Bis(neodecanoyloxy)dioctylstannane

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because a pre-natal developmental toxicity study is available

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to other study

Reference

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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

mouse

Strain:

Swiss

Details on test animals or test system 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

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.

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

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.

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

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.

Remarks on result:

not determinable due to adverse toxic effects at highest dose / concentration tested

Abnormalities:

not specified

Developmental effects observed:

not specified

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

 

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.

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 July 2014 to 12 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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

-TEST ANIMALS
- Age at study initiation: 5 - 6 months
- Weight at study initiation (GD 0): 2.50324 - 3.74756 kg
- Housing: during the pre-mating period animals were housed individually in stainless steel wire mesh cages (24 x 18 x 18 inches). During the mating period two rabbits (one male and one female) were housed together until visual confirmation of mating (GD 0). The mated rabbits were housed in their original cage.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6 - 22.6 °C
- Humidity (%): 50 - 63 % (relative)
- Air changes (per hr): 12 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

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 2 mL/kg. Control animals received vehicle only at a dose volume of 2 mL/kg.
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 6 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. The acceptance criteria for dose concentration test was ± 10 % recovery to the nominal concentration.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1 male/ 1 female
- Length of cohabitation: animals were cohoused until evidence of copulation was observed visually
- Proof of pregnancy: Visual observation of copulation; referred to as day 0 of pregnancy

Following the acclimatisation period of 4 - 6 female rabbits were paired per day with males.
In case of unsuccessful mating, females were returned to their original cages and given an opportunity to mate at a later time.

Duration of treatment / exposure:

Daily from gestation day 6 to gestation day 28 (inclusive).

Frequency of treatment:

Daily throughout treatment period.

Duration of test:

Does were sacrificed on gestation day 29.

No. of animals per sex per dose:

24 presumed-pregnant females per dose

Control animals:

yes

Details on study design:

- Dose selection rationale: Doses were selected upon consideration of available data on a closely related dioctyltin compound [dioctyltin iso-octyl thioglycolate; DOT[IOTG]; DOTI]. Two previous oral gavage developmental toxicity studies in the rabbit are available. Both used peanut oil as a vehicle and the test material utilised for both had approximately 80 % dioctyl and 20 % monooctyl components.
The first study with DOTI is a range-finding study using doses of 3, 10 and 30 mg/kg administered from gestation day 6 [GD6] to gestation day 18 [GD18]. Caesarean sections were done on GD28. In addition, within this study a dose of 100 mg/kg was added because there were no apparent maternal effects at doses up to and including 30 mg/kg. There were no observed developmental effects among the foetuses from treated does at doses up to and including 30 mg/kg. At the 100 mg/kg there was a decrease in maternal body weight and a possible increase in the incidence of dead foetuses.
The second study with DOTI is a definitive developmental toxicity study which followed the same methodology as the range-finding study. Doses were 1, 10, and 100 mg/kg. In the second study clear maternal toxicity was observed in the 100 mg/kg dose and developmental effects consistent with delayed development of the foetuses [low weight, delayed ossification]. The noted anomalies were classified as minor anomalies and the low incidence of major anomalies could not be definitively attributed to treatment with the test material.
In the definitive study with DOTI dosing was limited to GD6-GD18; there were 3 maternal deaths and 4 abortions at the 100 mg/kg dose. The maternal deaths could not definitively be ascribed to treatment, but the abortions were determined to be the result of treatment. These results are relevant to the selection of the high dose in the current study; the extended dosing regimen of GD6 - GD28 and the higher proportion of the dioctyl component may increase maternal toxicity beyond what was observed with DOTI and is considered to possibly exceed the upper bound dose for a study of this type. Therefore the maximum dose tested is 80 mg/kg bw/day.

- Rationale for animal assignment: Each day the body weight of mated rabbits 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.

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: Does were weighed on gestation days 0, 3, 6 and daily thereafter until gestation day 29.

FOOD CONSUMPTION: Yes
- Time schedule: Individual feed intake of mated females was recorded on gestation days 0 - 3, 3 - 6 and daily from day 6 to day 29.

WATER CONSUMPTION: No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
All the animals were sacrificed on gestation day 29 by intravenous injection of sodium thiopentone and subjected to detailed gross pathology. At necropsy the thymus was excised, weighed and placed in 10 % formalin for possible future histopathological examination. Furthermore, all gross lesions were excised 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: No data

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 body weight, food consumption, number of corpora lutea, uterus weight, number of implantations, number of live foetuses and body weight of live foetuses. Kruskal-Wallis test followed by Dunnett's post test was used for the percentage of visceral or skeletal malformations (or variations) for each litter, percentage of each individual malformation (or variation) for each litter using SPSS software version 2. All analyses and comparisons were evaluated at the 95 % level of confidence (P<0.05).

Indices:

Corrected body weight (g) = (Gestation day 29 body weight - Gestation day 6 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

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: biologically relevant depression [>10 %] in thymus weight at 80 mg/kg

Details on maternal toxic effects:
PREGNANCY DATA
A total number of 19, 21, 19 and 20 mated females were confirmed pregnant in groups G1, G2, G3 and G4, respectively (0, 4, 20 and 80 mg/kg, respectively). This is a pregnancy rate of 79, 88, 79 and 83 % at the time of caesarean section for groups G1, G2, G3 and G4, respectively.

MATERNAL DATA
- General Tolerability: No deaths or abortions were observed during the experimental period and there were no clinical signs recorded which were indicative of overt toxicity.
- Maternal Body weight, Body Weight Gain, and Corrected Body Weight Gain: There were no statistically significant differences in maternal body weights across the dose groups on any single gestation day, nor was there a dose-related pattern of reduced body weights beginning after GD6, the first day of dosing.There were no statistically significant differences in uncorrected maternal body weigh gain across the dose groups for any single gestational period. Treated does had higher weights than controls on GD29, this was considered to be a random occurrence and not related to test material administration.
- Food Consumption: No treatment related differences in average feed consumption were observed at any dose.
- Gross Pathology: There was a treatment-related macroscopic finding of reduced maternal thymus weight. The mean maternal thymus weights were -5.1, -9.6, and -12.8 % in the 4 mg/kg [low], 20 mg/kg [mid], and 80 mg/kg [high] dose groups, respectively when compared to controls. While these decreases were not statistically significant, the results were dose-dependent relative to controls and are consistent with data from other species [rat and mouse] which demonstrate the thymus is a target organ for octyltin compounds. 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.

REPRODUCTION DATA
No treatment related effects were noted in the number of corpora lutea, number of implantation sites, number of early or late resorptions, or percentage of post implantation loss across all the groups.
The mean gravid uterus weights were 230.1, 309.5, 253.7 and 196.9 g for groups G1 through G4, respectively. A statistically significant increase in the mean gravid uterus weight was noted at the low dose when compared to the vehicle control. This difference was attributed to the increase in the number of foetuses compared to the vehicle control group and other dose groups.
The mean pre-implantation losses were 0.9, 0.8, 2.3, and 4.9 % for groups G1, G2, G3 and G4, respectively. Mean post-implantation losses for these treatment groups were 3.1, 3.5, 6.4 and 5.7, respectively. These losses are within the historical control range and there is no clear evidence of a dose-response across dose groups.
There were no statistically significant differences in either the mean litter size or the number of live foetuses per doe across dose groups when compared to the vehicle control.
No treatment-related effect on the sex ratio of foetuses was noted at any dose compared to the vehicle controls.

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

80 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Dose descriptor:

LOAEL

Effect level:

80 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

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 36.6, 37.3, 35.5, and 32.3 g for groups G1, G2, G3 and G4, respectively. Mean foetal weights were not statistically significantly different across the dose groups when compared to controls. At the high dose the mean foetal body weight was -11.9 % relative to controls which suggests a biologically-relevant, but marginal effect on foetal maturation. However, the mean weight for this group was disproportianately affected by low foetal body weights in a single litter in this group. There did not appear to be a generalised impact on the entire group. In this case, the observations indicate no clear treatment-related foetal toxicity.

- 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.

- Crown rump length:
The mean foetal crown-rump length for both sexes was 92.1, 91.1, 89.3 and 82.3 mm for groups G1, G2, G3 and G4, respectively. A statistically significant reduction in the mean foetal crown-rump length was noted in the high dose group when compared to controls. This was -10.7 % relative to the vehicle control suggesting a marginal but biologically relevant effect on foetal maturation which correlated to the degree of skeletal ossification. One litter in this group had low crown-rump lengths which was found to disproportianately affect the mean. There was no apparent trend when considering all the litters of this group. In addition, there were no correlating statistically significant difference for any other parameter. These observations did not indicate a clear treatment-related effect.

- Skeletal Examination: The noted skeletal anomolies in the study are common findings for foetuses for this species and strain. The morphologic observations did not occur in a dose-dependent pattern, nor was the severity linked to an increase in dose. The incidences of absent sternum No. 5, a measure of delayed ossification were found to be higher in controls compared to any other treatment group. Other indicators of delayed ossification such as the absence of proximal phalanges, were found to be single incidences within a litter both in the control group and treatment groups. Poor ossification in sternum No. 5 and No. 6, an indicator of delayed ossification, wsa the only variation to be found to occur as multiple incidences within a litter. This variation however is considered to be less significant than the absence of sternum No. 5. The conflicting results indicate that these findings are incidental.

Remarks on result:

not determinable due to adverse toxic effects at highest dose / concentration tested

Abnormalities:

not specified

Developmental effects observed:

not specified

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: Mean Maternal Thymus Weights

Dose Group (mg/kg)	Thymus (g)	Thymus (% of control)	GD 6-29 (% of control)
G1: Control	2.2353	100.0	0.0
G2: 4	2.1214	94.9	-5.1
G3: 20	2.0209	90.4	-9.6
G4: 80	1.9499	87.2	-12.8

 

Table 2: Foetal Data

Dose Group (mg/kg)	Implantation Sites (mean)	Live Foetuses (mean)	Dead Foetuses (Litters)	Pre-implantation Loss (%)	Post-implantation Loss (%)	Early resorptions (mean)	Late resorptions (mean)
G1: Control	5.1	4.9	0 (0)	0.9	3.1	0.2	0.05
G2: 4	6.0	5.7	2 (2)	0.8	3.5	0.2	0.05
G3: 20	5.5	5.0	2 (2)	2.3	6.4	0.4	0.00
G4: 80	5.0	4.6	3 (1)	4.9	5.7	0.3	0.00

 

Table 3: Mean Foetal Body Weights

Dose Group (mg/kg)	Mean weight (g)	Average foetal crown-rump length (mm)
G1: Control	36.6	92.1
G2: 4	37.3	91.1
G3: 20	35.5	89.3
G4: 80	32.3	82.3*

 * P<0.05

Table 4: Summary of foetal skeletal observations (malformations)

Parameter	Group	G1 (control)	G2 (4 mg/kg)	G3 (20 mg/kg)	G4 (80 mg/kg)
	Number of litters	19	21	19	20*
	Numbers of foetuses	93	119	95	91
Sternum
Sternum No. 5 Absent	No.	7 (5)	4 (3)	4 (3)	5 (4)
	%	7.6	3.4	4.2	4.3
Fore limb
Proximal Phalanx No. 1 Absent	No.	2 (2)	1 (1)	1 (1)	5 (5)
	%	2.2	0.8	1.1	5.6
Proximal Phalanx No. 5 Absent	No.	2 (2)	1 (1)	1 (1)	5 (5)
	%	2.2	0.8	1.1	5.6

The number of litters affected are indicated in parentheses

*One doe litter had all dead foetuses

 

Table 5: Summary of foetal skeletal observations (variations)

Parameter	Group	G1 (control)	G2 (4 mg/kg)	G3 (20 mg/kg)	G4 (80 mg/kg)
	Number of litters	19	21	19	20*
	Numbers of foetuses	93	119	95	91
Skull
Parietal bones – poor ossification	No.	0 (0)	1 (1)	0 (0)	3 (3)
	%	0.0	0.8	0.0	2.9
Inter-parietal bones- poor ossification	No.	0 (0)	1 (1)	0 (0)	1 (1)
	%	0.0	0.8	0.0	1.3
Ribs
Rib at Lumbar vertebrae No. 1 – supplementary, bilateral	No.	0 (0)	1 (1)	2 (1)	1 (1)
	%	0.0	0.8	2.1	0.8
Rib at Lumbar vertebrae No. 1 – supplementary, unilateral	No.	0 (0)	2 (2)	0 (0)	0 (0)
	%	0.0	1.7	0.0	0.0
Sternum No. 5 – Poor ossification	No.	4 (3)	1 (1)	4 (3)	10 (6)
	%	4.4	0.8	4.2	8.7
Sternum No. 6 – Poor ossification	No.	4 (3)	1 (1)	3 (3)	10 (6)
	%	5.3	0.8	3.2	8.7

The number of litters affected are indicated in parentheses

*One doe litter had all dead foetuses

Conclusions:

Under the conditions of the study the NOAEL for maternal toxicity was determined to be 20 mg/kg based on a biologically relevant depression in thymus weight at the 80 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 80 mg/kg, the highest dose tested.

Executive summary:

The purpose of this study was to assess the effects of prenatal exposure of pregnant female New Zealand White rabbits and their developing foetuses to the test material when administered by oral gavage in a peanut oil vehicle at 2 mL/kg to the mated females from gestation day [GD] 6 to 28. 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 29 by iv injection of sodium thiopentone and subjected to detailed gross pathology; the gross pathology included a determination of thymus weight. 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 or abortions were observed during the experimental period and there were no clinical signs recorded which were indicative of overt toxicity and there were no treatment related differences in average feed consumption or body weights observed at any dose. A total number of 19 (79 %), 21 (88 %), 19 (79 %) and 20 (83 %) mated females were confirmed pregnant in groups G1, G2, G3 and G4, respectively. Gross necropsy revealed a treatment-related effect on the thymus. The mean maternal thymus weight was reduced 5.1, 9.6, and 12.8 % in the 4 mg/kg [low], 20 mg/kg [mid], and 80 mg/kg [high] dose groups, respectively. No other gross pathological findings were noted in any dose group.

Investigation of reproduction parameters revealed no treatment related effects in any of the following: mean gravid uterus weight, number of corpora lutea, number of implantation sites, number of early or late resorptions, percentage of pre and post implantation loss, sex ratio, mean litter size, foetal weight or the number of live foetuses per doe across all the groups.

The mean foetal weights [combined sexes] were 36.6, 37.3, 35.5, and 32.3 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. However, at the high dose the mean foetal body weight is -11.9 % relative to controls which suggests a biologically-relevant, but marginal effect on foetal maturation. This however, was found to be disproportionately affected by a single litter in this group who had low body weights. The other litters in this group did not follow the same trend.

Foetal crown-rump length was statistically significantly different from the controls in the highest dose group. This result was affected by one litter in this group who had disproportionately low crown-rump lengths when compared to the other litters of this group. In addition, there were no correlating statistically significant differences for any other developmental parameter.

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 20 mg/kg based on a biologically relevant depression in thymus weight at the 80 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 80 mg/kg, the highest dose tested.