Isobutylisopropyldimethoxysilane

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6 September to 30 October 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted in accordance with OECD Guideline with GLP certificate

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

see below

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: The United States Environmental Protection Agency (EPA) Health Effects Test Guidelines OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.

Deviations:

yes

Remarks:

see below

Principles of method if other than guideline:

List of protocol deviations:
1. Temporary deviations from the daily mean relative humidity occurred.
Evaluation: Laboratory historical data do not indicate an effect of the deviations.
2 During the lactation period, no clinical observations were registered in the computer for all pups of litter 49 (Control) and litter 79 (Group 4) on Day 5.
Evaluation: Sufficient data was available for evaluation.
3. Inadvertently, body weight of animal no. 69 was not determined at necropsy.
Evaluation: Sufficient data was available for evaluation.
4. No Salewski staining (to detect implantation sites) was performed for female no. 46 who did not mate.
Evaluation: An macroscopic examination of the implantation sites and corpora lutea was performed for this animal, and revealed no implantation sites or corpora lutea.
The study integrity was not adversely affected by the deviations.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Isobutylisopropyldimethoxysilane

EC Number:

402-580-4

EC Name:

Isobutylisopropyldimethoxysilane

Cas Number:

111439-76-0

Molecular formula:

Hill formula: C9H22O2Si

IUPAC Name:

dimethoxy(2-methylpropyl)(propan-2-yl)silane

Test material form:

other: liquid

Details on test material:

Identification: Isobutylisopropyldimethoxysilane
Molecular formula C9H22O2Si
Molecular weight 190.36
CAS Number 111439-76-0

Test animals

Species:

rat

Strain:

other: Wistar Han

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test system: Rat: Crl:WI(Han) (outbred, SPF-Quality). Nulliparous and non-pregnant females and untreated animals were used at initiation of the study.
Rationale: This species and strain of rat has been recognized as appropriate for reproduction toxicity studies. WIL Research Europe B.V. has reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Source F0: Charles River Deutschland, Sulzfeld, Germany.
Age at start F0-treatment: Approximately 11 weeks.
Number of F0-animals: 40 females and 40 males.
Acclimatization F0: At least 5 days prior to start of treatment.
Health inspection F0: Upon receipt of the animals.
Randomization F0: Prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.
Identification F0: Earmark and tattoo.

Animal husbandry
Room number A0.15.
Conditions: Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air
changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.
Accommodation: Pre-mating Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
Lactation: Pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm).
General: Sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment/nesting material (Enviro-dri, Wm.
Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied.

Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
Water: Free access to tap-water.
Diet, water, bedding and cage-enrichment/nesting material evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Vehicle: Corn oil, specific gravity 0.92 (Fagron, Nieuwerkerk a/d/ Ijssel, The Netherlands).
Rationale for vehicle: Based on trial formulations performed at WIL Research Europe and based on information provided by the Sponsor.
Method of formulation: Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity/density of the test substance and the vehicle. No correction was made for the purity/composition of the test substance.
Storage conditions: At ambient temperature.

Oral gavage, using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing.

Dose volume 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Details on mating procedure:

Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating occurred, the males and females were separated. A maximum of 14 days was allowed for mating, after which females who had not shown evidence of mating were separated from their males.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase (20 December 2012), according to a validated method (Project 500516). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

Duration of treatment / exposure:

Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 41-48 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy). Female nos. 49 and 50 (Group 1), 55 (Group 2), 63, 68 and 70 (Group 3) were not dosed during littering.

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.

Details on study schedule:

Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating occurred, the males and females were separated. A maximum of 14 days was allowed for mating, after which females who had not shown evidence of mating were separated from their males.
Parturition: The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes and placentas cleaned up, nest build up and/or feeding of pups started). Females that were littering were left undisturbed.
Number of pups: 429 pups.
Identification of pups: On Day 1 of lactation, all pups were individually identified by means of subcutaneous injection of Indian ink.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:
nominal conc.

No. of animals per sex per dose:

10 male + 10 female per dose

Control animals:

yes, concurrent vehicle

Details on study design:

Dose levels were based on a previous 28-Day study (IC 20/88) where animals were exposed to dose levels of 0, 100, 300 and 1000 mg/kg. For male animals at 300 and 1000 mg/kg an increased incidence of progressive nephropathy was seen. These findings are not uncommonly noted for male
rats. As this represents a rodent-specific condition with little relevance to the human condition, dose levels of 100, 300 and 1000 mg/kg were chosen for this study.

Positive control:

None

Examinations

Parental animals: Observations and examinations:

Mortality / Viability: At least twice daily.
Clinical signs: Daily from start of treatment onwards up to the day prior to necropsy, detailed clinical observations were made in all animals. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4.
Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.
Body weights: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.
Food consumption: Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 postcoitum and on Days 1 and 4 of lactation.
Water consumption: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

General reproduction data: Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Palpation was used to aid in confirmation of pregnancy. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of pregnant females was examined to detect signs of abortion or premature birth. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.

Oestrous cyclicity (parental animals):

Histological evaluation of the female reproductive tract was conducted.

Sperm parameters (parental animals):

Of the males of the control and high dose group, and all males suspected to be infertile, additional slides of the testes were prepared to examine staging of spermatogenesis. Additionally, the following organ weights were recorded from all F0-males on the scheduled day of
necropsy: Epididymides, Testes

Litter observations:

Each litter was examined to determine the following, if practically possible:
Mortality / Viability: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
Clinical signs: At least once daily, detailed clinical observations were made for all animals.
Body weights: Live pups were weighed on Days 1 and 4 of lactation.
Sex: Sex was determined for all pups on Days 1 and 4 of lactation.

Postmortem examinations (parental animals):

Necropsy parental animals
The animals were not deprived of food overnight. Animals surviving to scheduled necropsy were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands; supplemented with N2O when needed) and subsequently exsanguinated.
Necropsy was conducted on the following days:
Females which delivered: Day of necropsy was Lactation Day 5.
Females which failed to deliver (nos. 46, 69 and 72): Day of necropsy was post-coitum Days 25-27 (females with evidence of mating) or approximately 21 days after the last day of the mating period (females without evidence of mating).
Males: Day of necropsy was following completion of the mating period (a minimum of 28 days of dose administration).

Note: In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites (Salewski staining prepared at WIL Research Europe using Ammoniumsulfide-solution 20% (Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)).

All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.
The numbers of former implantation sites and corpora lutea were recorded for all paired females.

Samples of the following tissues and organs were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands).
Cervix, Preputial gland, Clitoral gland, Prostate gland, Coagulation gland, Seminal vesicles, Epididymides (2), Testes (2), Ovaries, Uterus, Vagina
Identification marks: not processed Vagina, All gross lesions

(2) Fixed in modified Davidson's solution (prepared at WIL Research Europe using Formaldehyde 37- 40%, Ethanol, Acetic acid (glacial) (all Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)) and transferred to formalin after fixation for at least 24 hours.

Organ weights:
The terminal body weight and kidney weights were recorded from all animals on the scheduled day of necropsy.
Additionally, the following organ weights were recorded from all F0-males on the scheduled day of necropsy:
Epididymides
Testes

Histotechnology
All organ and tissue samples, as defined under Histopathology (following section), were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands).
Of the males of the control and high dose group, and all males suspected to be infertile, additional slides of the testes were prepared to examine staging of spermatogenesis. The testes were processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin (Klinipath, Duiven, The
Netherlands).

Histopathology
A peer review on the histopathology data was performed by a second pathologist.
The following slides were examined by a pathologist:
- The kidneys, ovaries, testes and epididymides of the animals of Groups 1 and 4.
- The additional slides of the testes of the males of Groups 1 and 4, and all males suspected to be infertile to examine staging of spermatogenesis.
- All gross lesions of all animals (all dose groups).
- Kidneys of the 5 selected males (see Allocation) of Groups 2 and 3, based on (possible) treatment-related changes in this organ in Group 4.
- The reproductive organs* of all males that failed to sire (nos. 6, 29 and 32; Groups 1, 3 and 4, respectively) and all females that failed to deliver healthy pups (nos. 46, 69 and 72; Groups 1, 3 and 4).
* Reproductive organs includes the cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, and vagina.
All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.

Postmortem examinations (offspring):

Pups surviving to planned termination were killed by decapitation on Days 5 of lactation.
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated.

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (manyto-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (Ref. 3; many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Ref. 4) was applied to frequency data.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. 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.

Reproductive indices:

See under background material below

Offspring viability indices:

See under background material below

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Effect on kidneys in males at 300 and 1000 mg/kg bw/day

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

Mortality
No mortality occurred during the study period.

Clinical signs
No clinical signs of toxicity were noted during the observation period up to 1000 mg/kg.
Salivation was seen for all animals at 1000 mg/kg and all males at 300 mg/kg. Salivation was also noted for one male at 100 mg/kg (over 2 days). Salivation 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). This sign may be related to the taste or possible irritancy of the test substance.
Incidental findings seen for control and/or treated animals included scabs on the cheek and neck, a wound on the right cheek, swelling of the cervical region, and alopecia. These findings 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. At the incidence observed, these were not considered to be toxicologically relevant.

Body weights
No toxicologically relevant changes in body weights and body weight gain were noted up to 1000 mg/kg.
Body weights were significantly higher for females at 1000 mg/kg (absolute and body weight gains) and for males at 300 mg/kg (absolute only) on Day 8 of the premating period.
These changes were considered to be incidental since body weight loss would be expected if treatment related toxicity were evident, and for males, this occurred in the absence of a dose related distribution.
Body weight gains were significantly lower for females at 1000 mg/kg on Day 20 post coitum. This was attributable to low weights obtained for two females, and was therefore not considered to be toxicologically relevant. Female no. 75 did deliver live pups, and female no. 72 had implantation sites only. All other females at this dose level had normal body weights.

Food consumption
No toxicologically relevant changes in food consumption before or after allowance for body weight were noted.
Slightly higher relative food consumption was noted for males at 1000 mg/kg during the entire treatment period. This was not considered to be toxicologically relevant since lower food consumption would have been expected if treatment related toxicity were evident.
Absolute and relative food consumption was significantly lower for females at 1000 mg/kg from lactation Days 1-4. This was not considered to be toxicologically relevant because the difference from controls was attributable to very low food consumption for female no. 78.

Macroscopic examination
At 1000 mg/kg, enlarged kidneys were seen for one male (no. 36) and pale discoloration of the kidneys was noted for one male (no. 33), which was representative of hyaline droplets noted at the microscopic examination. Though the incidence of these findings was minimal, they were supportive
of relevant organ weight and microscopic changes of the kidneys (see below).
One female at 300 mg/kg was found with one mummified fetus in the cervix. This was not considered to be treatment related.
One female at 100 mg/kg (no. 51) was found with a large grey-white and greenish nodule (21x16 mm) in the back of the neck that was part firm and part soft. At the microscopic examination this was seen to be a well-differentiated adenocarcinoma. This was considered to be spontaneous in nature and not secondary to treatment-induced toxicity (for additional details see below)
Incidental findings noted for control and/or treated animals included yellowish, soft nodule on the epididymis tail or body, pelvic dilation of one or both kidneys, alopecia on the hindlegs, forelegs or left flank, scab formation, isolated tan focus/foci on the left clitoral gland and enlarged spleen. These
findings remained within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. These necropsy findings were therefore not considered to be toxicologically relevant.

Organ weights
There were relevant effects on kidney weights for males at 1000 mg/kg. Absolute and relative kidney weights were increased for animals of both sexes at 1000 mg/kg. Absolute weights were approximately 20% and 10% higher for males and females (respectively).
The difference noted for females was modest, remained within the range considered normal for animals of this age and strain and was not accompanied by any morphological changes. As such, it was not considered to be adverse for females.
Testes and epididymides weights and terminal body weights of treated males were similar to those of controls.
Control male no. 6 had massive seminiferous atrophy of the testes noted as the microscopic examination and was infertile (see below). This is also reflected in his macroscopic findings and very low testes and epididymis weights.

Microscopic examination
There were treatment-related microscopic findings in kidneys of male rats of all treated Groups:
− Cortical hyaline droplets were increased in incidence and severity in all treated groups of males. This was recorded in 2/10 males of Group 1 (0 mg/kg, control, 2: minimal), in 10/10 males of Group 2 (100 mg/kg, 2: minimal, 5: slight, 3: moderate), in 10/10 males of Group 3 (300 mg/kg,
1: minimal, 3: slight, 2: moderate, 4: marked) and in 10/10 males of Group 4 (1000 mg/kg, 5: moderate, 5: marked).
− Granular casts were recorded in 6/10 males of Group 3 (300 mg/kg. 1: minimal, 4: slight, 1: moderate) and in 10/10 males of Group 4 (1000 mg/kg, 2: minimal, 4: slight, 2: moderate, 1: marked).
− Tubular dilation at a minimal degree was recorded in 1/10 males of Group 3 (300 mg/kg) and in 3/10 males of Group 4 (1000 mg/kg).
− Corticomedullary tubular basophilia was increased in incidence and/or severity in all treated groups of males. This was recorded in 3/10 males of Group 1 (0 mg/kg, control, 3: minimal), in 9/10 males of Group 2 (100 mg/kg, (: minimal), in 8/10 males of Group 3 (300 mg/kg, 6: minimal, 2: slight) and in 10/10 males of Group 4 (1000 mg/kg, 5: minimal, 5: moderate).
− Lymphocytic, interstitial inflammation was recorded in 1/10 males of Group 1 (0 mg/kg, control, minimal), in 2/10 males of Group 2 (100 mg/kg, minimal), in 5/10 males of Group 3 (300 mg/kg, 5: minimal) and 6/10 males of Group 4 (1000 mg/kg, 6: minimal).
There was one pair of animals in Group 1 (male 6/female 46), Group 3 (male 29/female 69) and Group 4 (male 32/female 72) that failed to sire or deliver healthy offspring. For animal 6 (Group 1, control) the cause of infertility was massive, bilateral seminiferous atrophy of the testes. In the uterus of animal 69 a mummified fetus was present. No cause of infertility/delivery of healthy offspring could be established for these pairs in Group 3 and Group 4.

Spermatogenic staging profiles were normal for all males examined, except for infertile male 6 (0 mg/kg, control). In animal 6 no normal stages were present.
The females that failed to deliver healthy pups (46: did not mate, 69: had one mummified fetus and 72: had implantation sites only) all had a histologically normal cycling female reproductive tract.
There was one macroscopic/microscopic finding of note in female 51 (Group 2, 100 mg/kg):
− A gray-white/greenish, part firm/part soft nodule of 21x16 MM was recorded in the subcutis of the neck. This was a well differentiated adenocarcinoma of the mammary gland.
Adenocarcinomas of the mammary gland in female rats are seen as a spontaneous background finding at a low incidence.
All remaining microscopic findings recorded were within the normal range of background pathology encountered in Wistar Han rats of this age and strain.

Reproduction data
No toxicologically relevant effects on reproductive parameters were noted. The mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.

Developmental data
No toxicologically relevant effects on gestation index and duration, parturition, maternal care and early postnatal pup development (mortality, clinical signs, body weight and macroscopy) were observed.

Gestation
The gestation index and duration of gestation were unaffected by treatment up to 1000 mg/kg.

Parturition/maternal care
No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth and no deficiencies in maternal care were observed.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

Reproduction

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No reproduction toxicity was observed for treatment up to 1000 mg/kg.

2

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No treatment related adverse effects up to 1000 mg/kg bw/day.

3

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: see 'Remark'

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

Early postnatal pup development
Number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings.

Mortality
One pup of the control group and two, one and one pups of the 100, 300 and 1000 mg/kg groups were found dead or went missing during the first days of lactation. Missing pups missing were most likely cannibalized. 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.

Clinical signs
Incidental clinical symptoms of pups consisted of lean appearance, no milk in the stomach, discoloration of the snout, and scab on the left foreleg. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore not considered
to be toxicologically relevant.

Body weights
Body weights of pups were unaffected by treatment.

Macroscopy
Incidental macroscopic findings of pups that were found dead included beginning autolysis. Discoloration of the snout and no milk in the stomach were noted for individual pups surviving to the end of the scheduled necropsy. The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore not considered to be toxicologically relevant.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No developmental toxicity was observed for treatment up to 1000 mg/kg.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Analysis of dose preparations

The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%) and no test substance was detected in the Group 1 formulation.

The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 6 hours.

Applicant's summary and conclusion

Conclusions:

In conclusion, treatment with Isobutylisopropyldimethoxysilane by oral gavage in male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg revealed parental toxicity for males at 300 and 1000 mg/kg characterized by microscopic findings in the kidneys and (secondarily) increased kidney weights. The microscopic findings noted represent a condition unique to male rats and have little relevance for the human condition. No reproduction and developmental toxicity was observed for treatment up to 1000 mg/kg.
Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:
Parental NOAEL: 100 mg/kg (males) and 1000 mg/kg (females)
Reproduction NOAEL: at least 1000 mg/kg
Developmental NOAEL: at least 1000 mg/kg

Executive summary:

Isobutylisopropyldimethoxysilane was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 28 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 41-48 days).

Formulation analysis showed that the formulations were prepared accurately, were homogenous, and were stable for at least 6 hours at room temperature.

Parental results:

Treatment related findings were limited to renal effects for males at 300 and 1000 mg/kg and were primarily attributable to increased cortical hyaline droplets. Secondary to the increased hyaline droplets, tubular dilation, corticomedullary tubular basophilia and lymphophocytic, interstitial inflammation were also evident.

The hyaline droplets likely represented alpha2μglobulin, a normal protein in male rats which undergoes re-absorption in the proximal cortical tubules. A range of chemicals are known to increase hyaline droplet formation, leading ultimately to proximal cortical tubule cell injury, the formation of granular casts and increased cell turnover as manifested by tubular basophilia. The combination of renal lesions at 300 and 1000 mg/kg has been referred to as male rat progressive nephropathy. This protein is not present in female rats or in higher mammals, including man, and thus has little relevance to the human condition. An increased incidence and severity of cortical hyaline droplets was also seen for males at 100 mg/kg, though this was not accompanied by degenerative tubular alterations and was

thus not considered an adverse finding at this level. There were no relevant microscopic findings noted for females up to 1000 mg/kg.

Pale discoloration of the kidneys and an enlarged kidney were noted at the macroscopic examination for one male each at 1000 mg/kg. These incidental findings were secondary to the aforementioned effects: the pale discoloration was a secondary representation of the hyaline droplets, and the enlarged kidney is supportive of the increased kidney weights.

No treatment-related changes were noted in any of the remaining parental parameters investigated in this study (i.e. clinical appearance, body weight and food consumption).

Reproductive results:

No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg).

Developmental results:

No developmental toxicity was observed up to the highest dose level tested (1000 mg/kg).

In conclusion, treatment with Isobutylisopropyldimethoxysilane by oral gavage in male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg revealed parental toxicity for males at 300 and 1000 mg/kg characterized by microscopic findings in the kidneys and (secondarily) increased kidney weights. The microscopic findings noted represent a condition unique to male rats and have little relevance for the human condition. No reproduction and developmental toxicity was observed for treatment up to 1000 mg/kg.

Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:

Parental NOAEL: 100 mg/kg (males) and 1000 mg/kg (females)

Reproduction NOAEL: at least 1000 mg/kg

Developmental NOAEL: at least 1000 mg/kg