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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
5 October 1993 - 25 November 1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in accordance with recognised guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report Date:
1994

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Purity of test material: dibutyltin dichloride >98%

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Male and female rats of the Wistar Crl:CD (Wi) BR strain were obtained from a "specific pathogen free" colony at Charles River Wiga GmbH, 97633 Sulzfeld, Germany.
- Age at study initiation: 8-9 weeks old
- Weight at study initiation: Females: 179-249 g
- Housing: The female rats were housed individually in solid floor macrolone cages with stainless steel lids of type III (dimensions: 420 mm x 260 mm x 150 mm). In deviation from the study protocol, the animals were not housed in macrolone cages of type II.
Autoclaved sawdust was provided for bedding (supplied by J. Brandenburg, 49424 Goldenstedt, Germany). The cage bottoms and bedding were changed three times weekly. The bedding material is analysed every 6 months for specified contaminants.
- Diet (e.g. ad libitum): Throughout the study, the rats were allowed free access to food. The basic diet used was obtained in powdered form (Ssniff Spezialdiaten GmbH, 59494 Soest, Germany)
- Water (e.g. ad libitum): Tap water was available ad libitum from plastic water bottles attached to each cage
- Acclimation period: The animals used for the study were acclimatized to the laboratories for at least 7 days prior to the start of the mating.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 °C (except on seven occasions when 17 or 18 °C were recorded)
- Humidity (%): 30 to 70% (except on two occasions when 75 % was recorded)
- Photoperiod (hrs dark / hrs light): The animals were exposed to a constant artificial (fluorescent) light cycle of 12 hours light (6.00 to 18.00 hours) and 12 hours dark.


IN-LIFE DATES: From: 08.10.93 To: 05.11.93

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test article was prepared daily as a solution in the vehicle.


VEHICLE
- Justification for use and choice of vehicle (if other than water): nda
- Concentration in vehicle: Rats received dibutyltin dichloride by oral gavage at dosages of 1.0, 2.5, 5.0 and 10 mg/kg daily for 10 consecutive days from day 6 to 15 post-coitum, inclusive.
- Amount of vehicle (if gavage): 5 ml/kg/day
- Lot/batch no. (if required): 11748, supplied by Henry Lamotte, 28197 Bremen, Germany.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
During the first week of treatment (on 11.10.1993) and at the end of the treatment period (on 16.11.1993), samples of at least 10 ml were taken from each test article formulation together with a reference sample for the control group for determination of concentration, deep-frozen immediately after sampling and sent to Ciba Additive GmbH, D-68619 Lampertheim, Germany for analysis. Dibutyltin dichloride in olive oil was determined by flameless atomic absorption spectrophotometry using external standard calibration.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:4
- Length of cohabitation: Overnight
- Verification of same strain and source of both sexes: Yes
- Proof of pregnancy: Vaginal plug/sperm in vaginal smear referred to as day 0 of gestation
Duration of treatment / exposure:
The test and control articles were administered to the mated female rats once daily for 10 consecutive days from day 6 to 15 post-coitum.
Frequency of treatment:
Daily for 10 days
Duration of test:
20 days, on day 20 the rats were sacrificed
No. of animals per sex per dose:
25 females per dose to allow 20 pregnant females per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were selected by the study sponsor according to the results of previous studies of Ema et al.
- Rationale for animal assignment (if not random):
Immediately after mating female animals were allocated to treatment groups using a random table of the letters A to E representing the groups 1 to 5.
By use of this random table, the sequence in which successfully inseminated animals were assigned to treatment groups was predetermined.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were examined twice daily at the beginning and end of the working day for morbidity and mortality.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined daily for signs of ill health or overt signs of toxicity, and each finding was recorded.


BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each inseminated female rat was recorded on days 0, 6, 9, 12, 16 and 20 post-coitum.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Not a feeding study
- Food consumption: The food consumption of each inseminated female rat was recorded and evaluated for the intervals from day 0 to 6, 6 to 9, 9 to 12, 12 to 16 and 16 to 20 post-coitum.


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20 by carbon dioxide inhalation
- Organs examined: The following maternal organs and tissues were preserved in 10% formalin and histopathologically examined: thymus (weighed before fixation), liver, bile duct and mesenteric lymph nodes

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
The ovaries and uteri were removed and examined from all females and the following data recorded:
- pregnancy status
- number of corpora lutea in each ovary
- number and position of implantations subdivided into:
a. early resorptions
b. late resorptions
c. dead fetuses
d. live fetuses

Intra-uterine deaths were classified as follows:
- Early resorptions showed decidual or placental tissues only.
- Late resorptions showed embryonic or fetal tissue in addition to placental tissue but excluded fetuses dying in utero within approximately 2 days prior to the terminal kill.
- Dead fetuses included only the fetuses dying in utero within approximately the last 2 days.

The uteri of apparently non-pregnant females were immersed in a 10 per cent solution of ammonium sulphide to reveal evidence of implantation.
Fetal examinations:
The fetuses were killed by an intrapulmonal injection of Eutha 77 (pentobarbitone sodium, Coopers Tierarzneimittel GmbH, 30938 Burgwedel, Germany).
For each live fetus and, if possible, for each dead fetus, the following data were recorded:

- individual placental weight
- external fetal abnormalities
- individual fetal weight
- fetal sex
- skeletal or visceral fetal abnormalities

Approximately half of the fetuses from each litter were eviscerated and the carcasses processed for skeletal examination (Alizarin staining technique).
The remaining fetuses were fixed in ethanol and examined for visceral abnormalities using a modified Wilson-Barrow technique.

Dead fetuses were evaluated separately, if applicable. Structural deviations were classified as follows:

Malformation: rare and/or probably lethal, e.g. hydrocephaly
Variation: changes which regularly occur also in control groups and which are not of functional significance

Statistics:
The statistical evaluation was performed with the standard software package SAS (Statistical Analysis System, release 6.04) excluding the analysis for body weight, body weight change and food consumption, which was performed with the statistical package of the on-line data collection system TERASYS.
Indices:
Data were processed where appropriate to give mean values, group mean values, standard deviations and reproductive indices.
Group mean calculations were normally based on individual data, except for group mean fetal weights where calculations were based on litter means.Group mean values for implantations, intra-uterine deaths and post-implantation losses were calculated in two ways:
Value 1 - all surviving animals that provide evidence of pregnancy including those showing total intra-uterine deaths.
Value 2 - all animals with at least one live fetus at termination.

All values expressed as a percentage were first calculated within the litter and then summarized per group as the mean of individual litter percentages.
Historical control data:
No data available

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
No mortalities were observed in the study groups.
No treatment-related clinical signs were observed.
Minor changes such as thinness, bloody incrusted nose, injury at snout, extremities, trunk or skull/ear or hair loss were seen in a few animals of all dose groups on single days.
In group 5 (10.0 mg/kg), mean body weight gain was clearly reduced during the treatment period, in particular from day 9 to 16 of gestation. The differences from the control group were statistically significant from days 9 to 12 and 6 to 16 of gestation. This finding is considered to be related to treatment.
In group 4 (5.0 mg/kg), mean body weight gain was slightly lower than in the control group from day 9 to 12 post-coitum. This finding may be related to treatment although mean body weight gains from day 6 to 16 of gestation as well as from day 0 to 20 of gestation were comparable to the control group.
Mean body weight gains of groups 2 (1.0 mg/kg) and 3 (2.5 mg/kg) were generally comparable to the control group.
In group 5 (10.0 mg/kg), mean daily food consumption was reduced during the treatment period, in particular from day 9 to 16 of gestation. The differences from the control group were statistically significant from days 9 to 12 and 6 to 16 of gestation. This finding is considered to be related to treatment.
In groups 2 (1.0 mg/kg), 3 (2.5 mg/kg) and 4 (5.0 mg/kg) mean daily food consumption was comparable to the control group.
Necropsy did not reveal any treatment-related findings. Minor findings in the kidneys or liver were observed in a few animals of all study groups.
The mean weight of the thymus was significantly reduced in group 5 (10.0 mg/kg) and is considered to be related to treatment.
In group 4 (5.0 mg/kg), the mean weight of the thymus was slightly lower than in the control group. Although the difference was small and not statistically significant, a treatment-related effect cannot be excluded.
In group 3 (2.5 mg/kg) and group 2 (1.0 mg/kg), the mean weight of the thymus was slightly higher than or comparable to the control group, respectively.
Pre-implantation and post-implantation loss was not affected by treatment. The mean numbers of corpora lutea and implantations were comparable in all groups. One animal each of group 2 (1.0 mg/kg) and group 4 (5.0 mg/kg) showed 100 per cent intra-uterine deaths. If these animals are included in the calculation, post-implantation loss was slightly increased in these groups. This finding is considered to be incidental because of its isolated occurrence.
The mean number of fetuses per litter was comparable in all groups. The fetal sex distribution was similar in all groups.
The mean fetal weight and the mean placental weight were not affected by treatment.
In group 5 (10.0 mg/kg), a high number of animals showed a thymus atrophy which correlated with the clearly reduced weight of the thymus observed at necropsy.
A slightly increased incidence of thymus atrophy was also seen in group 4 (5.0 mg/kg) and group 3 (2.5 mg/kg), therefore an effect of the test article cannot be excluded.
No treatment-related histopathological findings were found in group 2 (1.0 mg/kg).

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
1 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
The incidence of fetuses showing malformations was increased in group 5 (10.0 mg/kg). Four fetuses of three litters had malformations. In one fetus an edema was observed. The remaining three fetuses showed more severe malformations. One showed externally ankyloglossia and viscerally an internal hydrocephaly, anophthalmia and diaphragmatic hernia. In the second fetus an agnathia was seen externally and at skeletal examination absence of mandibles and malformed zygomatic arches were observed. At skeletal examination, the third fetus, with a filamentous and curly tail, showed a scoliosis and due to the tail anomaly absence of sacral and caudal vertebrae and sacral vertebral arches.
This low incidence and the lack of a consistent type of malformation render this finding of equivocal toxicological significance.
In one fetus of group 4 (5.0 mg/kg), an edema was observed and one control fetus showed a visceral malformation as pulmonary valve atresia.
No malformations were observed in fetuses of the lower dose groups (1.0 and 2.5 mg/kg).
The incidence of fetuses with external/visceral and skeletal variations was similar in all study groups.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Administration of dibutyltin dichloride by oral gavage from day 6 to 15 of gestation at a dose level of 10 mg/kg elicited maternal toxicity (reduced body weight gain and food consumption, and thymus atrophy).

Although the incidence of fetuses with malformations was slightly increased at 10.0 mg/kg, this was due to three fetuses in two litters. This low incidence and the lack of a consistent malformation render this finding of equivocal toxicological significance.

Administration of dibutyltin dichloride at a dose level of 5.0 mg/kg elicited slight maternal toxicity (slightly reduced body weight gain and possible thymus atrophy), but did not elicit embyrotoxicity or teratogenicity.

Administration of dibutyltin dichloride at a dose level of 2.5 mg/kg revealed a slightly increased incidence of animals showing thymus atrophy at histopathological examination, but did not elicit embyrotoxicity or teratogenicity.

Administration of dibutyltin dichloride at a dose level of 1.0 mg/kg did not elicit maternal toxicity, embyrotoxicity or teratogenicity.

Applicant's summary and conclusion

Conclusions:
In the oral (gavage) teratogenicity study in the rat the test material was determined to have a NOAEL of 1.0 mg/kg bw/day for maternal toxicity and 5.0 mg/kg bw/day for teratogenicity.
Executive summary:

In the oral (gavage) teratogenicity study in the rat (HD project number: 380 -211) the test material was determined to have a NOAEL of 1.0 mg/kg bw/day for maternal toxicity and 5.0 mg/kg bw/day for teratogenicity.

This study was conducted in accordance with the "EEC Council Recommendations of 26.10.1983 Concerning Tests Relating to the Placing on the Market of Proprietary Medicinal Products (83/571/EEC)", the "OECD Guidelines for Testing of Chemicals, No. 414, May 12, 1981", the "OECD Principles of Good Laboratory Practice (1981)" and the "Good Laboratory Practice Regulations" as outlined in the "German Chemical Law (Bundesgesetzblatt I, 22.03.1990)".

No mortalities were observed in the study groups.

No treatment-related clinical signs were observed.

Minor changes such as thinness, bloody incrusted nose, injury at snout, extremities, trunk or skull/ear or hair loss were seen in a few animals of all dose groups on single days.

At 10 mg/kg body weight gain was clearly reduced during the treatment period, in particular from day 9 to 16 of gestation.

At 5.0 mg/kg, body weight gain was slightly lower than in the control group from day 9 to 12 of gestation.

At 10.0 mg/kg, mean daily food consumption was reduced during the treatment period, in particular from day 9 to 16 of gestation.

Necropsy did not reveal any treatment-related findings. Minor findings in the kidneys or liver were observed in a few animals of all study groups.

The mean weight of the thymus in dams was clearly reduced in group 5 (10.0 mg/kg) and slightly reduced in group 4 (5.0 mg/kg).

At histopathological examination, an increased number of dams showed a thymus atrophy at 10 mg/kg. A slightly increased incidence of thymus atrophy was also seen in group 3 (2.5 mg/kg) and group 4 (5.0 mg/kg), therefore, an effect of treatment with the test article cannot be excluded.

No effect of treatment was observed on implantation.

Post-implantation loss was not affected by treatment.

The mean number of fetuses per litter, mean fetal weights and fetal sex distribution, as well as the mean placental weight were not affected by treatment.

Although the incidence of fetuses with malformations was slightly increased at 10.0 mg/kg, this was due to three fetuses in two litters. This low incidence and the lack of a consistent malformation render this finding of equivocal toxicological significance.