2-ethylhexyl 10-ethyl-4-[[2-[(2-ethylhexyl)oxy]-2-oxoethyl]thio]-4-octyl-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:

key study

Study period:

27 February 2008-25 April 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

248-227-6

EC Name:

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

Cas Number:

27107-89-7

Molecular formula:

C38H74O6S3Sn

IUPAC Name:

2-ethylhexyl 10-ethyl-4-({2-[(2-ethylhexyl)oxy]-2-oxoethyl}sulfanyl)-4-octyl-7-oxo-8-oxa-3,5-dithia-4-stannatetradecan-1-oate

Constituent 2

Reference substance name:

2-ethylhexyl 10-ethyl-4-[[2-[(2-ethylhexyl)oxy]-2-oxoethyl]thio]-4-octyl-7-oxo-8-oxa-3,5-dithia-4-stannatetradecanoate = MOT(EHMA), purity 97.9%

IUPAC Name:

2-ethylhexyl 10-ethyl-4-[[2-[(2-ethylhexyl)oxy]-2-oxoethyl]thio]-4-octyl-7-oxo-8-oxa-3,5-dithia-4-stannatetradecanoate = MOT(EHMA), purity 97.9%

Details on test material:

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

Test substance name : Octyltin tris (2-ethylhexylmercaptoactate)
(Thermolite 9XX)
Chemical name : Octyltin tris(2-ethylhexylmercaptoactate)
Other name : MOTE, AR1211
Colour / appearance : colorless liquid
CAS reg. Number : 27107-89-7
Batch number : ESOC21.1314
Purity : 97.9%
Storage conditions : 2-10 °C in the dark in closed container
- TNO dispense reference nr : 0700C7
Total quantity : approx. 100 gram
Receipt at TNO : 14 August 2007
Expiry date : 15 June 2008
- TNO dispense reference nr : 080010
Total quantity : approx. 300 gram
Receipt at TNO : 17 January 2008
Expiry date : 15 June 2008

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga GmbH, Sulzfeld, Germany
- Age at study initiation: (P) 9 - 10 wks;
- Weight at study initiation: (P) Males: 321.90 - 326.26 g; Females: 187.76 - 188.88 g;
- Fasting period before study: N/A
- Housing: The animals were housed in macrolon cages with wood shavings (Lignocel, Type 3/4) as bedding material and strips of paper (Enviro-dri) as environmental enrichment. During the premating period, the animals were housed in groups of 4/sex. For mating, one male and one female were housed together. Mated females were housed individually in macrolon cages, which were placed in another cage rack. The location of the mated females in the new cage racks was determined by the date of mating (females found sperm-positive on the same date will be considered a "lot") and by the animal number (within each lot the mated females were housed in the order of animal number). After delivery, the cage containing the darn with litter was transferred to another cage rack, the location being determined by delivery date and animal number as described for sperm-positive females
- Use of restrainers for preventing ingestion (if dermal): N/A
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 ºC
- Humidity (%): 40 - 70 %
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES main study: From: 27th February 2008 To: 25th April 2008

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
A possible route of exposure for humans is oral and therefore this route of exposure of the rats was chosen. The test substance was administered at constant concentrations in the diet and remained constant for each group during the study.

DIET PREPARATION
- Rate of preparation of diet (frequency): Fresh batches of the experimental diets were prepared once for the first dose-range finding and once for the second dose-range finding study and twice for the main study.
- Mixing appropriate amounts with (Type of food): Experimental diets were prepared by mixing powdered Rat & Mouse No. 3 breeding diet, RM3 with the appropriate amounts of test substance. The diets were mixed in a mechanical blender (Lodige, Paderborn, Germany)
- Storage temperature of food: The experimental diets were stored in a freezer (<-18°C).

VEHICLE
N/A

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: until mating occurred up to a maximum of 14 days.
- Proof of pregnancy: sperm in vaginal smear] referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: NDA
- After successful mating each pregnant female was caged (how): individually
- Any other deviations from standard protocol: No

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical method for determination of octyltin tris(2-ethylhexylmercatoacctate) (MOT(EHMA)) in diet was based on derivatisation of the organotin test substance to the corresponding monooctyltin compound with three ethyl ligands, using sodium tetraethylborate, with simultaneous extraction of the derivatised compound into hexane. Subsequently, the concentration of the test substance in diet was determined by GC-MS of the hexane extracts. Calibration solutions were prepared by spiking rodent diet with a stock solution of the test substance, followed by derivatisation with sodium tetraethylborate and extraction with hexane.

Before the start of the study, the analytical method was validated in the matrix under examination (viz. RM3 diet), to conform to the following criteria:
The mean accuracy at each dose level of the test substance in diet should be between 80 % and 120 %.
The precision, relative standard deviation of mean accuracy per dose level (n-3), should be < 15 % for all dose levels.
The correlation coefficient r obtained after linear regression of the calibration graphs constructed from the detector response-(Q-value (ratio peak area test substance / peak area internal standard)) vs. concentration of calibration solutions of test substance should be at least 0.99.

The methods used for the quantitative analysis of MOT(EHMA) in diet met the preset validation criteria.
The test substance MOT(EHMA) was homogeneously distributed in the test diets at all dose levels.
MOT(EHMA) was considered to be stable in diet, when stored at room temperature in the animal room for 7 clays and when stored in the freezer (< -18°C) in a closed container for at least 4 weeks.
The content of MOT(EHMA)in diet, used in the main study, was close to the intended concentration at all dose levels, except for the 200 mg/kg level, where the content was 29.7% lower than normal.

Duration of treatment / exposure:

28 days for the males
At least 6 weeks exposure for the females (2 weeks premating, 2 weeks mating and 3 weeks gestation)

Frequency of treatment:

ad libitum

Details on study schedule:

N/A

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

200 mg/kg diet

Doses / concentrations 2

Dose / conc.:

500 mg/kg diet

Doses / concentrations 3

Dose / conc.:

1 250 mg/kg diet

No. of animals per sex per dose:

12 males and 12 females

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Based on the results of 2 range finding studies.
- First range finding study:
- Concentrations: 0, 10, 30, 100, 500 mg/kg diet
- number and sex of animals per dose level: 8 (4 males/4 females)

- Second range finding study:
- Concentrations: 0, 500, 1250 mg/kg diet
- Number and sex of animals per dose level: 8 (4 males/4 females)

- Result of range finding studies: Mean absolute and relative thymus weight of the male animals of the 500 mg/kg group were statistically significantly decreased compared to the control groups.

- Rationale for animal assignment (if not random): By computer randomisation and proportionately to body weight.

Positive control:

Not used

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- If necessary, animals handled to detect signs of toxicity. On working days, all cages were checked again in the afternoon for dead or moribund animals to minimize loss of animals from the study. On Saturdays, Sundays and public holidays only one check per day was carried out. All abnormalities, signs of ill health or reactions to treatment were recorded.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded at least once pretest. Body weight of males was recorded weekly during the premating and mating period until euthanized. Body weight of females was recorded weekly during the premating and mating period, during gestation on day 0, 7, 14 and 21, and during lactation on day 1 and 4.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): N/A
- Time schedule for examinations: N/A

Oestrous cyclicity (parental animals):

Vaginal smears were made daily to determine if sperm is present.

Sperm parameters (parental animals):

Parameters examined in P male parental generations:
testis weight and epididymis weight.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, physical or behavioural abnormalities.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were sacrificed after 28 days of exposure.
- Maternal animals: Females achieving pregnancy were sacrificed on or shortly after lactation day 4. Sperm-positive female 8135 of the low-dose group, that was not pregnant was sacrificed 25 days after copulation. Female A105 of the control group that did not show evidence of copulation after the end of the 14 day mating period was housed individually until sacrifice (16 days after the last day of the mating period).

GROSS NECROPSY
- Gross necropsy searched for pathological changes

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated below were prepared for microscopic examination and weighed, respectively.
- Kidneys
- Liver
- Spleen
- Testes
- Thymus

Postmortem examinations (offspring):

No grossly malformed pups were observed. A necropsy was performed on stillborn pups and pups dying during the study; macroscopic abnormalities were recorded.

Statistics:

The resulting data were analyzed using the methods mentioned below.

- Clinical findings were evaluated by Fisher's exact probability test
- Body weight, body weight gain, organ weights and food consumption data were subjected to one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison tests.
- Fisher's exact probability test were used to evaluate the number of mated and pregnant females and females with live pups,
- Number of corpora lutea, implantation sites, live and dead pups were evaluated by Kruskal-Wallis nonparametric analysis of variance followed by the Mann Whitney U test,
- Histopathological changes were evaluated by the Fisher's exact probability test.

All analyses were two-sided. Group mean differences with an associated probability of less thean 0.05 were considered to be statistically significant.

Reproductive indices:

- 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 if females placed with males) x 100
- Male fertility index = (number of males that become sire / 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 implantation sites - number of pups born alive
- Pre-implantation loss = [(number of corpora lutea -number of implantation sites/ number of corpora lutea] x 100
- 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 1 = (number of dead pups on day 1 / total number of pups on day 1) x 100
- Pup mortality day 4 = (number of dead pups on day 4 / total number of pups on day 4) x 100
- Sex ratio day 1= (number of live male pups on day 1 / number of live pups on day n) x 100
- Sex ratio day 4= (number of live male pups on day 4 / number of live pups on day n) x 100
- Viability index = (number of surviving pups on day 4 / number of live born pups) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs were observed in male and female parental animals.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortalities were observed in male and female parental animals.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean body weight of the male and female animals was comparable between the control and the Thermolite 9XX-treated groups. Apart from a statistically significant increase in body weight change of the male animals of the low-dose group during the entire study (day 0-14), no remarkable changes in growth were observed. This change was not considered to be related to treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Mean test substance intake during the premating, gestation and lactation period is summarized in Table C.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

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):

Microscopic examination of the sampled organs of the control and high-dose groups at the end of the main study did not reveal treatment-related histopathological changes. The histopathological changes observed are common findings in rats of this age and strain, they were about equally distributed amongst the groups or they occurred in only one or a few animals. Therefore, they were considered to be not related to treatment.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

In each group 12 females were placed with males. All females of the Thermolite 9XX¬treated groups were mated within 4 days. One control animal (A 105) was not mated within the mating period of 2 weeks. Pre-coital time was comparable for all groups. The number of pregnant females and the number of dams with livebom pup and the number of males that became sires amounted to 11, 11, 12 and 12 for the control, 200, 500 and 1250 mg Thermolite 9XX/kg diet groups, respectively. The mating index, the male and female fertility index, and the female fecundity index ranged from 92-100%. The gestation index was 100% in all groups. The duration of gestation was comparable between the groups. The number of corpora lutea, implantation sites and lost implantation and the calculated indices, post-and pre-implantation loss, were comparable between the control and the Thermolite 9XX-exposed groups.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

>= 1 250 mg/kg diet

Sex:

male/female

Basis for effect level:

other: Parental toxicity and fertility. This concentration is equivalent to 71.8 mg/kg bw/d in males and to 95.7 mg/kg bw/d in females.

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

No remarkable differences in the findings were observed in the control and the treated groups. The findings observed are normal for pups of this age and strain.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

The number of litters with liveborn pups was 11, 11, 12 and 12 for the control, 200, 500 and 1250 mg MOT(EHMA)/kg diet groups, respectively. There were no still born pups. The mean number of pups delivered per litter in the control, 200, 500 and 1250 mg MOT(EHMA)/kg diet groups was 9.7, 11.2, 10.6 and 11.3, respectively. The pup mortality (PN 1-4) of the control group, 21%, was higher than that of the exposed groups and for that reason a statistically significant decrease in pup mortality was observed in the MOT(EHMA)-exposed groups (2.4, 7.9 and 4.4%). One litter of the control group (A113) was lost entirely and darn A117 of the control group lost 10 of her 14 pups.
No difference was observed in the sex ratio between the groups.
In conclusion, no effects were observed on litter size parameters at any dose levels.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Pup weights and pup weight changes between PN 1 and PN 4 were comparable between the MOT(EHMA)-treated and the control groups.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic observation of the 10 dead pups of dam A117 of the control group did not reveal any abnormalities.

Histopathological findings:

not examined

Other effects:

not examined

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 1 250 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Developmental toxicity. This concentration is equivalent to 71.8 mg/kg bw/d in males and to 95.7 mg/kg bw/d in females.

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Table C. Test substance intake (mg MOT(EHMA)/kg bodyweight/day)

	control	200 mg/kg diet (low dose)	500 mg/kg diet (mid dose)	1250 mg/kg diet (high dose)
	Mean	Mean (range)	Mean (range)	Mean (range)
Premating period
Males	0	11.4 (11.1-11.7)	30.1 (29.9-30.3)	75.4 (73.4-77.4)
Period after mating (week 3-4)
Males	0	10.2	28.1	68.2
Premating period
Females	0	13.4 (13.1-13.8)	33.7 (32.8-34.6)	84.6 (81.5-87.8)
Gestation period
Females	0	13.1 (10.7-14.6)	32.3 (26.6-35.5)	81.7 (66.9-90.6)
Lactation period
Females	0	18.2	45.6	120.9
Overall intake males		10.8	29.1	71.8
Overall intake females		14.9	37.2	95.7

Applicant's summary and conclusion

Conclusions:

Under the condition of this reproduction and developmental toxicity screening study in rats, the oral administration of octyltin tris (2-ethylhexylmercaptoacetate) (MOT(EHMA)) at 200, 500 and 1250 mg/kg diet was well tolerated at all dose-levels.
The no adverse effect level (NOAEL) for parental toxicity and fertility toxicity is 1250 mg/kg diet [approx. 72 and 96 mg octyltin tris (2-ethylhexylmercaptoacetate) (Thermolite 9XX)/kg body weight/day for male and female animals, respectively].

Executive summary:

The objective of this study was to provide data on the possible effects of the test substance octyltin tris (2-ethylhexylmercaptoacetate) (MOT(EHMA)) (CAS reg. no. 27107-89-7) on the reproductive performance of male and female WistarCrl:WI(WU)rats and on the growth and development of the offspring after oral administration via the diet (OECD test guideline 421). Groups of 12 rats per sex were dosed via the diet with 0, 200, 500 and 1250 mg MOT(EHMA)/kg diet as follows:

-         for males, throughout the pre-mating period (2 weeks), during the mating and post-mating periods until sacrifice (in total 28 days)

-          for females, throughout pre-mating (2 weeks) and mating periods, during gestation and lactation, until lactation day 4 or shortly thereafter (approximately 6 weeks).

No mortalities and no remarkable clinical signs were observed in the male and female animals.

Mean body weights of male and female animals were comparable between the control and the MOT(EHMA)-treated groups during the premating and mating period of the males and during the premating, mating period, gestation and lactation periods of the females.

Mean food consumption of the male and female animals was comparable between the control and the MOT(EHMA)-treated groups during the entire study.

No effects were observed on the mating, fertility and gestation indices at all dose-levels.

The litter data and the development of the pups during lactation were not affected by the treatment at all dose levels.Pup weights and pup weight changes between postnatal day (PN) 1 and 4 were comparable between the MOT(EHMA)-treated and the control groups.  

In conclusion, no effect was recorded on fertility and developmental toxicity.

Mean absolute and relative organ weights (testes, epidydimides and thymus) of the male animals were comparable in all groups.

In pregnant females, no effect was observed in the 200 mg/kg diet group while a statistically significant decrease of absolute and relative thymus weight was observed in the 500 and 1250 mg/kg diet groups. This effect was most probably of less significance for the following reasons:

-          the values found for the control group were quite high and outside the historical control range

-          the absolute and relative thymus weights of the 500 mg/kg diet and the 1250 mg/kg diet group were not statistically significantly decreased compared to the 200 mg/kg diet group

-          at microscopic examination no effects were observed in the thymus of the pregnant female animals of the 1250 mg/kg group.

Macroscopic examination at necropsy did not reveal treatment-related findings.

Microscopic examination of the sampled organs (male: testes, epididymides, seminal vesicles, prostate, thymus; female: ovaries, uterus, thymus) of the control and high-dose groups at the end of the main study did not reveal treatment-related histopathological changes in the 1250 mg MOT(EHMA) group.

Under the condition of this reproduction and developmental toxicity screening study in rats, the oral administration of octyltin tris (2-ethylhexylmercaptoacetate) (MOT(EHMA)) at 200, 500 and 1250 mg/kg diet was well tolerated at all dose-levels.

The no adverse effect level (NOAEL) for parental toxicity and fertility toxicity is 1250 mg/kg diet [approx. 72 and 96 mg octyltin tris (2-ethylhexylmercaptoacetate/kg/day) (MOT(EHMA))/kg body weight/day for male and female animals, respectively].