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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:
From 12 January 2009 to 6 August 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study to GLP
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
Principles of method if other than guideline:
Not applicable.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Perform STi

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain NZ White (HsdIf)
- Source: Harlan UK Ltd, Bicester, Oxon, UK
- Age at study initiation: 3-5 months
- Weight at study initiation: 2.82-4.10 kg
- Fasting period before study: not applicable
- Housing: singly
- Diet (e.g. ad libitum): ad libitum
- Environmental enrichment: aspen blocks and small quantities of autoclaved hay were made available to the animals, as appropriate. A bunny block sugar treat produced by Bio-Serv, Frenchtown, NJ 08825, USA was also given to each animal as appropriate
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 4-6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 +/- 3
- Humidity (%): 55 +/- 15
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12:12

IN-LIFE DATES: from 8 January 2009 to 13 February 2009

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Dose levels were calculated in terms of test substance as supplied.
Formulations were prepared by adding the appropriate amount of test item to the required amount of vehicle (water for irrigation with 0.003% Citric Acid (to adjust for pH)). Formulations were mixed by means of manual inversion until visibly homogenous.
Formulations were prepared daily and used within the established stability period of 8 h (established in Charles River Laboratories Study No. 424830, Method No. 2483 for concentrations of 5-100 mg/mL). Formulations below 5 mg/mL were prepared from a dilution of a 5 mg/mL formulation; due to no clear established stability period at this concentration, these formulations were prepared in the animal unit and dosed immediately after preparation.
Control animals received vehicle alone (water for irrigation with 0.003% (w/v) citric acid)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of the formulations was undertaken with regard to concentration and homogeneity using a simple iodine titration. Formulations were analysed on two occasions during the study treatment period; from formulations prepared for use on Weeks 1 and 3 of the study.

A single sample was withdrawn from each formulation (including Control) and was analysed in triplicate. The samples were assayed at Charles River, using a method supplied by the Sponsor and previously validated in the Charles River laboratory under separate protocol and contract (Study No. 424830, Method No.2483).
Details on mating procedure:
- purchased timed pregnant
Duration of treatment / exposure:
Days 6 to 28 of gestation, inclusive, apart from two high dose animals for which dosing was stopped early, on GD 16 or 17, due to the severity of clinical signs.
Frequency of treatment:
once daily
Duration of test:
22 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
10 mg/kg bw/day
Dose / conc.:
40 mg/kg bw/day
Dose / conc.:
120 mg/kg bw/day
No. of animals per sex per dose:
24 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: determined in range-finding study

Examinations

Maternal examinations:
- Duration of observation period following administration: none
- Frequency of observations: twice daily
- Frequency of weighing: daily (except GD 5)
- Frequency of food consumption measurement: daily
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: early morning and late afternoon

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: GD 4, 6-29

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: Thoracic and abdominal cavities, liver, uterus

OTHER:
The following parameters related to possible liver toxicity were measured prior to dosing on GD 13 and 29:

Haematology
Haemoglobin
Red Blood Cell count

Coagulation
Activated Partial Thromboplastin Time
Prothrombin Time
Fibrinogen

Clinical Chemistry:
Gamma-glutamyltransferase
Aspartate Aminotransferase
Alanine Aminotransferase
Alkaline Phosphatase
Creatine Phosphokinase
Total Bilirubin
Cholesterol
Triglycerides
Lactate Dehydrogenase
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
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: [all per litter]
Statistics:
Statistical analysis was performed on body weight gain over Days 6-29 of gestation and on mean foetal weights and clinical pathology. Body weight gain and clinical pathology was subjected to analysis of variance and foetal weight was analysed by the Kruskal-Wallis test.
Liver weights were analysed by analysis of variance, and by analysis of covariance using the terminal body weight as the single covariate.
Histological data was analysed by the Fisher’s Exact test (two-tailed).
All statistical tests were 2-sided and performed at the 5% significance level using in-house software. Pairwise comparisons were only performed against the Control group.
Historical control data:
Not included

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Six animals at 120 mg/kg/day were killed prematurely on GD 15, 18, 20, 21(2), 22 due to severe clinical signs and anorexia.
At 120 mg/kg/day, signs of reaction to treatment included changes in faecal output; there was also an increase in the number of animals with red liquid on the tray. One animal (Animal 83) prematurely gave birth to 4 live pups and 4 dead pups on Day 29 of gestation.
At 120 mg/kg/day, necropsy findings in the animals that survived to Day 29 of gestation were mainly confined to liver findings, including pale and prominent lobulation. There was a statistically significant increase in the liver weights at this level.
Two animals at 10 mg/kg/day, and four at 40 mg/kg/day, had pale livers. Liver weights at these levels were similar to Control. At 40 mg/kg/day, there was also an increase in the changes to faecal output compared with Control. Other necropsy findings at these levels were not obviously associated with treatment.
At 120 mg/kg/day, there was a significant reduction in body weight gain compared with Control. The lower weight gain was more noticeable in those animals requiring premature sacrifice, but even those females that survived until Day 29 of gestation showed noticeably lower weight gain when compared with Control.
Group mean food consumption at 120 mg/kg/day was lower throughout the treatment period; this was most noticeable over ca Days 7-21 of gestation. The lower consumption was more noticeable in those females that did not survive until Day 29.
At 120 mg/kg/day, mild/moderate periportal hepatocyte vacuolation with vacuoles containing amorphous eosinophilic material was observed in all animals; of the 7 animals with moderate vacuolation, 3 were premature decedents and 4 were at terminal necropsy. Moderate/marked centrilobular hepatocyte vacuolation comprising of clear vacuoles was observed in 4 animals (all premature decedents) which correlated to the pale livers and prominent liver lobulation observed at necropsy. Mild to marked coagulative necrosis of the liver was observed in 4 animals, and 3 animals had mild/moderate periportal hepatocyte hypertrophy; all of these animals were premature decedents.
At 40 mg/kg/day, 6/24 animals had minimal periportal hepatocyte vacuolation with vacuoles containing amorphous eosinophilic material.
At 10 mg/kg/day, histological findings were similar to Control.
At 120 mg/kg/day on Day 13 of gestation (Day 8 of treatment), there was a significant increase in the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate deyhdrogenase (LDHIL) and a reduction in alkaline phosphatase (ALP), compared with Control; some of the increases in AST, ALT and LDHIL were attributed to Animals 83, 94 and 95 which were killed prematurely, however, these increases were still statistically significant when the premature decedents were excluded from the analysis. In addition to this, there was a significant increase in the fibrinogen (Fib) levels. On Day 29 of gestation, ALT levels were still significantly increased compared with Control although AST, LDHIL and ALP were within normal limits. However, by Day 29 of gestation, Cholesterol, Triglycerides and Gamma-Glytamyltransferase levels were also significantly increased. All of the effects on these parameters are consistent with the liver findings seen at this level. Haemoglobin and Red Blood Cell Counts were significantly reduced which may also be associated with the liver findings.
Slight intergroup differences in clinical pathology at 10 and 40 mg/kg/day, were too small to be attributed to treatment.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Basis for effect level:
other: maternal toxicity
Remarks on result:
not determinable
Remarks:
no NOAEL identified
Dose descriptor:
LOEL
Effect level:
10 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
At 120 mg/kg/day, there was an increase in the number of dead implants; these were mainly early embryonic deaths but there was also an increase in late deaths compared with Control. There was also a significant reduction in mean foetal weights compared with Control.
At 10 and 40 mg/kg/day, pregnancy performance and foetal weights were comparable with Control.
Major Foetal Abnormalities
At 120 mg/kg/day, there was a noticeable increase in the number of foetuses with a major foetal abnormality compared with Control (26 foetuses in 9 litters compared with 4 foetuses in 2 litters in Control). The majority of the major abnormalities (22 foetuses from 8 litters) were in the eyes and/or in the hindlimbs/digits. A total of 8 of the 16 foetuses that had major eye abnormalities also had major limb/digit abnormalities.
Abnormalities of the eyes included reduced eyes with aphakia and/or lens reduced with retinal folds, aphakia with retinal folds, reduced eyes/lens and retinal folds. It was noted the animals recorded as having major eye abnormalities (with the exception of one foetus) had their eyes examined by serial sectioning.
Limb abnormalities at this level included bilateral forelimb flexure, fore and hindlimb hyperflexion, mis-shapen limb bones, shortened limb bones, abnormally positioned fibulae, calcaneum and astragalus conncected/fused, incomplete ossification, digits poorly defined, digits missing, cartilage elements fused, hindpaws malrotated.
The type and distribution of major foetal abnormalities at 10 and 40 mg/kg/day were not considered to be associated with treatment.
Minor Visceral Findings
At 120 mg/kg/day, there was also a noticeable increase in the number of foetuses with a minor abnormality/variant compared with Control (49 foetuses in 13 litters compared with 10 foetuses in 8 litters).
At 120 mg/kg/day, there was an increase in the number of foetuses with eyes/lens reduced (with or without corneal and lenticular opacities, with or without retinal folds) with 18 foetuses (2 also with major eye abnormalities) from 7 litters affected compared with none in Control. Also, 10 foetuses in 6 litters (compared with none in Control) had haemorrhage in the orbital socket; these foetuses (with one exception) also had major eye defects.
At 120 mg/kg/day, there was also an increase in the number of foetuses with incisors not erupted and an increase in the number of small foetuses.
The type and distribution of minor visceral abnormalities at 10 and 40 mg/kg/day were not considered to be associated with treatment.
Minor Skeletal Findings
At 120 mg/kg/day, there was an increase in the number of foetuses with a reduced state of ossification (including skull bones, hyoid, odontoid process, pubes, limbs and digits). In addition there was an increase in the number of foetuses with the calcaneum and/or astragalus incompletely ossified/unossified. This reduced state of ossification is likely to be due to the fact that the foetuses were smaller and therefore the ossification was delayed.
At 120 mg/kg/day, there was also a greater incidence of foetuses with bilateral caudal displacement of the pelvic girdle (37% of foetuses affected, compared with 12%, 17% and 12% at 0, 10 and 40 mg/kg/day). Also, the proportion of foetuses with supernumerary ribs, and of complete 13th ribs, was much greater at this dose level; foetuses with pelvic girdle displacement generally had 13 complete ribs.
The type and distribution of minor skeletal abnormalities at 10 and 40 mg/kg/day were not considered to be associated with treatment.

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOEL
Effect level:
40 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Dose descriptor:
LOEL
Effect level:
120 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxcity, soft tissue and skeletal abnormalities

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Detailed information on pregnancy performance is shown in Table 7.8.2.1.

 

Table 7.8.2.1: Pregnancy Performance and Foetal Weight

 

Group/Dose Level (mg/kg/day)

1

(0)

2

(10)

3

(40)

4

(120)

Number of animals mated

24

24

24

24

Number pregnant

21

20

23

22

Number of premature decedents

1

0

0

6

Number of pregnant decedents

1

0

0

6

Number pregnant at Day 29 necropsy

20

20

23

14*

Pregnancy frequency as %

88

83

96

92

Total corpora lutea graviditatis

200

213

231

156

Total number of implants

164

180

195

130

Pre-implantation loss as %

18

15

16

17

Total live implants (%)

155 (95)

167 (93)

181 (93)

87 (67)

Total dead implants (%)

9 (5)

13 (7)

14 (7)

43 (33)

Total early embryonic deaths (%)

5 (3)

8 (4)

8 (4)

30 (23)

Total late embryonic deaths (%)

4 (2)

5 (3)

6 (3)

13 (10)

Total dead foetuses (%)

0

0

0

0

Mean corpora lutea graviditatis

10.0 ± 2.4

10.7 ± 1.5

10.0 ± 1.6

10.4 ± 1.7

Mean implants

8.2 ± 3.3

9.0 ± 2.5

8.5 ± 2.3

8.7 ± 2.6

Mean live implants

7.8 ± 3.3

8.4 ± 2.2

7.9 ± 2.2

5.8 ± 3.1

Mean dead implants

0.5 ± 0.8

0.7 ± 1.0

0.6 ± 0.9

2.9 ± 2.0

Mean early embryonic deaths

0.3 ± 0.7

0.4 ± 0.8

0.3 ± 0.7

2.0 ± 2.0

Mean late embryonic deaths

0.2 ± 0.5

0.3 ± 0.6

0.3 ± 0.4

0.9 ± 1.9

Mean dead foetuses

0

0

0

0

Total live male foetuses (%)

84 (54)

88 (53)

87 (48)

48 (55)

Total live female foetuses (%)

71 (46)

79 (47)

94 (52)

39 (45)

Live foetal sex ratio (male: female)

1:0.85

1:0.90

1:1.08

1:0.81

Mean total uterus weight (g)

521 ± 187

571 ± 116

524 ± 128

334 ± 85

Mean litter mean foetal weight (g) #

45.0 ± 5.1

45.1 ± 4.8

44.2 ± 5.3

31.4 ± 8.8***

 

Means are given+Standard Deviation

Note: Premature decedents excluded below double line

Animal 83 (Group 4) aborted litter on Day 29 of gestation, excluded below double line

*Excludes Animals 80 and 85(Group4) which only had early deaths, excluded from group calculations

# = Analysed statistically. Significantly different from the control, *P<0.05, **P<0.01, ***P<0.001

 

Applicant's summary and conclusion

Conclusions:
Developmental toxicity was demonstrated only at the severely maternally toxic high dose level. It was concluded not to be clear whether these foetal findings were directly related to Perform STi treatment or were secondary to the severe maternal toxicity evident at this dose level.
Executive summary:

The test substance, Perform STi, was administered to pregnant female New Zealand rabbits (HsdIf:NZW strain) to detect potential effects on pregnancy and foetal development from maternal exposure to the test item from implantation to termination.

 

Mated female New Zealand White rabbits were randomised into 3 test groups and 1 vehicle control group, each containing 24 animals. The animals were treated once daily over Days 6 to 28 of gestation, where the day of mating was designated Day 0. Dose levels were 0, 10, 40 and 120 mg/kg bw/day (product as supplied), equivalent to 6.23, 24.92 and 74.76 mg/kg be/day (active ingredient) at a dose volume of 5 mL/kg body weight.

 

The animals were monitored during gestation for clinical signs of toxicity and for alterations in body weight and food consumption. Blood samples for clinical pathology were taken from all animals on Days 13 and 29 of gestation. The animals were killed on Day 29 of gestation and the status of each implantation was recorded. The viable foetuses were weighed and examined for soft tissue abnormalities, then processed and stained with Alizarin Red and examined for skeletal abnormalities, including the state of ossification. Gross and microscopic examinations were conducted on the liver of all animals.

 

At 120 mg/kg/day, a marked degree of maternal toxicity of Perform STi was indicated by marked body weight loss and marked food consumption reduction, observed in almost all animals in this group, and reduced/altered faecal output, observed in all animals. Clinical signs including laboured breathing, subdued behaviour and signs of aborting litters required the premature sacrifice of 6/24 animals in this group. Liver weights were increased, and necropsy findings included an increased incidence of pale livers and prominent lobulation. Histopathology findings included periportal hepatocyte vacuolation (intracytoplasmic vacuoles containing amorphous eosinophilic material), observed in all animals in this group, with centrilobular vacuolation (intracytoplasmic clear vacuoles), hepatocyte necrosis and periportal hepatocyte hypertrophy also observed in premature decedents. These liver findings were preceded by increases in the levels of Aspartate Aminotransferase, Alanine Aminotransferase, Lactate Deyhdrogenase and a reduction in Alkaline Phosphatase, compared with Control on Day 13 of gestation and an increase in Alanine Aminotransferase, Cholesterol, Triglycerides, Gamma-Glytamyltransferase, and reduction in Haemoglobin and Red Blood Cell counts on Day 29. These clinical pathology effects are consistent with changes that would be associated with above liver findings.

There was an increase in the number of dead implants at 120 mg/kd/day; these were mainly early embryonic deaths but there was also an increase in late deaths compared with Control. Mean foetal weights at this level were lower than Control, and there was a marked increase in the incidences of foetal abnormalities and variants. The principal findings were eye (eye/lens reduced, retinal folds, lenticular/corneal opacities) and hindlimb/digit (absent/fused/poorly defined bones) abnormalities, with many foetuses having both eye and hindlimb/digit findings; haemorrhages in the orbital socket (usually in foetuses with eye effects); foetuses with incisors not erupted; reduced state of skeletal ossification, probably associated with the small size of the foetuses; greater incidences of foetuses with bilateral caudal displacement of the pelvic girdle and of foetuses with supernumerary ribs: foetuses with pelvic girdle displacement generally had 13 complete ribs. It is not clear if the above foetal findings were directly related to treatment or secondary to the maternal effects. The premature sacrifice of 6/24 animals at this dose level resulted in mortality higher than the 10% approximate recommended in the test guideline.

At 40 mg/kg/day, there was an increased incidence of females with reduced faecal output. The only necropsy findings that appeared to be associated with treatment were pale livers in 4 females; histopathology indicated 6 females with periportal hepatocyte vacuolation, but to a lesser degree than for the females at 120 mg/kg/day.

At 10 mg/kg/day, two animals had pale liver but there were no histological changes and no effects on liver weights at this level; it is not clear if these liver findings were incidental or related to treatment with Perform STi.

The type and distribution of foetal visceral and skeletal abnormalities at 10 and 40 mg/kg/day were not considered to be associated with treatment.

In conclusion, no clear maternal no observed effect level (NOEL) could be established, although findings at 10 mg/kg/day (6.23 mg/kg/day as A/I) were confined to an equivocal finding of pale livers of two animals. The foetal NOEL for this study was considered to be 40 mg/kg/day (24.92 mg/kg/day as A/I). It is not clear whether the foetal findings at 120 mg/kg/day (74.76 mg/kg/day as A/I) were directly related to treatment or were secondary to the severe maternal toxicity evident at this dose level.