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Effects on fertility

Description of key information

Under the condition of the 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].

Link to relevant study records
Reference
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
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
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
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
Dose / conc.:
200 mg/kg diet
Dose / conc.:
500 mg/kg diet
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
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
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: 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.
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.
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
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.
Key result
Reproductive effects observed:
no

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
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].

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
72 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available study was conducted under GLP conditions and in accordance with standardised guidelines. The quality of the database is therefore considered to be high.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

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.  No effects or abnormalities were observed in pups or litter size at any dose level.

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 weights was observed in the 500 and 1250 mg/kg diet groups.This effect was considered most probably of less significance because:

- 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 diet group

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

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

Effects on developmental toxicity

Description of key information

An OECD 414 guideline study is available (theuns-van Vliet, 2014). The dose-elevels were selected based on a range finding study. NOAEL (maternal toxicity) = 89 mg/kg bw/day; NOAEL (developmental toxicity) = 208 mg/kg bw/day. OECD 414, EU Method B.31. Thenus van-Vliet (2014)

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
7 July 2013 to 1 August 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
other: Wistar (RccHan:WIST)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: Approximately 13 weeks old at mating
- Weight at study initiation: On day 0 of gestation, the female group mean body weights were 217.92 to 219.41 g
- Fasting period before study: No
- Housing: The animals were kept in macrolon cages with wood shavings as bedding material and strips of paper and a wooden block as environmental enrichment. During the quarantine and acclimatisation periods, the animals were housed in groups of 4 per sex. For mating, one male and two females were housed together. Mated females were housed individually in macrolon cages, which were placed in another cage rack.
- Diet: Feed was provided ad libitum. The rats received a cereal-based (closed formula) rodent diet. The diets were provided as a powder to the rats in stainless steel cans, covered by a perforated stainless steel plate to prevent spillage.
- Water: Drinking water was provided ad libitum. Each cage was supplied with domestic mains tap-water in polypropylene bottles, which were cleaned weekly and filled as needed.
- Acclimation period: approximately 12 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 23 °C
- Humidity: at least 65 % (relative)
- Air changes: ca. 10 air changes per hour
- Photoperiod: Lighting was artificial with a sequence of 12 hours of light and 12 hours of dark.

IN-LIFE DATES: From: 26 June 2013 To: 1 August 2013
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Mixing: the test material was administered in the powder diet. The test material was incorporated in the basal diet by mixing in a mechanical blender.
- Storage temperature of food: <-18 °C. One batch of experimental diets was divided into portions that were stored in plastic bags in a freezer.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Five samples of each test diet (500, 1250 and 3000 mg/kg diet) prepared on 5 July 2013, taken at different locations in the feed container, were analysed.
The stability of the test material in the feed was determined for the concentration level of 3000 ppm only as for the other concentrations used (500 and 1250 ppm) the stability under similar conditions and at similar dietary levels was confirmed in a previous study.

The method used for the quantitative analysis of the test material in diet was validated for the high-dose 3000 mg/kg level; the method had already been validated for the 0, 500 and 1250 mg/kg levels. In addition, in this study the stability in diet was only tested for the high-dose 3000 mg/kg level as the test material had also previously found to be stable in diet up to the 1250 mg/kg level when stored at room temperature in the animal room for 7 days and at <-18 °C for at least 4 weeks.


TEST MATERIAL ANALYSIS IN THE FEED
The concentration of the test material in the diet was determined using the following procedure: after addition of an internal standard to the diet, samples were extracted with acetate buffer (pH 4.5). Derivatisation was performed by adding 20 % sodium tetraethyl borate (STEB) solution in tetrahydrofuran (THF). After addition of hexane, the mixture was shaken and incubated at 60 °C. The hexane extract was cleaned up by adding 2 M HCl and shaking. Samples were analysed with Gas Chromatography – Mass Spectrometry (GC-MS). Quantitation was obtained by comparison of Q values (ratio peak area test material / peak area internal standard) in the sample extracts with those in calibration solutions containing known amounts of the test material.

VALIDATION CRITERIA
Before analysis of study samples, the analytical method was validated by analysing three spiked samples of the 3000 mg/kg dose level, to conform to the following criteria:
> Linearity: the correlation coefficient of the calibration curve should be greater than or equal to 0.99;
> Recovery: the mean recovery of the test material from the diet should be between 80 and 120 %;
> Repeatability: the relative standard deviation in the percentage recovery, when the recovery test is performed three times, should be less than 15 %.
With respect to specificity: the signal obtained for samples should be corrected for the signal obtained with blank samples in case the signal obtained for blank samples is ≥5 % of the signal obtained for low-dose samples.

- Preparation of validation samples
Validation samples with nominal concentration of 3000 mg/kg diet were prepared in triplicate by adding an aliquot of the test material in methanol stock solution to 1 g diet. All validation and blank samples were extracted as described above and analysed as described below.

SAMPLE PREPARATION
Study samples were analysed in duplicate. Test material was extracted from the diet using the following method:
Approximately 2 g diet for the 0, 500 and 1250 mg/kg level and 1 g diet for the 3000 mg/kg level was accurately weighed and 50 μL internal standard solution (approximately 0.6 mg/mL methanol), 20 mL methanol, 5 mL acetate buffer pH 4.5 (approximately 80 g sodium acetate/L milliQ water, acidified to pH 4.5 using acetic acid), 2.0 mL 20 % STEB in THF and 20.0 mL hexane was added. The mixture was shaken at 200 rpm for 45 minutes and placed in a water bath at 60 °C for 45 minutes. To approximately 3 mL hexane extract, approximately 3 mL 2 M HCl was added and the mixture was shaken at 200 rpm for 30 minutes. Sample hexane extracts were diluted with blank diet hexane extract to obtain expected concentrations within the calibrated concentration range.
The following dilutions were prepared: 500 mg/kg samples were diluted 40 times and 1250 and 3000 mg/kg samples were diluted 100 times.

CHROMATOGRAPHY
The concentration of test material in the diluted extracts of the validation samples and the study samples was determined using GC-MS. The GC-MS conditions were as follows:
- Injection: 1 μL, splitless time 1.2 min, PTV (programmable temperature vaporisation) 60 °C x 14.5 °C/min; 300 °C (5 min)
- Carrier gas: Helium
- Column: HP 5MS, 30 m, 0.25 mm ID, 0.25 μm film
- Column temperature: 45 °C (3 min) x 5 °C/min x 80 °C (0 min) x 15 °C/min x 260 °C (15 min)
- Detection: Mass Spectrometry (MS). Quantification: internal standard m/z 277; test material m/z 291. Qualifiers: internal standard m/z 179; test material m/z 179.
- Source temperature: 250 °C
- Interface temperature: 200 °C

CALIBRATION
On each day that the concentration of the test material in the diet was analysed, 2 stock solutions of the test material were prepared by accurately weighing approximately 100 mg into 20 mL volumetric flasks and bringing to volume with methanol. The stock solutions were diluted 50 times with methanol. From the 2 diluted stock solutions 6 calibration samples were prepared by adding 50, 100, 200, 300, 400 and 500 μL from the stock solutions to approximately 2 grams of diet, in an alternating fashion. The calibration samples were extracted and analysed as described previously. Calibration graphs were constructed by plotting the Q value (ratio peak area test material/peak area internal standard) against the concentration of the test material.

CALCULATION
The concentration of the test material in the sample extracts was calculated using the calibration graph. The concentration in the samples was calculated using the following formula:
C = A * D * B
Where:
C = concentration of test material in diet samples (mg/kg)
A = concentration of test material determined in diet sample extracts (expressed as mg/kg)
D = dilution factor for hexane extract
B = correction factor (2 g/weight of sample analysed (g))

DETERMINATION OF HOMOGENEITY AND CONTENT OF TEST MATERIAL IN DIET
- Homogeneity: The homogeneity was assessed in the batch of diets prepared on 5 July 2013. Five samples of each test diet (500, 1250 and 3000 mg /kg diet), taken at different locations in the feed container, were analysed.
For each concentration level, a one-way analysis of variance (ANOVA) was performed using the sample location (1 to 5) as grouping factor. An associated F-value with probability p<0.01 was considered to be significant (i.e. the mean concentrations differ significantly at the five locations in the feed container). The test material was considered to be homogeneously distributed in the diet if p ≥0.01 and/or if the relative standard deviation (RSD) between the mean concentrations at the five locations was less than or equal to 15 %.
- Stability: The stability in diet under simulated experimental conditions was examined in the batch of diets prepared on 5 July 2013. Samples were analysed at t = 0, after storage at ambient temperature in the animal room for 4 days and after storage in the freezer (< -18 °C) for 4 weeks.
For the 3000 ppm concentration level, a one-way analysis of variance (ANOVA) was performed using time as grouping factor. An associated F-value with probability p<0.01 was considered to be significant (i.e. the difference between the determined concentrations on the first day (t = 0) and the day on which the analyses were repeated, was significant). The test material was considered to be stable in the diet 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.
- Content: The content of test material was determined in the batches of diets prepared on 5 July 2013.
The content of the test material in diet was considered to be “close to intended” if the mean measured concentration was between 80 and 120 % of the intended concentration.

RESULTS AND DISCUSSION
VALIDATION OF THE ANALYTICAL METHOD
- Linearity: All calibration graphs had a correlation coefficient of ≥0.99 and were therefore considered to be linear.
In total 2 calibration curves were recorded, which had correlation coefficients which had correlation coefficients of 1.000 (homogeneity/content/validation series, 5 July 2013) and 0.999 (stability series, 2 August 2013).
- Specificity: The chromatogram of the extract of blank diet did not show a peak at the position of the test material.
- Recovery: The mean recovery obtained for the 3000 mg/kg level was 88.8 %, which met the validation criteria set for recovery.
- Repeatability: The relative standard deviations (RSD) in the mean recovery was 2.3 %, which met the criterion set for the RSD in the percentage recovery.

HOMOGENEITY, STABILITY AND CONTENT OF TEST MATERIAL IN DIET AND QUALITY CONTROL
- Homogeneity: The RSD between the mean concentrations at five different locations was <15 % for all dose levels (500, 1250 and 300 mg/kg diet), i.e. the test material was considered to be homogeneously distributed in the diets.
- Stability: The mean determined concentration after storage at room temperature for 4 days and after storage at <-18 °C for 4 weeks was between 80 and 120 % of the mean determined concentration at t = 0. Therefore the test material was considered to be stable under these storage conditions at a concentration level of 3000 ppm.
- Content: The concentration of the test material was close to intended (80 to 120 %) for all diets at all dose levels.

CONCLUSIONS
The method used for the quantitative analysis of the test material in diet met the pre-set validation criteria. The test material was homogeneously distributed in test diets prepared for all dose levels. The test material was stable in diet for 4 days at room temperature and for 4 weeks at <-18 °C. The content of the test material in the diet was close to the intended concentration at all dose levels.
Details on mating procedure:
- Impregnation procedure: Cohoused
- M/F ratio per cage: Two females were caged with one male
- Length of cohabitation: Until a sperm positive smear was detected
- Proof of pregnancy: Every consecutive morning, vaginal examinations were made to ascertain copulation by detection of sperm cells in the vaginal smear. The day on which sperm was detected in the vaginal smear was defined as gestation day 0.
Duration of treatment / exposure:
From gestation day 6 up to and including gestation day 19.
Frequency of treatment:
Continuous in diet
No. of animals per sex per dose:
24 mated females per dose group
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Doses were selected on the basis of a range-finding study.
One control group and three test groups, each comprising 5 mated females, were administered different dose levels of the test material by diet from gestation day 6 up to gestation day 19.
The animals received diets containing 0, 500, 1250 and 3000 mg/kg. On gestation day 21 the animals were sacrificed and caesarean section was performed.
In-life parameters included morbidity, mortality, body weight and food consumption. At sacrifice thymus weight, uterus weight, ovary weight, number of corpora lutea, number of implantation sites, early and late resorptions, number of live and dead foetuses, foetus weight and placenta weight were recorded. In addition, foetuses were examined for external abnormalities and dams were observed for gross anatomical changes.
There were no treatment-related clinical signs. A reduced body weight gain was seen in the low- and high-dose groups from gestation day 19 to 21. A non-statistically significant reduced food consumption was observed in the low- and high-dose groups from gestation day 19 to 21.
A statistically significant decreased absolute and relative thymus weight was noted in the low- and high-dose groups. There were no treatment related effects on the number of corpora lutea, implantation sites, live and dead foetuses, the number of resorptions or on pre- and post-implantation loss.
Decreased foetal weight was observed in the dosing groups (possibly due to the lower mean number of foetuses as observed in the control group). There were no treatment-related foetal external observations.
Based on the food consumption the mean test material intake during the gestation period was 0, 40.14, 103.64 and 240.75 mg/kg body weight/day for the control, low-, mid- and high-dose groups, respectively.
It was concluded that dietary administration of 0, 500, 1250 and 3000 mg/kg from gestation day 6 up to gestation day 19 resulted in maternal toxicity in the treated pregnant animals as evidenced by a decreased thymus weight.
Maternal examinations:
CAGE SIDE AND DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Each selected, mated female was observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. On working days all cages were checked again in the afternoon for dead or moribund animals to minimise loss of animals from the study. All abnormalities, signs of ill health or reactions to treatment were recorded.
The observations included, but were not restricted to, the following: general, respiration, mouth, abdomen, faeces, behaviour, skin/fur, head, nose, eyes, ears, penis, perineum, extremities (legs), tail, testes, urethra and urine.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights of the selected, mated females were recorded on gestation days (GD) 0, 6, 10, 14, 17 and 21.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: The food consumed for each mated female was measured over the periods: gestation days 0 to 6, 6 to 10, 10 to 14, 14 to 17 and 17 to 21. The results are expressed in g per animal per day and g per kg body weight per day.

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
The females were killed by decapitation after CO₂/O₂ anaesthesia on gestation day 21 and examined for gross abnormalities. At autopsy, maternal tissues showing severe macroscopic abnormalities were sampled and fixed in a neutral, aqueous phosphate buffered 4 % solution of formaldehyde. It was decided not to examine this material microscopically.
At necropsy the thymus of all dams was preserved in a neutral aqueous phosphate-buffered 4 % solution of formaldehyde. The thymus was weighed after fixation. The thymus was not microscopically examined for any animal.
Ovaries and uterine content:
The uteri (including the foetuses), ovaries and placentas of all females killed on GD 21 were examined for the following parameters:
- number of corpora lutea
- number of implantation sites ( if necessary made visible following Salewski)
- number of early and late resorptions
- gross evaluation of placentas
The following were weighed:
- uterus, containing placentas and foetuses
- uterus, empty
- ovaries
- live foetuses (individually) and corresponding placentas
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: No data

The uteri (including the foetuses) of all females killed on GD 21 were examined for the following parameters:
- number of live and dead foetuses
- sex of the foetuses
- number of grossly visible malformed foetuses and foetuses with external abnormalities

FOETOPATHOLOGICAL EXAMINATION
Foetuses were sacrificed by hypothermia. Subsequently, half of the foetuses of each litter of the study were fixed in Bouin's fixative, examined for soft tissue anomalies according to a method modified after Barrow and Taylor (1969) and then discarded.
The other half of the foetuses were fixed in 70 % alcohol, subsequently partly eviscerated, and then cleared in potassium hydroxide and stained with Alizarin Red S modified after Dawson (1926) and examined for skeletal abnormalities and then retained.
Statistics:
Tests were generally performed as two-sided tests with results taken as significant where the probability of the results was p <0.05 or p <0.01.
Continuous data were subjected to the ‘Decision tree for continuous data’ and dichotomous data to the ‘Decision tree for dichotomous data’.
Indices:
REPRODUCTIVE PERFORMANCE
For each group the following indices were calculated:
- female fertility index = (no. of pregnant females / no. of inseminated females) x 100
- pre-implantation loss = [(no. of corpora lutea – no. of implantation sites) / no. of corpora lutea] x 100
- post-implantation loss = [(no. of implantation sites – no. of live foetuses) / no. of implantation sites] x 100
- gestation index = (no. of females with live foetuses / no. of females pregnant) x 100
- sex ratio = (no. of live male foetuses / no. of live foetuses) x 100
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Animals in all groups including the control group showed piloerection in the last days of gestation. This is considered a normal physiological condition related to parturition and was not considered to be related to treatment.
Dermal irritation (if dermal study):
not examined
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Initial body weight gain from the start of the treatment (day 6) until day 10 of gestation was statistically significantly decreased in the high dose group as compared to the control group (40 %, p<0.01). Mean body weights were however, comparable in all groups.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In the high dose group food intake was statistically significantly lower in the intervals from gestation day 6 to 10 (10 %, p <0.01) and from day 10 to 14 (7 %, p<0.05) as compared to the control group both if expressed per animal and per kg body weight.
This was considered to be related to the start of the treatment and the presence of test material in the diet. Thereafter, food consumption in the high dose group became comparable to the control group. No effects on food consumption were observed in the low and mid dose groups.
The test material intake during gestation was calculated from the nominal concentrations of the test- and control substances in the diets and the food consumption and body weight of the animals.
The daily mean test material intake from day 6 to 19 was 36 mg/kg body weight in the low dose group, 89 mg/kg body weight in the mid dose group and 208 mg/kg body weight in the high dose group.
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:
effects observed, treatment-related
Description (incidence and severity):
Mean ovary weight, mean full and empty uterus weight were comparable in all groups. Placenta weight was comparable in all groups.
A statistically significantly lower carcass weight was observed in females of the low dose group as compared to the control group. However, in absence of a dose response relationship, this was not considered to be treatment-related.
The mean absolute and relative thymus weights showed a dose-related decrease. Although this finding did not reach statistical significance, the decrease in absolute and relative thymus weight in the mid and high dose group was more than 10 %. In the low-dose group the decrease was only slight (≤5 %) and therefore not considered adverse.
Although the decreases in the mid and high dose group did not reach statistical significance, in view of the dose response relation it was considered to be an adverse and test material related effect.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
One female in the low dose group showed a yellow nodule in the abdominal cavity and one animal in the mid dose group showed a nodule with a diameter of 2 centimetres in the right axillary region of the mammary gland.
One animal in the mid dose group showed a uterus horn filled with red fluid and one animal in the high dose group showed a swollen uterus.
One animal in the low dose and one animal in the high dose group showed red discolouration and partly haemorrhage of the thymus, respectively.
Several animals distributed over all groups showed discolouration or ulcer of the stomach.
Based on the incidence and distribution, these observations are not considered to be treatment related.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on maternal toxic effects:
REPRODUCTIVE PERFORMANCE
Twenty-four animals per group were mated with males, resulting in 23, 21, 20 and 22 pregnant females in the control group, low dose, mid dose and high dose group, respectively.
One female in the control group started delivery before Caesarean section was performed (animal number 19, reported as “delivered early”). The animal was included in mean body weight and food consumption data, but excluded from Caesarean section data and pups were excluded from foetal evaluations.

CAESAREAN SECTION DATA
The mean number of corpora lutea, the mean number of implantation sites, the mean number of early and late resorptions were comparable in all groups.
No treatment related effects were observed on pre-implantation loss and post-implantation loss.
Dose descriptor:
NOAEL
Effect level:
35 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
208 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Fetal body weight changes:
no effects observed
Description (incidence and severity):
No effect on foetus weight was observed in the low dose group. Mean foetus weight was slightly, but statistically significantly, lower in the mid dose group (9 %, p<0.05) and the high dose group (9 %, p <0.05) as compared to the control group. For female foetuses the mean foetus weight was statistically significantly lower in the mid dose group (11 %, p <0.01) as compared to the control group. However, as compared to the historical background, the foetus weight in the control group was relatively high (historical control values are between 4.25 ± 0.5 and 4.7 ± 0.3). Based on the absence of a dose response relationship and because the mean pup weight was within the historical control data range at each dose level and for each sex, this was not considered to be treatment-related. The statistically significant difference is considered to be related to the relatively high mean pup weight in the control group.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There were no dead foetuses. The mean number of live foetuses were comparable in all groups.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The mean percentages of male littermates were comparable in all groups.
Changes in litter size and weights:
no effects observed
Anogenital distance of all rodent fetuses:
not examined
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
- Malformations: One foetus in the mid dose group showed acephalostomia. Based on the incidence and distribution this was not treatment related.
No other external malformations were observed.
- Variations: Several foetuses (1, 1, 4 and 4 in the control, low dose, mid dose and high dose group, respectively) showed subcutaneous haemorrhages. One foetus in the high dose group was small. Based on the incidence and distribution this was not treatment related.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
- Malformations: One foetus in the mid dose group showed microcephaly and one foetus in the mid dose group showed scoliosis. Based on the incidence and distribution this was not related to treatment.
No other skeletal malformations were observed.
- Variations: Skeletal variations observed included variations in ossification status of the bones, supernumerary ribs, wavy ribs and irregular shape of the sternebra. Based on the incidence and distribution of the external, visceral and skeletal malformations and variations observed, no test material related effects were observed. At the dose levels tested the test material showed no teratogenic potential.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
- Malformations: Three foetuses in the control group, 1 foetus in the mid dose group and 2 foetuses in the high dose group showed a misshapen lens. Based on the incidence and distribution these findings were not treatment related.
No other visceral malformations were observed.
- Variations: Visceral variations observed included a folded retina, blood-filled pericardium, dilation of the renal pelvis, distended bladder and several variations in the ureters. These observations were noted in foetuses from all groups. One foetus in the low dose group showed dark content in the mouth. A discolouration of the stomach content was observed in foetuses from all groups and was considered to be related to the fixating and processing of the foetuses.
Other effects:
not examined
Remarks on result:
not measured/tested
Remarks:
In the absence of developmental effects the NOAEL for prenatal developmental toxicity in the rat was 3000 mg/kg in diet (corresponding to a daily mean test material intake of 208 mg/kg body weight).
Abnormalities:
effects observed, non-treatment-related
Key result
Developmental effects observed:
no

Table 1: Absolute and Relative maternal Thymus weights

 

Dose Group (mg/kg diet)

0

500

1250

3000

Terminal body weight (g)

Mean

SD

N

301.81

20.49

22

288.39

15.93

21

290.26

17.56

20

290.41

17.14

22

Thymus weight (g)

Mean

SD

N

0.2413

0.0949

22

0.2286

0.0891

20

0.2114

0.0580

19

0.2096

0.0658

22

Relative thymus weight (%)

Mean

SD

N

0.0810

0.0367

22

0.0792

0.0337

20

0.0726

0.0179

19

0.0720

0.0221

22

 

Table 12: Foetal and Placental Weights

Parameter

Dose Group (mg/kg diet)

0

500

1250

3000

Foetus weight (g)

Mean

SD

N

4.7

0.3

21

4.3

0.5

20

4.3*

0.4

20

4.3*

0.5

22

Foetus weight - male foetuses (g)

Mean

SD

N

4.7

0.4

21

4.4

0.4

19

4.4

0.4

20

4.4

0.6

22

Foetus weight - female foetuses (g)

Mean

SD

N

4.6

0.3

21

4.3

0.5

20

4.1**

0.4

20

4.3

0.5

22

Placenta weight (g)

Mean

SD

N

0.5

0.1

21

0.5

0.1

20

0.5

0.1

20

0.5

0.1

22

Placenta weight - male foetuses (g)

Mean

SD

N

0.5

0.1

21

0.5

0.0

19

0.5

0.1

20

0.5

0.1

22

Placenta weight - female foetuses (g)

Mean

SD

N

0.5

0.0

21

0.5

0.2

20

0.5

0.1

20

0.5

0.1

22

Conclusions:
Under the conditions of this study, the NOAEL for maternal toxicity was 500 mg/kg diet (corresponding to a daily mean test material intake of 36 mg/kg body weight). In the absence of developmental effects the NOAEL for prenatal developmental toxicity in the rat was 3000 mg/kg in diet (corresponding to a daily mean test material intake of 208 mg/kg body weight).
Executive summary:

A developmental toxicity study was carried out with the test material under GLP conditions and in accordance with the standardised guidelines OECD 414 and EU Method B.31.

The objective was to provide data on the possible effects of the test material on pregnant female Wistar rats and the development of the embryo and foetus consequent to continuous oral administration in the diet from gestation day (GD) 6 until GD 19.

Rationale for dose-level selection:

In a dose range finding study with MOTE (TNO report V20347, 2014) in pregnant females, dose-levels were 500, 1250 and 3000 ppm. Decreased in thymus weight (known organotin target organ) were observed at all dose-levels but due to low number of pregnant animals (3 -5 per group), the effect was not dose-related but important (-48%, -19%, -37%, in the low, mid and high-dose level, respectively). In the main study the same dose-levels were selected (see below) as based on the range finding study the observed decreased in thymus weight at all dose-levels was a suficient indicator of maternal toxicity.

The test material was given at constant concentrations of 0 (control), 500 mg/kg diet (low-dose), 1250 mg/kg diet (mid-dose), and 3000 mg/kg diet (high-dose). During the in-life phase clinical signs, maternal body weight and food consumption were recorded. At Caesarean section, females and foetuses of all groups were macroscopically examined. Foetuses, placentas and reproductive organs were weighed. Foetuses were further processed for visceral and skeletal examination.

It was determined that the test material was homogeneously distributed at all dose levels and was stable in the diet after storage in the animal room for four days or in a freezer (<-18 °C) for four weeks. The content of the test material was close to intended at all levels.

Administration via the diet did not result in mortalities or morbidities. No effect on food consumption and body weight was observed in the low and mid-dose levels. Maternal toxicity was susbtanciated by a transient decrease in body weight gain (-40 %, p <0.01) and food consumption on days 6 to 10 (10 %, p <0.01) and 10 to 14 (7 %, p <0.01) were observed in the high dose group as compared to the control group during the first week of the treatment period.

Mean test material intake was 36, 89 and 208 mg/kg body weight/day for the animals fed 500, 1250, and 3000 mg/kg diet, respectively.

No differences were observed on reproductive performance. The mean number of implantation sites, resorptions and live foetuses were comparable in all groups.

No adverse effect on thymus weight was observed at the low dose level. The mean absolute and relative thymus weights were decreased (>10 %) in the mid and high dose group. Immunotoxic compounds containing octyltin are known to affect thymus weight in pregnant/lactating females. Therefore, although the decrease in thymus weight in the mid and high dose group did not reach statistical significance, it was considered to be an adverse and test material-related effect.

Statistically significant decreased mean foetal weight in both sexes at the mid and high dose levels were considered to be related to a relatively high mean pup weight in the control group and were therefore not considered treatment related. Foetal external, visceral, and skeletal examinations did not reveal any treatment-related effects.

Daily administration of the test material at dose levels of 0, 500, 1250 or 3000 mg/kg diet to pregnant rats from gestation day 6 up to and including gestation day 19, resulted in slight maternal toxicity as evidenced by transient decreases in body weight gain and food consumption at 3000 mg/kg diet, and a decreased thymus weight at 1250 and 3000 mg/kg diet. No developmental toxicity was observed.

The NOAEL for maternal toxicity was therefore 500 mg/kg diet (corresponding to a daily mean test material intake of 36 mg/kg body weight). In the absence of developmental effects the NOAEL for prenatal developmental toxicity in the rat was 3000 mg/kg in diet (corresponding to a daily mean test material intake of 208 mg/kg body weight).

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
208 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available study was conducted under GLP conditions and in accordance with standardised guidelines. The quality of the database is therefore considered to be high.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

A developmental toxicity study was carried out with the test material under GLP conditions and in accordance with the standardised guidelines OECD 414 and EU Method B.31.

The objective was to provide data on the possible effects of the test material on pregnant female Wistar rats and the development of the embryo and foetus consequent to continuous oral administration in the diet from gestation day (GD) 6 until GD 19.

Rationale for dose-level selection:

In a dose range finding study with MOTE (TNO report V20347, 2014) in pregnant females, dose-levels were 500, 1250 and 3000 ppm. Decreased in thymus weight (known organotin target organ) were observed at all dose-levels but due to low number of pregnant animals (3 -5 per group), the effect was not dose-related but important (-48%, -19%, -37%, in the low, mid and high-dose level, respectively). In the main study the same dose-levels were selected (see below) as based on the range finding study the observed decreased in thymus weight at all dose-levels was a suficient indicator of maternal toxicity.

The test material was given at constant concentrations of 0 (control), 500 mg/kg diet (low-dose), 1250 mg/kg diet (mid-dose), and 3000 mg/kg diet (high-dose). During the in-life phase clinical signs, maternal body weight and food consumption were recorded. At Caesarean section, females and foetuses of all groups were macroscopically examined. Foetuses, placentas and reproductive organs were weighed. Foetuses were further processed for visceral and skeletal examination.

It was determined that the test material was homogeneously distributed at all dose levels and was stable in the diet after storage in the animal room for four days or in a freezer (<-18 °C) for four weeks. The content of the test material was close to intended at all levels.

Administration via the diet did not result in mortalities or morbidities. No effect on food consumption and body weight was observed in the low and mid-dose levels. Transient decreases in body weight gain (40%, p <0.01) and food consumption on days 6 to 10 (10%, p <0.01) and 10 to 14 (7%, p <0.01) were observed in the high dose group as compared to the control group during the first week of the treatment period.

Mean test material intake was 36, 89 and 208 mg/kg body weight/day for the animals fed 500, 1250, and 3000 mg/kg diet, respectively.

No differences were observed on reproductive performance. The mean number of implantation sites, resorptions and live foetuses were comparable in all groups.

No effect on thymus weight was observed at the low dose level. The mean absolute and relative thymus weights were decreased in the mid and high dose group (-10.4 and -11% relative weight, for the mid and high-dose group, respectively, compared to the controls), without attaining statistical significance. Compounds containing octyltin are known to affect thymus weight in pregnant/lactating females and therefore, although the decrease in thymus weight in the mid and high dose group did not reach statistical significance, it was considered to be a test material-related effect.

Statistically significant decreased mean foetal weight in both sexes at the mid and high dose levels were considered to be related to a relatively high mean pup weight in the control group and were therefore not considered treatment related. Foetal external, visceral, and skeletal examinations did not reveal any treatment-related effects.

In conclusion:

The following treatment-related effects were observed in the main study :

At 3000 ppm:

- Maternal toxicity was substantiated by the following treatment related effects :

A transient decrease in maternal body weight gain in the high dose-group from day 6-10 (-40%, p<0, 01)
A slightly decrease in maternal food consumption in the high dose-group from days 6-10 (-10%, p<0, 01) and 10-14 (-7%, p<0, 05),
A decrease in mean absolute and relative maternal thymus weight (-10%)
At 1250 ppm:
A decrease in mean absolute and relative thymus weight (-10%).
At  500 ppm:
No treatment-related effect.
ØAs recommended in the guideline graduated effects were observed in the study.

Daily administration of the test material at dose levels of 0, 500, 1250 or 3000 mg/kg diet to pregnant rats from gestation day 6 up to and including gestation day 19, resulted in some maternal toxicity as evidenced by transient decreases in body weight gain and food consumption, and decreased thymus weights at 1250 and 3000 mg/kg diet which, in the absence of histopathological examination, could be considered adverse. No developmental toxicity was observed.

According to the study report, the NOAEL for maternal toxicity was therefore 500 mg/kg diet (corresponding to a daily mean test material intake of 89 mg/kg body weight). In the absence of developmental effects the NOAEL for prenatal developmental toxicity in the rat was 3000 mg/kg in diet (corresponding to a daily mean test material intake of 208 mg/kg body weight).


Justification for selection of Effect on developmental toxicity: via oral route:
Only one study available

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the substance does not require classification with respect to fertility or developmental toxicity.

Additional information