2-ethylhexyl 10-ethyl-4-[[2-[(2-ethylhexyl)oxy]-2-oxoethyl]thio]-4-methyl-7-oxo-8-oxa-3,5-dithia-4-stannatetradecanoate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted on read across material. This study was a part of the original dossier for MMTE. It remains in the dossier to provide continuity to reviewers and an historical perspective of the changes which are being made to the dossier. New data on in vitro metabolism, cited in section 7.1.1 of this dossier, caused a change in the interpretation of the data cited. The new in vitro metabolism data support the conclusion that MMTE does not metabolise to MMTC under simulated mammalian gastric conditions [pH ~2 and 37 °C as was formerly believed. It then follows that the toxicology of MMTE via the oral route in mammalian species cannot be accurately predicted based on studies conducted with MMTC via the oral route in mammalian species. The implications are clear: (a) dietary feeding and oral gavage studies conducted with MMTC cannot be read-across to MMTE, and (b) for studies conducted with MMTC, their relevance for hazard classification of MMTE must be reduced or eliminated. Therefore, the Klimisch score of this study has been reduced to Klimisch 3 because the read-across strategy from this study to MMTE is no longer valid (though the study itself is still considered to be reliable).

Justification for type of information:

Read-across test result from source substance MMTC to target substance MMT(EHTG).
This data requirement is fulfilled by read-across. Justification for the read-across of this test result is provided in Section 7.1 Toxicokinetics. The simulated gastric hydrolysis study shows rapid and complete conversion of MMT(EHTG) to MMTC. Based on this result MMTC fulfils the requirements of being a source compound for studies required for MMT(EHTG) where the endpoint is based on oral exposure. The study on MMTC can be applied both quantitatively and qualitatively for the end point for MMT(EHTG).

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

other: Read across target

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Study conducted on read across material

Remarks:

This study was a part of the original dossier for MMTE. It remains in the dossier to provide continuity to reviewers and an historical perspective of the changes which are being made to the dossier. New data on in vitro metabolism, cited in section 7.1.1 of this dossier, caused a change in the interpretation of the data cited. The new in vitro metabolism data support the conclusion that MMTE does not metabolise to MMTC under simulated mammalian gastric conditions [pH ~2 and 37 °C as was formerly believed. It then follows that the toxicology of MMTE via the oral route in mammalian species cannot be accurately predicted based on studies conducted with MMTC via the oral route in mammalian species. The implications are clear: (a) dietary feeding and oral gavage studies conducted with MMTC cannot be read-across to MMTE, and (b) for studies conducted with MMTC, their relevance for hazard classification of MMTE must be reduced or eliminated. Therefore, the Klimisch score of this study has been reduced to Klimisch 3 because the read-across strategy from this study to MMTE is no longer valid (though the study itself is still considered to be reliable).

Justification for type of information:

Read-across test result from source substance MMTC to target substance MMT(EHTG).
This data requirement is fulfilled by read-across. Justification for the read-across of this test result is provided in Section 7.1 Toxicokinetics. The simulated gastric hydrolysis study shows rapid and complete conversion of MMT(EHTG) to MMTC. Based on this result MMTC fulfils the requirements of being a source compound for studies required for MMT(EHTG) where the endpoint is based on oral exposure. The study on MMTC can be applied both quantitatively and qualitatively for the end point for MMT(EHTG).

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Key result

Dose descriptor:

NOAEL

Remarks:

fertility and developmental effects

Effect level:

150 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Key result

Dose descriptor:

NOAEL

Remarks:

maternal toxicity

Effect level:

150 mg/kg diet

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

other: 150 mg/kg diet (equivalent to 6.2 - 11.7 mg/kg bw/day in females) was considered to be the NOAEL for maternal toxicity.

Key result

Dose descriptor:

NOAEL

Remarks:

postnatal toxicity

Generation:

F1

Effect level:

150 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on the incidences of missing pups in the 750 mg/kg group, 150 mg/kg can be considered as a NOAEL for postnatal toxicity.

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

750 mg/kg diet

Treatment related:

yes

Relation to other toxic effects:

not specified

Dose response relationship:

no

Relevant for humans:

not specified

Conclusions:

The NOAEL for general sub-chronic toxicity and maternal toxicity, fertility and developmental effects was placed at 150 mg trichloromethylstannane per kg diet.

Executive summary:

The toxicity of trichloromethylstannane [CAS # 993-16-8] in Wistar rats was examined using continuous administration via the diet for 13 consecutive weeks (OECD Test Guideline 408). In satellite groups of female rats a reproduction/developmental screening test (OECD Test Guideline 421) was performed to provide initial data on possible reproductive and developmental effects of trichloromethylstannane. The main study used four groups of 10 rats/sex (13 -week study) and the satellite study used four groups of 10 female rats (reproduction/developmental screening study). For both studies the control group was kept on control diet and three test groups received experimental diets containing 30, 150 and 750 mg/kg [ppm] of the test substance. The dose levels used in both studies were based on the results of a preceding dose range finding study.

In the satellite study female rats were fed their respective test diets beginning 2 weeks prior to the mating period, and continued on test diets through mating, gestation, and up to PN 4 or shortly thereafter. Male rats from the main study were mated after a premating period of 10 weeks with female rats of the satellite groups which were fed the same dose of test diets.

Clinical observations, growth, food consumption, food conversion efficiency, neurobehavioural testing, ophthalmoscopy, haematology, clinical chemistry, renal concentration test, urinalysis, organ weights and gross examination at necropsy, microscopic examination of various organs and tissues and assessment of various reproductive and developmental parameters were used as criteria for detecting the effects of treatment. 

The calculated doses during the pre-mating, gestation and lactation periods for the females receiving 30, 150, or 750 mg/kg trichloromethylstannane in the diet ranged from 1.2-2.0, 6.2-11.7 and 26.5-45.8 mg/kg body weight, respectively.

During the study one animal of the 750 mg/kg group was found dead on GD22. Most probably haemothorax caused by dystocia was the cause of death. No other mortalities or treatment-related clinical signs were observed in the female animals of the satellite groups.

Mean body weight, body weight change and food consumption was similar in the control and the 30 and 150 mg/kg groups. Mean body weight on PN 4 and mean body weight change PN 1-4 of the 750 mg/kg group was decreased, although not statistically significantly.

During the premating and gestation periods, mean food consumption of the female animals was similar in the control, 30 and 150 mg/kg groups. During the lactation period food consumption of the 750 mg/kg group was decreased (not statistically significantly).

No treatment-related effects on reproduction and development of the pups were observed in the 30 and 150 mg/kg groups.

In the 750 mg/kg group, a number of changes were seen indicative of a reproductive and developmental effect. One animal was found dead on GD22 (death was most probably caused by haemothorax caused by dystocia), post-implantation loss was 43 %, the number of pups delivered was 7.1 versus 11.2 in the control group, 3 females lost all pups between PN 1-4, and pup mortality between PN 1-4 in the 750 mg/kg group was 65 versus 16 % in the control group.

No treatment-related effects on pup weight and pup abnormalities were observed.

Absolute and relative organ weights (uterus, ovary and thymus) were similar among the groups. At microscopic examination of these organs no treatment-related histopathological changes were observed.

Based on reproductive and developmental effects (decreased number of pups delivered and increased post implantation loss and pup mortality) observed after mating of female animals of the 750 mg/kg satellite group with male animals of the main study, the mid-dose level 150 mg Trichloromethylstannane/kg diet (equivalent to 9.8 mg/ kg body weight/day in males and 6.2-11.7 mg/kg body weight for females) can be considered as a NOAEL for fertility and developmental effects.

Based on the effects on body weight and food consumption in the 750 mg/kg group, 150 mg Trichloromethylstannanel kg diet (equivalent to 6.2-11.7 mg/kg body weight/day) can be considered as a NOAEL for maternal toxicity.

The NOAEL for general sub-chronic toxicity and maternal toxicity, fertility and developmental effects was placed at 150 mg trichloromethylstannane per kg diet.

Cross-reference 2

Reason / purpose for cross-reference:

read-across: supporting information

Reference

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other:

Remarks:

This study was a part of the original dossier for MMTE. It remains in the dossier to provide continuity to reviewers and an historical perspective of the changes which are being made to the dossier. New data on in vitro metabolism, cited in section 7.1.1 of this dossier, caused a change in the interpretation of the data cited. The new in vitro metabolism data support the conclusion that MMTE does not metabolise to MMTC under simulated mammalian gastric conditions [pH ~2 and 37 °C as was formerly believed. It then follows that the toxicology of MMTE via the oral route in mammalian species cannot be accurately predicted based on studies conducted with MMTC via the oral route in mammalian species. The implications are clear: (a) dietary feeding and oral gavage studies conducted with MMTC cannot be read-across to MMTE, and (b) for studies conducted with MMTC, their relevance for hazard classification of MMTE must be reduced or eliminated. Therefore, the Klimisch score of this study has been reduced to Klimisch 3 because the read-across strategy from this study to MMTE is no longer valid (though the study itself is still considered to be reliable).

Objective of study:

metabolism

Qualifier:

no guideline followed

Principles of method if other than guideline:

The test substance was tested under low pH conditions (0.07 N HCl) at 37 °C in order to simulate the possible hydrolytic action on mammalian gastric contents. The hypothesis is that in the hydrochloric acid solution the tin-EHMA bond breaks leading to formation of the corresponding alkyltin chloride and simultaneous liberation of the ligand, 2-Ethylhexylmercaptoacetate (EHMA). Under these conditions EHMA, that contains an ester group, can (partially) hydrolyse to thioglycolic acid (TGA) and 2-ethylhexanol (EH).

A stock solution of the test substance in acetonitrile (2.87 mg/mL) was freshly prepared by dissolving 340.81 mg of the test substance in 100.0 mL acetonitrile. A correction for the percentage of the test substance (84.14 %) was used; the amounts of free EHTG and EH present in the test substance were taken into account in the calculations. Into a series of 4 Teflon vessels, 175 µL of a stock solution of the test substance (2.87 mg/mL) was added to 50 mL of 0.07 N HCl. In this way, the concentration of the test substance in the final 0.07 N HCl solution was 10.0 mg/L. The solution was stirred for predetermined periods at 37 °C. The temperature was maintained using an oven. A sample was taken from one of the Teflon vessels after 0.5, 1.0, 2.0 and 4.0 hours, respectively. Once a vessel had been sampled, no other sample was collected from that vessel. Fifty mL of the sample (0.07 N HCl solution) was extracted with 25 mL of heptane. The amount of EHMA and EH in the heptane layer was analysed by GC-FID. The experiments were performed in duplicate.

GLP compliance:

no

Radiolabelling:

no

Metabolites identified:

yes

Details on metabolites:

The recovery of EHMA spiked to 0.07N HCl at a level of 10.0 mg/L was 125 ± 3 %.
The recovery of EH at a level of 12.7 mg/L was 92 ± 3 %.

It was observed that the simulated gastric hydrolysis of the test substance to EHMA and EH was rapid, to a level of 93.9 % after 0.5 h. Looking at the later time points, the measured amounts of EHMA and EH decreased resulting in a calculated percentage of hydrolysis of 78.0 % after 4 hours.

Based on the percentage of completion at 0.5 hours, the half-life time was estimated to be 0.27 hours.

Results (as % conversion to MMTC) for MMT(2-EHMA), by sample collection time:

0.5 h: 94 %

1 h: 91 %

2 h: 85 %

4-h: 78 % 

t1/2 (estimated) = 0.27 hours 

 

The data show that within 0.5 hours, most of the available EHMA ligands have been released and there is greater than 90 % hydrolysis of the test substance. Following the reaction for an additional amount of time it is evident that the amount of free EHMA in solution decreases from the initial level. In previous studies, reanalysing the samples in reverse order confirmed that this trend was real and not caused by the GC drifting out of calibration. This effect was thought to be due to a loss of free EHMA by adsorption, oxidation or the formation of other hydrolysis or reaction products.

Conclusions:

These results support the use of monomethyltin chloride as an appropriate surrogate for mammalian toxicology studies of monomethyltin (ethylhexylthioglycolate) via the oral route.

Executive summary:

These results support the use of monomethyltin chloride as an appropriate surrogate for mammalian toxicology studies of monomethyltin (ethylhexylthioglycolate) via the oral route.

The percentage of hydrolysis of monomethyltin (ethylhexylthioglycolate) under simulated gastric hydrolysis conditions (0.07 N HCl at 37 °C) reached a level of 93.9 % after 0.5 hours. The corresponding half-life time of the test substance was estimated to be 0.27 hours.

 

The chemistry of the alkyl organotins has been well studied. For organotins, like MMT(EHTG), the alky groups are strongly bound to tin and remain bound to tin under most reaction conditions. However, other ligands, such as carboxylates or sulfur based ligands (EHTG), are more labile and are readily replaced under mild reaction conditions. To assess the reactivity of MMT(EHTG) under physiological conditions simulating the mammalian stomach, an in-vitro hydrolysis test was performed. This in vitro test provides chemical information that strongly suggests both the probable in vivo metabolic pathway and the toxicokinetics of the MMT(EHTG) substance. This result verifies that under physiological conditions MMT(EHTG) is rapidly and essentially completely converted to the corresponding monomethyltin chloride, MMTC.

 

Specifically, in the simulated gastric hydrolysis studies at low pH (0.07 N HCl) the EHTG ligands are rapidly displaced from tin and replaced by chloride ligands; the methyl group remains attached to tin. For MMT(EHTG), > 90 % hydrolysis of the test compound occurred within 0.5 hours, and the estimated half-life was 0.27 hours. The replacement of all three EHTG ligands under these conditions is therefore a rapid and complete reaction with respect to the overall metabolic timescale.   

 

This same type of simulated gastric hydrolysis study was also performed on other monomethyltin and dimethyltin compounds having sulfur based ligands with the same result, rapid hydrolysis to release the sulfur based ligands and generate the corresponding methyltin chloride compounds. These studies were all conducted as part of the OECD HPV program covering the monomethyltin and dimethyltin chemicals.

 

Since this hydrolysis was done under simulated gastric conditions, the result is entered into this dossier as fulfilling a toxicokinetic endpoint and as a justification for read-across. As there is rapid conversion of MMT(EHTG) to MMTC, this dossier uses studies on MMTC as the source substance to fill certain specific endpoints for MMT(EHTG), the target substance, by read-across. This read-across is justified because oral exposure to MMT(EHTG) places it in the gastro-intestinal tract where, based on this study, it is hydrolysed to MMTC as the initial metabolic action. Therefore, MMTC studies can be used to fulfil the REACH requirements for MMT(EHTG) related to exposure via the oral route, in particular the mammalian toxicology endpoints of repeated dose, reproduction, developmental and in vivo toxicity. Use of studies on MMTC in a “read-across manner” to cover these specific MMT(EHTG) endpoints is fully supported both by the ECHA guidelines on when and how to apply read-across (see below). In addition, the read-across of MMTC data to MMT(EHGT) was also accepted by the OECD under the HPV program for all methyltin substances on the basis of the simulated gastric hydrolysis.

 

The ECHA document “Guidance on Information Requirements and Chemical Safety Assessment, Part B: Hazard Assessment”, Version 2.1, 2011, discusses the role of toxicokinetics in part B.6.2.1 “Guidance on Toxicokinetics”. This section notes the role of toxicokinetics as an important component in providing information regarding metabolism and absorption of chemicals. It comments further that data on the toxicokinetic behaviour of a substance should be considered in conducting the human health hazard assessment. In this regard, the toxicokinetics directly identify MMTC as the principal and sole organotin metabolite of MMT(EHTG) via oral exposure. 

 

The ECHA document “Guidance on Information Requirements and Chemical Safety Assessment, Chapter R.7.c: Endpoint Specific Guidance” section R.7.12 notes that toxicokinetics can be used as a means to assist testing strategies, study design, and the application of read-across for building substance categories. It further notes that the appropriateness or applicability of toxicokinetic studies needs to be made on a case-by-case basis, because the toxicokinetic parameters will affect the hazard profile in determining the concentration of the ultimate toxicant at the target site.  

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Principles of method if other than guideline:

The toxicity of trichloromethylstannane [CAS # 993-16-8] in Wistar rats was examined using continuous administration via the diet for 13 consecutive weeks (OECD Test Guideline 408). In satellite groups of female rats a reproduction/developmental screening test (OECD Test Guideline 421) was performed to provide initial data on possible reproductive and developmental effects of trichloromethylstannane.

The method of analysis involved derivatisation. This method only measures the amount of the alkyltin moiety, MMT, present and does not identify the other ligands attached to the tin. Currently there is no analytical method available that can quantify the actual named substance, i.e., the entire organotin compound with its associated chloride ligand.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Trichloromethylstannane

EC Number:

213-608-8

EC Name:

Trichloromethylstannane

Cas Number:

993-16-8

Molecular formula:

CH3SnCl3

IUPAC Name:

Methyltin trichloride

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): Trichloromethylstannane
- Molecular formula (if other than submission substance): CH3Cl3Sn
- Molecular weight (if other than submission substance): 240.8 g/mol
- Smiles notation (if other than submission substance): Cl[Sn](Cl)(Cl)C
- InChl (if other than submission substance): IUPAC : Stannane, trichloromethyl
- Analytical purity: 82.85 %
- Impurities (identity and concentrations):
- Composition of test material, percentage of components:
- Alkyl group distribution (% (mass/mass))
Monomethyltin trichloride: 82.85
Dimethyltin dichloride: 9.29
Trimethyltin chloride: 0.02
Tin tetrachloride: 4.68
Me2ClSnCH2SnCl3: 0.59
MeCl2SnCH2SnCl3: 1.78
Cl3SnCH2SnCl3: 0.80
- Lot/batch No.: 82420
- Expiration date of the lot/batch: December 1, 2003
- Storage condition of test material: <-18 °C, in the dark

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: (P) 15-16 weeks
- Weight at study initiation: (P) Males: 113.9 - 13.8 g (mean 126.3g); Females: 178.8 - 213.5 g (mean 199.7 g)
- Fasting period before study: not reported
- Housing: 3 or 4 per group per cage; during gestation and lactation they were housed individually
- Use of restrainers for preventing ingestion (if dermal): not applicable
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 % not exceeding 70 %
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 hours light/ 12 hours dark

IN-LIFE DATES: From: May 20, 2003 To: June 26- July 14, 2003

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance was incorporated into the basal diet by mixing in a mechanial blender

DIET PREPARATION
- Rate of preparation of diet (frequency): shortly before the start of the studies and every 6 weeks thereafter
- Mixing appropriate amounts with (Type of food): Rat and Mouse No. 3 Breeding Diet, RM3
- Storage temperature of food: stored in a freezer until use

VEHICLE- no vehicle

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: During the mating period one male and one female of the same dose group were caged until copulation occurred or two weeks had elapsed.
- Proof of pregnancy: The day a vaginal smear was detected sperm positive was considered gestation day 0. During the mating period every consecutive morning vaginal smears were made to ascertain copulation by detection of sperm cells in the smear.
- After unsuccessful pairing, replacement of first male by another male with proven fertility: Yes. If a male died before or during the mating period before the female was found sperm positive, the female was mated with another, proven male of the same group (i.e. a male which already had a successful copulation, sperm positive smear with another female).
- After successful mating each pregnant female was caged (how): During the premating period females were housed 3 or 4 per group per cage. Male rats of the 13 week study were mated after a premating period of 10 weeks with the female rats. During the gestation and lactation periods the females were housed individually.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

ANALYTICAL METHODS: GC-MS was used to determine the achieved concentration, homogeneous distribution and stability of the test substance in diet samples. The method of analysis involved derivatisation. This method only measures the amount of the alkyltin moiety, MMT, present and does not identify the other ligands attached to the tin. Currently there is no analytical method available that can quantify the actual named substance, i.e., the entire organotin compound with its associated chloride ligand.
From each diet sample, 2.0 g was transferred into a 50 mL Greiner tube. An aliquot of the internal standard solution was added. Subsequently, methanol, acetate buffer solution, 20 % aqueous NaBEt4 solution and hexane were added to each sample and this mixture was shaken and heated to 60 °C. Prior to GC/MS analysis, the hexane layer was washed with 2 mol/L HCl in order to remove the ethylboron compounds. The concentration of each test substance in feed was determined by GC-MS analysis of the hexane extracts.

Analysis of the test substance in diet samples revealed that the test substance dose was close to the nominal level for all diets. Mean measured concentrations ranged from 96 to 113 % of nominal concentrations. Homogeneity: The test substance was considered to be homogenously distributed in all diets. Stability: The test substance was considered to be stable in the diets upon storage at room temperature for 7 days and upon storage at < -18 °C for 6 weeks.

Duration of treatment / exposure:

13 week study; treated for 2 consecutive weeks during the pre-mating period, daily during gestation (up to 26 days) and up to euthanasia at or shortly after postnatal day (PN) 4.

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

30 mg/kg diet

Doses / concentrations 2

Dose / conc.:

150 mg/kg diet

Doses / concentrations 3

Dose / conc.:

750 mg/kg diet

No. of animals per sex per dose:

10 females per dose level for satellite group

Control animals:

yes, concurrent no treatment

Details on study design:

The test substance was administered at constant concentrations in the diet for 13 consecutive weeks. The test substance was administered via the diet two weeks premating, during gestation and up to euthanasia at or shortly after postnatal day 4.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Clinical signs and mortality were observed at least once daily (in the morning) and on working days also once in the afternoon.

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: Once during the acclimatisation period, once at initiation of the study prior to introduction of feed. The females of the satellite groups were weighed 4 days before gestation, on gestation days 0, 7, 14 and 21 and on postnatal days 1 and 4. Furthermore, all animals were weighed on the day of necropsy in order to determine their correct organ to body weight ratios.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Measured per cage over weekly periods by weighing the feeders (in g/animal/day).

WATER CONSUMPTION: No

Sperm parameters (parental animals):

Parameters examined in P male parental generations: The reproductive organs of the males of the 30 and 150 mg/kg groups that failed to sire (did not mate or female was not pregnant) were microscopically examined.

Litter observations:

At the end of the gestation period, females were examined twice daily for signs of parturition. The litters were examined only daily for dead pups. The total litter size and numbers of each sex as well as the number of stillbirths, live and dead pups and grossly malformed pups were evaluated on days 1 and 4 of lactation. Mean pup weights were calculated as litter weight/number pups. The number of runts (< 2 sd from the litter mean) were noted and reported as well.

Postmortem examinations (parental animals):

GROSS NECROPSY
Organs weighed included ovaries, uterus (after counting of the implantation sites), thymus and all gross lesions.

HISTOPATHOLOGY / ORGAN WEIGHTS
Microscopic examination of the ovaries, uterus and thymus of the control and 750 mg/kg groups was performed. Examination was extended to the thymus of the females of the 30 and 150 mg/kg groups because of the effects observed in the 750 mg/kg group for this tissue/organ. Furthermore, the reproductive organs of the males of the 30 and 150 mg/kg groups that failed to sire (did not mate or female was not pregnant) and the reproductive organs of females of the 30 and 150 mg/kg groups that were non-mated or non-pregnant were microscopically examined.

Postmortem examinations (offspring):

SACRIFICE
Pups were killed by hypothermia at < -18 °C.

GROSS NECROPSY
A necropsy was performed on stillborn pups and pups dying during the study; pups were examined externally for gross abnormalities and macroscopic abnormalities were recorded.

Statistics:

- Test substance analysis: Homogeneity: one way analysis of variance (Anova) using the sample location (1-5) as grouping factor. The test substance was considered to be homogeneously distributed in the diets if p > 0.01 and/or if the relative standard deviation (RSD) between the sample means was less than or equal to 15 %. Stability: one way analysis of variance (Anova) using time as grouping factor. The test substance was considered to be stable in the diets if p > 0.01 and/or if the mean concentration on the last day was between 80 and 120 % of the mean concentration on the first day (t =0). Achieved concentration: for each concentration level, the mean of the concentrations, as measured in the diet samples used for the assessment of the homogeneity, was considered to represent the achieved concentration. The content of the test substance in the diet was considered to be 'close to intended' if the mean measured concentration was between 80 and 120 % of the intended concentration.
- Body weight: one way analysis of covariance (covariate: body weight on day 0) followed by Dunnett's multiple comparison tests.
- Food consumption and food efficiency: one way analysis of variance (Anova) followed by L.S.D. tests.
- Fisher's exact probability test was used to evaluate the number of mated and pregnant females and females with live pups. Numbers of implantation sites and live and dead pups were evaluated by Kruskal-Wallis non-parametric analysis of variance followed by the Mann-Whitney U-test.
- The litter was used as the statistical unit for calculation of foetal values.
- Histopathological changes: Fisher's exact probability test. All tests were two-sided. Probability values of p<0.05 were considered significant.

Reproductive indices:

With regard to fertility and reproductive performance, the following parameters were calculated:
-Pre-coital time = time between the start of mating and successful copulation
- Duration of gestation = time between gestation day 0 and day of delivery
- Mating index = (number of females mated/number of females placed with males) x 100
- Male fertility index = (number of males that became sires/number of males placed with females) x 100
- Female fertility index = (number of pregnant females/number of females placed with males) x 100
- Female fecundity index = (number of pregnant females/number of females mated) x 100
- Gestation index = (number of females with live pups/number of females pregnant) x 100
- Number of lost implantations = number of implantations sites - number of pups born alive
- Post-implantation loss = [(number of implantation sites - number of pups born alive)/number of implantation sites] x 100

Offspring viability indices:

- Live birth index = (number of pups born alive/number of pups born) x 100
- Pup mortality day n = (number of dead pups on day n/total number of pups on day n) x 100
- Viability index day 1-4 = (number of pups surviving 4 days/total number of live pups on day 1) x 100
- Sex ratio day n = (number of male pups on day n/ total number of pups) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

not specified

Mortality:

mortality observed, treatment-related

Description (incidence):

One animal of the 750 mg/kg group was found dead on GD 22 (i.e. 37 days after the start of exposure).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

An increased body weight change from GD 7-14 of the females of the 30 mg/kg group was noted, which was considered a chance finding. Mean body weights (change) of the females were similar among the control, 30 and 150 mg/kg group during the entire study. Mean body weight (changes) between PN 1-4 of the 750 mg/kg group was decreased; however, no statistical significance was reached for these findings.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption of the female animals of the 750 mg/kg group was decreased (not statistically significantly) during the lactation period. During the premating and gestation periods food consumption of the females was similar in the control, 30, 150 and 750 mg/kg groups.

The test substance intake of the female animals of the 30, 150 and 750 mg/kg dose groups was respectively:
- Premating period days 0-7: 1.8, 9.0 and 44.5 mg/kg bw/day
- Premating period days 7-14: 1.8, 8.8 and 43.9 mg/kg bw/day
- Gestation period GD 0-7: 1.9, 9.6 and 44.5 mg/kg bw/day
- Gestation period GD 7-14: 2.0, 9.6 and 45.8 mg/kg bw/day
- Gestation period GD 14-21: 1.2, 6.2 and 35.9 mg/kg bw/day
- Lactation period PN 1-4: 1.7, 11.7 and 26.5 mg/kg bw/day

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No treatment related histopathological changes were observed in the uterus, ovary and thymus of the female animals of the control and 750 mg/kg groups.

Histopathological findings: neoplastic:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

- Pre-coital time: comparable among the control and the treated groups.
- Mating index: 90 -100 %
- Number of pregnant females per dose level: 9, 8, 9 and 8 for the control, 30, 150 and 750 mg/kg groups, respectively.
- Female fecundity index: comparable among the control and the treated groups.
- Female fertility index: comparable among the control and the treated groups.
- Male fertility index: comparable among the control and the treated groups.
- Number aborting: No data available
- Number of resorptions: No data available
- Mean number of implantations: 11.2 (control group), 10.8 (30 mg/kg), 11.6 (150 mg/kg), 10.5 (750 mg/kg).
- Gestation index: 89, 100, 100 and 88 % in the control, 30, 150 and 750 mg/kg groups, respectively.
- Live birth index: 98, 97, 100 and 96 % in the control, 30, 150 and 750 mg/kg groups, respectively.
- Number of pups born (number of litters): 90 (8), 86 (8), 99 (9) and 50 (7) for the control, 30, 150 and 750 mg/kg groups, respectively.
- Number of stillborn pups (number of litters): 2 (1), 3 (2), 0 and 2 (2) for the control, 30, 150 and 750 mg/kg groups, respectively.
- Post implantation losses [total implantation sites minus total live births at the first observation]: 13 (18.6 %), 16 (15.3 %), 5 (4.7 %) and 36 (42.9 %) for the control, 30, 150 and 750 mg/kg groups, respectively. Interpretation of these data was complicated by the incidence of missing pups across groups. A variable incidence of pups "missing" after birth was recorded. The incidence of missing pups was statistically significantly higher than controls in the high-dose group, statistically significantly lower than controls in the mid-dose group, and not statistically different than controls in the low-dose group. The missing pups were presumed to have been cannibalised by the dams, but it is not known if the missing pups were alive or dead. It is also not known if some pups were cannibalised prior to being counted for litter size. This could account for the slightly lower number of recorded live births and the slightly higher post-implantation loss in the high-dose versus controls. The reason for missing pups cannot be determined on the basis of the data within the study. Missing pups could be due solely to a toxic behavioural effect on dams which caused a lack of, or abnormal, nurturing. It is clear that no malformations were noted at any observation point for any of the missing pups and no overt behavioural effects were noted; however some other effect could have caused the dam to eat them.
- Number of corpora lutea: No data available
- Duration of Pregnancy: 21 -23 days

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

fertility and developmental effects

Effect level:

150 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

2

Key result

Dose descriptor:

NOAEL

Remarks:

maternal toxicity

Effect level:

150 mg/kg diet

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

other: 150 mg/kg diet (equivalent to 6.2 - 11.7 mg/kg bw/day in females) was considered to be the NOAEL for maternal toxicity.

Results: F1 generation

General toxicity (F1)

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

- Pup mortality: 2.2, 3.5, 0 and 4 % for the control, 30, 150 and 750 mg/kg groups, respectively (PN 1); 16, 25, 3 and 65 % for the control, 30, 150 and 750 mg/kg groups, respectively (PN 4).
- Number viable: The viability index (PN 1-4) was 84, 75, 97 and 35 % in the control, 30, 150 and 750 mg/kg groups, respectively.
- Number live pups per litter: 11.0, 10.4, 11.0 and 6.9 for the control, 30, 150 and 750 mg/kg groups, respectively (PN 1); 10.6, 7.8, 10.7 and 4.2 for the control, 30, 150 and 750 mg/kg groups, respectively (PN 4).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

- Litter weight: The mean pup weights and pup weight changes were similar in the treated groups when compared to the control group.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- Grossly visible abnormalities: In the 30 mg/kg group, the number of cold pups was statistically significantly increased on PN 1 and 4. In addition on PN 4, the number of runts, pale pups and pups with no milk in the stomach was statistically significantly increased in the 30 mg/kg group. In the 150 mg/kg group the number of pale pups on PN 4 was statistically significantly increased. In the 750 mg/kg group the number of cold pups was statistically significantly increased on PN 1. As most effects were observed in the 30 mg/kg group only, these effects were not considered to be treatment related. Macroscopic observation of the stillborn pups revealed no abnormalities of the pups.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

- Litter size: The mean number of pups delivered per litter amounted to 11.2, 10.8, 11.0 and 7.1 for the control, 30, 150 and 750 mg/kg groups, respectively.
- Sex ratio: No difference was observed in the sex ratio between the groups.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

postnatal toxicity

Generation:

F1

Effect level:

150 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on the incidences of missing pups in the 750 mg/kg group, 150 mg/kg can be considered as a NOAEL for postnatal toxicity.

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

750 mg/kg diet

Treatment related:

yes

Relation to other toxic effects:

not specified

Dose response relationship:

no

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

The NOAEL for general sub-chronic toxicity and maternal toxicity, fertility and developmental effects was placed at 150 mg trichloromethylstannane per kg diet.

Executive summary:

The toxicity of trichloromethylstannane [CAS # 993-16-8] in Wistar rats was examined using continuous administration via the diet for 13 consecutive weeks (OECD Test Guideline 408). In satellite groups of female rats a reproduction/developmental screening test (OECD Test Guideline 421) was performed to provide initial data on possible reproductive and developmental effects of trichloromethylstannane. The main study used four groups of 10 rats/sex (13 -week study) and the satellite study used four groups of 10 female rats (reproduction/developmental screening study). For both studies the control group was kept on control diet and three test groups received experimental diets containing 30, 150 and 750 mg/kg [ppm] of the test substance. The dose levels used in both studies were based on the results of a preceding dose range finding study.

In the satellite study female rats were fed their respective test diets beginning 2 weeks prior to the mating period, and continued on test diets through mating, gestation, and up to PN 4 or shortly thereafter. Male rats from the main study were mated after a premating period of 10 weeks with female rats of the satellite groups which were fed the same dose of test diets.

Clinical observations, growth, food consumption, food conversion efficiency, neurobehavioural testing, ophthalmoscopy, haematology, clinical chemistry, renal concentration test, urinalysis, organ weights and gross examination at necropsy, microscopic examination of various organs and tissues and assessment of various reproductive and developmental parameters were used as criteria for detecting the effects of treatment. 

The calculated doses during the pre-mating, gestation and lactation periods for the females receiving 30, 150, or 750 mg/kg trichloromethylstannane in the diet ranged from 1.2-2.0, 6.2-11.7 and 26.5-45.8 mg/kg body weight, respectively.

During the study one animal of the 750 mg/kg group was found dead on GD22. Most probably haemothorax caused by dystocia was the cause of death. No other mortalities or treatment-related clinical signs were observed in the female animals of the satellite groups.

Mean body weight, body weight change and food consumption was similar in the control and the 30 and 150 mg/kg groups. Mean body weight on PN 4 and mean body weight change PN 1-4 of the 750 mg/kg group was decreased, although not statistically significantly.

During the premating and gestation periods, mean food consumption of the female animals was similar in the control, 30 and 150 mg/kg groups. During the lactation period food consumption of the 750 mg/kg group was decreased (not statistically significantly).

No treatment-related effects on reproduction and development of the pups were observed in the 30 and 150 mg/kg groups.

In the 750 mg/kg group, a number of changes were seen indicative of a reproductive and developmental effect. One animal was found dead on GD22 (death was most probably caused by haemothorax caused by dystocia), post-implantation loss was 43 %, the number of pups delivered was 7.1 versus 11.2 in the control group, 3 females lost all pups between PN 1-4, and pup mortality between PN 1-4 in the 750 mg/kg group was 65 versus 16 % in the control group.

No treatment-related effects on pup weight and pup abnormalities were observed.

Absolute and relative organ weights (uterus, ovary and thymus) were similar among the groups. At microscopic examination of these organs no treatment-related histopathological changes were observed.

Based on reproductive and developmental effects (decreased number of pups delivered and increased post implantation loss and pup mortality) observed after mating of female animals of the 750 mg/kg satellite group with male animals of the main study, the mid-dose level 150 mg Trichloromethylstannane/kg diet (equivalent to 9.8 mg/ kg body weight/day in males and 6.2-11.7 mg/kg body weight for females) can be considered as a NOAEL for fertility and developmental effects.

Based on the effects on body weight and food consumption in the 750 mg/kg group, 150 mg Trichloromethylstannanel kg diet (equivalent to 6.2-11.7 mg/kg body weight/day) can be considered as a NOAEL for maternal toxicity.

The NOAEL for general sub-chronic toxicity and maternal toxicity, fertility and developmental effects was placed at 150 mg trichloromethylstannane per kg diet.