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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:
08 August 2014 to 11 November 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or methodological deficiencies, which do not affect the quality of the relevant results.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Appearance: clear, pale-yellowish liquid
- Storage condition of test material: ambient (21 - 29 °C)

Test animals

Species:
mouse
Strain:
Swiss
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: In-house bred animals
- Age at study initiation: 10 - 12 weeks
- Weight at study initiation (GD 0): 24.01 - 33.20 g
- Housing: during the pre-mating period a maximum of three animals were housed in a standard polypropylene cage (290 x 220 x 140 mm) with stainless steel mesh top grill. Clean sterilised paddy husk was provided as bedding material. During the mating period two mice (one male and one female) were housed together until confirmation of mating (GD 0) at which point the females were housed individually in polypropylene cages and males were housed with their former cage mates.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6 - 23.1 °C
- Humidity (%): 50 - 59 % (relative)
- Air changes (per hr): 12 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
peanut oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test material was administered by oral gavage in peanut oil at a dose volume of 5 mL/kg bw. Control animals received vehicle only at a dose volume of 5 mL/kg bw.
Fresh dosing solutions were prepared daily before administration.

VEHICLE
- Justification for use and choice of vehicle: Peanut oil was selected since it has been utilised in previous toxicology studies with related test materials and is a routinely used vehicle.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulation analysis was conducted to enable verification of dose concentration by analysing total tin content. Analyses were conducted during week 1 and week 2 of the dosing period. The dose samples were collected in duplicate (2 x 5 mL) for each dose formulation including vehicle control. One set of samples were analysed while the other set was stored and analysed later, if required.
Details on mating procedure:
- Impregnation procedure: Cohoused
- If cohoused:
- M/F ratio per cage: 1 male / 1 female
- Length of cohabitation: Until copulation was confirmed or for 2 weeks (maximum)
- Proof of pregnancy: Vaginal plug and/or vaginal smear, referred to as day 0 of pregnancy

Females not mated within 14 days of pairing with the first male were placed with a second proven male if presence of sperm in the vaginal smear and/or vaginal plug was not confirmed.
Duration of treatment / exposure:
Females received test material daily from gestation day 5 to gestation day 17 (inclusive).
Frequency of treatment:
Daily throughout treatment period.
Duration of test:
Dams were sacrificed on gestation day 18.
No. of animals per sex per dose:
25 pregnant females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were selected upon consideration of the following available data.
In a published report of an oral gavage developmental toxicity study in mice [Faqi, 2001] with a closely related dioctyltin material [dioctyltin isooctyl thioglycolate; DOT[IOTG]; DOTI], the test material utilised had approximately 80 % dioctyl and 20 % monooctyl components. Doses were 20, 30, and 45 mg/kg body weight [Phase I], and 67 and 100 mg/kg body weight [Phase II]. Dosing was done from gestation day 6 [GD6; day of vaginal coital plug = GD1] to gestation day 17 [GD17]. Caesarean sections were done on GD18.
Adjusted maternal weight gain [GD18 weight minus GD1 weight minus gravid uterine weight] at the 100 mg/kg dose was 56 % of the control. High variability in the maternal weight data, as evidenced by the high SD of the mean, obscured the statistical significance of this biologically relevant maternally toxic outcome. One maternal death occurred at this dose further supporting the fact that this is a maternally toxic dose. The maternal weight effect was accompanied by a statistically significant decrease in maternal thymus weight. Maternal liver weight, on both an absolute and relative basis, was also statistically significantly decreased at this dose.
Relative to the control mice, maternal thymus weight and liver weight trended downward at 67 mg/kg, suggesting that this dose is an upper bound in order to preclude an unacceptable level of maternal toxicity and generate a valid assessment of developmental toxicity in a new study.
The number of dams maintaining pregnancy in the treated groups up to the 45 mg/kg dose was not different from controls. The number of implantation sites, number of viable foetuses, and foetal weight were unaffected by treatment. There was a suggestion of an increase in the number of resorptions at 45 mg/kg, but this was a not statistically significant and the number of viable foetuses was comparable to controls at this dose.
The number of dams maintaining pregnancy in the control group for Phase II of the study was decreased relative to all other groups in the study. The number of dams maintaining pregnancy in the 67 and 100 mg/kg groups was not statistically different than the Phase II control. As noted above, the 67 mg/kg dose appears to be the upper bound dose for a developmental toxicity study because foetal weight at the 67 mg/kg dose is significantly less than controls, the number of resorptions is increased, and the number of resorptions per implantation is statistically significantly increased.
There is evidence of foetal toxicity, expressed as delayed ossification, at 45 mg/kg and higher which further supports 67 mg/kg as an upper bound for any further investigation of developmental toxicity.
In the study reported by Henninghausen, (1979) a single intramuscular dose of dioctyltin chloride [DOTC] at 60 mg/kg reduced thymus weight to 48 % of controls and thymocyte count to 64 % of controls. A single oral gavage dose of 300 mg/kg resulted in thymus weight at 65 % of controls and thymocyte count at 70 % of controls. The repeated-dose test paradigm for developmental toxicity studies makes it highly unlikely that pregnancy in mice could be sustained.
Therefore, the high dose chosen for this study is 60 mg/kg, to reflect a dioctyltin dose with minimal maternal and foetal toxicity as the upper bound. It is anticipated that this dose will meet the developmental toxicity test guideline criteria of producing some maternal toxicity without compromising the survival of the pregnant dam, the integrity of pregnancies to Day 18, or the survival of the developing foetuses.
The low dose chosen (15 g/kg) was selected to reflect the NOEL in the published study.

- Rationale for animal assignment: Each day the body weight of mated mice was recorded. The mated females were distributed to all groups based on their body weights so as to maintain comparable mean body weights across all groups.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: animals were observed twice daily for mortality and morbidity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: animals were observed once daily for clinical signs of toxicity.

BODY WEIGHT: Yes
- Time schedule for examinations: Dams were weighed on gestation days 0, 3, 5 and daily thereafter up to, and including, gestation day 18.

FOOD CONSUMPTION: Yes
- Time schedule: individual feed intake of mated females was recorded on gestation days 0 - 3, 3 - 5, 5 - 7, 7 - 9, 9 - 11, 11 - 14, 14 - 17, and 17 - 18.

WATER CONSUMPTION: No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 18
All the animals were sacrificed on gestation day 18 by carbon dioxide exposure and subjected to detailed gross pathology. At necropsy the thymus was excised, weighed and placed in 10 % formalin for possible future histopathological examination.
Ovaries and uterine content:
The ovaries and uterine content were 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
- Number of live and dead foetuses: Yes
Fetal examinations:
- External examinations: Yes
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
- Head examinations: Yes

Other examinations included:
- Sex, number and weight of live foetuses
- Crown rump length (measured after sacrifice)
Statistics:
Raw data was subjected to computer statistical processing. One-way ANOVA with Dunnett's post test was performed for data (body weight, food consumption, number of corpora lutea, uterus weight, number of implantations, number of live foetuses, body weight of live foetuses), using SPSS software. All analyses and comparisons were evaluated at the 95 % level of confidence (P<0.05).
Indices:
Corrected body weight (g) = (Gestation day 18 body weight – Gestation day 5 body weight) – Gravid uterus weight
Pre-implantation loss (%) = ((Number of corpora lutea – Number of implants)/Number of corpora lutea) x 100
Post-implantation loss (%) = ((Number of implants – Number of viable foetuses)/Number of Implants) x 100
Male/Female sex ratio = Number of live male foetuses/Number of live female foetuses
Male/female foetuses (%) = (Number of live male foetuses/Total number of foetuses) x 100 or (Number of live female foetuses/Total number of foetuses) x 100
Foetal incidence (%) = (Number of foetuses with a particular observation/Total number of foetuses in a group) x 100

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: Reduction in thymus weight at 30 mg/kg

Details on maternal toxic effects:
MATERNAL DATA
- Clinical Signs of Toxicity and Morbidity/Mortality
The animals did not reveal any clinical signs of toxicity and mortality at any of the tested doses throughout the experiment.
- Maternal Body Weight
There were no statistically significant differences in maternal body weights across the dose groups on any single gestation day. However, there was a clear dose-related pattern of reduced body weights beginning after GD6, the first day of dosing, and continuing for the duration of the study. The high dose was the most severely affected, though a dose-related decrement relative to the control body weights can be seen across all doses particularly from GD16 to GD18. At 30 mg/kg the maternal weight effect was marginal, but maternal body weight gain in the 60 mg/kg high dose group was 11.5 % [uncorrected] and 26.6 % [corrected] less than the vehicle control, a clear adverse effect. The corrected body weight gain in the low (15 mg/kg) and mid (30 mg/kg) dose groups were -16.7 % and - 17.7 % when compared to the controls.
- Feed Consumption
No treatment related differences in average feed consumption were observed at any dose.
- Gross Pathology [Maternal]
There was a treatment-related macroscopic finding of reduced maternal thymus weight.
The mean maternal thymus weight was statistically significantly reduced in the 30 mg/kg [mid] and 60 mg/kg [high] dose groups. The mean maternal thymus weight in the low dose mice was reduced relative to controls, but was not statistically significant. These observations are indicative of a treatment-related specific target organ toxicity resulting from exposure to the test material. No other gross pathological findings were noted in any dose group.

PREGNANCY DATA
A total number of 21 (84 %), 21 (84 %), 20 (80 %) and 20 (80 %) mated females were confirmed pregnant at the time of caesarean section for groups G1, G2, G3 and G4, respectively.

UTERINE OBSERVATIONS
There were no statistically significant differences in these gravid uterus weights, number of implantation sites, pre- and post-implantation loss or early or late resorptions across dose groups when compared to the vehicle control.

REPRODUCTION DATA
No treatment related effects were noted in mean gravid uterus weight, no. of corpora lutea, no. of implantations in all the groups, no. of early or late resorptions and percentage of post implantation loss.
There were no statistically significant differences in the sex ratio, mean litter size or the number of live foetuses per dam across dose groups when compared to the vehicle control.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Dose descriptor:
LOAEL
Effect level:
30 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
FOETAL DATA
- Foetal Weight
The mean foetal weights [combined sexes] were 1.35, 1.37, 1.30 and 1.31 grams for groups G1, G2, G3 and G4, respectively. Mean foetal weights were not statistically significantly different across the dose groups when compared to controls.

- External Examination
No external abnormalities were noted during gross examinations of foetuses at any dose.

- Visceral Examination
No treatment-related abnormalities were observed during visceral examinations of foetuses at any dose. The noted findings [pale coloured kidneys and dilated renal pelvis] are common findings for foetuses of this species and strain. The observations were not dose dependent, nor was the severity of the anomaly increased with dose. This result supports the conclusion that the findings are incidental and that the test material did not produce an adverse effect during foetal development of the soft tissues.

- Skeletal Examination
The noted anomalies (poorly ossified frontal, parietal and inter-parietal bones; ossification site at first lumbar vertebrae; supplementary ribs) are common findings for foetuses of this species and strain. These morphologic observations did not occur in a dose-dependent pattern, nor was the severity of the anomaly increased with dose. The findings were therefore considered to be incidental, and not indicative of a teratogenic effect.

- Crown-rump length
The average crown-rump lengths were 23.2, 24.0, 23.3 and 22.9 mm for groups, G1 through G4, respectively. There were no statistically significant differences in length across all dose groups when compared to the control.

Effect levels (fetuses)

Remarks on result:
not determinable due to adverse toxic effects at highest dose / concentration tested

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Dose Formulation Analysis

The results for chemical homogeneity and dose concentration verification are within the acceptance limits of ± 10 % of the nominal concentrations.

Table 1: Maternal Body Weights (g)

Dose Group (mg/kg)

Gestation Day

0

3

5

6

7

8

9

10

11

12

13

14

15

16

17

18

G1: 0

27.41

28.25

29.03

29.49

30.06

30.54

31.41

32.68

34.03

35.59

37.52

39.34

41.31

43.81

46.23

49.10

G2: 15

27.08

27.71

28.33

28.90

29.59

30.21

30.74

31.73

33.20

34.80

36.73

38.81

41.12

43.96

46.02

48.21

G3: 30

27.23

28.11

28.67

29.20

29.59

30.42

31.04

32.24

33.72

35.13

36.80

38.76

40.78

43.35

45.71

48.17

G4: 60

26.93

27.69

28.58

29.12

29.52

30.11

30.74

31.74

33.06

34.61

36.15

37.81

40.15

42.63

44.30

46.35

 

Table 2: Maternal Body Weight Gain

Dose Group (mg/kg)

GD 5-18 (g)

GD 5-18 (% of control)

GD 5-18 (% gain compared to control)

G1: 0

20.07

100.0

0.0

G2:15

19.18

95.6

-4.4

G3: 30

19.5

97.2

-2.8

G4: 60

17.77

88.5

-11.5

 

Table 3: Litter Data

Dose Group

No. of Corpora Lutea (mean)

Implantation Sites (mean)

Live Foetuses (mean)

No. of Early Resorptions (mean)

No. of Late Resorptions (mean)

Mean Pre-implantation Loss (%)

Mean Post-implantation Loss (%)

G1: 0

10.1

10.1

10.1

0.0

0.0

0.0

0.0

G2: 15

10.7

10.7

10.1

0.1

0.0

0.0

0.9

G3: 30

10.7

10.6

10.5

0.1

0.1

0.5

1.5

G4: 60

10.0

10.0

9.7

0.1

0.2

0.0

2.6

 

Group & Dose (mg/kg)

Mean Thymus weight (g)

G1 (0 mg/kg)

0.0400

G2 (15 mg/kg)

0.385

G3 (30 mng/kg)

0.0306*

G4 (60 mg/kg)

0.0260*

* p < 0.05

 

Table 4: Summary of Foetal Examinations

Parameters

Group (Dose mg/kg)

G1 (0 mg/kg)

G2 (15 mg/kg)

G3 (30 mg/kg)

G4 (60 mg/kg)

External Examinations

Malformations

No. of foetuses

0

0

0

0

%

0.0

0.0

0.0

0.0

Variations

No. of foetuses

0

0

0

0

%

0.0

0.0

0.0

0.0

Visceral Examinations

Malformations

No. of foetuses

0

0

0

0

%

0.0

0.0

0.0

0.0

Variations

No. of foetuses

4

5

3

6

%

4.0

4.8

3.0

6.5

Skeletal

Malformations

No. of foetuses

0

0

0

2

%

0.0

0.0

0.0

2.0

Variations

No. of foetuses

6

3

2

9

%

5.4

2.6

2.0

8.8

 

Applicant's summary and conclusion

Conclusions:
Under the conditions of the study the NOAEL for maternal toxicity was determined to be 15 mg/kg based on a biologically relevant depression in thymus size at the 30 mg/kg dose. However, no treatment-related effects were noted in any of the developmental toxicity parameters investigated; therefore the NOAEL for developmental toxicity was concluded to be 60 mg/kg, the highest dose tested.
Executive summary:

The purpose of this study was to assess the effects of prenatal exposure of pregnant female Swiss mice and their developing foetuses to the test material when administered by oral gavage in a peanut oil vehicle at 5 mL/kg to the mated females from gestation day [GD] 5 to 17. In life examinations included checks for mortality and clinical signs of toxicity, body weight measurements and an evaluation of food consumption. All the animals were sacrificed on gestation day 18 by carbon dioxide exposure and subjected to detailed gross pathology; the gross pathology included a determination of thymus size. The gravid uterus was collected by hysterectomy and foetuses were removed by caesarean section. Foetuses were subjected to external, soft tissue and skeletal examination.

No deaths were observed during the experimental period and there were no clinical signs recorded which were indicative of overt toxicity. There were no statistically significant differences in maternal body weights across the dose groups on any single gestation day. However, there was a clear dose-related pattern of reduced body weights beginning after GD6, the first day of dosing, and continuing for the duration of the study. The high dose was the most severely affected and a dose-related decrement relative to the control body weights can be seen across all doses particularly from GD16 to GD18. At 30 mg/kg the maternal weight effect was marginal, but maternal body weight gain in the 60 mg/kg high dose group was 11.5 [uncorrected] and 26.6 % [corrected] less than the vehicle control, a clear adverse effect. There was a treatment-related macroscopic finding of reduced maternal thymus weight.

The mean maternal thymus weight was statistically significantly reduced in the 30 mg/kg [mid] and 60 mg/kg [high] dose groups. The mean maternal thymus weight in the low dose mice was reduced relative to controls, but was not a statistically significant difference. No other gross pathological findings were noted in any dose group.

No treatment related effects were noted in mean gravid uterus weight, no. of corpora lutea, no. of implantations in all the groups, no. of early or late resorptions and percentage of post implantation loss.

No external abnormalities were noted during gross examinations of foetuses at any dose and no treatment-related abnormalities were observed during visceral examinations of foetuses at any dose.

Therefore, under the conditions of the study the NOAEL for maternal toxicity was determined to be 15 mg/kg based on a biologically relevant depression in thymus size at the 30 mg/kg dose. However, no treatment-related effects were noted in any of the developmental toxicity parameters investigated; therefore the NOAEL for developmental toxicity was concluded to be 60 mg/kg, the highest dose tested.