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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:
10 June 2014 to 05 September 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical state: Clear, beige liquid
- Storage conditions of test material: Room temperature, protected from light

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Hra:(NZW) SPF
- Age at study initiation: 5 to 5.5 months
- Weight at study initiation: 2.56 to 3.63 kg
- Housing: Individually housed in suspended, stainless steel, slatted floor cages. Animal enrichment was provided.
- Diet: Limited to 50 g/animal/day on arrival (GD 0) and increased on second day of acclimation to approximately 170 g/animal/day for the remainder of the study. Enrichment food was offered on occasion.
- Water: tap water was available ad libitum via an automatic watering system
- Acclimation period: 1 day

ENVIRONMENTAL CONDITIONS
- Temperature: 61-72 °F
- Humidity: 30 to 70 %
- Photoperiod: 12 hour light/dark cycle

IN-LIFE DATES: From: 10 June 2014 To: 11 July 2014

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test material in vehicle was prepared weekly and stored refrigerated at 2 to 8 °C (protected from light).

VEHICLE
- Justification for use and choice of vehicle (if other than water): The vehicle was selected based on information from a pilot study.
- Concentration in vehicle: 60, 120 and 240 mg/mL
- Amount of vehicle (if gavage): 0.5 mg/mL
- Lot/batch no.: 2CK0155
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dosing samples were evaluated for homogeneity and concentration. 1 mL samples were collected for homogeneity during week 1 from the top, middle and bottom of the 30 and 120 mg/kg formulations. Samples from all concentrations were taken during weeks 1 and 4 from the middle stratum for analysis of concentration. The dose formulations were analysed using HPLC/UV.

HPLC/UV CONDITIONS
Column: Agilent Poroshell 120 SB-C8 column, 3.0 x 50 mm
Gradient flow: 0.05 % trifluoroacetic acid in water (mobile phase A) and acetonitrile (mobile phase B) at a flow rate of 1.0 mL/minute.
Wavelength: 230 nm
Derivatisation: Prior to analysis, duplicate samples were derivatised in hexane and phenylmagnesium bromide then diluted with isopropyl alcohol to within the range of the calibration curve. Vehicle samples were also derivitised and then diluted using a dilution factor of 100.

RESULTS
- Homogeneity:
Analysis of the low- and high-dose formulations (60 and 240 mg/mL, respectively) used for dosing in the first week of study confirmed they were homogeneous as prepared, meeting the laboratory’s acceptance criteria of 100 ± 15 % of nominal, and percent relative standard deviation (RSD) ≤10.
- Concentration:
Mean concentrations of formulations used for dosing in Weeks 1 and 4 of study ranged between 96.7 and 104.7 % of nominal with percent RSDs ranging between 0.523 and 2.649, confirming that animals were receiving the appropriate dose levels when the formulation was administered at 0.5 mL/kg. No test material was detected in the control samples.
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
Duration of treatment / exposure:
Gestation day 1 to 28
Frequency of treatment:
Once daily
Duration of test:
29 days
Doses / concentrationsopen allclose all
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
60 mg/kg bw/day (nominal)
Dose / conc.:
120 mg/kg bw/day (nominal)
No. of animals per sex per dose:
25 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were selected based on information from a pilot study.
In the pilot study, dose levels of 30, 65, 140 and 300 mg/kg/day were evaluated. However, due to reduced food consumption levels in several groups by GD 5, the dose levels were adjusted to 30 (unchanged), 50, 100 and 200 mg/kg/day. On GD 8, the 200/300 mg/kg/day dose group was terminated due to continued low food consumption, weight loss and being in a state of in extremis. In all remaining groups there was variability in food consumption as expected with the corn oil vehicle administered at 1 mL/kg/day. One animal died in the 100 mg/kg/day dose group (cause undetermined from necropsy observations) and one animal aborted in each of the 30 and 100 mg/kg/day dose groups. The aborted pregnancies were preceded by lengthy periods of inappetence. The maternal toxicity observed in the pilot study was considered largely attributed to the 1 mL/kg corn oil vehicle treatment and in the main study this volume was reduced to 0.5 mL/kg. No effect of treatment to 100 mg/kg/day was observed on uterine implantation data or foetal external examinations. There was about a 10 % reduction in mean foetal body weight at the 100 mg/kg/day dose level relative to controls.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- Cage side observations: Morbidity, mortality, injury and availability of food and water.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily
- Examinations: Animals were removed from the cage and given a detailed clinical examination. On occasion, clinical observations were recorded at unscheduled intervals. The observations included, but were not limited to, evaluation of the skin, fur, eyes, ears, nose, oral cavity, thorax, abdomen, external genitalia, limbs and feet, as well as evaluation of respiration.

BODY WEIGHT: Yes
- Time schedule for examinations: Gestation days 0, 1, 3, 6, 9, 12, 15, 18, 21, 24, 27 and 29. Body weight change was calculated for gestation days 0-1, 1-3, 3-6, 6-9, 9-12, 12-15, 15-18, 18-21, 21-24, 24-27, 27-29 and 1-29. Adjusted body weight (minus gravid uterus) and adjusted bodyweight change were also calculated.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: Food consumption recorded daily and reported on the corresponding body weight days.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29. Each surviving female was euthanised by injection with euthanasia solution, followed by exsanguination from the femoral vessels and immediately subjected to a laparohysterectomy.
- Organs examined: Does were subjected to a complete necropsy. Special emphasis was placed on structural abnormalities or pathologic changes that may have influenced pregnancy.
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: all per litter
- Head examinations: Yes: half per litter
Statistics:
All parental in-life data, gravid uterine weights, corpora lutea/doe, total implantations/doe, litter size/doe, viable foetuses/doe, total number of resorptions/doe, number of early resorptions/doe and number of late resorptions/doe were assessed using group pair-wise comparisons. Levene's test was used to assess homogeneity of group variances. If Levene's test was not significant (p≥0.01), a pooled estimate of variance (Mean Square Error) was computed from a one-way analysis of variance (ANOVA) and utilised by a Dunnett's comparison of each treatment group with the control. If the Levene's test was significant (p<0.01), then Welch's t-test was used with a Bonferroni correction for comparisons with the control group. All endpoints were analysed using two-tailed tests.
Foetal sex ratio (% males/litter), % preimplantation loss and % postimplantation loss were analysed using Arcsin-Square-Root Transformation. Percent values were transformed using the arcsin of the square root and analysed in accordance with the group pair-wise comparisons.
Pregnancy index, malformations and variations were all evaluated using Fisher’s exact test. An overall test for association between response and treatment was performed. If this test was significant (p<0.05) and there were more than two groups, then each treatment group was compared to the control group. All endpoints were analysed sing two-tailed tests. Overall testing and follow-up pair-wise testing was conducted for the control and all treatment groups that had sufficient sample size (n≥3).
Mean foetal bodyweights were analysed by covariate analysis by comparing treatment groups for each endpoint (mean of foetal bodyweights per female). The covariate was litter size. Each treatment satisfying the sample size assumption was compared to the control using Dunnett's test under the analysis of covariance model. Endpoints were analysed using two tailed tests.
The number of non-viable foetuses/doe was analysed by descriptive statistics.
Indices:
- Viable foetuses
- Postimplantation loss = ((No. implantations - No. viable foetuses) / No. implantations) x 100
- Preimplantation loss = ((No. corpora lutea - No. implantations) / No. corpora lutea) x 100
- Pregnancy index = (No. pregnant females - No. females with evidence of mating) x 100
- Foetal sex ratio
Historical control data:
A large database of historical control data for the rabbit was available for comparison.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- In the 30 and 60 mg/kg bw/day dose groups, no clinical effects were noted at post-dose examination. There was a slight decrease in defecation in the 30 and 60 mg/kg bw/day dose groups. This generally correlated with late gestation inappetence which is common in rabbits and not considered to be test material related.
- In the 120 mg/kg bw/day group, findings attributed to the test material included 2/25 animals with decreased activity, abnormal shaped faeces in 4/25 animals, discoloured urine (orange) in 13/25 animals and thin appearance in 11/25 animals. There was also a decrease in defecation in these animals (19/25 animals) appearing from around gestation day 4 and lasting throughout gestation. The decreased defecation in this group correlated with low food consumption and was considered test material related. Brown/red material was found in the sub-cage pan of 7 animals in the 120 mg/kg bw/day. This was related to failed pregnancies in 5 of these animals (two aborted pregnancies, one early delivery and two litters with resorbing foetuses). No further treatment related clinical findings were observed in the 120 mg/kg bw/day group.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
- At all dose levels tested, there were no effects observed on maternal survival. For all animals that died or were euthanised, the cause of death was attributed to dosing injuries, and were not considered to be test material related.
- A single animal in the 30 mg/kg bw/day group died on gestation day 29 prior to euthanasia. The death of this animal was attributed to a dosing event.
- An animal in the 60 mg/kg bw/day group was noted to have difficulty breathing on gestation day 6 and was euthanised in extremis; this was also attributed to a dosing event and was not considered test material related.
- An animal in the 120 mg/kg bw/day group was found dead on gestation day 24. Apart from being noted as thin on gestation day 3, the animal was found to be clinically unremarkable thereafter. Findings at necropsy indicated that this was also the result of a dosing event.
- All other animals survived until scheduled sacrifice.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- No adverse effects on bodyweight and bodyweight change were noted at 30 and 60 mg/kg bw/day.
- In the 120 mg/kg bw/day group, mean bodyweights from gestation day 6 to 29 were 5 to 7 % lower than controls and were found to be statistically significant. Statistically significant differences in bodyweight change were noted in this group when compared to controls over gestation days 3 to 6 and over the entire gestation period. These observed differences correlated with reduced food consumption and were attributed to test material administration.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- No adverse effects were observed at 30 and 60 mg/kg bw/day on food consumption. Throughout the entirety of gestation, food consumption in the 30 mg/kg bw/day group was found to be comparable to controls and in the 60 mg/kg bw/day group with the exception of gestation days 27 to 29. As food consumption in the 60 mg/kg bw/day group was comparable to the control group for gestation as a whole and in the absence of an effect on food consumption earlier in gestation, this was not considered to be a toxicological response to test material administration.
- In the 120 mg/kg bw/day group, the mean food consumption was 17 to 54 % lower than control values for the majority of the intervals recorded. Most of these differences were found to be statistically significant, and overall throughout the entirety of gestation food consumption was found to be 41 % lower than controls in this group. The lower food consumption in this group correlated with effects noted on bodyweight and bodyweight change and was determined to be test material related.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
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:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
- At necropsy, no effect of treatment was observed at any dose level tested. The findings observed were found to be of low incidence and not related to test material administration.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
effects observed, treatment-related
Description (incidence and severity):
- One animal in the 60 mg/kg bw/day group and two animals in the 120 mg/kg bw/day group aborted.
Abortion can occasionally occur in rabbits, and a single abortion at the mid dose is within normal variability; this is also supported by the Historical control data included in the report. However, body weight and food consumption data for this specific mid dose female (No. 257) which aborted indicate signs of toxicity (reduced food consumption and body weight loss).
The two females of the high dose group that aborted (No. 287 and No. 281) also showed significant deficits of food consumption and weight gain, even relative to the group mean values for the top dose group.
Consistent with the results of the dose ranging study, the principal maternal effect was a reduction in food consumption; a clear treatment-related effect is evident on both food consumption and group mean body weight at the top dose level and, to a lesser extent, for the single female of the mid dose group which aborted. Maternal well-being was insufficient to maintain pregnancy.
In summary, all abortions were preceded by signs of toxicity (clinical signs, low weight gain or weight loss, and extended periods of inappetence) and were considered to be related to treatment with the test material, but secondary to maternal toxicity and not due to a direct effect of the test substance.
Exclusive of these comprised pregnancies, there were 25, 23, 23, and 18 GD 29 pregnancies with viable fetuses for evaluation of developmental toxicity in the control, 30, 60, and 120 mg/kg/day dose groups, respectively.
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
- There were two females in the 120 mg/kg bw/day group with only resorbing foetuses in utero (100 % post implantation loss).
In the 120 mg/kg bw/day group, an increase in post-implantation loss was recorded. The mean post-implantation loss was higher at 15.32 % compared to 2.09 % in controls, though this was not found to be statistically significant. When these two females with only resorbing fetuses are excluded from the calculation of the group mean, there is no clear increase.
The increase in post-implantation loss was considered to be secondary to maternal toxicity evidenced through significant deficits of food consumption and weight gain in affected animals.
- At 30 and 60 mg/kg bw/day, no effect was noted on uterine implantation.
Total litter losses by resorption:
effects observed, treatment-related
Description (incidence and severity):
- There were two females in the 120 mg/kg bw/day group with only resorbing foetuses in utero (100 % post implantation loss).
In the 120 mg/kg bw/day group, the mean number of early resorptions and total resorptions were statistically higher, 1.3 and 1.4, respectively, when compared to control values of 0.1 and 0.2, respectively. This increase was considered to be secondary to maternal toxicity evidenced through significant deficits of food consumption and weight gain in affected animals.
- At 30 and 60 mg/kg bw/day, no effect was noted on uterine implantation.
Early or late resorptions:
effects observed, treatment-related
Description (incidence and severity):
- At 30 and 60 mg/kg bw/day, no effect was noted on uterine implantation. In the 120 mg/kg bw/day group, the mean number of early resorptions and total resorptions were statistically higher, 1.3 and 1.4, respectively, when compared to control values of 0.1 and 0.2, respectively. This increase was considered to be secondary to maternal toxicity evidenced through significant deficits of food consumption and weight gain in affected animals.
Dead fetuses:
no effects observed
Changes in pregnancy duration:
effects observed, treatment-related
Description (incidence and severity):
One animal in the 120 mg/kg bw/day group delivered early. This animal showed marked toxicity in this study. The litter of 7 kits appears otherwise normal from the report and accounted for all implantation sites; no effect on development can be inferred. Early delivery, even if plausibly treatment-related, however is insufficient evidence for classification in this study since it is confined to a single animal.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Pregnancy indices were 100, 96, 100 and 96 % in the control, 30, 60 and 120 mg/kg bw/day groups, respectively.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Mean gravid uterine weights and adjusted weight change in the low and mid dose groups were comparable to the control group. In the 120 mg/kg bw/day group, the mean adjusted weight change over the gestation period was statistically significant as an overall loss of 0.137 kg was observed compared to a 0.039 kg weight gain in controls. The mean gravid uterine weight and adjusted bodyweight in this group were comparable to mean control values.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOEL
Effect level:
30 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
number of abortions
pre and post implantation loss
total litter losses by resorption

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
- No effect of the test material was observed on foetal bodyweight with administration of the test material at 30 and 60 mg/kg bw/day to the mothers.
- In the 120 mg/kg/day dose group, mean foetal weights, distinguished by sex and for the combined sexes were 11 to 13 % lower than mean control values. These however mirrored maternal weight change, and can be attributed to the maternal weight effect.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No effect was observed on foetal sex ratio with the administration of the test material at any dose level. These mean ratios ranged from 49.9% to 53.4% in the treated groups and were comparable to the 51.2% in controls.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
No effects were observed in litter size, at any dose level. Litter weight was not determined.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
- No effect of treatment was observed at all dose levels evaluated with respect to external examinations.
- The external malformations observed in the mid and high dose group foetuses were dissimilar, occurred at low incidence (single foetus affected) or with similar frequency as the controls. These were therefore considered to be unrelated to administration of the test material.
- No external malformations were observed in the 30 mg/kg bw/day group.
- No external developmental variations were observed among foetuses in the control or treated groups.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
- No effect of treatment was observed in the skeletal examinations at all dose levels tested.
In the 60 mg/kg bw/day group, the litter incidence of unossified sternebrae, a common developmental variation, was 73.9 %; this was statistically different from the 28 % observed in controls, and just outside the maximum incidence commonly observed in the historical control data. With the absence of a similar increase in incidence of this variation in the 120 mg/kg bw/day group (50%), this was considered to be an incidental occurrence and not test material related.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
- No effect of treatment at the dose levels evaluated was observed from the foetal visceral examinations.
- The few visceral malformations and developmental variations observed in the treated groups occurred at low incidence or with similar frequency as in controls and were considered unrelated to the test material.
Other effects:
not examined

Effect levels (fetuses)

Key result
Dose descriptor:
NOEL
Effect level:
60 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reduced foetal weight

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
120 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes

Any other information on results incl. tables

Results of the study indicate an increase in adverse pregnancy outcomes (secondary to maternal toxicity), an increase in post-implantation loss, increases in total and early resorptions, and lower foetal bodyweights at the top dose level. At all dose levels, the number of viable litters at term was adequate for evaluating any possible developmental effects; no teratogenic effects were noted.

A summary of pregnancy outcomes is presented here below.

Key pregnancy outcomes with individual animal identifiers

 

Dose, mg/kg bw/day

0

30

60

120

Number of females

25

25

25

25

Non-pregnant

0

1

0

1

Died or euthanized

0

1 (247)

1 (252)

1 (282)

Abortion

0

0

1 (257)

2 (278, 281)

Early delivery

0

0

0

1 (288)

Litters entirely resorbed in utero

0

0

0

2 (280, 292)

Viable litters at term

25

24

23

18

Individual animal ID numbers of affected rabbits given in parentheses

 

For all animals that died or were euthanized, the cause of death was attributed to dosing injuries, unrelated to test material administration.

Abortion can occasionally occur in rabbits, and a single abortion at the mid dose is within normal variability; this is also supported by the Historical control data included in the report.

Historical control developmental toxicity data New Zealand White rabbit (test facility 08.01.2009 to 08.01.2014)

 

Number of studies

32

Number of females

701

Number pregnant

650

Number not pregnant

51

Number that died or pregnant euthanised moribund

4

Number of abortions

2

Number of early deliveries

3

Number with all resorptions

2

Number pregnant by stain

0

Number of females with viable fetuses day 29 gestation

639

 

Overall, the mid dose mothers did not show any signs of toxicity, and no significant difference from the control group, particularly for effects on food consumption and/or body weight were recorded. Body weight and food consumption data for the mid dose female (No. 257) which aborted however indicate severe effects, possibly related to treatment.

A comparison of rabbit food consumption and weight gain in all unsuccessful pregnancies in the study is presented in the table below.


Comparison of rabbit food consumption and weight gain in unsuccessful pregnancies (Schroeder 2014a)

 

 

Study Period (days)

 

 

0

1

3

6

9

12

15

18

21

24

27

29

Control group mean

BW

3.13

3.19

3.25

3. 31

3.36

3.40

3.50

3.52

3.55

3.60

3.63

3.65

BW gain

 

60

60

60

50

40

100

20

30

50

30

20

FC

 

52.04

150.76

156.20

149.92

147.13

132.65

142.91

142.44

111.73

93.23

88.22

Mid dose group mean

BW

3.11

3.15

3.21

3.28

3.33

3.39

3.48

3.52

3.54

3.58

3.59

3.60

BW gain

 

40

60

80

30

50

100

30

30

40

20

-10

FC

 

53.04

143.04

147.16

137.32

140.54

122.61

136.44

135.56

110.54

80.69

50.54*

High dose group mean

BW

3.13

3.15

3.17

3.15*

3.16**

3.18**

3.26**

3.26**

3.31**

3.36**

3.40*

3.42*

BW gain

 

30

20

-20**

10

10

80

0

60

50

30

-10

FC

 

49.75

109.29**

90.65**

71.67**

67.02**

63.58**

70.64**

88.00**

91.40

77.41

59.00

Individual values

257 – aborted (mid dose)

BW

3.25

3.28

3.29

3.38

3.32

3.38

3.34

3.31

3.23

3.22

3.24

NDC

BW gain

 

30

10

90

-60

60

-40

-30

-80

-10

20

NDC

FC

 

55.0

125.5

140.7

77.0

84.7

10.0

3.7

11.7

25.3

34.0

NDC

278 – aborted

BW

3.47

3.58

3.51

3.38

3.27

3.26

3.28

3.34

3.33

3.25

NDC

 

BW gain

 

110

-70

-130

-110

-10

20

60

-10

-80

NDC

 

FC

 

50.0

134.0

40.7

0.7

6.0

0.7

0.3

0.3

0.7

0.0

NDC

280 – all resorbed

BW

3.24

3.24

3.27

3.17

3.14

2.98

2.97

2.88

2.76

2.99

3.11

3.08

BW gain

 

0

30

-100

-30

-160

-10

-90

-120

230

120

-30

FC

 

36.0

114.0

60.7

0.3

5.3

1.0

2.0

2.3

127.0

155.0

97.5

281 - aborted

BW

3.31

3.39

3.33

3.21

3.15

3.11

3.15

3.00

3.25

NDC

 

 

BW gain

 

80

-60

-120

-60

-40

40

-150

250

NDC

 

 

FC

 

47.0

35.0

18.7

2.5

3.5

0.0

0.0

33.7

0.0

NDC

 

288 – premature delivery

BW

2.58

2.65

2.63

2.73

2.77

2.74

2.79

2.73

2.74

2.74

2.83

NDC

BW gain

 

70

-20

100

40

-30

50

-60

10

0

90

NDC

FC

 

59.0

103.5

104.3

84.0

71.3

21.3

2.7

1.7

1.3

4.0

NDC

292 – all resorbed

BW

3.16

3.10

3.17

3.15

3.06

2.96

3.18

3.35

3.35

3.41

3.50

3.52

BW gain

 

-60

70

-20

-90

-100

220

170

0

60

90

20

FC

 

30.0

105.5

66.7

0.0

16.0

100.0

107.0

108.3

123.0

169.3

152.5

 

BW: bodyweight (kg); BW gain: body weight gain (g) (presented in kg in the report); FC: food consumption (g/animal/day during the period between bodyweight measurement). Values in bold show clear deficits from normal.

NDC: no data collected. Level of statistical significance: * p >0.05, ** p> 0.01 in comparison with control.


The above data clearly demonstrate that all the affected animals showed significant deficits of food consumption and weight gain, even relative to the group mean values for the top dose group. Consistent with the results of the dose ranging study, the principal maternal effect was a reduction in food consumption; a clear treatment-related effect is evident on both food consumption and group mean body weight at the top dose level and, to a lesser extent, for the single female of the mid dose group which aborted. Maternal well-being was insufficient to maintain pregnancy.

As mentioned above, developmental effects were all considered secondary to maternal toxicity and consisted in an increase in post-implantation loss, statistically higher mean number of early and total resorptions and increased, although not statistically significant, mean post-implantation loss at 120 mg/kg/day. All other uterine implantation parameters (corpora lutea, implantation sites, preimplantation loss, viable fetuses, nonviable fetus, and litter size) were comparable to mean control values.

In addition, mean fetal weights, distinguished by sex and for the combined sexes were 11% to 13% lower than mean control values at 120 mg/kg/day.

Endpoint

0

30

60

120

Number of females on study

25

25

25

25

Non-pregnant

0

1

0

1

Number pregnant

25

24

25

24

Pregnancy index percentage

100.0

96.0

100.0

96.0

Number died/ euthanised moribund

0

1

1

1

Number of abortions

0

0

1

2

Number of early deliveries

0

0

0

1

Number of females with all resorptions

0

0

0

2

Number of females with viable fetuses (day 29 gestation)

25

23

23

18

Corpora lutea (mean per animal)

9.5

10.0

9.9

8.8

Implantation sites (mean per animal)

8.6

8.9

9.1

9.1

Preimplantation loss (mean per animal), %

9.04

11.31

7.63

5.64

Viable fetuses (mean per animal)

8.4

8.7

8.9

7.8

Fetal sex ratio (mean % males per animal)

51.2

53.4

51.8

49.9

Postimplantation loss (mean per animal), %

2.09

4.61

3.06

15.32

Nonviable fetuses (mean per animal)

0.0

0.0

0.0

0.0

Litter size (mean per animal)

8.4

8.7

8.9

8.6

Resoprtions (early + late) (mean per animal)

0.2

0.3

0.3

1.4*

Early resorptions (mean per animal)

0.1

0.1

0.2

1.3*

Late resorptions (mean per animal)

0.1

0.1

0.0

0.1

 * p >0.05

The “increased post-implantation loss” was attributable to two top dose litters showing complete resorption, in turn a consequence of marked maternal toxicity. When these two values are excluded from the calculation of the group mean, there is no clear increase.

Dose, mg/kg bw/day

0

30

60

120

Post implantation loss, % (report p64)

2.09

4.61

3.06

15.32

Excluding 2 litters with all resorptions

 

 

 

6.6

 

The “lower foetal weights” were significant at the high dose only. These however mirrored maternal weight change, and can be attributed to the maternal weight effect.

Dose, mg/kg bw/day

0

30

60

120

Final maternal bodyweight, mean (kg)

3.6

3.6

3.6

3.4**

Maternal adjusted weight gain (g)

40

30

10

-130*

Foetal bodyweight, mean (g)

40.0

40.0

37.9

35.6**

Level of statistical significance: * p >0.05, ** p > 0.01 in comparison with control

 

There were no teratogenic effects, and no dose-related increase in the number of variants either on a fetal or litter basis, at external, visceral and skeletal examinations.

Summary of external malformations and developmental variations

Observation

0

30

60

120

Number of litters evaluated

25

24

23

18

Number of fetuses evaluated

211

206

204

155

Total malformations - number of litters (%)

1 (4.0)

0 (0.0)

2 (8.7)

1 (5.6)

Total malformations - number of fetuses (%)

1 (0.5)

0 (0.0)

2 (1.0)

1 (0.6)

Total variations - number of litters (%)

0 (0.0)

0 (0.0)

0 (0.0)

0 (0.0)

Total variations - number of fetuses (%)

0 (0.0)

0 (0.0)

0 (0.0)

0 (0.0)

 

Summary of visceral malformations and developmental variations

Observation

0

30

60

120

Number of litters evaluated

25

24

23

18

Number of fetuses evaluated

211

206

204

155

Total malformations - number of litters (%)

2 (8.0)

1 (4.2)

1 (4.3)

1 (5.6)

Total malformations - number of fetuses (%)

2 (0.9)

1 (0.5)

1 (0.5)

1 (0.6)

Total variations - number of litters (%)

10 (40.0)

3 (12.5)

6 (26.1)

5 (27.8)

Total variations - number of fetuses (%)

14 (6.6)

5 (2.4)

8 (3.9)

9 (5.8)

 

Summary of skeletal malformations and developmental variations

Observation

0

30

60

120

Number of litters evaluated

25

24

23

18

Number of fetuses evaluated

211

206

204

155

Total malformations - number of litters (%)

10 (40.0)

6 (25.0)

2 (8.7)

3 (16.7)

Total malformations - number of fetuses (%)

12 (5.7)

8 (3.9)

2 (1.0)

3 (1.9)

Total variations - number of litters (%)

23 (92.0)

23 (95.8)

23 (100.0)

15 (83.3)

Total variations - number of fetuses (%)

101 (47.9)

85 (41.3)

99 (48.5)

59 (38.1)

 

The maternal effects seen in the affected rabbits are typical of the substance-related toxicity evident in this study. The particular does showing individual adverse pregnancy outcomes are shown to be particularly severely affected, even within the group. Further, the nature of pregnancy loss – affecting the entire litter, not individual pups – is suggestive of an effect on the mother, not on the foetus. It can therefore be concluded that the test material has no direct effect on the developing organism.

Applicant's summary and conclusion

Conclusions:
Under the conditions of the test, the No-Observed-Effect Level (NOEL = no difference from control) for maternal toxicity was determined to be 30 mg/kg bw/day based on clinical findings, lower gestation bodyweights, lower bodyweight gain and lower food consumption seen at 120 mg/kg bw/day and aborted pregnancies at 60 and 120 mg/kg bw/day (secondary effects to maternal toxicity). The single abortion occurring at 60 mg/kg/day is within HCD, although it is noted that the affected female showed severe effects on food consumption and reduced body weight gain or even body weight losses.
The NOEL for developmental toxicity was determined to be 60 mg/kg bw/day based on increases in post-implantation loss, increases in total and early resorption sites and lower foetal body weights at 120 mg/kg bw/day however, these effects were all considered to be secondary to maternal toxicity (low weight gain, prolonged inappetance and clinical signs).
No teratogenic effects were observed.
In conclusion, all developmental effects noted in this study are secondary and directly attributable to maternal toxicity.
Executive summary:

The developmental toxicity potential of the test material was investigated in the rabbit in a study performed in accordance with the standardised guidelines OECD 414 and US EPA OPPTS 870.3700 under GLP conditions.

The test material was administered to timed-mated New Zealand White rabbits via oral gavage in corn oil from gestation day 1 to gestation day 28 at doses of 0, 30, 60 and 120 mg/kg bw/day. Doses were selected based on a range-finding study, in which the dose-limiting toxicity was maternal inappetance; a dose group starting at 300 mg/kg bw/day was reduced to 200 mg/kg bw/day then terminated within 8 days. In the range-finding study, dosing at 200 mg/kg bw/day was therefore clearly not tolerated on the basis of maternal toxicity.

In the main study, observations of the animals included clinical signs, body weights and food consumption. Animals were sacrificed on gestation day 29. The does underwent ovarian and uterine examinations and necropsy. The foetuses were examined for external, skeletal and visceral malformations and variations.

No test material-related mortality was observed.

At the top dose, fewer does successfully completed pregnancy compared to controls; the number of viable litters at term (18) was sufficient to evaluate any possible developmental effects. In this group, one female was non-pregnant, and one female died with cause of death attributed to a dosing injury.  Neither of these can be attributed to the test material.

Consistent with the results of the dose ranging study, the principal maternal effect was a reduction in food consumption, and a clear treatment-related effect is evident on both food consumption and group mean bodyweight at the top dose level.  Individual values of animals being particularly affected within a dose group show a “clear deficit” when there is any weight loss from the previously recorded bodyweight; or a food consumption value less than half of the mean value of the top dose group.

Three animals aborted (one at 60 mg/kg bw/day and two at the top dose), and one (at the top dose) delivered prematurely.  The abortions are attributed to maternal toxicity (low weight gain, prolonged inappetance and clinical signs).  The affected animals showed significant deficits of food consumption and weight gain, even relative to the group mean values for the top dose group. These failures of pregnancy can be directly attributed to the maternal effects; maternal well-being was insufficient to maintain pregnancy.

One animal of the top dose delivered early.  The litter of 7 kits appears otherwise normal and hence no effect on development can be inferred.

Two animals (both in the top dose group) showed total litter resorptions in-utero.  These were reported as “early” resorptions.  Once again, each of these animals showed prolonged periods of markedly deficient food consumption and weight gain.  

Increased post-implantation loss was attributable to the two litters showing complete resorption, in turn a consequence of marked maternal toxicity.  When these two values are excluded from the calculation of the group mean, there is no clear increase.

The reported increases in total and early resorptions are again attributable to these two litters, both of which were reported as early resorptions.  A post-implantation loss rate of 4.6% is reported at 30 mg/kg bw/day and considered the no-effect level (i.e., no differenec from controls); the high dose rate at 6.6% is not considered biologically different.  

Lower foetal weights were significant at the high dose only. These however mirrored maternal weight change, and can be attributed to the maternal weight effect.

Under the conditions of the test, the No-Observed-Effect Level (NOEL) for maternal toxicity was determined to be 30 mg/kg bw/day based on clinical findings, lower gestation bodyweights, lower bodyweight gain and lower food consumption seen at 120 mg/kg bw/day and aborted pregnancies at 60 and 120 mg/kg bw/day (secondary effects to maternal toxicity).

The NOEL for developmental toxicity was determined to be 60 mg/kg bw/day based on increases in post-implantation loss, increases in total and early resorption sites and lower foetal body weights at 120 mg/kg bw/day however, these effects were all considered to be secondary to maternal toxicity (low weight gain, prolonged inappetance and clinical signs).

In conclusion, all developmental effects noted in this study are secondary and directly attributable to maternal toxicity.

At all dose levels tested, the test material was found not to be teratogenic in the rabbit.