139*

70/131*

Non ossification of phalanx 3 (1st to 5th hindpaw toe

1/123

7/117*

3/139

18/131*

*, statistically significant difference from control

Applicant's summary and conclusion