Tetrakis(2-butoxyethyl) orthosilicate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 March 2016 to 23 May 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

2015

Deviations:

yes

Remarks:

Overt toxicity was observed shortly after administration of 250 mg/kg bw/day, therefore the high dose was reduced to 175 mg/kg bw/day. Three animals from HD group were not dosed on premating days 5, 6 and 7 due to overt toxicity at 250 mg/kg bw/day.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Tetrakis(2-butoxyethyl) orthosilicate

EC Number:

242-560-0

EC Name:

Tetrakis(2-butoxyethyl) orthosilicate

Cas Number:

18765-38-3

Molecular formula:

C24H52O8Si

IUPAC Name:

tetrakis(2-butoxyethyl) orthosilicate

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Expiration date of the lot/batch: 03/2017
- Purity test date: No data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, protected from light and moisture
- Stability under test conditions: Stable under test conditions.
- Solubility and stability of the test substance in the solvent/vehicle: Test item formulation was prepared in dried corn oil. The vehicle was selected based on the test item's characteristics and testing guideline.
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was weighed into a tared plastic vial on a suitable precision balance and the vehicle was added to give the appropriate final concentration of the test item and further vortexing it for 2-3 minutes. To protect from hydrolysis by humidity, the test item container and formulation vials were purged with nitrogen after the preparation before closing. The test item formulation was prepared freshly every day. Homogeneity of the test item in the vehicle was maintained by vortexing the prepared formulation thoroughly before every dose administration. The vehicle was also used as a control item.
- Preliminary purification step (if any): no data

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han) (Full Barrier)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Germany
- Age at study initiation: 10 - 11 weeks old
- Weight at study initiation: 245 - 282 g for males and 164 - 195 g for females
- Fasting period before study: none
- Housing: The animals were housed in groups of 2 animals/sex/cage in IVC cages during the premating period for both males and females and during postmating period for males depending on the mating status. During the mating period males and females were housed together in ratio 1:1. After confirmation of mating, females were housed individually during gestation and lactation period and each male was returned to their original cages. Recovery group animals were housed in groups of 2/sex/cage.
- Diet: Altronin 1324 maintenance diet for rats and mice, ad libitum
- Water: sulphur acidified to a pH of approximately 2.8 tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 55 +/- 10 %
- Air changes (per hr): 10x/hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: dried corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Test item formulation was prepared freshly daily. The test item was suspended in dried corn oil. Dose volumes were adjusted individually based on weekly body weights measurements. The administration volume was 3 mL/kg body weight.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Dried corn oil was the selected vehicle. The vehicle was selected based on the test item's characteristics and testing guideline.
- Concentration in vehicle: not specified
- Amount of vehicle (if gavage): not specified
- Lot/batch no. (if required): MKBV2080V
- Purity: not specified

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation analysis was made prior to initiation of dosing which included stability analysis and homogneity investigation. Study prestart stability analysis included samples from high dose and low dose groups and the investigation was made for 0 h, 6 h, 10 day (2-8 °C), 10 day (- 20 °C) and freeze-thaw stability. Prestart homogeneity investigation included samples collected from various levels (top, middle and bottom) of the high dose and the low dose groups. During the study samples were collected for the investigation of homogeneity and nominal substance concentration. Samples for homogeneity analysis were taken from the top, middle and bottom of the high and the low dose formulations in study weeks 1 and 5. Samples for the nominal concentration verification were taken in study week 1 (first day of premating period), 3 (first week of mating), 5 (gestation) and in the last week of the study (gestation / lactation) from all groups. All formulation samples collected during the study were analysed on the same day.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused: yes
- M/F ratio per cage: 1:1
- Length of cohabitation: maximum 14 days
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility. Not applicable
- Further matings after two unsuccessful attempts: no
- 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 group animals: the animals were treated for up to 54 days.
Main group females: the animals were treated during the 14-day premating period, maximum 14-day mating period for both males and females, during gestation and up to post-natal day 3.
Main group males: males were dosed after the mating period until the minimum total dosing period of 28 days.
Recovery group males: treated for 28 days.
Recovery group females: treated up to the first scheduled necropsy of dams and were subjected to necropsy 14 days later.

Frequency of treatment:

7 days a week

Duration of test:

Up to 54 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

25 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Remarks:

The dose was reduced from 250 mg/kg bw/day to 175 mg/kg bw/day from day 3 (for 6 male and 6 female animals of the high dose group and all animals of the high dose recovery group) or from day 2 (for 4 male and 4 female animals of the high dose group).

Doses / concentrations 4

Dose / conc.:

175 mg/kg bw/day (actual dose received)

Remarks:

The dose was reduced from 250 mg/kg bw/day to 175 mg/kg bw/day from day 3 (for 6 male and 6 female animals of the high dose group and all animals of the high dose recovery group) or from day 2 (for 4 male and 4 female animals of the high dose group).

No. of animals per sex per dose:

Main males and main females: 10 males and 10 females per dose
Recovery males and females: 5 males and 5 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses were selected based on toxicity data from a dose-range finding study and in consultation with the sponsor. The highest dose level (250 mg/kg bw/day) was chosen with the aim of inducing toxic effects, but no death or severe suffering. However, mortality or morbidity were observed at 250 mg/kg bw/day. Therefore, the highest dose was reduced to 175 mg/kg bw/day from day 3 for 6 males and 6 females of the high dose main group and all animals of the high dose recovery group or from day 2 for 4 males and females of the high dose main group.
- Rationale for animal assignment (if not random): random
- Other: Rationale for selecting satellite groups: In order to allow detection of possible delayed occurrence or persistence of or recovery from toxic effects, additional animals in recovery groups were observed following the last administration. Animals of the recovery groups were not mated.
- Post-exposure recovery period in satellite groups: 14 days
- Section schedule rationale (if not random): random

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily clinical observations were performed preferably at the same time each day. Twice daily all animals were observed for morbidity and mortality except on weekends and public
holidays.
- Cage side observations included: The health condition of the animals was recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before the first exposure, and once a week thereafter, detailed clinical observations were made in all the animals of main and recovery groups. The observations were performed
outside their home cage in a standard arena. Clinical observations included: spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes
in the skin and fur, eye and mucous membranes, piloeretion and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or p
rolonged parturition or bizarre behaviour were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: The animals were weighed once before the assignment to the experimental groups, on the first day of dosing, and weekly thereafter, as well as at the end of the study. During pregnancy females were weighed on gestation days 0, 7, 14 and 20 and within 24 hours of parturition as well as on day 4 post-partum along with the pups. Any animal prematurely sacrificed was w
eighed prior to sacrifice.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice of female animals on post-natal day # 4.
- Organs examined: See Table No. 1 for external and internal examinations including the cervical, thoracic, and abdominal viscera

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:

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: Yes: [all per litter]

Statistics:

A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical biochemistry and absolute and relative organ weights were performed for each gender by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. statistical comparisons of data acquired during the recovery period were performed with a Student’s t-Test or Mann-Whitney U-Test when appropriate.

Indices:

Reproductive indices: copulation index, fertility index, delivery index, viability index
Offspring viability indices: viability index

Historical control data:

Historical control data was used.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No adverse clinical signs were seen in males and females from low dose group. Red nasal discharge was observed in three males from mid dose group, which was considered to have an adverse effect. Adverse clinical signs were observed in the high dose group during the treatment period. In the surviving males and females of the high dose (HD) group and the high dose recovery (HDR) group haematura, reddish nasal discharge, slight to moderate piloerection, moving the bedding and reduced spontaneous activity were noted. Additionally, in females kyphosis, and moderate salivation were recorded. The reported signs were seen mostly up to the end of the premating period and for the remainder of the study no clinical signs were observed.
Reduced spontaneous activity was observed a day before one high dose male was found dead. Haematuria, reddish nasal discharge, moderate piloerection, sunken flanks, half eyelid closure and kyphosis were observed in one male from high dose recovery group before the animal was euthanised. Moderate to severe salivation, moderate to severe piloerection, half eyelid closure, reduced spontaneous activity, kyphnosis, haematuria, sunken flanks and dehydration were observed in one high dose group female before euthanization.

Mortality:

mortality observed, treatment-related

Description (incidence):

Test item treatment related mortalities were observed at the initial high dose of 250 mg/kg bw/day. One male from high dose group was found dead on premating day 3. One male of the high dose recovery group and one female of the high dose group were euthanized due to animal welfare reasons on premating day 7. These animals showed adverse clinical signs and weight loss. Due to the observed toxic effects at 250 mg/kg bw/day, the high dose was reduced to 175 mg/kg bw/day from day 3 (for 6 males and 6 females of the high dose group and all animals of the high dose recovery group) or from day 2 (for 4 males and 4 females of the high dose group).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

In males and females, the mean absolute body weight was not statistically significantly altered in test item -treated groups compared to the control during the treatment and recovery period except for the statistically significantly lower body weight on premating day 7 in females of the HD group. However, body weight gain was statistically significantly lower in the first week of treatment in both male and female HD and HD recovery groups compared to the corresponding controls. This was due to the treatment with the higher 250 mg/kg bw/day dose. After lowering the high dose to 175 mg/ kg body weight, the weight gain in the second week of premating was statistically higher in both male and female HD and HD recovery groups compared to the corresponding controls. In addition, during the mating and post-mating periods including the gestation period of females and also during the recovery period, no statistically significant changes were noted between the test item treated and control groups. There was a lower weight gain in female HD group during the lactation period, which correlated with lower food consumption in the HD group.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In males (main group) at the end of treatment, statistically significantly lower RBC, HB, HCT and MCHC were observed in the HD group and statistically significantly higher MCV and MCH were observed in the HD group compared to the corresponding control group. Statistically significant increase in MCV and MCH in the HD recovery group males was also observed.
In females (main group) at the end of treatment, statistically significantly lower RBC were observed in the MD and HD groups compared to the control. Statistically significantly higher MCV and MCH were observed in the HD group compared to the control. Additionally, the reticulocyte count was higher in the female LD, MD and HD groups compared to the control in a dose response pattern, but without statistical significance. The mean reticulocyte count in the MD and HD groups were above the range of historical control data. These changes could be due to possible bleeding during parturition. Moreover, this corresponded to a statistically significantly lower mean RBC in the MD and HD groups and was related to test item treatment except in the LD group where the mean RBC was not statistically significant and was considered non-adverse. This finding was related to test item treatment.
The haematological findings in males and females at the end of treatment in the HD group were due to hematotoxicity of test item as evident from the treatment-related pathological changes characterized by hemolysis-related findings including hemoglobinuric nephrosis and increased erythroid hemopoiesis as well as splenic infarction in survivors and decedents, and by the disseminated thrombosis in decedents.
In both males and females (main and recovery groups), there were no effects of test- item on coagulation parameters including prothrombin time (PT) and activated prothrombin time (aPTT).

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

In males and females (main and recovery groups), the mean of biochemistry parameters were not statistically significantly different between the dose and corresponding control groups. However, there were higher TBA in male HD group, higher mean ASAT and Na in female LD and HD groups and higher mean ALAT, AP, CREA in all dose groups (LD, MD and HD) without attaining statistical significance. These findings, in the absence of statistically significant differences, were not considered to be an adverse effect of test item treatment.

Urinalysis findings:

no effects observed

Description (incidence and severity):

In males and females at the end of the treatment, there were no significant differences in urine parameters of dose groups compared to the corresponding control. At the end of recovery one male and one female of the HD group had a higher leucocyte count. These findings were attributed to tubular degeneration, mononuclear foci and cyst and/or hyaline cast of grade-1 observed histopathologically in
kidneys. These findings were in single isolated animals of the group and therefore, were not considered adverse.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no effects on body temperature, rearing supported, rearing not supported, urination and defecation in male and female rats. There were no significant changes in neurological assessment bet
ween the test- item -treated and control groups during the treatment and the recovery period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In males at the end of the treatment, statistically significantly higher kidney weight relative to body weight was observed in the HD group. In males at the end of recovery period, there was statistically significantly higher absolute kidney and liver weight, statistically significantly higher spleen weight (absolute and relative to body weight) and statistically significantly higher pituitary gland weight (absolute and relative to body weight) in HD recovery group compared to the control. In females at the end of the treatment, statistically significantly higher spleen weight relative to body we
ight was observed in the HD group, which did not reverse after the 14 day recovery period. Statistically significantly higher spleen weight (absolute and relative to body weight) was noted in the HD recovery group compared to the corresponding control. Additionally in females, there were statistically significantly higher brain weights relative to body weight in the HD group and statistically significantly higher liver weight relative to body weight in the MD group compared to the control. At the end of recovery, there was also statistically significantly higher absolute kidney weight compared to the corresponding controls.
The observed changes in organ weight correlated histopathologically with observed structural changes in the kidneys and spleen of both sexes, and those were the changes related to hematotoxic effects of the test item.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Black and marbled discoloration in kidneys; pale discoloration in liver, pituitary gland, adrenal glands and brain were observed in one male and one female animals that were euthanised on study day 7 for ethical reasons. In addition, pink discolouration in the testes was observed for the male animal. No macroscopic abnormalities were observed in one male animal that died on study day 2. Haemoglobin casts and/or tubular necrosis/degeneration were considered to be the cause for black and marbled discolouration of kidneys. Pale discoloration of spleen (no. 51) and adrenal glands (no. 87) were deemed to correlate microscopically with infarction of the spleen and increased vacuolation (fatty change) of zona fasciculata of the adrenal glands, respectively. Pink discoloration of testes (no. 51) was deemed to correlate microscopically with hemorrhage in the interstitium. It was considered that macroscopic findings in spleen, kidneys and testes corresponded to histologic lesions being attributed to treatment with the test item.
In the survivors, small spleen was noted in two HD males. Pale discolouration of the spleen was noted in another male. These changes correlate microscopically with infraction of the spleen caused by the test item.
Fluid-filled uterus, red discolouration in the thymus, haemorrhage in the cecum, absent left testes and epididymis were also observed in the survivors, however these changes were normal physiological change, congenital anomaly, or incidental findings that were not related treatment with the test article.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The test item produced haematotoxicity, and the treatment-related pathological changes were characterised by hemolysis-related findings, including hemoglobinuric nephrosis and increased erythroid hemopoiesis as well as splenic infarction in survivors and decedents, and the disseminated thrombosis in decedents. In main group survivours, haematotoxicity related findings were observed in kidneys, spleen and bone marrow. Increased severity of tubular degeneration accompanied by hemoglobin casts, granular casts, tubular hemosiderin deposits, and/or tubulo-interstitial inflammation in kidneys were seen in mid-dose females and mid and high dose males and females. These changes were not seen after a 14-day recovery period. In the spleen, infraction was observed in high dose males and an increased severity of erythrocytic extramedullary hemopoiesis in high dose females. Hemosiderin deposits were found in both sexes from high dose groups. These changes were not seen after a 14-day recovery period In the bone marrow, an increased incidence and/or severity of erythroid hypercellularity was observed in high dose females. These changes were not seen after a 14-day recovery period.
Histological changes related to hematotoxicity were also observed in decedents of the high dose group, which included thrombi in heart, lungs, liver, spleen, kidneys (glomeruli), testes, and/or spinal cord (meningeal vessels); hemoglobin casts, tubular necrosis/degeneration, tubulo-interstitial inflammation and/or tubular dilation in the kidney; infarction and/or increased severity of erythrocytic extramedullary hemopoiesis in the spleen; erythroid hypercellularity in the bone marrow; hemopoietic foci, multifocal necrosis and/or centrilobular vacuolation in the liver; alveolar edema in the lung; hemorrhage in the interstitium of testes; and necrosis with gliosis and meningeal hemorrhage in the cervical segment of the spinal cord.
Hemoglobinuric nephrosis related findings including hemoglobin casts, tubular necrosis/ degeneration and/or tubulo-interstitial inflammation, which were recorded at moderate to marked severity, as well as disseminated thrombosis and/or splenic infarction, were considered the major causes of animal’s death and morbidity.
The findings in lungs, liver and spinal cord recorded in decedents were copnsidered to be caused by local circulation disturbance related to haematotoxicity including hemolysis and/or thrombosis. Stress-related changes in decedents included lymphoid depletion in lymph nodes, atrophy of thymus, diffuse cortical hypertrophy and fatty change in zona fasciculata of adrenal glands and erosion in stomach and/or duodenum.
Indicators of toxicity in the kidneys were observed in the kidneys of females at 100 mg/kg bw/day consisting of two cases of granular casts and an increased (severity only) tubular degeneration/
regeneration. Especially the granular casts that represent tubular cell necrosis are deemed to be of adverse nature. It may be considered that these findings are secondary to hemoglobinuric nephrosis.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Food consumption: Lower food consumption was observed in HD and HD recovery male and female groups when compared to the corresponding controls during the first week of treatment, which was due to the adverse effects of the test item-treatment at 250 mg/kg bw/day. After the high dose was reduced to 175 mg/kg bw/day, the food consumption measures were comparable to corresponding controls. During the lactation period, statistically significantly lower food consumption was observed in the HD group females, which correlated with lower body weight gain in the HD group.

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The number of Corpora lutea and the number of implantation sites were not affected by the test item. There was higher percentage of pre-implantation losses in the LD and HD groups, but no dose response relationship or statistical significance were observed.

Total litter losses by resorption:

not examined

Early or late resorptions:

not examined

Dead fetuses:

no effects observed

Description (incidence and severity):

There was markeldly higher percentage of post-implantation loss in the HD group, which was not statistically significant when compared to control.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Precoital interval and duration of gestation were not affected by the test item.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): The precoital interval and the duration of gestation were not affected by the test item.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

There was no effect on copulation index. The fertility index was lower in the HD group.

Other effects:

not specified

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: The delivery and viability indices were lower in HD group.

Maternal abnormalities

Abnormalities:

effects observed, treatment-related

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): The pup mean weight on PND 0 and 4 was not statistically significantly different in test-item treated groups compared to the corresponding control group.

Reduction in number of live offspring:

effects observed, treatment-related

Description (incidence and severity):

On PND 0, the mean number of pups born and the mean number of live pups were lower in the HD group but without statistical significance compared to the corresponding control. Given the higher implantation losses in the HD group, these findings were secondary. There was a higher mean number of still births in the LD and HD groups compared to the control, but without statistical significance or a dose response relation. There were no runts observed in any of the groups. Due to the missing significance no adverse effects can be derived from the litter data on PND 0.
On PND 4, there was statistically significantly lower number of live pups in the HD group compared to the control. Sex specific evaluation also revealed a markedly lower number of male and female live pups on PND 4 in the HD group compared to the control, but without statistical significance. Higher pup mortality in the HD group during the lactation period (between PND 0 and PND4) resulted in lower number of live pups and lower number of male and female live pups in the HD group and was considered to be related to test-item treatment. There was markedly higher pup mortality in the HD group, which was due to pup mortality observed in two females and was related to test-item treatment. These pups were cold and showed lack of suckling, which was related to nursing behaviour of female and therefore, is secondary to maternal toxicity.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex-ratio on PND 0 and PND 4 was higher in the MD group and lower in the HD group compared to the corresponding control without attaining statistical significance and a dose response relationship.

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

The pup mean weight on PND 0 and 4 were not statistically significantly different in test-item treated groups compared to the corresponding control group.
The total litter weight and male litter weight on PND 0 was lower in the HD group without attaining statistical significance and without a dose response relationship.
On PND 4 the total litter weight and male and female litter weight were slightly lower in the LD and MD groups and markedly lower in the HD group compared to the corresponding control without attaining statistical significance. The markedly lower total litter weight and total male and total female litter weights in the HD group was attributed to higher pup mortality during the lactation period (between PND 0 and PND 4). This finding was considered to be an effect of test- item treatment.

Changes in postnatal survival:

effects observed, treatment-related

Description (incidence and severity):

On PND 4, there was statistically significantly lower number of live pups in the HD group compared to the control. Sex specific evaluation also revealed a markedly lower number of male and female live pups on PND 4 in the HD group compared to the control, but without statistical significance. Higher pup mortality in the HD group during the lactation period (between PND 0 and PND4) resulted in lower number of live pups and lower number of male and female live pups in the HD group and was considered to be related to test-item treatment. There was markedly higher pup mortality in the HD group, which was due to pup mortality observed in two females and was related to test-item treatment. These pups were cold and showed lack of suckling, which was related to nursing behaviour of female and therefore, is secondary to maternal toxicity.

External malformations:

effects observed, treatment-related

Description (incidence and severity):

There were no adverse pup external findings up to the MD group. In the HD group, all pups of dams 84 and 92 were cold and showed lack of suckling. Additionally, one pup from dam no. 84 was blueish and one pup from dam no. 90 had haematoma on neck. All these pups were found dead or missing by up to PND 3. The missing pups were considered to be cannibalized by dams. Cold pups including the blueish one with no indication of suckling were directly related to nursing behaviour of females. Therefore, these pup findings were considered secondary to maternal toxicity.

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

changes in postnatal survival

external malformations

Fetal abnormalities

Abnormalities:

effects observed, treatment-related

Localisation:

other: gross external findings of hypothermia and lack of suckling

Overall developmental toxicity

Developmental effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

Any other information on results incl. tables

Table 1: Individual Reproductive Indices

Group	Animal No.	Copulation Index (%)	Fertility Index (%)	Delivery Index (%)	Viability Index (%)
C	53	100	100	100	100.00
C	54	100	100	100	100.00
C	55	100	100	100	100.00
C	56	100	100	100	100.00
C	57	100	100	100	100.00
C	58	100	100	100	100.00
C	59	100	100	100	100.00
C	60	100	100	100	100.00
C	61	100	100	100	100.00
C	62	100	100	100	100.00
LD	63	100	100	100	100.00
LD	64	100	100	100	100.00
LD	65	100	100	100	100.00
LD	66	100	100	100	100.00
LD	67	100	100	100	100.00
LD	68	100	100	100	100.00
LD	69	100	100	100	40.00
LD	70	100	100	100	100.00
LD	71	100	100	100	100.00
LD	72	100	100	100	100.00
MD	73	100	100	100	100.00
MD	74	100	100	100	100.00
MD	75	100	100	100	100.00
MD	76	100	100	100	100.00
MD	77	100	100	100	100.00
MD	78	100	100	100	100.00
MD	79	100	100	100	100.00
MD	80	100	100	100	100.00
MD	81	100	100	100	100.00
MD	82	100	100	100	100.00
HD	83"	100	88.9	87.5	-
HD	84	100	88.9	87.5	88.89
HD	85	100	88.9	87.5	100.00
HD	86	100	88.9	87.5	100.00
HD	87**	100	88.9	87.5	-
HD	88#	100	88.9	87.5	-
HD	89	100	88.9	87.5	100.00
HD	90	100	88.9	87.5	100.00
HD	91	100	88.9	87.5	100.00
HD	92	100	88.9	87.5	0.00

Copulation Index (%) = (No. of rats copulated / No. of pairs) X 100

Fertility Index (%) = (No. of females pregant / No. of females copulated) X 100

Delivery Index (%) = (No. of dams with live newborns / No. of pregnant dams) X 100

Viability Index (%) = (No. of live offspring at day 4 / No. of live offspring at birth) X 100

# = non pregnant; ** = euthanised for animal welfare reasons; " = pub was cannibalised

Applicant's summary and conclusion

Conclusions:

In the Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test, conducted according to OECD TG 422 and in compliance with GLP, the reported NOAEL for tetra kis(2-butoxyethyl) orthosilicate was 100 mg/kg bw/day for developmental toxicity based on lower delivery and viability indices in females and pups of the high dose group.