2,11-Dioxa-3,10-disiladodecane, 3,3,10,10-tetramethoxy-

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:

2009-09-28 to 2009-12-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study was conducted according to the appropriate OECD test guideline and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 001

Cas Number:

87135-01-1

Molecular formula:

C12H30O6Si2

Constituent 2

Reference substance name:

3,3,10,10-tetramethoxy-2,11-dioxa-3,10-disiladodecane

EC Number:

617-969-6

Cas Number:

87135-01-1

Molecular formula:

(CH3O)3Si(CH2)6Si(OCH3)3 C12 H30 O6 Si2

IUPAC Name:

3,3,10,10-tetramethoxy-2,11-dioxa-3,10-disiladodecane

Test material form:

other: liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: no details
- Age at study initiation: 9 wks
- Weight at study initiation: Males: 270.2-352.6 g; Females: 199.2-233.1 g
- Housing: 1/suspended wire mesh cage (except during mating, gestation, lactation and urine analysis). During mating 1 female:1 male in the home cage of the male. After positive evidence of mating - shoebox cages. During lactation dams were housed with their litters. For urine collection (the day prior to necropsy) - stainless steel metabolism cages (without feed).
- Use of restrainers for preventing ingestion (if dermal): no
- Diet: standard diet ad libitum
- Water: drinking water ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.7-22.8
- Humidity (%): 14->70 (guideline indicates 30-70 except during cleaning). One occurrence each of 14% and 29% RH were recorded - these were brief and not considered to affect the overall findings of this study.
- Air changes (per hr): approximately 13.1
- Photoperiod (hrs dark / hrs light): 12 h/12 h

IN-LIFE DATES: From: 2009-09-28 presumably for up to 55 days

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Prepared 4 times during study, analysed for dose concentration (GC/FID). The lowest dose was analysed for stability and homogeneity during this study, the two higher concentrations were analysed for stability and homogeneity in a similar previous study with the same test material at similar concentrations.

VEHICLE
- Justification for use and choice of vehicle (if other than water): corn oil was considered an appropriate vehicle for oral administration.
- Amount of vehicle (if gavage): total administered volume 3 ml/kg bw
- Lot/batch no. (if required): not given
- Purity: not given

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: continuously until evidence of mating
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

GC/FID (no further details given in the 105 pages of this report that have been seen).

Duration of treatment / exposure:

daily, 7 days/week for 29 consecutive days to males and 55 consecutive days to females.

Frequency of treatment:

daily

Details on study schedule:

Single generation study, F1 animals not mated.

Doses / concentrations

Remarks:

Doses / Concentrations:
30, 300, 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: none identified in the report.
- Rationale for animal assignment: weight-stratified randomization.

Positive control:

No

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to first dose and weekly thereafter.
Observations included: changes in skin, fur, eyes, mucous membranes, secretions, excretions, autonomic activity (e.g. lacrimation, piloerection, pupil size, respiratory pattern), changes in gait and posture, response to handling, presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), bizarre behaviour, difficult parturition.

BODY WEIGHT: Yes
- Time schedule for examinations: prior to randomization, at the first dose, then at least weekly.

FOOD CONSUMPTION AND COMPOUND INTAKE :
- Food consumption for each animal determined: at least weekly.

WATER CONSUMPTION AND COMPOUND INTAKE: No

OTHER:
Duration of gestation.

Functional Observational battery and Motor Activity evaluations: on all rats prior to dosing and during the 4th week for males and on post-natal day (PND) 4 for females. These comprised: cage side observations (abnormal muscle movements or behaviour, posture, resistance to removal from the cage), hand-held observations (including pupil size and reactivity, salivation, muscle tone, reactivity to handling), open field observations (level of activity, responsiveness to sharp noise, touch or tail pinch, gait, urine and faecal pellets voided), categorical observations, measurement/counts (rectal temperature, grip strength, landing foot splay), motor activity (using Cage Rack Activity System).

Urinalysis: Physical examination (colour, appearance, volume, specific gravity), pH, bilirubin, nitrite, blood, glucose, protein, leukocytes, ketones, urobilinogen.

Haematology and clinical chemistry (see table 1).

Oestrous cyclicity (parental animals):

not examined

Sperm parameters (parental animals):

not examined

Litter observations:

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

GROSS EXAMINATION OF DEAD PUPS: yes, for external abnormalities.

Postmortem examinations (parental animals):

GROSS NECROPSY
All adult animals - complete gross necropsy (external surfaces, orifices, cranial, thoracic and abdominal cavities)

Females - number of corpora lutea, implantation sites.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 2 from the control and high dose groups were prepared for microscopic examination and/or weighed

In the case of the kidney, identified as a likely target organ, tissues were taken from all male dose groups.

The following tissues were also taken from mid-dose males: urinary bladder, prostate, heart, stomach and liver; and mid-dose females: urinary bladder, kidney, stomach and liver.

IMMUNOHISTOCHEMISTRY
The microscopic findings in the kidney were not indicative of alpha 2U globulin accumulation and therefore tissues were not sent for immunohistochemical staining

Postmortem examinations (offspring):

Dead pups and pups terminated on PND4 were only examined for external gross abnormalities.

Statistics:

ANOVA: body weight changes, organ weights, food consumption, haematology, clinical chemistry, prothrombin times. Also log or rank transformation, Kruskal-Wallis test, Dunnett’s Test (SAS v9.1.3).
Statistical analysis of urinalysis data, FOB, motor activity, reproductive parameters and histopathology (SAS v9.1.3).

Reproductive indices:

Corpora lutea, total implants, pre-implantation loss, post-implantation loss, gestation duration, total pups post-natal day (PND) 0, sexes of pups.

Offspring viability indices:

Viable pups PND0 and PND4, litter weight PND0 and PND4, average pup weight PND0 and PND4, post-natal loss%

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

see details below

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

see details below

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

see details below

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

see details below

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

see details below

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

see details below

Behaviour (functional findings):

not examined

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):

see details below

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Test substance intake: see details below

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

see details below

Details on results (P0)

Treatment-related effects – results for controls (0), 300, 3000 and 1000 mg/kg bw/day groups, number of affected animals out of groups of 10 in each case:

CLINICAL SIGNS
Muzzle soiling: 0, 3, 7,10 (m); 5, 2, 8, 10 (f)
Repetitive paw pressing (transient after dosing): 0, 5, 5, 10 (m); 0, 2, 7, 10 (f)

BODY WEIGHT
Statistically significant decreases in body weight (12%) and body weight gain (32%) in 1000 mg/kg bw/day males compared to controls on day 29, considered test article related. No such clear effect in females.

FOOD CONSUMPTION
No treatment-related effect on mean food consumption in males, but a statistically significant increase in food consumption in 1000 mg/kg bw/day females during study days 1-8 (13.4%), gestational days (GDs) 0-7 (10.5%) and in overall food consumption (2.7%). An increase in food consumption was seen in 30 mg/kg bw/day females during GDs 0-7 (10.6%). No toxicological significance was attributed to this effect.

FUNCTIONAL OBSERVATIONAL BATTERY AND MOTOR ACTIVITIES
No treatment-related statistically significant difference in FOB categories or motor activity data in either sex.

CLINICAL CHEMISTRY
1000 mg/kg bw/day males had increased mean cholesterol levels (29.5% compared to controls) that were within the historical range for the laboratory, but 3/10 were above the historical range. Kidney function parameters blood urea nitrogen (BUN) and creatinine were increased in 1000 mg/kg bw/day males (183% and 200%, respectively, compared to controls; above the historical range). These finding were consistent with microscopic findings in the kidney.

Top dose females also had increased cholesterol (33% compared to concurrent controls) – the mean and individual values were within the historical range. The finding was considered not toxicologically significant.

No other treatment-related clinical chemistry effects in either sex.

HAEMATOLOGY
Males treated with 1000 mg/kg bw/day had increased neutrophils, measured as percentage and absolute, (%-66% increase) and monocytes (%-70% increase; abs-77% increase) compared to concurrent controls; the mean values for monocytes were slightly above historical controls. The decrease in lymphocytes (%-24% decrease) in 1000 mg/kg bw/day males was outside the historical range. These effects were considered secondary to kidney effects.

No significant treatment-related haematological effects in female rats and no differences in prothrombin times in either sex.

URINALYSIS
Females in the 30 and 1000 mg/kg bw/day groups had reduced urinary pH (5% and 11%, respectively) compared to concurrent controls. This effect was not considered toxicologically significant. There were no treatment-related effects on urine in males.

ORGAN WEIGHTS
1000 mg/kg bw/day males had increased relative kidney weight (36.4%), relative brain weight (12.4%) and relative testis weight (12.2%). Mean absolute brain and testis weight were similar to controls, but the kidneys were enlarged and their weight increased (18.5%, without statistical significance). Absolute liver and prostate weights were decreased (15.3% and 29.8%, respectively) but the relative weights of these organs were similar to controls or slightly but not significantly lower. Apart from the kidney weight changes these effects are probably related to the overall reduction in body weights.

30 and 1000 mg/kg bw/day females had increased absolute kidney weight. 1000 mg/kg bw/day females had increased absolute liver weight. No other absolute or relative changes in organ weights.

REPRODUCTIVE PERFORMANCE
One 300 mg/kg bw/day females was non-gravid despite evidence of mating, one 1000 mg/kg bw/day female failed to mate. Other females produced litters similar in all respects to those of controls (number of live born pups, litter weight, survival to PND4). No treatment-related reproductive or developmental effects were identified.

GROSS PATHOLOGY
Kidney effects: discoloured (pale and or mottled) kidneys (1, 4, 0, 6 (m); 0, 0, 3, 5 (f)); enlarged kidneys (0, 2, 0, 6 (m)). Two male rats at 1000 mg/kg bw/day had dilated kidneys and one had a kidney nodule. All other gross findings were sporadic and considered not treatment-related.

HISTOPATHOLOGY
Clear lesions were reported in the kidneys of all males and one female at 1000 mg/kg bw/day; the lesions varied but were described as obstructive nephropathy. The two most severely and bilaterally affected animals had other lesions (mineralization of the stomach and/or aorta) indicating they were uremic (had renal failure). The characteristic kidney lesions included: foci of pale staining globular material in the cortex or medulla, multinucleated phagocytic cells, necrotic epithelial cells, dilated tubules filled with necrotic cells and exudate, interstitial infiltration of neutrophils and lymphocytes, papillary necrosis, hyperplasia and inflammation of renal pelvis.

Hyperplasia of the urinary bladder epithelium was reported in most treated rats; statistically significant in all treated males and in females at >=300 mg/kg bw/day. In the more severely affected (typically at higher doses) the hyperplasia was associated with inflammation of the submucosa, with granulomatous inflammation and pale globular deposits (similar to that in the kidneys), in one case. The globular deposit was recorded as a calculus and considered to have caused acute urinary obstruction. Combining the various forms of inflammation, this effect was statistically significant at >=300 mg/kg bw/day in both sexes.

Inflammation of the prostate in males at 1000 mg/kg bw/day (combined incidence of minimal-moderate inflammation 2, 4, 4, 7) was considered a local effect of a material excreted in the urine that had also caused urinary bladder irritation.

Significant minimal centrilobular hepatocyte hypertrophy in 9/10 females at 10000 mg/kg bw/day was considered an adaptive change to the treatment. Decreased periportal vacuolation in 1000 mg/kg bw/day males was considered in the report probably due to the depletion of glycogen stores under stress.

Diffuse gastric gland dilation (significant-mild) occurred in all females at 1000 mg/kg bw/day with occasional single necrotic cells in these dilated glands. 6/10 males were minimally affected at this dose; controls were not affected.

Other findings were sporadic and/or typical of rats of this age and stock, and considered not to be treatment-related.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Absence of treatment-related reproductive effects at the highest dose tested

Remarks on result:

other: Generation not specified (migrated information)

2

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

< 30 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Effects on the urinary bladder epithelium at 30 mg/kg bw/day in males treated for 29 days

Remarks on result:

other: Generation not specified (migrated information)

3

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Effects on the urinary bladder epithelium in females treated with 300 mg/kg bw/day for up to 55 days

Remarks on result:

other: Generation not specified (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

external examination only

Histopathological findings:

not examined

Details on results (F1)

One adult female at 300 mg/kg bw/day was found to be non-gravid despite positive evidence of mating. One adult female in the 1000 mg/kg bw/day group failed to mate. The remaining females produced litters similar to controls in respect of all evaluated parameters (number of live pups, litter weight, survival). No treatment-related reproductive effects (see table 3).

NOAEL (reproductive) >1000 mg/kg bw/day

Effect levels (F1)

Key result

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: Overall effects

Remarks on result:

other:

Remarks:

Overall effects

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 3: Summary of reproductive/developmental measurements

Parameter	Dose in mg/kg bw/day
	0	30	300	1000
Corpora Lutea counts	17.7	19.3	19.3	17.7
Total implants	14.8	15.6	13.5	14.1
Pre-implantation loss%	15.6	17.8	32.6	17.9
Post implantation loss%	8.9	5.1	9.5	8.3
Gestation days	22	22	22	22
Total pups PND0	13.7	14.9	13.7	14.8
Viable pups PND0	13.6	14.8	13.6	14.4
Males/females PND0	1.0	1.5	1.6	1.1
Litter weight PND0	87.2	95.8	85.7	93.3
Average pup weight PND0	6.5	6.5	6.4	6.5
Total pups PND4	13.6	14.8	13.4	14.4
Viable pups PND4	13.6	14.8	13.4	14.4
Males/females PND4	0.97	1.37	1.24	1.01
Litter weight PND4	134.6	150.6	135.1	140.6
Average pup weight PND4	10.2	10.2	10.2	9.9
Postnatal loss%	0.0	0.0	0.8	0.0

PND: post-natal day

None of the above data were said to indicate any statistically significant treatment-related effect.

Applicant's summary and conclusion

Conclusions:

A well reported combined repeated dose toxicity study with reproductive/developmental toxicity screening test, conducted according to the current guideline (OECD 422) and GLP, found that gavage administration of up to 1000 mg/kg bw/day to male and female rats from 2 weeks prior to mating was not associated with reproductive toxicity. Systemic effects were reported in males at 30 and females at 300 mg/kg bw/day. The NOAEL for reproductive toxicity is >1000 mg/kg bw/day based on the screening element of this study.