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Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

No reproductive toxicity studies are available for the registered substance, trimethoxy(methyl)silane and its reaction products with 3-aminopropyltriethoxysilane and [3-(2,3-epoxypropoxy)propyl]trimethoxysilane (EC 701 -410 -9). The requirement for new reproductive toxicity testing will be reconsidered when the results of the proposed OECD 408 test on the registered substance are available, after approval from ECHA. As an interim measure, available reproductive toxicity data for the constituents are considered.


Block A

Trimethoxy(methyl)silane (CAS 1185-55-3)

 

In the oral Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test with trimethoxy(methyl)silane (CAS 1185-55-3), conducted according to OECD Test Guideline 422 and in compliance with GLP, the NOAEL for reproductive toxicity was at least 1000 mg/kg bw/day, the highest dose tested (Dow Corning Corporation, 2005).

 

Triethoxy(methyl)silane (CAS 2031-67-6)

 

In the oral Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test with triethoxy(methyl)silane (CAS 2031-67-6), conducted according to OECD Test Guideline 422 and in compliance with GLP, there were no treatment-related effects apparent for any of the reproductive endpoints. All females bred successfully and delivered live litters. Litter sizes were comparable for all groups. Differences in group mean values for the treated groups relative to the control group were small and none were found to be statistically significant. Therefore, exposure to triethoxy(methyl)silane was not associated with reproductive toxicity and a NOAEL of 750 mg/kg bw/day was identified (Bozo Research Center, 2012).


Trimethoxy(methyl)silane and its reaction products with [3-(2,3-epoxypropoxy)propyl]trimethoxysilane and 3-(trimethoxysilyl)propylamine (EC No. 701 -408 -8)

 

In the combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test conducted according to OECD Test Guideline 422 and in compliance with GLP, no reproductive or developmental toxicity was observed up to the maximum dose of 250 mg/kg bw/day. The NOAEL for reproductive and developmental effects was concluded to be greater than 250 mg/kg bw/day for trimethoxy(methyl)silane and its reaction products with [3-(2,3-epoxypropoxy)propyl]trimethoxysilane and 3-(trimethoxysilyl)propylamine.

Link to relevant study records

Referenceopen allclose all

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
19.11.2003 to 19.05.2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Qualifier:
according to guideline
Guideline:
other: USEPA OPPTS 870.3650
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: No data
- Age at study initiation: Nine weeks
- Weight at study initiation: Males: 294.2-351.5; Females: 200.2-260.2 g
- Fasting period before study: None
- Housing: individually housed in suspended wire-mesh cages (pregnant rats in shoebox cages)
- Diet (e.g. ad libitum): Ad libitum (except during FOB)
- Water (e.g. ad libitum): Ad libitum (except during FOB)
- Acclimation period: Six days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.2-22.5
- Humidity (%): 36.0-62.0
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 09.02.2004 To: 19.04.2005
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Conducted over nitrogen atmopshere. Test substance was placed into a volumetric flask and corn oil added to achieve the desired volume. The weight of the test substance added to the flask was used to calculate nominal dose solution concentrations. Dosing solutions were prepared at least once every two weeks consistent with the previously determined 15-day stability. The concentration, homogeneity and stability of the test substance in vehicle for at least 15 days.

VEHICLE
- Justification for use and choice of vehicle (if other than water): No data
- Concentration in vehicle: Various
- Amount of vehicle (if gavage): Up to 3 ml/kg
- Lot/batch no. (if required): 122K0131
- Purity: Considered 100%
Details on mating procedure:
A 1:1 mating ratio was used. After dosing on study day 14, the animals were paired by placing the lowest numbered ear tag reproductive group female within each group in the home cage of the male with the lowest numbered ear tag from the same group. Female animals were housed continuously with the same male until evidence of copulation was obtained. Females were evaluated daily for evidence of copulation, as indicated by either a vaginal copulatory plug or sperm in the vaginal smear. Day 0 of gestation was defined as the day evidence of copulation was obtained, at which time the female was returned to her home cage (shoebox cage).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of methyltrimethoxysilane (MTMS) in corn oil dosing solutions was determined prior to the beginning of the definitive study.
Duration of treatment / exposure:
Toxicity group females and males were treated for 28 and 29 days, respectively. Reproductive group females were treated for 14 days prior to the mating period, during the mating period, and then up to and including post partum day 3, for a total of up to 51 days.
Frequency of treatment:
Daily, seven days/week
Details on study schedule:
No further relevant details.
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
corn oil
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on a range-finding study
Parental animals: Observations and examinations:
Mortality/Morbidity: Animals were observed at least twice daily in their cages for moribundity and mortality throughout the in-life phase of the study.
Clinical observations:
Daily Observations: General clinical examinations were made at least once a day and were conducted immediately after dosing. The examinations included, but were not limited to, changes in the skin, fur, eyes, mucous membranes, respiratory, circulatory, autonomic and central nervous system functions, motor activity and behavior patterns. Findings were recorded for individual animals. General clinical examinations were not performed on days when detailed physical examinations were performed.
Detailed Physical Examinations: All animals received a detailed physical examination once before the first dose administration (to allow for within-subject comparisons), and weekly thereafter. Examinations were made outside the home cage in a standard arena at approximately the same time each day. Observations were detailed and carefully recorded. Examinations included, but were not limited to, changes in skin, fur eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity. Changes in gait, posture and response to handling as well as the presence of clonic or tonic movement, stereotypies, difficult or prolonged parturition or bizarre behavior were recorded. The presence or absence of findings was recorded for individual animals.

Body weights and food consumption were recorded weekly. Additional body weights for reproductive group females were obtained on gestational day 0, 7, 14, and 20, and within 24 hours of parturition, and on postnatal day 4. Individual food consumption was determined for each group following group specific schedules. In addition, detailed clinical observations (functional observational battery [FOB] conducted out of the home cage) and locomotor activity were evaluated for all adult male and toxicity phase females once prior to the start of test article administration (baseline evaluations) and again during the last week of the test article administration. Blood samples were collected from males and toxicity group females on the day of scheduled termination for analysis of hematology and serum chemistry parameters.
Litter observations:
All reproductive phase females were allowed to deliver and rear their offspring to lactation day 4; surviving dams and pups were euthanized and examined on lactation day 4.On the day parturition was initiated (PND 0), the pups were sexed and examined for gross malformations, and the numbers of still born and live pups were recorded. Individual gestation length was calculated using the date delivery started. Abnormal behavior of the offspring was recorded. The dam and litter remained together until PND 4.

Mean measured parameters were calculated for:
Days of gestation
Undetermined sex
Male pups/litter
Female pups/litter
Males/Females per litter
Total pups/litter
Viable (live) pups/litter
Viable/Total pups per litter

Initial litter weight at parturition (g)
Initial average pup weight at parturition (g)
Final litter weight at PND 4 (g)
Final average pup weight at PND 4 (g)

Total number of implants
Corpora counts
Postmortem examinations (parental animals):
Clinical pathology assessments (hematology and serum chemistry) and macroscopic and microscopic examinations (including organ weights) were also performed on the appropriate groups of adult males and toxicity phase females. For females that delivered or had macroscopic evidence of implantation, the numbers of former implantation sites and corpora lutea were recorded. Recognizable fetuses for the females euthanized in extremis were examined externally and preserved in 10% neutral-buffered formalin. For females that failed to deliver, a pregnancy status was determined. Uteri with no macroscopic evidence of implantation were opened and subsequently placed in a 10% ammonium sulfide solution for detection of early implantation loss.
Postmortem examinations (offspring):
Intact offspring dying from PND 0 to 4 were necropsied. Cannibalized pups were discarded without necropsy. Tissues were preserved in 10% neutral-buffered formalin for possible future histopathologic examination only as deemed necessary by gross findings. The carcass of each pup was then discarded.
Statistics:
Reproductive parameters with the exception of litter size were analyzed using an ANCOVA (Analysis of Covariance) with liter size as the covariate. Litter size was analyzed using an ANOVA.
Reproductive indices:
Male (Female) Mating Index (%)
Male Fertility Index (%)
Male Copulation Index (%)
Female Fertility Index (%)
Female Conception Index (%)
Offspring viability indices:
On the day parturition was initiated (PND 0), the pups were sexed and examined for gross malformations, and the numbers of still born and live pups were recorded.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Thirty percent of the animals in the 50 mg/kg bw/day dose group and 100 % of the animals in the 250 and 1000 mg/kg bw/day dose groups exhibited a transient period of salivation and/or abnormal inactivity at least once over the course of treatment immediately after dosing.
Mortality:
mortality observed, treatment-related
Description (incidence):
There were two unscheduled deaths (one female each at 0 and 50 mg/kg  bw/day); both were related to dosing errors.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant decreases in body weight gain and food consumption were noted for 1000 mg/kg bw/day group males. 
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No statistically significant differences in treatment group maternal food consumption relative to control group animals.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
A marked increase in prothrombin time was observed for males at 250 and 1000 mg/kg bw/day whereas females were unaffected. Exposure was also associated with increased blood platelet concentration for males and females, and increased red blood cell concentration in males at 1000 mg/kg bw/day.
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Exposure to MTMS was associated with organ weight and/or histomorphological changes in males (liver, thymus, thyroid, duodenum, jejunum) and females (liver, thyroid, duodenum, jejunum, and adrenal gland) at dose levels at or above 250 mg/kg bw/day.  
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
No treatment-related effects were observed in any of the reproductive parameters evaluated. All females bred successfully and delivered live litters. 
"Number pregnant per dose level: 10
"Number aborting: 0
"Number of resorptions, early/late if available: None detected.
"Number of implantations: Group Mean (standard deviation) Control: 15 (2.2); 50 mg/kg: 16 (2.0); 250 mg/kg: 16 (1.4); 1000 mg/kg: 16 (1.9)
"Number of corpora lutea: Group Mean (standard deviation)
Control: 19(4.7); 50 mg/kg: 19(3.1); 250 mg/kg:18(2.0); 1000 mg/kg: 17(3.9)
"Duration of Pregnancy: Group Mean (standard deviation)
Control: 21 (0.5); 50 mg/kg: 21(0.5); 250 mg/kg: 22(0.5); 1000 mg/kg: 22(0.5)
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No reproductive effects at the highest dose tested.
Critical effects observed:
no
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
No gross abnormalities were found for any of the pups, with the exception of a single runt in the 50 mg/kg bw/day group.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects at the highest dose tested.
Critical effects observed:
no
Reproductive effects observed:
no
Conclusions:
In the oral Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test with trimethoxy(methyl)silane (CAS 1185-55-3), conducted according to OECD Test Guideline 422 and in compliance with GLP, the concluded NOAEL for reproductive toxicity was at least 1000 mg/kg bw/day.
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
3 Oct 2011 - 28 Mar 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
Dams with offspring and pups were terminated on Day 5 post-partum. Litter weights, anogenital distance and nipple retention were not measured. Thyroid hormones of pups were not assessed.
Qualifier:
according to guideline
Guideline:
other: Circular on Test Methods of New Chemical Substances (Japan)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In the refrigerator
- Solubility and stability under test conditions: confirmed by IR at the end of the administration period
- Stability of the test substance in the solvent/vehicle: The test substance is stable for 8 days in the refrigerator and 24 hours at room temperature.
Species:
rat
Strain:
other: Crl:CD (SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc., Atsugi, Japan
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks
- Weight at study initiation: 371 - 432 g (males), 223 - 274 g (females)
- Housing: individual animal in cage in steel cages before mating. During mating, one female and one male were in the same metal cage. From Day 17 in gestation period to Day 4 in the lactation period, one dam and its pups were in the plastic cage with white wood chip as bedding.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 2 weeks

DETAILS OF FOOD AND WATER QUALITY:
- Diet: NMF food (Oriental Yeast Co., Ltd., Itabashi-ku, Japan) was provided ad libitum during the acclimation period and throughout the study. Food was removed the afternoon/evening prior to necropsy.
- Water: Quality of tap water was analysed quarterly.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 24
- Humidity (%): 40 - 56
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 24 Oct To: 19 Dec 2011
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Each dosing formulation was prepared every 8 days. Stability of formulation was determined and stable for 8 days in the refrigerator and 24 hours at room temperature. Dosing solutions were prepared by adding the appropriate amount of the test article to a tared container and adding the appropriate amount of corn oil to yield the desired dose level. Dosing solutions were administered by oral gavage with a flexible stomach tube. The test article was administered at a dose volume of 5 mL/kg bw and was calculated from the most recent body weight. Until administration, stock solution was stored in a dark bottle and in a refrigerator (4 - 8°C).

VEHICLE
- Justification for use and choice of vehicle (if other than water): According to the preliminary test by using GC and IR due to the physical and chemical properties of the test substance, the test substance was stable in the corn oil.
- Concentration in vehicle: 6, 30 and 150 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg b.w.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Study Day 15 up to Day 28
- Proof of pregnancy: Females rats were evaluated daily for evidence of copulation, by confirming the presence of either vaginal plug or sperm in vaginal smear referred to as Day 0 of pregnancy.
- After successful mating each pregnant female was caged (how): Day 0 of gestation was defined as the day where copulation was observed, at which time the female was individually caged.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses of the dosing formulations were conducted using GC to confirm the concentrations in all dose groups before and at the end of the administration period. The results of dose concentration analyses were determined to be 97.7 - 107.7%. These results were within the acceptable limits (100.0 ± 10.0% of nominal values).

Homogeneity of formulations was not determined.
Duration of treatment / exposure:
Males:
14 days before mating
14 days during mating
14 days after mating

Females:
14 days before mating
27 - 37 days during mating, gestation and lactation period

Males and females for recovery group:
42 days administration and 14 days post-exposure observation period
Frequency of treatment:
once daily, 7 days/week
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
750 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Male treatment group:
7 (control and 750 mg/kg bw/day)
12 (30 and 150 mg/kg bw/day)

Male recovery group:
5 (control and 750 mg/kg bw/day)

Female
12 (treatment group)
10 (recovery group)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dose levels were based on the results of a range-finding study, in which animals were orally exposed to 250, 500 and 1000 mg/kg bw/day for 14 days. No death occurred in all groups and effects on general clinical signs and body weight were not observed. Males exposed to 1000 mg/kg bw/day showed slightly decreased food consumption at the beginning of the study, decreased red blood cell count, haemoglobin and haematocrit, increased platelet and total protein, increased liver and thyroid weight and decreased testis weight. Males exposed to 500 mg/kg bw/day showed increased total protein and liver weight. Male 250 mg/kg bw/day group showed increased liver weight. Females exposed to 1000 mg/kg bw/day group showed decreased food consumption during the administration period, increased total protein, increased liver, thyroid and ovary weight. Therefore, 30, 150 and 750 mg/kg bw/day were selected as the dose levels for the main study.
- Post-exposure recovery period in satellite groups: 14 days
Positive control:
Not performed
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: three times a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Males in the treatment and recovery group and females in the recovery group were obsereved once prior to exposure, once a week during the administration period and recovery period.
Females in the treatment group were observed once prior to the exposure, once a week during mating period, on gestation Days (GD) 1, 7, 14, and 20, and on Day 4 post-partum.

BODY WEIGHT: Yes
- Time schedule for examinations: Males in the treatment and recovery groups and females in the recover y group were weighed on Day 1, 8, 15, 22, 29, 36 and 42 during administration period, on Day 1, 8 and 14 during recovery period, and before termination.
Parental females were weighed on Day 1, 8 and 15 during administration period (non-pregnant animals on Day 22), on GD 0, 7, 14 and 20, on Day 0 and 4 post-partum, and before termination.

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: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No



Oestrous cyclicity (parental animals):
Performed dduring the pre-mating period
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, presence of gross anomalies, postnatal mortality, weight gain

GROSS EXAMINATION OF DEAD PUPS:
no

Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals at the end of the administration or at the end of the observational period
- Maternal animals: All surviving animals were euthanised by isoflurane on post-partum Day 4.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
Tissues and organ taken for histopathological examination:
Adrenal, bone, bone marrow, femoral, cerebellum (pons), cerebrum, epididymis, eye, heart, duodenum, jejunum, ileum (Peyer's patch), cecum, colon, rectum, kidney, mesenteric and submandibular lymph nodes, liver, parathyroid, pancreas, pituitary, salivary gland submandibular, sciatic nerve, spleen, stomach, skeletal muscle, femoral, seminal vesicle (coagulating gland), spinal cord, thoracic, testis, thymus, trachea, thyroid, urinary bladder, ovary, uterus, vagina

The following organs were weighed:
Brain, pituitary, thyroids, thymus, heart, liver, spleen, kidneys, adrenals, ovaries, uterus, seminal vesicles, prostate, testis, epididymis,
Statistics:
Bartlett test, Dunnett's test, Steel test, F test, Student's t test, Aspin-Welch's t test, Fischer's test
Reproductive indices:
Copulation index, fertility index, gestation index, implantation index, and delivery index
Offspring viability indices:
Live birth index, viability index on postnatal Day 4, sex ration of total number of pups at birth, sex ration of live pups on Day 4
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
One male in the 750 mg/kg bw/day group died on Day 12 of administration. Before death, the male showed reddish urine and smudge of lower abdomen on Day 10, decreased spontaneous movement on Day 11, and prone/lateral position and pale skin on day 12. Reddish urine was also observed in one animal in the 750 mg/kg bw/day group on Week 1, 2 and 3.
One female in the 150 mg/kg bw/day treatment group died on Day 1 of the lactation period. However, there was no abnormal general condition reported.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One male in the 750 mg/kg bw/day treatment group died on Day 12 of administration.
One female in the 150 mg/kg bw/day treatment group died on Day 1 post-partum.
All animals in the recovery groups survived the duration of the study.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
1) At the end of administration period
Males in the 750 mg/kg bw/day treatment group showed increased platelet numbers and fibrinogen value and prolonged prothrombin time and activated partial thromboplastin time (APTT). The mode of action is not clear but this can be caused by the test substance.
Females in the 750 mg/kg bw/day treatment group showed prolonged APTT, decreased relative and a bsolute neutrophil counts, and increased relative lymphocyte and eosinophil counts. The forementioned changes were considered as incidental due to the slight changes.
Females in the 750 mg/kg bw/day recovery group showed prolonged PT and APTT and increased MCV. Although the relative neutrophil counts were decreased, the absolute counts were not changed. This change was considered to be incidental.
No changes were found in the 30 and 150 mg/kg bw/day treatment group in comparison with the control group.

2) At the end of the recovery period
Males in the 750 mg/kg bw/day recovery group showed increased platelet. No changes were observed in females after recovery period in comparison with the control group.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
1) At the end of the administration period
Males in the 750 mg/kg bw/day treatment group showed increased γ-GTP, decreased total bilirubin, decreased potassium, increased calcium, increased total protein, and decreased A/G ratio. Decreased AST was not considered as toxicological effect. Total bilirubin was also decreased in male 150 mg/kg bw/day treatment group.
Females in the 750 mg/kg bw/day treatment group showed increased γ-GTP, decreased total bilirubin, increased BUN and decreased A/G ratio.
Females in the 750 mg/kg bw/day recovery group showed increased γ-GTP, increased total protein, and decreased A/G ratio. Decreased AST was not considered as toxicological effect.
No significant changes in clinical biochemistry parameters were observed in the 30 and 150 mg/kg bw/day treatment group compared to the control group.

2) At the end of the recovery period
Males in the 750 mg/kg bw/day recovery group showed decreased total bilirubin. Slight increased albumin was considered as the physiological variation because this change was not observed at the end of the administration period.
Females in the 750 mg/kg bw/day recovery group didn’t show the any differences as compared with the control group.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
1) At the end of administration period
Yellow urine was observed in 5 males and 3 females in the 750 mg/kg bw/day tratment group.
Increased urine volume and decreased osmotic pressure were observed in male in the 150 mg/kg bw/day treatment group. These changes regarding urine volume and osmotic pressure were not dose-depending.

2) At the end of the recovery period
Potassium value was increased in males in the 750 mg/kg treatment group. This change was regarded to be incidental because it was only a slight change and no abnormality was found at the end of the administration.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
1) During administration
Hearing was decreased in 30 mg/kg bw/day treatment group on Week 6 of administration. However, this change was not dose-depending. Since the dead male animal in the 750 mg/kg bw/day treatment group was not able to walk on Day 11 of administration, detailed clinical signs were not tested.
Females in the 750 mg/kg bw/day recovery group showed also decreased hearing in Week 1 of administration. This was regarded to be incidental because no changes were found afterward in this group and in females of the treatment group at the same time.

2) During recovery period
The increased hearing in females in the 750 mg/kg bw/day recovery group in Week 1 of recovery period was also regarded to be incidental because no change was observed at other time point and general condition was not abnormal.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Exposure of the test material to male and female rats resulted in effects being observed in the following organs: kidney, urinary bladder, liver, and thyroid. In the kidney, there were minimal tubular regeneration (4/6 males in the 750 mg/kg bw/day treatment group), eosinophilic body found in tubular cell (minimal 3/6, mild 1/6 males in the 750 mg/kg bw/day treatment group) and minimal transitional hyperplasia/hypertrophy observed (1/6 males in the 750 mg/kg bw/day treatment group). In the urinary bladder, there was transitional hyperplasia/hypertrophy seen (minimal 2/6 and moderate 1/6 males in the 750 mg/kg bw/day treatment group, minimal 3/12 females in the 750 mg/kg bw/day treatment group). The observed transitional hyperplasia/hypertrophy in male rats in the 30 mg/kg bw/day treatment group was concluded to be not treatment-related effects but instead caused by incidental inflammation of the urinary bladder. In the liver, there was centrilobular hepatocytic hypertrophy observed (minimal 3/6 males in the 750 mg/kg bw/day treatment group, minimal 9/11 and mild 2/12 females in the 750 mg/kg bw/day treatment group, minimal 4/9 females in the 150 mg/kg bw/day treatment group, and minimal 4/6 and mild 1/6 females in the 750 mg/kg bw/day recovery group). In the thyroids, there was hypertrophy in follicular cell (minimal 2/12 males in the 150 mg/kg bw/day treatment group, mild 6/6 males in the 750 mg/kg bw/day treatment group, minimal 10/12 and mild 1/12 females in the 750 mg/kg bw/day treatment group, and minimal 1/5 and mild 4/5 females in the 750 mg/kg bw/day recovery group). The effect observed in the liver is concluded to be associated with induction of drug metabolising enzyme. It is well known that a substance which induces drug metabolising enzyme also promotes metabolism of thyroid hormone and causes hypertrophy of follicular cell through the hypothalamus-hypophysis system as a secondary effect. Effects observed in the transitional epithelium of the kidneys were also noted in the urinary bladder. The mode of action causing this effect was not clear according to this test result. However, the effects on the transitional epithelium in the kidneys and urinary bladder were reversed by the end of the recovery period; these effects were therefore concluded to be reversible.
Histopathological findings: neoplastic:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
There were no statistically significant differences between controls and treatment groups in the mean body weights on any of the test groups.
There were no differences in the average daily food consumption between controls and the males groups for any of the measured time periods. In the reproductive group females, at 30 and 750 mg/kg bw/day showed a significant increase (p< 0.05 and p<0.01, respectively) in food consumption compared to control at day 2 during the lactation period. But this change was not regarded as treatment related due to the lack of dose response.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
There was no statistically significant difference across treatment groups for sperm measures.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There was no statistically significant difference across treatment groups for estrous cycle, mean days until copulation for males and females, copulation index, insemination index, fertility index, delivery index, gestation length in days, number of corpora lutea, number of implantation sites, implantation index, live birth index, and viability index on postnatal day 4.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No remarkable changes were found in organ weights of reproductive organs in all treatment groups compared to the control group.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No remarkable changes were found in reproductive organs in all treatment groups compared to the control group.
Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no reproductive toxicity observed
Critical effects observed:
not specified
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality:
not examined
Body weight and weight changes:
not examined
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
There were no statistically significant differences between controls and treatment groups in live birth index, viability index on postnatal Day 4, sex ratio of delivered pups and liveborns, and sex ratio of live pups on Day 4.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No significant difference was found in any treated groups from control group.
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
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no abnormal findings in male and female pups.
Although one pup had a vestigial tail in 30 mg/kg bw/day, this was considered as effect not associated with the test item.
Histopathological findings:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
VIABILITY (OFFSPRING)
There were no statistically significant differences between controls and treatment groups in live birth index, viability index on postnatal Day 4, sex ratio of delivered pups and liveborns, and sex ratio of live pups on Day 4.

BODY WEIGHT (OFFSPRING)
No significant difference was found in any treated groups from control group.

GROSS PATHOLOGICAL FINDINGS
There were no abnormal findings in male and female pups. Although one pup had a vestigial tail in 30 mg/kg bw/day, this was considered as effect not associated with the test item.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
750 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no reproductive toxicity observed
Critical effects observed:
no
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
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
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Reproductive effects observed:
no

Table 2. Summary of mean reproductive parameters for reproductive group female rats

 

 

Control (0 mg/kg bw/day)

30 mg/ kg bw/day

150 mg/ kg bw/day

750 mg/ kg bw/day

Corpora lutea counts

mean

16.6

16.8

16.2

16.8

S.D.

1.8

2.0

2.4

2.7

N

12

12

10

12

Total implants

mean

15.3

15.6

15.2

14.9

S.D.

1.9

1.4

1.8

2.2

N

12

12

10

12

Days gestation

mean

22.5

22.0

22.3

22.0

S.D

0.5

0.3

0.5

0.3

N

12

12

10

12

Total live pups day 0

mean

13.4

14.3

14.4

13.3

S.D.

1.7

1.8

1.7

2.4

N

12

12

10

12

Male pups

mean

6.8

7.0

7.2

6.6

S.D.

2.4

1.5

2.1

2.2

N

12

12

10

12

Female pups

mean

6.6

7.3

7.2

6.7

S.D

2.1

1.9

2.1

2.8

N

12

12

10

12

Delivered males/Delivered pups (%)

mean

0.50

0.49

0.51

0.51

S.D

0.16

0.10

0.13

0.16

N

12

12

10

12

Day 4 viable pups

mean

13.2

14.1

14.4

13.2

S.D

1.7

1.6

1.7

2.4

N

12

12

9 a)

12

Viable/total liveborns (%)

mean

98.2

99.1

99.3

99.4

S.D.

3.3

3.2

2.2

1.9

N

12

12

10

12

Initial Male Average Pup Weight (g) [Live Pups only]

mean

6.7

6.6

6.7

6.7

S.D.

0.3

0.4

0.5

0.6

N

12

12

10

12

Initial Female Average Pup Weight (g) [Live Pups only]

mean

6.3

6.2

6.3

6.4

S.D.

0.6

0.4

0.4

0.5

N

12

12

10

12

Final Average Male Pup (g)

mean

10.3

10.2

10.0

10.2

S.D.

0.9

0.9

0.9

1.5

N

12

12

9 a)

12

Final Average Female Pup (g)

mean

9.6

9.8

9.4

10.0

S.D.

1.1

0.7

0.9

1.5

N

12

12

9 a)

12

 

a)     One dam died on lactation Day 1

Conclusions:
Based on the results of this study, the NOAEL for reproductive toxicity was set at 750 mg/kg bw/day, the highest dose tested. No adverse effects on development of offspring were observed up to and including 750 mg/kg bw/day.
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Deve lopmental Toxicity Screening Test
Version / remarks:
2000
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD 421, Reproduction/Developmental Toxicity Screening Test
Version / remarks:
2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EC No 440/2008, B.7 Repeated Dose (28 days) Toxicity (oral)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3050, Repeated Dose 28-day Oral Toxicity Study in Rodents
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, protected from moisture/water as well as heat and ignition sources
- Stability under test conditions: Not stable above 180°C (potential formaldehyde release). Stable under test conditions.
- Solubility and stability of the test substance in the solvent/vehicle: not applicable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was dosed undiluted. The required amount of test item for daily dosing was transferred into a container and stored at room temperature.
- Preliminary purification step: No correction factor required
- Final dilution of a dissolved solid, stock liquid or gel: The test item was used undiluted.
- Final preparation of a solid: not applicable
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River,
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks old males and 13 weeks old females
- Weight at study initiation: 260 and 295g for males and 194 and 249g for females
- Fasting period before study: none
- Housing: On arrival and following the pre-test (females only) and pre-mating period, main animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages. Recovery males and females were group housed during the entire study period. During the mating phase, Main males and females were cohabitated on a 1:1 basis in Macrolon plastic cages. During the post-mating phase, Main males were housed in their home cage with a maximum of 5 males/cage. Main Females were individually housed in Macrolon plastic cages. During the lactation phase, Main females were housed in Macrolon plastic cages. Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water.
- Diet: Pelleted rodent diet, ad libitum
- Water (e.g. ad libitum): Municipal tap water was freely available to each animal via water bottles.
- Acclimation period: 6 days

DETAILS OF FOOD AND WATER QUALITY: The feed and water were analyzed by the supplier for nutritional components and environmental contaminants. It was considered that there were no known contaminants in the feed that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C (target) and 20 to 22°C (actual)
- Humidity (%): 40 to 70% (target) and 49 to 73% (actual)
- Air changes (per hr): 10x/hour
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark cycle
Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test material was administered undiluted.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused: for 14 days
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: not specified
- Further matings after two unsuccessful attempts: not specified
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: none
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test substance administered neat. No analytical verification conducted as not needed.
Duration of treatment / exposure:
Main and Recovery Males were treated for 29 days. Females that delivered were treated for 50-63 days, i.e. 14 days prior to mating, the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver or had a total litter loss were treated for 41 or 53 days. Recovery females (not participating in the reproduction part of the study) were treated for 50 days.
Frequency of treatment:
7 days per week
Details on study schedule:
Not applicable
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Test animals: 10 males and 10 females
Recovery groups: 5 males and 5 females for high dose and control groups
Control animals:
yes
Details on study design:
- Dose selection rationale: The dose levels were selected based on the results of a 14-day oral dose range finder with oral administration of Reaction mass of trimethoxy(aminoalkyl)-silanes and modified alkylether oligomers in rats, and in an attempt to produce graded responses to the test item.
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: Recovery groups were selected for low and high dose treatment groups.
- Post-exposure recovery period in satellite groups: 14 days
- Section schedule rationale (if not random): random
Positive control:
Not used
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day.
- Cage side observations checked included: general health/mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: hree times daily, shortly before, immediately after and 1 to 2 hours after dosing. During the recovery period, animals were observed at least once daily up to the day prior to necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated main females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD CONSUMPTION:
- Food consumption was quantitatively measured weekly, except for Main males and Main females which were housed together for mating and for Main females without evidence of mating. Food consumption of mated Main females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.
Thyroid hormone:
Blood samples were processed for serum for possible analysis for the thyroid hormone parameters total thyroxine (T4) and/or thyroid-stimulating hormone (TSH). These serum samples were stored until (possible) analysis in a freezer (≤-75°C).
Samples for T4 of F0-males and PND 13-15 pups were analysed.
Samples for T4 of F0-females and PND 4 pups and samples for TSH of F0-males, F0-females and PND 13-15 pups were not analysed.
Oestrous cyclicity (parental animals):
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.
Daily vaginal lavage was performed for all females (Main and Recovery) during 14 days prior to treatment (pre-test period) and the first 14 days of treatment. For Main females, daily vaginal lavage was continued during mating until evidence of copulation was observed.
On the day of necropsy, a vaginal lavage was also taken from Main females to determine the stage of estrous. This was done for all Main females, except for females with total litter loss.
Sperm parameters (parental animals):
Parameters examined in P male parental generations: testis weight, 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, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups

GROSS EXAMINATION OF DEAD PUPS: yes
Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development.
In addition, blood was collected from two pups per litter, and the thyroid from two pups per litter (if possible one male and one female pup) was preserved in 10% buffered formalin. The pups selected for blood sampling were the same pups as selected for thyroid preservation


ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were euthanised following completion of the mating period (a minimum of 28 days of administration)
- Maternal animals: All surviving animals that delivered were euthanised on PND 14-16.

GROSS NECROPSY
- Gross necropsy consisted of a full post mortem examination, with special attention being paid to the reproductive organs

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [# 1] were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 13-15 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of sex determination both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development.

HISTOPATHOLOGY / ORGAN WEIGTHS
Not performed.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Reproductive indices:
Mating index, precoital index, gestation index, fertility index, duration of gestations, post-implantation survival index, live birth index, percentage of live males at first litter check, percentage of live females at first litter check, viability index, lactation index.
Offspring viability indices:
viability index
Clinical signs:
no effects observed
Description (incidence and severity):
Rales were observed in several 75 and 250 mg/kg bw/day treated main and recovery animals predominantly in the first two weeks of treatment. In one female at 250 mg/kg bw/day the rales were accompanied by laboured respiration. Rales were also observed in a single 25 mg/kg bw/day treated male and female on one day in the first week of treatment. The rales were always observed temporarily, lasting between one observation to maximally three days.
Piloerection was observed in one 250 mg/kg bw/day treated female (no.94) for two days in the first week of mating.
No specific clinical signs were noted in the animals of all dose groups during the weekly arena observations.
During the treatment period, salivation was observed among animals of the 75 and 250 mg/kg bw/day dose group immediately after dosing on one or more occasions. Salivation was observed on a single occasion in a 25 mg/kg bw/day treated female. Dose response relationship was observed. Salivation observed in this study was considered not toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity.
Other clinical signs noted during the treatment period, including alopecia, scales and/or scabs, occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
In the morning of Day 3 (prior to dosing) male no.42 (Group 4) was found moribund (gasping) and died shortly thereafter. Macroscopic examination of this animal revealed an enlarged mandibular lymph node (unilateral) foamy contents in the trachea, dark red foci in the thymus and reddish foci in the lungs. Based on the time of occurrence and the absence of similar signs in the other animals, it was considered an accidental event, rather than indicative of test item related toxicity.
No further mortality occurred during the study period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Lower body weights and body weight gain were observed in main and recovery males at 250 mg/kg bw/day, achieving levels of statistical significance for body weights on days 15 and 22 of treatment and on all occasions during treatment for body weight gain when compared to controls. At the end of treatment, a body weight difference of only approximately 4% between controls and males at 250 mg/kg bw/day was noted.

During the recovery period, the difference in body weights persisted during the recovery period, achieving levels of statistical significance on all occasions. The body weight gain data indicated that growth of the recovery males ran parallel. The body weight gain (when compared to study day 1) in high dose recovery males at start of recovery, was statistically significantly lower than in control recovery males. The increase in difference in mean body weights between control and high dose males over one day (from end of treatment to start of recovery) was due to relatively high body weights of the control males that continued in the recovery period compared to those of the whole group. Since the body weight gain over the recovery period was comparable for the control and high dose males, no toxicological significance was attached to this finding.

Body weights and body weight gain in 25 and 75 mg/kg bw/day treated males were considered not to be affected by treatment.
Body weights and body weight gain in female rats were considered to have been unaffected by treatment.
Over the recovery period, body weights and body weight gain were also unaffected by cessation of treatment in female rats.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption before or after correction for body weight in main and recovery male and recovery (not-mated) female rats was similar to the control level over the treatment period and recovery period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
At the end of the treatment period, haematological parameters of treated rats and recovery rats were considered not to have been affected by treatment.
The statistical significance for the mean number of lymphocytes in 75 mg/kg bw/day treated males and for the mean corpuscular haemoglobin concentration (MCHC) in 75 mg/kg bw/day treated females were fortuitous findings. In the absence of a treatment-related distribution or corroborative findings these changes were considered to be of no toxicological significance.
Coagulation parameters of treated rats were considered not to be different from controls at the end of treatment as well as after a subsequent fourteen-day recovery period.
The lower prothrombin time (PT) seen in 250 mg/kg be/day treated males, achieving a level of statistical significance was considered not to be of toxicological relevance as the opposite effect (i.e. an increase) would be expected in case of target organ toxicity.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
At the end of treatment, lower mean levels for bile acids were observed in 250 mg/kg bw/day treated males and recovery females, achieving a level of statistical significance in recovery females. The high dose animals did not recover from this difference and the levels of bile acids remained lower at the end of the subsequent fourteen-day treatment free period, now achieving a level of statistical significance in 250 mg/kg be/day treated males.
In main females, the mean levels for bile acids did not indicate an effect by treatment and were comparable between the treated and control animals at the end of treatment. However, it should be noted that a greater individual variation was observed and the mean level for bile acids was approximately two times higher than in the recovery females. This was likely to be related to the difference in physiological status between the primiparous and nulliparous females.
The statistical significances for the mean level for inorganic phosphate in 75 and 250 mg/kg bw/day treated males and for the mean level of calcium in 250 mg/kg bw/day treated main females occurred by chance. As the changes in these parameters were minimal (<10%) and the values for these parameters remained within the historical range for rats of this strain and age, these findings were considered to be of no toxicological significance.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Urinalysis parameters of treated rats were considered not to be different from controls at the end of treatment as well as after a subsequent fourteen-day recovery period.
The pH value of the urine in 75 mg/kg bw/day treated (main) females achieving a level of statistical significance when compared to controls, was considered to have arisen as a result of slightly low control value and in the absence of a treatment-related distribution considered to be of no toxicological significance.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
The functional observation parameters, hearing ability, pupillary reflex, static righting reflex and grip strength, were considered not to be affected by treatment.

The group mean data for motor activity, comprising total movements and ambulation, showed a large variation between the groups in both males and females.

Whereas the group mean values for total movements and ambulation in the 25 and 75 mg/kg bw/day treated main and recovery males were approximately 20% lower than controls, these group mean values in the 250 mg/kg bw/day treated males were similar to that of controls. In the absence of clear dose response relationship and since the motor activity data of individual animals of all groups were within the historical control range for male rats of this strain, age and used in this type of studies, it was concluded that the variation in motor activity between groups was not treatment related. Moreover, a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period was observed in all groups.
In lactating (main) females, the motor activity was higher in the 25 and 75 mg/kg bw/day treated animals, whereas a marked decrease was observed in the 250 mg/kg bw/day treated females in comparison with controls. The increases in group mean total movements were 19% and 16% and for ambulation were 36% and 27% for the 25 and 75 mg/kg bw/day treated females, respectively. In the 250 mg/kg bw/day treated females, the group mean values were 43% lower for total movements, achieving a level of statistical significance, and 40% lower for the ambulation, when compared to controls.

The motor activity in not-mated (recovery) females at 250 mg/kg bw/day was in general higher than in lactating females, but a similar difference between control and high dose females was observed with a 30% lower value for total movements and a 40% lower value for ambulation in the latter animals, but not achieving a level of statistical significance in comparison with controls.
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):
There were no test item-related microscopic observations.
All of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Thyroid hormone
At the end of treatment, the serum levels of T4 in F0-males were comparable between the treated and control animals and considered not to be affected by treatment.
At the end of the subsequent fourteen-day recovery period, a difference in levels of T4 between the high dose males and controls was observed, achieving a level of statistical significance. The difference was considered to have arisen as a result of slightly high control value and since the T4 values of the high dose males were well within the historical control range for rats of this strain and age no toxicological significance was attached to this finding.

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Length and regularity of the estrous cycle were considered not to have been affected by treatment.
All females had regular cycles of 4 days. During the mating period, extended di-estrus occurred in two control females (nos.58 and 59) until mating. Both females delivered normal litters. Extended di-estrus was also observed in one 250 mg/kg bw/day treated female no.88 during the mating period.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Testis and epididymis weight were unchanged following treatment.
Reproductive performance:
no effects observed
Description (incidence and severity):
Mating index was considered not to be affected by treatment. One 250 mg/kg bw/day treated female showed no evidence of mating, which was considered an incidental finding and not related to treatment.
Key result
Dose descriptor:
NOAEL
Remarks:
Systemic toxicity
Effect level:
>= 75 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on reduced motor activity in main and recovery females at 250 mg/kg bw/day.
Key result
Dose descriptor:
NOAEL
Remarks:
Reproductive toxicity
Effect level:
>= 250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effect on reproductive parameters
Key result
Critical effects observed:
yes
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs occurred among pups that were considered to be related to treatment.
A pale and cold appearance and a wound on the right leg was observed for the pup of control litter no.51 before it went missing on Day 5.
From Day 9-10 of lactation onwards all pups in litter no.93 (at 250 mg/kg bw/day) showed alopecia.
The nature and incidence of these and other clinical signs remained within the range considered normal, and were therefore considered not to be toxicologically relevant.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
For the treated females, the number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was considered not affected by treatment.
One pup (of litter 72 at 25 mg/kg bw/day) was found dead on Day 2. For control females, 6 pups (out of three litters, nos.51, 52 and 59) were found dead or went missing between lactation Days 2 and 4. The missing pups were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
At birth, body weights of male and female pups in litters at 250 mg/kg bw/day were higher (slightly over 10%) in comparison with that in the other groups, achieving levels of statistical significance when compared to controls. During lactation this difference remained, but had reduced to less than 10% on lactation Day 13.
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
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment.
No milk in the stomach was noted for all stillborn pups in litter 84.
The nature and incidence of the other macroscopic findings remained within the range considered normal for pups of this age, were related to the death or confirmed the in-life observation of the pup.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Serum T4 levels in male and female PND 13-15 pups were considered not to be affected by treatment.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effect observed at any dose level.
Critical effects observed:
no
Reproductive effects observed:
no
Conclusions:
In the combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test conducted according to OECD Test Guideline 422 and in compliance with GLP, no reproductive or developmental toxicity was observed up to the maximum dose of 250 mg/kg bw/day. The NOAEL for reproductive and developmental effects was concluded to be greater than 250 mg/kg bw/day for trimethoxy(methyl)silane and its reaction products with [3-(2,3-epoxypropoxy)propyl]trimethoxysilane and 3-(trimethoxysilyl)propylamine.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
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 registered substance is a UVCB substance which is the reaction mass of tri-(methoxy/ethoxy)methylsilane and amino-functional (methoxy/ethoxy)-silanes. The identity and concentration range of the constituents have been determined by GC analysis. A detailed description of the composition can be found in Section 1.2 of the Chemical Safety Report.

 

No reproductive toxicity studies are available for the registered substance, trimethoxy(methyl)silane and its reaction products with 3-aminopropyltriethoxysilane and [3-(2,3-epoxypropoxy)propyl]trimethoxysilane. The requirement for new reproductive toxicity testing will be reconsidered when the results of the proposed OECD 408 test on the registered substance are available, after approval from ECHA. As an interim measure, available reproductive toxicity data and ongoing testing for the constituents are considered.

 

Block A

Trimethoxy(methyl)silane (CAS 1185-55-3)

 

In the oral Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test with trimethoxy(methyl)silane (CAS 1185-55-3), conducted according to OECD Test Guideline 422 and in compliance with GLP, the NOAEL for reproductive toxicity was at least 1000 mg/kg bw/day (Dow Corning Corporation, 2005).

 

There is an ongoing OECD 443 test on trimethoxy(methyl)silane.

 

Triethoxy(methyl)silane (CAS 2031-67-6)

 

In the oral Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test with triethoxy(methyl)silane (CAS 2031-67-6), conducted according to OECD Test Guideline 422 and in compliance with GLP, there were no treatment-related effects apparent for any of the reproductive endpoints. All females bred successfully and delivered live litters. Litter sizes were comparable for all groups. Differences in group mean values for the treated groups relative to the control group were small and none were found to be statistically significant. Therefore, exposure to triethoxy(methyl)silane was not associated with reproductive toxicity and a NOAEL of 750 mg/kg bw/day was identified (Bozo Research Center, 2012).

 

Block B

3-Aminopropyl(triethoxy)silane (CAS 919-30-2)

 

There are no reproductive toxicity data for 3-aminopropyl(triethoxy)silane; however, the dossier for this substance includes a testing proposal for an OECD 443 test, which will be conducted after ECHA approval.

 

No effects on reproductive organs, oestrus cycle or sperm parameters were evident at the highest tested dose in the 90-day oral repeated dose toxicity study, conducted according to OECD Test Guideline 408 and in compliance with GLP, with 3-aminopropyl(triethoxy)silane (CAS 919-30-2) (WIL Research Laboratories, 2001)

 

3-(Trimethoxysilyl)propylamine (CAS 13822-56-5)

 

There is an ongoing OECD 443 test on 3-(trimethoxysilyl)proplamine.

 

No effects on reproductive organs were reported in the 90-day oral repeated dose toxicity study, conducted according to OECD Test Guideline 408 and in compliance with GLP, with 3-(trimethoxysilyl)propylamine (CAS 13822-56-5) (Charles River, 2018).

 

Trimethoxy(methyl)silane and its reaction products with [3-(2,3-epoxypropoxy)propyl]trimethoxysilane and 3-(trimethoxysilyl)propylamine (EC No. 701 -408 -8 )

 

In the key combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test conducted according to OECD Test Guideline 422 and in compliance with GLP for trimethoxy(methyl)silane and its reaction products with [3-(2,3-epoxypropoxy)propyl]trimethoxysilane and 3-(trimethoxysilyl)propylamine, undiluted test material was administered by oral gavage to 10 male and female Wistar Han rats respectively per dose groups (Charles River Laboratories, 2018). The administered concentrations were 25, 75 or 250 mg/kg bw/day with a treatment period for a minimum of 28 days. A control group was also included that received water. A 14-day recovery period was included for 5 additional male and female rats respectively, from the high and low dose groups. The recovery animals (used to study the potential reversibility of possible toxic effects) were not mated and consequently were not used for the assessment of reproduction/ developmental toxicity.

No toxicological significant clinical signs were observed in male and female rats treated up to 250 mg/kg bw/day.  The motor activity in male and female rats showed a large variation between the groups. Despite the fact that the motor activity recorded in the animals of all dose groups was within the historical control range, there was no indication of a treatment related change in males, but it could not be ruled out that the marked decrease in group mean values for total movements and ambulation in both main and recovery females at 250 mg/kg bw/day was a treatment related effect. Any corroborating evidence from other possibly related parameters was not observed in the current study, e.g. no other behavioural changes, no effects on grip strength and absence of histopathological alterations in nerve and muscle tissue in these females. Nevertheless, in case the changes in motor activity in females at 250 mg/kg bw/day were treatment related the magnitude of the decrease of approximately 40% lower group mean values for total movements and ambulation should be an indication of an adverse effect.

Slightly reduced body weight gain was observed in males at 250 mg/kg bw/day during treatment, resulting in approximately 4% lower group mean body weight compared to that of controls. During the recovery period, the body weight gain was similar between these two groups and the difference in body weight continued until termination of the recovery males. Based on the magnitude of the changes in body weight the effect of treatment was considered to be non-adverse.

No treatment-related changes were noted in the other parameters investigated in this study (i.e. food consumption, haematology, coagulation and (male) T4 thyroid hormone levels, macroscopic and microscopic examination) at the end of treatment.

Furthermore, the fourteen-day recovery period after cessation of treatment did not induce changes between the 250 mg/kg bw/day treated males and females compared to their concurrent controls in any of the parameters investigated. 

No reproduction toxicity was observed up to the highest dose level tested (250 mg/kg bw/day) and the NOAEL was concluded to be at least 250 mg/kg bw/day.

 

This source substance is the methoxy analogue of the registered methoxy/ethoxy substance (target). The reproductive toxicity study for this analogue are included to cover Blocks C to G, and unspecified minor constituents, for which there are no reproductive toxicity data. The only difference between the target and source substances is one of the three starting materials. The source substance has a starting material, 3-aminopropyl(trimethoxy) silane [237-511-5, 13822-56-5] and the target has the starting material that is the triethoxy analogue, 3-aminopropyl(triethoxy)silane [213-048-4, 919-30-2]. Therefore, the target substance has additional ethoxy constituents. However, whether ethoxy or methoxy, the target and source substance constituents hydrolyse to the same respective silanol hydrolysis products. The only difference is the non-silicon hydrolysis product; the target releases methanol and ethanol, whereas the source substance releases methanol only. The small molecule alkoxysilanes of Block A, Block B and those of Block U1 have similar physicochemical properties as the starting materials. Together they constitute about 60-70% of the substance. Blocks C-G and U2 constitute approximately 20-30% of the substances. These are oligomers formed by combination of the monomer starting materials. Under acidic conditions (pH <4) as experienced in the stomach and relevant to oral studies, the hydrolysis of the source and target substances is rapid and will form the same hydrolysis products

Effects on developmental toxicity

Description of key information

No developmental toxicity studies are available for the registered substance, trimethoxy(methyl)silane and its reaction products with 3-aminopropyltriethoxysilane and [3-(2,3-epoxypropoxy)propyl]trimethoxysilane (EC 701 -410 -9). As an interim measure, available developmental toxicity data for the constituents are considered.

 

A prenatal developmental toxicity study in rats (OECD Test Guideline 441) has been proposed with the registered substance. After approval by ECHA the test will be conducted and the risk characterisation updated using results from the new test data.

 

Block A

Trimethoxy(methyl)silane (CAS 1185-55-3)

 

In the key oral Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test with trimethoxy(methyl)silane (CAS 1185-55-3), conducted according to OECD Test Guideline 422 and in compliance with GLP, the NOAEL for developmental toxicity was at least 1000 mg/kg bw/day, the highest dose tested (Dow Corning Corporation, 2005).

 

Triethoxy(methyl)silane (CAS 2031-67-6)

 

In the oral prenatal developmental toxicity test with triethoxy(methyl)silane (CAS 2031-67-6), conducted according to OECD Test Guideline 414 and in compliance with GLP, there were no treatment-related effects apparent for any of the developmental endpoints, including foetal parameters and the NOAEL for maternal and embryo-foetal toxicity was at least 1000 mg/kg bw/day (Covance, 2020).

 

Block B

3-Aminopropyl(triethoxy)silane (CAS 919-30-2)

 

A well-reported study conducted according to OECD Test Guideline 414 and in compliance with GLP reported maternal toxicity (increased incidences of mortality, clinical observations, and slight decreases in body weight gain and food consumption) at 600 mg/kg bw/day. The occurrence of maternal toxicity was accompanied by slight foetal toxicity (increased minor skeletal variations). No significant maternal or developmental effects were observed at 20 or 100 mg/kg bw/day. The maternal and developmental NOAEL was 100 mg/kg bw/day.

 

3-(Trimethoxysilyl)propylamine (CAS 13822-56-5)

 

In the prenatal developmental toxicity study, conducted according to the appropriate OECD Test Guideline 414 and in compliance with GLP in the rat, the reported NOAEL for maternal toxicity was 300 mg/kg bw/day based on clinical effects of systemic toxicity. The reported NOAEL for developmental toxicity was greater than 1000 mg/kg bw/day based on no adverse effects observed.

 

Trimethoxy(methyl)silane and its reaction products with [3-(2,3-epoxypropoxy)propyl]trimethoxysilane and 3-(trimethoxysilyl)propylamine (EC No. 701 -408 -8)

 

In the combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test conducted according to OECD Test Guideline 422 and in compliance with GLP, no reproductive or developmental toxicity was observed up to the maximum dose of 250 mg/kg bw/day. The NOAEL for reproductive and developmental effects was concluded to be greater than 250 mg/kg bw/day for trimethoxy(methyl)silane and its reaction products with [3-(2,3-epoxypropoxy)propyl]trimethoxysilane and 3-(trimethoxysilyl)propylamine.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
19.11.2003 to 19.05.2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Qualifier:
according to guideline
Guideline:
other: USEPA OPPTS 870.3650
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: No data
- Age at study initiation: Nine weeks
- Weight at study initiation: Males: 294.2-351.5; Females: 200.2-260.2 g
- Fasting period before study: None
- Housing: individually housed in suspended wire-mesh cages (pregnant rats in shoebox cages)
- Diet (e.g. ad libitum): Ad libitum (except during FOB)
- Water (e.g. ad libitum): Ad libitum (except during FOB)
- Acclimation period: Six days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.2-22.5
- Humidity (%): 36.0-62.0
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 09.02.2004 To: 19.04.2005
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Conducted over nitrogen atmopshere. Test substance was placed into a volumetric flask and corn oil added to achieve the desired volume. The weight of the test substance added to the flask was used to calculate nominal dose solution concentrations. Dosing solutions were prepared at least once every two weeks consistent with the previously determined 15-day stability. The concentration, homogeneity and stability of the test substance in vehicle for at least 15 days.

VEHICLE
- Justification for use and choice of vehicle (if other than water): No data
- Concentration in vehicle: Various
- Amount of vehicle (if gavage): Up to 3 ml/kg
- Lot/batch no. (if required): 122K0131
- Purity: Considered 100%
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of methyltrimethoxysilane (MTMS) in corn oil dosing solutions was determined prior to the beginning of the definitive study.
Details on mating procedure:
A 1:1 mating ratio was used. After dosing on study day 14, the animals were paired by placing the lowest numbered ear tag reproductive group female within each group in the home cage of the male with the lowest numbered ear tag from the same group. Female animals were housed continuously with the same male until evidence of copulation was obtained. Females were evaluated daily for evidence of copulation, as indicated by either a vaginal copulatory plug or sperm in the vaginal smear. Day 0 of gestation was defined as the day evidence of copulation was obtained, at which time the female was returned to her home cage (shoebox cage).
Duration of treatment / exposure:
Toxicity group females and males were treated for 28 and 29 days, respectively. Reproductive group females were treated for 14 days prior to the mating period, during the mating period, and then up to and including post partum day 3, for a total of up to 51 days.
Frequency of treatment:
Daily, 7 days/week
Duration of test:
51 days
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
corn oil
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
No further relevant details.
Maternal examinations:
Mortality/Morbidity: Animals were observed at least twice daily in their cages for moribundity and mortality throughout the in-life phase of the study.

Daily Observations: General clinical examinations were made at least once a day and were conducted immediately after dosing. The examinations included, but were not limited to, changes in the skin, fur, eyes, mucous membranes, respiratory, circulatory, autonomic and central nervous system functions, motor activity and behavior patterns. Findings were recorded for individual animals. General clinical examinations were not performed on days when detailed physical examinations were performed.

Detailed Physical Examinations: All animals received a detailed physical examination once before the first dose administration (to allow for within-subject comparisons), and weekly thereafter. Examinations were made outside the home cage in a standard arena at approximately the same time each day. Observations were detailed and carefully recorded. Examinations included, but were not limited to, changes in skin, fur eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity. Changes in gait, posture and response to handling as well as the presence of clonic or tonic movement, stereotypies, difficult or prolonged parturition or bizarre behavior were recorded. The presence or absence of findings was recorded for individual animals.

Body weights and food consumption were recorded weekly. Additional body weights for reproductive group females were obtained on gestational day 0, 7, 14, and 20, and within 24 hours of parturition, and on postnatal day 4. Individual food consumption was determined for each group following group specific schedules.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #
- Organs examined:

OTHER:
Ovaries and uterine content:
For females that delivered or had macroscopic evidence of implantation, the numbers of former implantation sites and corpora lutea were recorded. Recognizable fetuses for the females euthanized in extremis were examined externally and preserved in 10% neutral-buffered formalin. For females that failed to deliver, a pregnancy status was determined. Uteri with no macroscopic evidence of implantation were opened and subsequently placed in a 10% ammonium sulfide solution for detection of early implantation loss.
Fetal examinations:
Each litter was examined following delivery to determine the number and sex of the pups, stillbirths, live births, runts, sex ratio, and the presence of any gross abnormalities. Litter weights were taken within 24 hours of parturition and on day 4 postpartum.
Statistics:
Reproductive parameters with the exception of litter size were analyzed using an ANCOVA (Analysis of Covariance) with liter size as the covariate. Litter size was analyzed using an ANOVA.
Statistically significant probabilities are reported at either the p<0.05 or p<0.01 levels.
Indices:
On the day parturition was initiated (PND 0), the pups were sexed and examined for gross malformations, and the numbers of still born and live pups were recorded.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical signs included transient inactivity or salivation following dosing. 
Mortality:
mortality observed, treatment-related
Description (incidence):
There were two unscheduled deaths (one female each at 0 and 50 mg/kg  bw/day); both were related to dosing errors.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No statistically significant differences in treatment group maternal body weight relative to control group animals.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No statistically significant differences in treatment group maternal food consumption relative to control group animals.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Exposure was associated with increased blood platelet concentration for females at 1000  mg/kg bw/day.
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Increased liver weight was observed at 250 and 1000 mg/kg bw/day.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Exposure to MTMS was associated with organ weight and/or histomorphological changes in females (liver, thyroid, duodenum, jejunum, and adrenal gland) at dose levels at or above 250 mg/kg bw/day. 
Histopathological findings: neoplastic:
no effects observed
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Description (incidence and severity):
None detected.
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
"Number pregnant per dose level: 10
"Number aborting: 0
"Number of resorptions, early/late if available: None detected.
"Number of implantations: Group Mean (standard deviation) Control: 15 (2.2); 50 mg/kg: 16 (2.0); 250 mg/kg: 16 (1.4); 1000 mg/kg: 16 (1.9)
"Number of corpora lutea: Group Mean (standard deviation)
Control: 19(4.7); 50 mg/kg: 19(3.1); 250 mg/kg:18(2.0); 1000 mg/kg: 17(3.9)
"Duration of Pregnancy: Group Mean (standard deviation)
Control: 21 (0.5); 50 mg/kg: 21(0.5); 250 mg/kg: 22(0.5); 1000 mg/kg: 22(0.5)
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Abnormalities:
no effects observed
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Litter sizes were comparable for all groups. 
External malformations:
no effects observed
Description (incidence and severity):
No external malformations observed.
Skeletal malformations:
no effects observed
Description (incidence and severity):
No skeletal malformations observed.
Visceral malformations:
no effects observed
Description (incidence and severity):
No soft tissue malformations observed.
Details on embryotoxic / teratogenic effects:
Overall, there were no developmental effects. There were no gross abnormalities were found for any of the pups, with the exception of a single runt in the 50 mg/kg/day group.
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No developmental effect at the highest dose tested.
Abnormalities:
no effects observed
Developmental effects observed:
no
Conclusions:
In the oral Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test with trimethoxy(methyl)silane (CAS 1185-55-3), conducted according to OECD Test Guideline 422 and in compliance with GLP, the concluded NOAEL for developmental toxicity was at least 1000 mg/kg bw/day.
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
August 2019 through November 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
25 June 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Department of Health of the Government of the United Kingdom
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 70 to 79 days old
- Weight at study initiation: 231 to 294 g
- Fasting period before study:
- Housing: Housed up to 4 animals per cage during acclimazation; housed one stock male and one female during mating; individually housed during gestation. Cages comprised of a polycarbonate body with a stainless steel mesh lid. Solid (polycarbonate) bottom cages were used during the acclimatization and gestation periods.
- Diet: SDS VRF1 Certified pelleted diet (manufactured in Witham, Essex, England), available ad libitum
- Water: Water from the public, available ad libitum
- Acclimation period: Six days before commencement of pairing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Air changes (per hr): at least 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light : 12 hours dark
Route of administration:
oral: gavage
Vehicle:
other: Dried and de-acidified corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The required amount of test item was added to the required volume of vehicle. The formulation was stirred using a magnetic stirrer under a fume hood until uniformly mixed and then transferred to final containers, via syringe, while magnetically stirring. A series of formulations at the required concentrations were prepared by dilution of individual weighing of the test item in ascending order of concentration.

VEHICLE
- Preparation of vehicle: 50% silica, 25% aluminum oxide neutral and 25% aluminum oxide activated acidic (40g, 20g and 20g respectively per liter of oil) were mixed. The dry mixture was placed in a vented oven set to 250 ºC overnight or for at least eight hours. The oil and dry materials were mixed in a suitable container, using a stir bar on a magnetic stirring plate, for at least two hours under a nitrogen purge and then allowed to dry to room temperature. The oil and dry materials mix was allowed to settle for a minimum of 30 minutes. The oil was filtered with 0.22 µm cellulose acetate filter system with vacuum to remove the alumina and silica.
- Amount of vehicle (if gavage): 4 mL/kg body weight.
Analytical verification of doses or concentrations:
yes
Remarks:
The mean concentrations of formulation samples taken from the first and last preparation were within 3% of the nominal concentration, confirming the accuracy of formulation. The difference from mean remained within 3%, confirming precise analysis.
Details on analytical verification of doses or concentrations:
Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 1 and 250 mg/mL were analyzed to assess the stability and homogeneity of the test item in the liquid matrix. Stability was confirmed as one day at ambient temperature (15 to 25 ºC) and fifteen days at refrigerated temperature (2 to 8 ºC).

Samples of each of the first and last preparation formulations were analyzed for achieved concentration of the test item.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1:1
- Verification of same strain and source of both sexes: yes; a colony of stud males was maintained specifically for the purpose of mating; these animals were not part of the study and were maintained as stock animals.
- Proof of pregnancy: evidence of vaginal plug and sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
From Day 6 to Day 19 (inclusive) after mating
Frequency of treatment:
Daily
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
20 females per dose
Control animals:
yes, concurrent no treatment
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed observations were recorded daily during the treatment period at the following times in relation to dose administration: pre-dose observation; one to two hours after completion of dosing; as late as possible in the working day. A detailed physical examination was performed on each animal on Days 0, 5, 12, 18 and 20 after mating to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each adult was recorded on Days 0, 3 and 6-20 after mating.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: See Table 1

OTHER: A thyroid hormone analysis was performed on all animals, which occurred as scheduled termination. Animals were no fasted. Samples were analyzed for serum thyroxine (T4) and triiodothyronine (T3) levels, as well as thyroid-stimulating hormone (TSH).
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
Statistics:
The following sequence of statistical tests was used for body weight, gravid uterus weight, food consumption, corpora lutea, implantations, pre/post implantation loss, live young, sex ratio - percentage male, placental, litter and fetal weights and organ weight data:

A parametric analysis was performed if Bartlett's test for variance homogeneity was not significant at the 1% level. For pre-treatment data, analysis of variance was used to test for any group differences. Where this was significant (p<0.05) inter group comparisons using t-tests, with the error mean square from the one-way analysis of variance, were made. For all other analyses the F1 approximate test was applied. If the F1 approximate test for monotonicity of dose-response was not significant at the 1% level, Williams' test for a monotonic trend was applied. If the F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test was performed instead.

A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. Forpre-treatment data, Kruskal-Wallis’ test was used to test for any group differences. Where this was significant (p<0.05) inter group comparisons using Wilcoxon rank sum tests were made. For all other analyses the H1 approximate test, the non-parametric equivalent of the F1 test described above, was applied. If the H1 approximate test for monotonicity of dose-response was not significant at the 1% level, Shirley's test for a monotonic trend was applied. If the H1 approximate test was significant, suggesting that the dose-response was not monotone, Steel's test was performed instead.

For organ weight data, analysis of covariance was performed using terminal body weight as covariate, unless non-parametric methods were applied.
Indices:
Pre-implantation loss (%) = ((Number of copora lutea - Number of implantations)/Number of corpora lutea) X 100
Post-implantation loss (%) = ((Number of copora lutea - Number of implantations)/Number of implantations) X 100
Historical control data:
Historical Control data was included and used for the evaluation of both fetal and litter incidences.
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day group mean food consumption was slightly lower, and attained statistical significance, during Days 6-18 of gestation when compared with the group mean food consumption of the control 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
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):
The mean gravid uterine weight for females treated with triethoxy(methyl)silane was comparable to the mean gravid uterine weight from the control females, as was adjusted body weight gain.

The weight of the liver was higher in females treated with 1000 mg/kg bw/day when compared with the controls (136% of the controls, respectively) and slightly higher in females receiving 300 mg/kg bw/day (109% of the controls, respectively). The weight of the thyroids and parathyroids were higher in females receiving 1000 mg/kg bw/day triethoxy(methyl)silane (129% of the controls, respectively). The weight changes stated above all attained statistical significance.
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In the thyroids, minimal/slight follicular cell hypertrophy was seen in all treated groups, with a clear relationship to dose. The hypertrophy was considered to account for the statistically significant higher than control group mean bodyweight adjusted thyroid weights for females that received 1000 mg/kg bw/day. This effect is likely secondary to the increase in TSH, which is a compensatory feedback mechanism to increase thyroid hormone production by the thyroid gland and part of a homeostatic response by the hypothalamic-pituitary-thyroid axis.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
The analysis of serum TSH concentrations performed at scheduled termination on Day 20 of gestation revealed that the TSH level at 1000 mg/kg bw/day was found to be statistically significantly higher compared to the control group at the 0.1% (p < 0.001) level.

There was a decrease in T4 and T3 concentration in females receiving 1000 mg/kg bw/day triethoxy(methyl)silane, both were found to be statistically significantly lower compared to the control group at the 0.1% (p < 0.001) level.

It was concluded that there was an effect of triethoxy(methyl)silane on serum T3, T4 and TSH concentrations in pregnant rats at a dose level of 1000 mg/kg bw/day, administered via oral gavage. It is possible that the change in TSH and thyroid hormones were secondary to liver enzyme induction and enhanced metabolism and clearance of thyroid hormones, which is consistent with increased liver weights.
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No adverse effects observed
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day there was a slight increase in incidence of cranial interparietal fissure(s) compared to concurrent control and was outside historical control data range. In isolation and at such low incidence this was not considered adverse.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
At 1000 and 300 mg/kg bw/day there was a slight increase in the incidence of partially undescended thymus compared to concurrent control but was within historical control data range and therefore considered unrelated to treatment. This is a transient stage in fetal development and therefore not considered adverse.
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Conclusions:
Based on the results of this study, no adverse effects on development of offspring were observed up to and including 1000 mg/kg bw/day, the highest dose tested.
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1997-04-14 to 1997-05-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EPA OTS 798.4900 (Prenatal Developmental Toxicity Study)
Version / remarks:
(Similar to OECD 414)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Charles River Crl:CD VAF/Plus
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Labs, Portage, MI, USA
- Age at study initiation: not stated
- Weight at study initiation: 235-240 g (day 0 of study)
- Housing: 1/suspended stainless steel cage
- Diet: standard diet ad libitum
- Water: drinking water ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72-75 deg F
- Humidity (%): 44-56
- Photoperiod (hrs dark / hrs light): 12 h/12 h

IN-LIFE DATES: From: 1997-04-14 To: 1997-04-28
Route of administration:
oral: gavage
Vehicle:
peanut oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: 0.3, 1.5 or 9 g of TS were added to 30 ml vehicle (peanut oil), mixed with magnetic stir bar. Solution said to be stable for 12 h; prepared daily. A constant volume of 2 ml/kg bw of these solutions or the vehicle were administered daily. No tests were conducted on the on homogeneity or stability of prepared solutions.

DIET PREPARATION
no details given

VEHICLE
- Justification for use and choice of vehicle (if other than water): None given (TS hydrolyses in water)
- Concentration in vehicle: 0.3, 1.5 or 9 g of TS in 30 ml vehicle
- Amount of vehicle (if gavage): 2 ml/kg bw
- Lot/batch no.: Sigma Peanut Oil (P-2144); lot 83H0848
- Purity: not stated
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
doses: 20, 100 and 600 mg/kg bw/day
target concentrations: 10, 50, 300 mg/ml
measured average concentration: 9.34, 51.2, 299 mg/ml
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: until copulatory plug or vaginal smear was present.
- Proof of pregnancy: copulatory plug or vaginal smear confirmed mating
Duration of treatment / exposure:
Day 6 of gestation to day 17 of gestation [NB the SIAR (2003) report of this study notes treatment from GD 6 to 20]
Frequency of treatment:
once per day
Duration of test:
Observations from gestation day (GD) 6 to GD 20.
No. of animals per sex per dose:
30 females
Control animals:
yes, concurrent vehicle
Details on study design:
Sex: female
Duration of test: Through day 20 of gestation
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily GD 6-20

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: GDs 0, 6, 9, 12, 15, 18, 20

FOOD CONSUMPTION : Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg bw/day: Yes. determined on GDs 0, 6, 9, 12, 15, 18, 20

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on GD 20
- Organs examined: laparaohysterectomic examination and necropsy

Ovaries and uterine content:
The ovaries and uterine content were examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half of the fetuses from 0 and 600 mg/kg bw /day groups
- Skeletal examinations: Yes: half per litter from all dose groups
- Head examinations: yes
Other: weight and sex determination
Statistics:
One-way analysis of variance (ANOVA) was used to analyze mean maternal gestation body weights, body weight changes, and food consumption, mean number of corpora lutea, implantation sites, live foetuses(male and female), postimplantation losses, resorptions (early and late), mean fetal weights (male and female), gravid uterine weights, carcass weights, and net weight change from day 0. If the ANOVA was significant, pairwise comparisons to the vehicle control were performed using Dunnett's test. Pairwise comparison with vehicle control (Dunnet, 1964) if ANOVA significant.
A Kruskal-Wallis test was used to analyze mean percent preimplantation losses and live fetuses (male and female) per animal, mean percent postimplantation losses, dead fetuses, and resorptions (early and late) expressed as percentages of implantations per animal, mean percent affected fetuses per litter for external, visceral, and skeletal malformations and developmental variations, and mean percent affected fetuses per liter for external, visceral, and skeletal malformations and developmental variations. If the Kruskal-Wallis test was significant, pairwise comparisons to the vehicle control were made using a Mann-Whitney U test.

A Pearson chi-square test was used to analyze fetal and litter incidence of fetal external, visceral and skeletal malformations and developmental variations, as well as litter incidence of total fetal external, visceral and skeletal malformations and developmental variations. If the chi-square test was significant, pairwise comparisons to the vehicle control were performed using a Fischer's exact test.
Indices:
No data given as indices (see REMARKS ON RESULTS INCLUDING TABLES AND FIGURES for details of reproductive/developmental findings).
Historical control data:
Full historical control data given (Charles River CD).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical signs although not restricted to the animals that died, were predominantly observed in these animals exposed to 600 mg/kg/day and included hypoactivity, cold to the touch, body surface stained and material around the mouth and nose. Additionally, respiratory signs including laboured breathing, gasping and rales in the 600 mg/kg/day dose group. No signs were observed in the two lower dose (100 and 20 mg/kg bw/day) groups.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
At 600 mg/kg bw/day, 5/30 deaths occurred. Furthermore, 2 rats were found dead on gestation day 7, one rat died on gestation days 13, 15 and 17 respectively.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 600 mg/kg/day, a slight decrease (not statistically significant) in the body weight gain was observed during gestation days 6 to 9. Since this decrease was consistent with significant decreases in the food consumption, it was considered to be treatment-related observations. No other significant treatment-related effects on the body weight gain were observed at any dose level.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 600 mg/kg/day dose level, a statistically significant decrease occurred in the food consumption during gestation days 6 to 9.
A statistically significant decrease in the food consumption were observed at gestation day 19 to 20 at 100 mg/kg/day. However, since no effect was observed at the 600 mg/kg/day dose level in the latter incidence, it was considered to be not treatment-related. No other significant treatment-related effects on the food consumption were observed at any dose level during the treatment period.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified
Number of abortions:
not specified
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
A statistically significant increase in the mean number of corpora lutea was observed at 600 mg/kg/day, however, it was considered to not be treatment related as ovulation and corpora lutea formation occurred prior to exposure to the test article.
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Key result
Dose descriptor:
LOAEL
Effect level:
600 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Key result
Dose descriptor:
LOAEL
Effect level:
600 mg/kg bw/day
Basis for effect level:
other: developmental toxicity
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not specified
External malformations:
no effects observed
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
At 600 mg/kg/day, statistically significant increases in the incidences of the variations 27 presacral vertebrae and sternebrae unossified were observed.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
At 100 mg/kg/day, a statistically significant increase in the incidence of the variation extra pair of full ribs was observed, however, since no effects were observed at the 600 mg/kg/day, it was considered to not be treatment-related indices.
Other effects:
not specified
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: teratogenicity
Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: sternum
skeletal: vertebra
Description (incidence and severity):
At 600 mg/kg/day, statistically significant increases in the incidences of the variations 27 presacral vertebrae and sternebrae unossified were observed
Developmental effects observed:
yes
Lowest effective dose / conc.:
600 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
not specified
Dose response relationship:
yes
Relevant for humans:
not specified

Mortality and day of death: 

Dose (mg/kg bw/day)                 No. Dead   Day of Death (gestation day)

0                                              0/30                              -

20                                             0/30                              -

100                                           0/30                              -

600                                           5/30                              7,7,13,15,17

Number pregnant per dose level:

Dose (mg/kg bw/day)                 No. Pregnant

0                                              29/30

20                                             25/30

100                                           26/30

600                                           22/30

Number aborting: none

Number of resorptions: 

Dose (mg/kg bw/day)                 No. Resorptions (early + late)

0                                              34

20                                             25

100                                           38

500                                           25

Number of implantations:
Dose (mg/kg bw/day)                             No. Implantations 

0                                                          437

20                                                         368

100                                                       361

600                                                       358

Pre and post implantation loss:

Dose (mg/kg bw/day)                  Preimplantation loss       Postimplantation loss

0                                                          50                                 34

20                                                         67                                 25

100                                                       74                                 38

600                                                       60                                 25

Number of corpora lutea:

Dose (mg/kg bw/day)                 No. Corpora lutea 

0                                                          487

20                                                         435

100                                                       435

600                                                       418

Duration of Pregnancy: 20 days

Dose (mg/kg bw/day)                           Mean body weight, grams (GD 20)

0                                                                      404.7   

20                                                                     405.1

100                                                                   390.4

600                                                                   407.4

Conclusions:
A well reported study conducted according to generally accepted scientific standards and in compliance with GLP reported maternal toxicity (increased incidences of mortality, clinical observations, and slight decreases in body weight gain and food consumption) at 600 mg/kg bw/day. The occurrence of maternal toxicity was accompanied by slight fetal toxicity (increased minor skeletal variations). No significant maternal or developmental effects were observed at 20 or 100 mg/kg bw/day. The maternal and developmental NOAEL was 100 mg/kg bw/day.
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
9th February 2018 to 13th March 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2001
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Den Bosch
- Age at study initiation: not specified
- Weight at study initiation: body weights within ± 20% of the mean for each set of animals
- Fasting period before study: not specified
- Housing: housed individually in Macrolon plastic cages
- Diet (e.g. ad libitum): Pelleted rodent diet, ad libitum. The feed was analyzed by the supplier for nutritional components and environmental contaminants.
- Water (e.g. ad libitum): Municipal tap water was freely available to each animal via water bottles. Periodic analysis of the water was performed.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 to 22°C
- Humidity (%): 28 to 51%
- Air changes (per hr): 12 hour light/12 hour dark cycle
- Photoperiod (hrs dark / hrs light): Ten or greater air changes per hour with 100% fresh air

Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
Dehydrated and deacidified corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a solution and dosed within 1 hour after completion of the preparation of the formulation. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle (0.92) and test item (1.0140). No correction was made for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Stability analyses performed previously in conjunction with the method development and validation study demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples were collected for analysis. During week 1 of treatment all groups were analysed for concentration and low dose and high dose groups were analysed for homogeneity. As the analytical results of low dose group were outside the acceptance criteria, a second concentration and homogeneity analysis was included during week 1. For concentration analysis duplicate sets of samples (approximately 500 mg) for each sampling time point were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration. For the homogeneity analysis duplicate sets of samples (approximately 500 mg) for each sampling time point were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was < 10%. Stability analyses performed previously in conjunction with the method development and validation study demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
Details on mating procedure:
Not specified.
Duration of treatment / exposure:
single oral administration
Frequency of treatment:
once daily, 7 days a week
Duration of test:
Day 6 to Day 20 post-coitum, gestation days
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
22
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on the results of a 14-day dose range finder (DRF) in non-pregnant rats, and in an attempt to produce graded responses to the test item. In this DRF, females treated up to 1000 mg/kg were noted with flat gait and rales on single occasions. Body weight gain, food consumption and organ weights were normal, and in one female reddish isolated foci were noted on the glandular mucosa of the stomach. Based on these minor effects, a top dose level of 1000 mg/kg was selected for this prenatal developmental toxicity study in pregnant rats.
- Rationale for animal assignment (if not random): random
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked included: general health/mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on Days 2, 6, 9, 12, 15, 18 and 21 post-coitum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption was quantitatively measured for Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum.

WATER CONSUMPTION: No
- Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: All animals (including animals found dead or sacrificed before planned necropsy) were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution). No organs (except for the (gravid) uterus) were weighed. The nasal cavity including the nasopharynx, larynx incl. ventral pouch and lungs were collected at scheduled necropsy from all surviving animals and fixed in 10% buffered formalin for possible further examination of the respiratory difficulties. No histopathological examination was conducted.


Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other:
• The number and distribution of live and dead fetuses.
• The number and distribution of embryo-fetal deaths.
• The sex of each fetus based on the ano-genital distance.
Fetal examinations:
- External examinations: Yes: [all per litter ]
- Soft tissue examinations: Yes: [half per litter ]
- Skeletal examinations: Yes: [half per litter ]
- Head examinations: Yes: [half per litter ]
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels.
Indices:
Not calculated
Historical control data:
Charles River Den Bosch has historical data on the background incidence of fetal malformations and developmental variations in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of developmental toxicants.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Signs of respiratory distress were observed among animals treated with test item surviving until scheduled necropsy as well as in those sacrificed in extremis. Reflux directly after dosing was seen for three surviving high-dose females (nos. 70, 72 and 88) on one or two occasions.
Rales (slight to moderate) was observed in 4, 8 and 9 females at 100, 300 and 1000 mg/kg, respectively, at a dose-related increased frequency and duration. Three out of 9 high-dose animals showing rales (nos. 70, 72 and 81) were additionally noted with other transient respiratory abnormalities, such as gasping and/or slight laboured and shallow respiration, discharge form nose and cough on one or three days during treatment. These breathing problems were considered to be related to the reflux of test item after administration via oral gavage.
Among animals showing rales, transient abnormal postures such as hunched or flat posture and flat gait were observed in one female at 300 mg/kg (no. 46) and in 5 females at 1000 mg/kg (nos. 70, 72, 79, 87 and 88) on one or two consecutive days during treatment.
Piloerection was noted in a few animals at 100 and 300 mg/kg, and in 8 females at 1000 mg/kg.
Transient and slight chromodacryorrhoea in the snout was observed in one female (no. 33) at 100 mg/kg and 2 females at 1000 mg/kg (nos. 72 and 88). Those two females at 1000 mg/kg presented with slight to moderate ptosis on one single day.
Salivation (slight to moderate) observed in one control female and in several females at 1000 mg/kg was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing).
Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Some of the clinical signs observed (respiratory effects, piloerection and abnormal posture), and body weight loss and reduced food consumption variations, may have been related to the respiratory distress and reflux caused by the administration of the test item via oral gavage; however, it cannot be excluded that some of these effects are related to systemic toxicity of the test item. Therefore, the conservative NOAEL for maternal effects is based on these findings.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No mortality occurred during the study that was considered to represent a systemic test item effect.
A total of four females dosed at 1000 mg/kg spontaneously died or were sacrificed in extremis between Days 8 and 16 post-coitum:
Two females at 1000 mg/kg (nos. 86 and 77) spontaneously died on Day 8 and 14 post-coitum, respectively, without showing any signs of toxicity. At necropsy, a discoloured lung (dark red left lobe) was noted for female no. 86, which might be due to a bleeding and could be related to the oral gavage procedure. For female no. 77, no macroscopic abnormalities were observed at necropsy. Although no clear cause of mortality could be established for these two spontaneous deaths, they were considered unrelated to treatment since there were no relevant findings either during in-life or at necropsy that indicated a poor condition of these two females due to a systemic toxicological test item effect.
Moreover, two females at 1000 mg/kg (nos. 73 and 76) were sacrificed in extremis on Day 13 and 16 post-coitum, respectively for animal welfare reasons. The main clinical signs for these two females were related to respiratory distress. Female no. 73 was noted with slight rales, piloerection and gasping in the morning of Day 13 post-coitum (before dosing) and had an overnight body weight loss of about 17%. For female no. 76 reflux was observed directly after dosing on two occasions (i.e. Days 14 and 16 post-coitum) and this animal presented with hunched posture, slight rales and piloerection from Day 10 post-coitum onwards and slight laboured respiration from Day 14 post-coitum onwards. On one single day, flat gait, slight ptosis and moderate salivation was also noted. No body weight loss was observed for this female, but food consumption over Days 9-12 post-coitum was slightly lower than normal. No macroscopic findings were noted for these two females sacrificed in extremis. These premature decedents were considered to be related to reflux and respiratory distress, rather than a systemic effect of the test item.
No mortality occurred in the other groups.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean body weights, body weight gain and weight gain corrected for gravid uterus of treated animals remained in the same range as controls.
The slightly lower mean body weight and body weight gain (not statistically significant) observed in females at 300 mg/kg at the end of the gestation period (Days 18 and 21 post-coitum) were attributed to female no. 45 that presented with a body weight loss of 11% between Days 12 and 15 post-coitum and female no. 52 that presented with absence of body weight gain over Days 18 to 21 post-coitum. As these changes occurred in absence of a dose-related trend and were considered to be caused by individual animals, this was considered not to be toxicologically significant.
At 1000 mg/kg, some individual surviving animals (nos. 70 and 81) were noted with a body weight loss of 7% between Days 9 and 12 post-coitum followed by a normal to slightly reduced body weight gain until the end of the study period. In both females, this decrease in body weight was accompanied by a reduction in food intake. Another female at 1000 mg/kg (no. 72) was noted with a slight body weight loss of 2% between Days 6 and 9 post-coitum followed by normal body weight gain during the remaining days of treatment.
The slightly (not statistically significantly) lower mean body weight gain after correction for gravid uterus observed in females at 1000 mg/kg when compared to concurrent control mean (23.6 vs 30.9 gram) was mainly attributed to the 3 females (no. 70, 72 and 81) that presented with body weight loss during the study period.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
At 1000 mg/kg, mean absolute and relative food consumption was statistically significantly decreased over Days 12-15 post-coitum and Days 12-18 post-coitum, respectively. Relative food consumption was reduced up to 11% when compared to control means. This reduction was mainly attributed to three females (nos. 70, 76 and 81) that presented with a decreased food intake from Day 12 post-coitum onwards. At the end of the treatment period both mean absolute and relative food consumption values were similar to those observed in the concurrent control group.
The statistically significantly higher food consumption at 300 mg/kg on Days 6-9 post-coitum occurred incidentally and in absence of a dose-related trend, and was therefore considered to be unrelated to treatment.
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic observations at scheduled necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
No adverse effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
One female each at 100 and 300 mg/kg (nos. 34 and 58) was not pregnant. This finding was not considered to be test item-related as no dose-response was observed and as treatment started from Day 6 post-coitum onwards. All four females that did not survive until planned necropsy were pregnant and had normal implantation sites in development. All pregnant females surviving to scheduled necropsy had litters with viable fetuses.
Other effects:
no effects observed
Description (incidence and severity):
No toxicologically relevant changes were noted in the numbers of corpora lutea.
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
clinical signs
Remarks on result:
other: Clinical signs may have been due to the respiratory distress and reflux caused by oral gavage. It cannot be excluded that some of these effects are related to systemic toxicity.
Abnormalities:
effects observed, treatment-related
Fetal body weight changes:
not examined
Reduction in number of live offspring:
not examined
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There were no toxicologically relevant effects on fetal body weights (both sexes) noted by treatment up to 1000 mg/kg. Mean combined (male and female) fetal body weights were 5.3 gram for the 100 mg/kg group and 5.2 gram for the other groups.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on litter size of any group.
Mean litter sizes were 10.3, 10.8, 9.1 and 11.5 fetuses/litter for the control, 100, 300 and 1000 mg/kg groups, respectively.
The slightly lower mean litter size at 300 mg/kg was related to a slightly higher litter incidence of early resorptions and pre-implantation loss in this group when compared to controls. No statistical significance was reached and all values in the treatment group remained within the historical control range. As no dose-related response was observed, these changes were not considered to be test item-related.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on external morphology following treatment up to 1000 mg/kg.
Only one external malformation occurred in this study. A trunk omphalocele was observed in fetus from litter of a dam treated with 100 mg/kg. The single occurrence of this malformation in the low-dose group did not indicate a relationship to treatment.
External variations were not observed in any group.
Skeletal malformations:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on skeletal morphology following treatment up to 1000 mg/kg.
Only one fetus (A038-06) at 100 mg/kg was observed with skeletal malformations; sternoschisis and rib anomaly. Due to the single occurrence of these malformations in one single fetus of the lowest dose group, they were considered to be unrelated to treatment.
Skeletal variations occurred at an incidence of 66.4%, 76.9%, 71.0% and 58.6% per litter in the control, 100, 300 and 1000 mg/kg groups, respectively.
There was a statistically significantly lower litter incidence of bent ribs at 1000 mg/kg. Litter incidences were 14.3, 14.5, 7.7 and 1.1% in the control, 100, 300 and 1000 mg/kg groups, respectively. Since the other chondrodystrophy (namely scapula bent) parameters were not affected, and as a similar low litter incidence of bent ribs occasionally occur in untreated controls, this skeletal variation was considered not to be toxicologically relevant.
Also noteworthy was the higher incidence of 7th cervical ossification sites at 1000 mg/kg. Litter incidences were 3.3, 6.5, 2.9 and 10.5% in the control, 100, 300 and 1000 mg/kg groups, respectively. Although the mean percentage was outside the historical control data, no dose-relationship could be established, and since it is known that these cervical ossification sites will be incorporated into the ventral arch of the 7th cervical vertebra during skeletal development, the increased incidence of this finding in the highest dose group was not considered to be toxicologically relevant.
All other variations noted were considered unrelated to treatment as they occurred in the absence of a dose-related trend, infrequently and/or at frequencies that were within the range of available historical control data.
Visceral malformations:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on visceral morphology following treatment up to 1000 mg/kg.
One fetus at 100 mg/kg was noted with visceral malformations. This fetus (A028-8) was observed with a total situs inversus and the presence of one lung lobe at both sides. Due to the single occurrence of these malformations, and as these were also earlier noted in the current historical control data, these findings were considered not to be related to treatment.
During the soft tissue cephalic examination on fixed heads, two control fetuses (A006-05 and A018-04), two fetuses of the 100 mg/kg group (A030-05 and A040-05), one fetus of the 300 mg/kg group (A051-11) and two fetuses of the 1000 mg/kg group (A085-06 and A085-10) were observed with small eyes. Since this malformation was equally distributed in all groups, including controls, and as this malformation was earlier noted among the current historical control data, it was considered that the occurrence of small eyes was not treatment-related and, therefore, was considered to be a chance finding.
Visceral variations were observed in 6.2%, 3.1%, 2.1% and 3.9% of fetuses per litter in the control, 100, 300 and 1000 mg/kg groups, respectively. All variations (i.e. small supernumerary liver lobe and convoluted ureter) occurred in the absence of a dose-related incidence, infrequently and/or at frequencies that were within the range of available historical control data.
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed.
Abnormalities:
no effects observed
Developmental effects observed:
no

See attachment for summary result tables and individual animal results tables.

Conclusions:
In the prenatal developmental toxicity study, conducted according to the appropriate OECD 414 Test Guideline and in compliance with GLP in the rat, the reported NOAEL for maternal toxicity was 300 mg/kg bw/day based on clinical effects of systemic toxicity. The reported NOAEL for developmental toxicity was greater than 1000 mg/kg bw/day based on no adverse effects observed.
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Version / remarks:
2000
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD 421, Reproduction/Developmental Toxicity Screening Test
Version / remarks:
2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EC No 440/2008, B.7 Repeated Dose (28 days) Toxicity (oral)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3050, Repeated Dose 28-day Oral Toxicity Study in Rodents
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
yes
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, protected from moisture/water as well as heat and ignition sources
- Stability under test conditions: Not stable above 180°C (potential formaldehyde release). Stable under test conditions.
- Solubility and stability of the test substance in the solvent/vehicle: not applicable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was dosed undiluted. The required amount of test item for daily dosing was transferred into a container and stored at room temperature.
- Preliminary purification step: No correction factor required
- Final dilution of a dissolved solid, stock liquid or gel: The test item was used undiluted.
- Final preparation of a solid: not applicable
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River,
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks old males and 13 weeks old females
- Weight at study initiation: 260 and 295g for males and 194 and 249g for females
- Fasting period before study: none
- Housing: On arrival and following the pre-test (females only) and pre-mating period, Main animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages. Recovery males and females were group housed during the entire study period. During the mating phase, Main males and females were cohabitated on a 1:1 basis in Macrolon plastic cages. During the post-mating phase, Main males were housed in their home cage with a maximum of 5 males/cage. Main Females were individually housed in Macrolon plastic cages. During the lactation phase, Main females were housed in Macrolon plastic cages. Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water.
- Diet: Pelleted rodent diet, ad libitum
- Water (e.g. ad libitum): Municipal tap water was freely available to each animal via water bottles.
- Acclimation period: 6 days

DETAILS OF FOOD AND WATER QUALITY: The feed and water were analyzed by the supplier for nutritional components and environmental contaminants. It was considered that there were no known contaminants in the feed that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C (target) and 20 to 22°C (actual)
- Humidity (%): 40 to 70% (target) and 49 to 73% (actual)
- Air changes (per hr): 10x/hour
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark cycle
Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test material was administered undiluted.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test substance administered neat. No analytical verification conducted as not needed.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused: for 14 days
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: not specified
- Further matings after two unsuccessful attempts: not specified
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: none
Duration of treatment / exposure:
Main and recovery males were treated for 29 days. Females that delivered were treated for 50-63 days, i.e. 14 days prior to mating, the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver or had a total litter loss were treated for 41 or 53 days. Recovery females (not participating in the reproduction part of the study) were treated for 50 days.
Frequency of treatment:
7 days per week
Duration of test:
up to 50 days
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Test animals: 10 males and 10 females
Recovery groups: 5 males and 5 females for high dose and control groups
Control animals:
yes
Details on study design:
- Dose selection rationale: The dose levels were selected based on the results of a 14-day oral dose range finder with oral administration of Reaction mass of trimethoxy(aminoalkyl)-silanes and modified alkylether oligomers in rats, and in an attempt to produce graded responses to the test item.
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: Recovery groups were selected for low and high dose treatment groups.
- Post-exposure recovery period in satellite groups: 14 days
- Section schedule rationale (if not random): random
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day.
- Cage side observations checked included: general health/mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Three times daily, shortly before, immediately after and 1 to 2 hours after dosing. During the recovery period, animals were observed at least once daily up to the day prior to necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated main females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD CONSUMPTION:
- Food consumption was quantitatively measured weekly, except for Main males and Main females which were housed together for mating and for Main females without evidence of mating. Food consumption of mated Main females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

Thyroid hormone:
Blood samples were processed for serum for possible analysis for the thyroid hormone parameters total thyroxine (T4) and/or thyroid-stimulating hormone (TSH). These serum samples were stored until (possible) analysis in a freezer (≤-75°C).
Samples for T4 of F0-males and PND 13-15 pups were analysed.
Samples for T4 of F0-females and PND 4 pups and samples for TSH of F0-males, F0-females and PND 13-15 pups were not analysed.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes [all per litter]
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: Yes [all per litter]
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Indices:
Mating index, precoital index, gestation index, fertility index, duration of gestations, post-implantation survival index, live birth index, percentage of live males at first litter check, percentage of live females at first litter check, viability index, lactation index.
Clinical signs:
no effects observed
Description (incidence and severity):
Rales were observed in several 75 and 250 mg/kg bw/day treated main and recovery animals predominantly in the first two weeks of treatment. In one female at 250 mg/kg bw/day the rales were accompanied by laboured respiration. Rales were also observed in a single 25 mg/kg bw/day treated male and female on one day in the first week of treatment. The rales were always observed temporarily, lasting between one observation to maximally three days.
Piloerection was observed in one 250 mg/kg bw/day treated female (no.94) for two days in the first week of mating.
No specific clinical signs were noted in the animals of all dose groups during the weekly arena observations.
During the treatment period, salivation was observed among animals of the 75 and 250 mg/kg be/day dose group immediately after dosing on one or more occasions. Salivation was observed on a single occasion in a 25 mg/kg bw/day treated female. Dose response relationship was observed. Salivation observed in this study was considered not toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity.
Other clinical signs noted during the treatment period, including alopecia, scales and/or scabs, occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
In the morning of Day 3 (prior to dosing) male no.42 (Group 4) was found moribund (gasping) and died shortly thereafter. Macroscopic examination of this animal revealed an enlarged mandibular lymph node (unilateral) foamy contents in the trachea, dark red foci in the thymus and reddish foci in the lungs. Based on the time of occurrence and the absence of similar signs in the other animals, it was considered an accidental event, rather than indicative of test item related toxicity.
No further mortality occurred during the study period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Lower body weights and body weight gain were observed in main and recovery males at 250 mg/kg bw/day, achieving levels of statistical significance for body weights on days 15 and 22 of treatment and on all occasions during treatment for body weight gain when compared to controls. At the end of treatment, a body weight difference of only approximately 4% between controls and males at 250 mg/kg bw/day was noted.

During the recovery period, the difference in body weights persisted during the recovery period, achieving levels of statistical significance on all occasions. The body weight gain data indicated that growth of the recovery males ran parallel. The body weight gain (when compared to study day 1) in high dose recovery males at start of recovery, was statistically significantly lower than in control recovery males. The increase in difference in mean body weights between control and high dose males over one day (from end of treatment to start of recovery) was due to relatively high body weights of the control males that continued in the recovery period compared to those of the whole group. Since the body weight gain over the recovery period was comparable for the control and high dose males, no toxicological significance was attached to this finding.

Body weights and body weight gain in 25 and 75 mg/kg bw/day treated males were considered not to be affected by treatment.
Body weights and body weight gain in female rats were considered to have been unaffected by treatment.
Over the recovery period, body weights and body weight gain were also unaffected by cessation of treatment in female rats.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption before or after correction for body weight in main and recovery male and recovery (not-mated) female rats was similar to the control level over the treatment period and recovery period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
At the end of the treatment period, haematological parameters of treated rats and recovery rats were considered not to have been affected by treatment.
The statistical significance for the mean number of lymphocytes in 75 mg/kg bw/day treated males and for the mean corpuscular haemoglobin concentration (MCHC) in 75 mg/kg bw/day treated females were fortuitous findings. In the absence of a treatment-related distribution or corroborative findings these changes were considered to be of no toxicological significance.
Coagulation parameters of treated rats were considered not to be different from controls at the end of treatment as well as after a subsequent fourteen-day recovery period.
The lower prothrombin time (PT) seen in 250 mg/kg bw/day treated males, achieving a level of statistical significance was considered not to be of toxicological relevance as the opposite effect (i.e. an increase) would be expected in case of target organ toxicity.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
At the end of treatment, lower mean levels for bile acids were observed in 250 mg/kg bw/day treated males and recovery females, achieving a level of statistical significance in recovery females. The high dose animals did not recover from this difference and the levels of bile acids remained lower at the end of the subsequent fourteen-day treatment free period, now achieving a level of statistical significance in 250 mg/kg bw/day treated males.
In main females, the mean levels for bile acids did not indicate an effect by treatment and were comparable between the treated and control animals at the end of treatment. However, it should be noted that a greater individual variation was observed and the mean level for bile acids was approximately two times higher than in the recovery females. This was likely to be related to the difference in physiological status between the primiparous and nulliparous females.
The statistical significances for the mean level for inorganic phosphate in 75 and 250 mg/kg bw/day treated males and for the mean level of calcium in 250 mg/kg bw/day treated main females occurred by chance. As the changes in these parameters were minimal (<10%) and the values for these parameters remained within the historical range for rats of this strain and age, these findings were considered to be of no toxicological significance.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Urinalysis parameters of treated rats were considered not to be different from controls at the end of treatment as well as after a subsequent fourteen-day recovery period.
The pH value of the urine in 75 mg/kg bw/day treated (main) females achieving a level of statistical significance when compared to controls, was considered to have arisen as a result of slightly low control value and in the absence of a treatment-related distribution considered to be of no toxicological significance.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
The functional observation parameters, hearing ability, pupillary reflex, static righting reflex and grip strength, were considered not to be affected by treatment.

The group mean data for motor activity, comprising total movements and ambulation, showed a large variation between the groups in both males and females.

Whereas the group mean values for total movements and ambulation in the 25 and 75 mg/kg bw/day treated main and recovery males were approximately 20% lower than controls, these group mean values in the 250 mg/kg bw/day treated males were similar to that of controls. In the absence of clear dose response relationship and since the motor activity data of individual animals of all groups were within the historical control range for male rats of this strain, age and used in this type of studies, it was concluded that the variation in motor activity between groups was not treatment related. Moreover, a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period was observed in all groups.
In lactating (main) females, the motor activity was higher in the 25 and 75 mg/kg bw/day treated animals, whereas a marked decrease was observed in the 250 mg/kg bw/day treated females in comparison with controls. The increases in group mean total movements were 19% and 16% and for ambulations were 36% and 27% for the 25 and 75 mg/kg bw/day treated females, respectively. In the 250 mg/kg bw/day treated females, the group mean values were 43% lower for total movements, achieving a level of statistical significance, and 40% lower for the ambulation, when compared to controls.

The motor activity in not-mated (recovery) females at 250 mg/kg bw/day was in general higher than in lactating females, but a similar difference between control and high dose females was observed with a 30% lower value for total movements and a 40% lower value for ambulation in the latter animals, but not achieving a level of statistical significance in comparison with controls.

Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
At the end of treatment, a dose related increase for the splenic weight was observed in Main male rats, achieving a level of statistical significance for the relative spleen weight in high dose males when compared to controls. In Main females, a dose related decrease was observed for the adrenal glands, achieving levels of statistical significance for the absolute and relative adrenal gland weight in high dose females when compared to controls.
No test item-related alterations were observed in the other organ weights at the end of treatment.
At the end of recovery, in the males a difference in absolute liver weights and relative weights of the brain and seminal vesicles was observed between control and high dose males, achieving levels of statistical significance. These differences were considered to be related to the differences in terminal body weight and considered not related to treatment and of no toxicological significance.
In high dose females at the end of recovery, minimal higher weights of the heart, liver and kidneys were observed, achieving levels of statistical significance for absolute and relative heart weight and relative liver and kidney weights when compared to controls. Since all these organ weights remained within the historical control range for female rats of this strain and age and no treatment related effects were observed in main females at the end of treatment no toxicological significance was attached to these findings
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related gross observations.
All of the recorded macroscopic findings at the end of treatment in main males and females and at the end of recovery in recovery males and females were within the range of background gross observations encountered in rats of this age and strain.

Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test item-related microscopic observations.
All of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Thyroid hormone
At the end of treatment, the serum levels of T4 in F0-males were comparable between the treated and control animals and considered not to be affected by treatment.
At the end of the subsequent fourteen-day recovery period, a difference in levels of T4 between the high dose males and controls was observed, achieving a level of statistical significance. The difference was considered to have arisen as a result of slightly high control value and since the T4 values of the high dose males were well within the historical control range for rats of this strain and age no toxicological significance was attached to this finding.

Number of abortions:
not examined
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Number of implantation sites was considered not to be affected by treatment.
One 250 mg/kg bw/day treated female (no. 90) had a relatively low number of implantation sites, i.e. 7 compared to the group mean value of 12.7. Since it was observed in a single female only this finding was considered to have occurred by chance and considered to be unrelated to treatment.
Total litter losses by resorption:
not examined
Early or late resorptions:
no effects observed
Description (incidence and severity):
The total number of offspring born compared to the total number of uterine implantations was considered not to be affected by treatment.
For female no.82 (at 75 mg/kg bw/day), the number of pups was higher than the number of implantations, i.e. 13 living pups compared to 12 implantation sites. This phenomenon is observed from time to time and is caused by normal resorption of these areas during (the 14 days of) lactation. No toxicological relevance was attached to this finding in the current study.
Dead fetuses:
no effects observed
Description (incidence and severity):
The number of live offspring on Day 1 after littering compared to the total number of offspring born was considered not to be affected by treatment.
Female no.84 delivered eleven dead pups, which was regarded as total litter loss. One pup in a control group litter (no.51) was found dead at first litter check. No toxicological relevance was attributed to these dead pups/total litter loss since the mortality incidence did not show a dose-related trend and remained within the range considered normal.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Gestation index and duration of gestation were considered not to be affected by treatment.
One control female (no.54) had implantations only and a 75 mg/kg bw/day treated female (no.84) delivered (eleven) dead pups only, regarded as total litter loss. Moreover, the duration of gestation in the latter female was also slightly longer than normal, i.e. 23 days compared to 21-22 days in the other females. In the absence of a dose effect relationship, these events were considered not related to treatment and of no toxicological significance.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Fertility index was considered not to be affected by treatment. All impregnated females, except one 75 mg/kg bw/day treated female (no.81) developed a pregnancy.
Since the case of non-pregnancy occurred in a single female no.81 only and showed no dose-related incidence across the dose groups, this event was considered not to be related to treatment.
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Remarks:
General toxicity
Effect level:
>= 75 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: Based on reduced motor activity in main and recovery females at 250 mg/kg bw/day
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
At birth, body weights of male and female pups in litters at 250 mg/kg bw/day were higher (slightly over 10%) in comparison with that in the other groups, achieving levels of statistical significance when compared to controls. During lactation this difference remained, but had reduced to less than 10% on lactation Day 13.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
For the treated females, the number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was considered not affected by treatment.
One pup (of litter 72 at 25 mg/kg) was found dead on Day 2. For control females, 6 pups (out of three litters, nos. 51, 52 and 59) were found dead or went missing between lactation Days 2 and 4. The missing pups were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Sex ratio was considered not to be affected by treatment.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Litter size was considered not affected by treatment.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
For the treated females, the number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was considered not affected by treatment.
One pup (of litter 72 at 25 mg/kg bw/day) was found dead on Day 2. For control females, 6 pups (out of three litters, nos.51, 52 and 59) were found dead or went missing between lactation Days 2 and 4. The missing pups were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
External malformations:
no effects observed
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment.
No milk in the stomach was noted for all stillborn pups in litter 84.
The nature and incidence of the other macroscopic findings remained within the range considered normal for pups of this age, were related to the death or confirmed the in-life observation of the pup.
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment.
Treatment up to 250 mg/kg bw/day had no effect on areola/nipple retention. Serum T4 levels in male and female PND 13-15 pups were considered not to be affected by treatment.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed at any dose level.
Abnormalities:
no effects observed
Developmental effects observed:
no
Conclusions:
In the combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test for trimethoxy(methyl)silane and its reaction products with [3-(2,3-epoxypropoxy)propyl]trimethoxysilane and 3-(trimethoxysilyl)propylamine, no reproduction and developmental toxicity was observed up to treatment at 250 mg/kg bw/day. The NOAEL for reproductive and developmental effects was concluded to be greater than 250 mg/kg bw/day (the highest dose tested).
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Species:
rat
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

The registered substance is a UVCB substance which is the reaction mass of tri-(methoxy/ethoxy)methylsilane and amino-functional (methoxy/ethoxy)-silanes . The identity and concentration range of the constituents have been determined by GC analysis. A detailed description of the composition can be found in Section 1.2 of the Chemical Safety Report.

 

No developmental toxicity studies are available for the registered substance, trimethoxy(methyl)silane and its reaction products with 3-aminopropyltriethoxysilane and [3-(2,3-epoxypropoxy)propyl]trimethoxysilane. As an interim measure, available developmental toxicity data and ongoing testing for the constituents are considered.

 

A prenatal developmental toxicity study in rats (OECD Test Guideline 441) has been proposed with the registered substance. After approval by ECHA the test will be conducted and the risk characterisation updated using results from the new test data.

 

Block A

Trimethoxy(methyl)silane (CAS 1185-55-3)

 

In the key oral Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test with trimethoxy(methyl)silane (CAS 1185-55-3), conducted according to OECD Test Guideline 422 and in compliance with GLP, the NOAEL for developmental toxicity was at least 1000 mg/kg bw/day, the highest dose tested (Dow Corning Corporation, 2005).

 

There is an ongoing OECD 414 test in rabbits for this constituent.

 

Triethoxy(methyl)silane (CAS 2031-67-6)

 

In the oral prenatal developmental toxicity test with triethoxy(methyl)silane (CAS 2031-67-6), conducted according to OECD Test Guideline 414 and in compliance with GLP, there were no treatment-related effects apparent for any of the developmental endpoints, including foetal parameters and the concluded NOAEL for maternal and embryo-foetal toxicity was at least 1000 mg/kg bw/day (Covance, 2020). Pregnant female Sprague-Dawley rats were treated with 100, 300 and 1000 mg/kg bw/day by oral gavage from gestation days 6 to 19. A control group received dried and de-acidified corn oil at the same volume dose as the treated groups. Animals were killed on gestation day 20 for reproductive assessment and fetal examination.

The clinical condition of females receiving 100, 300 or 1000 mg/kg bw/day triethoxy(methyl)silane was unaffected by treatment. The overall group mean body weight performance and overall gain (Day 6-20) of females treated with triethoxy(methyl)silane at 100, 300 or 1000 mg/kg bw/day was comparable to that of the control group. The mean gravid uterine weight for females treated with triethoxy(methyl)silane was comparable to the mean gravid uterine weight from the control females, as was adjusted body weight gain. The group mean food consumption of females treated with 100 or 300 mg/kg bw/day triethoxy(methyl)silane was generally similar to the controls during gestation Days 6-20. At 1000 mg/kg bw/day, mean food consumption was slightly and statistically significantly lower during Days 6-18 of gestation compared to the control group. At macroscopic examination there were no adverse findings in the females related to treatment with triethoxy(methyl)silane. The weight of the liver was slightly higher in females receiving 300 mg/kg bw/day, and the weight of the liver and thyroids were higher in females receiving 1000 mg/kg bw/day triethoxy(methyl)silane, when compared with the control group, all attaining statistical significance. In the thyroids, minimal/slight follicular cell hypertrophy was seen in all treated groups, with a clear relationship to dose. The hypertrophy was considered to account for the statistically significantly higher than control group mean bodyweight adjusted thyroid weights for females that received 1000 mg/kg bw/day. This effect is likely secondary to the increase in TSH, which is a compensatory feedback mechanism to increase thyroid hormone production by the thyroid gland and part of a homeostatic response by the hypothalamic-pituitary-thyroid axis. At scheduled termination, serum samples taken for thyroxine (T4), triiodothyronine (T3) and thyroid stimulating hormone (TSH) revealed that the T4 and T3 concentrations were slightly lower and the TSH levels were higher, when compared with the controls in females receiving 1000 mg/kg bw/day. Statistical evaluation displayed significant difference between mean T3 and T4, and TSH concentration in samples from control females and females receiving 1000 mg/kg bw/day. It was concluded that there was an effect of triethoxy(methyl)silane on serum T3, T4 and TSH concentrations in pregnant rats at a dose level of 1000 mg/kg bw/day, administered via oral gavage. It is possible that the change in TSH and thyroid hormones were secondary to liver enzyme induction and enhanced metabolism and clearance of thyroid hormones, which is consistent with increased liver weights. On Day 20 of gestation, all animals were found to be pregnant at macroscopic examination, with the mean number of corpora lutea, implantations, resorptions, the number of live young and sex ratio considered to be unaffected by treatment with triethoxy(methyl)silane. Group mean placental, litter and foetal weights were all considered to be unaffected by treatment with triethoxy(methyl)silane. There was no effect on the ano-genital distance of foetuses whose mother were treated with triethoxy(methyl)silane. There were no major foetal abnormalities considered to be related to treatment. At 1000 mg/kg bw/day, there was a slight increase in the incidence of the minor finding cranial interparietal fissure(s) compared with concurrent control and outside the historical control data (HCD) range. In isolation and at such low incidence this was not considered adverse. It was therefore concluded that the no observed adverse effect level (NOAEL) for maternal toxicity and embryo-foetal toxicity is 1000 mg/kg bw/day.

 

Block B

3-Aminopropyl(triethoxy)silane (CAS 919-30-2)

 

The key developmental toxicity study was an oral gavage study in male and female rats, conducted according to a protocol similar to OECD Test Guideline 414 and in compliance with GLP. The study identified maternal toxicity at 600 mg/kg bw/day, which included increased mortality, clinical observations and reduced body weight gain compared with the control group. The NOAEL for maternal toxicity was therefore 100 mg/kg bw/day. The study authors also identified a statistically significant increase in the number of minor skeletal variations (27 presacral vertebrae and sternebrae unossified), indicating minor foetotoxicity, in litters of the 600 mg/kg bw/day group. In the case of the vertebrae variation, at the highest dose it was only observed in 4/22 litters (in a total of eight animals) and therefore, considered to be of no biological significance. The sternebrae variation was observed at all doses, and in the control group, at quite high levels, but there was a very clear increase in the highest dose group. It is not possible to conclude that the effect was secondary to maternal toxicity because it occurred in several litters from dams with little or no apparent toxicity. However, the variation can result from maternal stress and is considered to be minor, and not a threat to normal functioning of the animals, and therefore not of significance in deriving a DNEL for humans. The maternal and developmental NOAEL was concluded to be 100 mg/kg bw/day. However, the slight fetotoxicity is considered to be secondary to maternal toxicity. 

 

The dossier for this constituent includes a testing proposal for an OECD 414 test in rabbits.

 

3-(Trimethoxysilyl)propylamine (CAS 13822-56-5)

 

The developmental toxicity study for 3-(trimethoxysilyl)propylamine was an oral (gavage) study in male and female rats conducted according to OECD 414 and in compliance with GLP (Charles River Laboratories, 2018). The test substance was administered at doses of 0, 100, 300 and 1000 mg/kg bw/day. Treatment at 1000 mg/kg bw resulted in unscheduled deaths of 4 rats. Two females were found dead, without notice of any signs of toxicity.  One of these females was noted with a discoloured lung, possibly due to bleeding and perhaps related to the oral gavage procedure.  As no toxicologically relevant findings were noted that indicated a poor condition in either of these two females, these deaths were considered to be unrelated to a systemic toxicological effect of the test item.  Two other high-dose females were sacrificed in extremis on Days 13 and 16 post-coitum for animal welfare reasons.  The main clinical signs in these two animals and in surviving animals were related to respiratory distress, in a dose-related manner. Reflux was also observed in 3 of the high dose animals, breathing problems shown by these females were considered to be related to the reflux of test item after administration via oral gavage.  Rales (slight to moderate severity) were observed in 4, 8 and 9 females at 100, 300 and 1000 mg/kg, respectively, at a dose-related increased frequency and duration. There were no effects attributable to treatment on uterine parameters.

Some of the clinical signs observed (respiratory effects, piloerection and abnormal posture), and body weight loss and reduced food consumption variations, may have been related to the respiratory distress and reflux caused by the administration of the test item via oral gavage; however, it cannot be excluded that some of these effects are related to systemic toxicity of the test item, so the maternal NOAEL was considered to be 300 mg/kg bw/day.

There were no effects attributable to treatment on foetal parameters, so the developmental NOAEL was considered to be at least 1000 mg/kg bw/day.

 

An OECD 414 pre-natal developmental toxicity study is in progress for this substance.

    

Trimethoxy(methyl)silane and its reaction products with [3-(2,3-epoxypropoxy)propyl]trimethoxysilane and 3-(trimethoxysilyl)propylamine (EC No. 701 -408 -8)

 

In the key combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test conducted according to OECD Test Guideline 422 and in compliance with GLP for trimethoxy(methyl)silane and its reaction products with [3-(2,3-epoxypropoxy)propyl]trimethoxysilane and 3-(trimethoxysilyl)propylamine, undiluted test material was administered by oral gavage to 10 male and female Wistar Han rats respectively per dose groups (Charles River Laboratories, 2018). The administered concentrations were 25, 75 or 250 mg/kg bw/day with a treatment period for a minimum of 28 days. A control group was also included that received water. A 14-day recovery period was performed to 5 additional male and female rats respectively, from the high and low dose groups. The recovery animals (used to study the potential reversibility of possible toxic effects) were not mated and consequently were not used for the assessment of reproduction/ developmental toxicity.

No toxicological significant clinical signs were observed in rats treated up to 250 mg/kg bw/day.  

The motor activity in male and female rats showed a large variation between the groups. Despite the fact that the motor activity recorded in the animals of all dose groups was within the historical control range, there was no indication of a treatment related change in males, but it could not be ruled out that the marked decrease in group mean values for total movements and ambulation in both main and recovery females at 250 mg/kg bw/day was a treatment related effect. Any corroborating evidence from other possibly related parameters was not observed in the current study, e.g. no other behavioural changes, no effects on grip strength and absence of histopathological alterations in nerve and muscle tissue in these females. Nevertheless, in case the changes in motor activity in females at 250 mg/kg bw/day were treatment related the magnitude of the decrease of approximately 40% lower group mean values for total movements and ambulation should be an indication of an adverse effect.

Slightly reduced body weight gain was observed in males at 250 mg/kg bw/day during treatment, resulting in approximately 4% lower group mean body weight compared to that of controls. During the recovery period, the body weight gain was similar between these two groups and the difference in body weight continued until termination of the recovery males. Based on the magnitude of the changes in body weight the effect of treatment was considered to be non-adverse.

At the end of treatment, lower mean levels for bile acids were observed in 250 mg/kg bw/day treated males and recovery females. These high dose animals had not recovered from this difference and the levels of bile acids remained lower at the end of the subsequent fourteen-day treatment free period in the recovery animals.

At the end of treatment, a dose related increase in splenic weight (in males) and decrease in adrenal weight (in females) was observed. The changes in weight of these organs in high dose animals were still within the historical control range and there were no microscopic correlates for these organ weight changes. In addition, the splenic weights in control and high dose recovery males and the adrenal weights in control and high dose recovery females were comparable at the end of the fourteen-day treatment free period. Therefore, these changes in organ weights at the end of treatment were considered to be non-adverse after treatment up to 250 mg/kg bw/day. 

No treatment-related changes were noted in the other parameters investigated in this study (i.e. food consumption, haematology, coagulation and (male) T4 thyroid hormone levels, macroscopic and microscopic examination) at the end of treatment.

Furthermore, the fourteen-day recovery period after cessation of treatment did not induce changes between the 250 mg/kg bw/day treated males and females compared to their concurrent controls in any of the parameters investigated. 

No developmental toxicity was observed up to the highest dose level tested (250 mg/kg bw/day).

Higher pup weights in the litters at 250 mg/kg bw/day were observed at birth (+12%), for which the difference had slightly decreased on PND 13-15 (+8%) when compared to controls. Although a relation to treatment cannot be ruled out, this change in body weight was considered to be non-adverse based on the magnitude and direction of difference (a slight increase in pup weight compared to controls) and the fact that the overall development of the pups was normal. The NOAEL for developmental toxicity was concluded to be at least 250 mg/kg bw/day.

This source substance is the methoxy analogue of the registered methoxy/ethoxy substance (target). Developmental toxicity study for this analogue is included to cover Blocks C to G, and unspecified minor constituents, for which there are no developmental toxicity data. The only difference between the target and source substances is one of the three starting materials. The source substance has a starting material, 3-aminopropyl(trimethoxy) silane [237-511-5, 13822-56-5] and the target has the starting material that is the triethoxy analogue, 3-aminopropyl(triethoxy)silane [213-048-4, 919-30-2]. Therefore, the target substance has additional ethoxy constituents. However, whether ethoxy or methoxy, the target and source substance constituents hydrolyse to the same respective silanol hydrolysis products. The only difference is the non-silicon hydrolysis product; the target releases methanol and ethanol, whereas the source substance releases methanol only. The small molecule alkoxysilanes of Block A, Block B and those of Block U1 have similar physicochemical properties as the starting materials. Together they constitute about 60-70% of the substance. Blocks C-G and U2 constitute approximately 20-30% of the substances. These are oligomers formed by combination of the monomer starting materials. Under acidic conditions (pH <4) as experienced in the stomach and relevant to oral studies, the hydrolysis of the source and target substances is rapid and will form the same hydrolysis products. 

Justification for classification or non-classification

Based on the available data for the constituents of trimethoxy(methyl)silane and its reaction products with 3-aminopropyltriethoxysilane and [3-(2,3-epoxypropoxy)propyl]trimethoxysilane, the substance does not require classification for reproductive or developmental toxicity according to Regulation (EC) No 1272/2008.

Additional information