1,1,5,5,5-hexamethyl-3,3-bis[(trimethylsilyl)oxy]trisiloxane

Endpoint summary

Description of key information

In an oral combined repeated dose toxicity study with the reproduction/developmental toxicity screening test conducted according to OECD Test Guideline 422 and in compliance with GLP, the NOAEL for 1,1,1,5,5,5-hexamethyl-3,3-bis[(trimethylsilyl)oxy]trisiloxane (CAS 3555-47-3, EC 222-613-4) for systemic parental toxicity in male and female rats was at least 1000 mg/kg bw/day based on no adverse systemic effects observed (Charles River Laboratories, 2021, reliability 1).

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-04-17 to 2021-02-08

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/Developmental Toxicity Screening Test

Version / remarks:

2000

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and 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.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: Males: 10 to 11 weeks old; Females: 11 to 12 weeks old
- Weight at study initiation: Males: 250 to 325 g; Females: 194 to 244 g
- Fasting period before study: No
- Housing: Main animals were randomly group housed (up to five animals of the same sex and same dosing group together) in Makrolon polycarbonate cages on arrival and during the pre-mating period. Main males and females were cohabitated on a 1:1 basis in Makrolon plastic cages during the mating phase. Main males and females were housed in Makrolon plastic cages during the post-mating phase, with a maximum of five/cage for males and individually for females. During the lactation phase, pups were housed with the dam in Makrolon plastic cages except during locomotor activity monitoring of the dams. Each cage was clearly labeled with a colour-coded cage card indicating Test Facility Study No., group, animal number(s), and sex.
- Diet: ad libitum, except during locomotor activity
- Water: ad libitum, except during locomotor activity
- Acclimation period: Males: six days before commencement of dosing; Females: 7 days prior to start of the pretest period

DETAILS OF FOOD AND WATER QUALITY: Pelleted rodent diet was provided ad libitum throughout the study, except during designated procedures. During motor activity measurements, animals had no access to food for a maximum of 2 hours. The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study. Municipal tap water was freely available to each animal via water bottles. During motor activity measurements, animals had no access to water for a maximum of 2 hours. Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 22°C
- Humidity (%): 46 to 78%
- Air changes : 10 per hour minimum
- Photoperiod : 12-hour light/12-hour dark

Route of administration:

oral: gavage

Details on route of administration:

The oral route of administration was selected because this is an acceptable route of exposure for studies of this type.

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was administered as received. An adequate amount of the test item was dispensed into daily aliquots, which were stored the same as for the bulk test item until use. 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 test item (0.87). No correction was made for the purity/composition of the test item.

Analytical verification of doses or concentrations:

no

Remarks:

The test item was used as received from the Sponsor; therefore, samples for dose formulation analysis were not collected by the Test Facility.

Duration of treatment / exposure:

At least 29 days. Main males and Recovery males were treated for 29 days including a minimum of 14 days prior to mating and during the mating period for Main males. Females that delivered offspring were treated for two weeks prior to mating, during mating, during post-coitum, and at least 13-15 days of lactation (for 52-65 days). Females that failed to deliver pups were treated for 41-52 days. Recovery females were treated during the same period as Main females, until at least the first scheduled necropsy of Main females (55 days).

Frequency of treatment:

Daily, 7 days a week

Dose / conc.:

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

Remarks:

Group 1 (control group)

Dose / conc.:

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

Remarks:

Group 2

Dose / conc.:

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

Remarks:

Group 3

Dose / conc.:

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

Remarks:

Group 4

No. of animals per sex per dose:

15 males and females for Group 1 and 4 (water and 100 mg/kg/day of undiluted test item, respectively); 10 males and 10 females for Group 2 and 3 (300 and 1000 mg/kg/day of undiluted test item, respectively)

Control animals:

yes

Details on study design:

- Dose selection rationale: Based on the results of a 14-day Dose Range Finder (Test Facility Reference No. 20226169)
- Rationale for animal assignment: random
- Fasting period before blood sampling for clinical biochemistry: Yes for main and recovery males, not for main females
- Rationale for selecting satellite groups: The recovery animals, euthanised following a 14-day treatment-free recovery period, were used to study the potential reversibility of possible toxic effects and were not mated.
- Post-exposure recovery period in satellite groups: Yes

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed
from the cage during observation, unless necessary for identification or confirmation of possible findings.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
1. Once daily, beginning during the first administration of the test item and lasting throughout the dosing and recovery periods up to the day prior to necropsy. During the dosing period, these observations were performed after dosing at no specific time point, but within a similar time period after dosing for the respective 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 1, 2, 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.
2. Arena observations were also conducted beginning before the first administration of the test item and then once weekly throughout treatment and recovery. These observations were conducted after dosing.

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. A terminal weight was recorded on the day of scheduled necropsy.

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

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION : Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On the day of scheduled necropsy in main and recovery animals
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes for main and recovery males, not for main females
- How many animals: All animals except for animals which were found dead or died during the blood sampling procedure
- Parameters checked in table were examined.: yes, see table 1

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On the day of scheduled necropsy in main and recovery animals
- Animals fasted: Yes for main and recovery males, not for main females
- How many animals: All animals except for animals which were found dead or died during the blood sampling procedure
- Parameters checked in table were examined.: yes, see table 1

URINALYSIS: No

FUNCTIONAL OBSERVATIONAL BATTERY: Yes
- Time schedule for examinations: Functional tests were performed on the five selected Main males and all Recovery males during Week 4 of treatment and the five selected Main females during the last week of lactation (i.e. PND 6-13), and all Recovery females were tested on the first day a Main female was tested. These tests were performed after completion of clinical observations (including
arena observation, if applicable).
- Battery of functions tested: Hearing ability, Pupillary reflex, statis righting reflex, Fore-and hind-limb grip strenght, locomotor activity.
As the above-mentioned measurements did not reveal test item-related effects, the functional observation tests were not performed at the end of the recovery phase.r:

IMMUNOLOGY: No

Sacrifice and pathology:

At the end of the treatment period 10 surviving rats from all groups were sacrificed and subjected to complete necropsies. These animals are identified as Main Group animals. The remaining 5 males and 5 females from Groups 1 and 4 were sacrificed and subjected to complete necropsies following a 14-day treatment free period. These animals are identified as Recovery Group animals.

GROSS PATHOLOGY: Yes, see table 2
Animals surviving until scheduled euthanasia were weighed, and deeply anesthetized using isoflurane and subsequently exsanguinated and subjected to a full postmortem examination, with special attention being paid to the reproductive organs. The organs identified (see table ) were weighted at necropsy for all scheduled euthanasia animals.

Scheduled necropsies were conducted on the following days:
- Main Males (which sired or failed to sire): Following completion of the mating period (a minimum of 28 days of administration).
- Recovery males: After the recovery period of at least 14 days, which is at least 14 days after the scheduled necropsy of Main males.
- Main Females which delivered: PND 14-16.
- Main Females which failed to deliver: With evidence of mating: Post-coitum Days 26-27 (Nos. 69, 70, 78, 83 and 85); Without evidence of mating: Approximately 24 days after the last day of the mating
period (No. 51). Note that mating was overlooked, as 1 implantation site was recorded at necropsy.
- Recovery females: After the recovery period of at least 14 days, which is at least 14 days after the first scheduled necropsy of Main females.


HISTOPATHOLOGY: Yes, see table 2
Histopathologic examination was performed on an extensive list of organs and tissues from five selected Main Group 1 and 4 animals as well as the lung from selected Main Group 2 and 3 and Recovery Group 1 and 4 rats and all organs with macroscopic findings from all rats. The reproductive organs were examined from all males that failed to sire and all females that failed to deliver healthy pups.

Other examinations:

None reported

Statistics:

Numerical data collected on scheduled occasions for the variables were analysed according to sex and occasion. Descriptive or Inferential statistical methods were used.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations.
Any 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. No toxicologically relevant clinical signs were observed in Recovery animals.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No mortality occurred during the study period that was considered to be related to treatment with the test item.
Female No. 59 (control) died at blood sampling on the day of scheduled necropsy. No clinical signs were noted. Slightly low body weight gain was recorded on the days preceding its death. At necropsy many reddish foci on the thymus were recorded. As this was a control animal which died during the blood sampling procedure, this death was considered not treatment-related.
Female No. 80 (300 mg/kg/day) was found dead on Day 15 post-coitum. No clinical signs or body weight changes were noted on the days preceding death. At macroscopy reddish fluid in the trachea, not collapsed lungs and reddish discoloration of the lungs were recorded, suggesting that death was related to the gavage procedure.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights and body weight gain of treated animals up to 1000 mg/kg/day remained in the same range as controls over the treatment period (Main and Recovery animals) and in the recovery period thereafter (Recovery animals only).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption before or after correction for body weight was similar to the control level over the treatment period up to 1000 mg/kg/day (Main and Recovery animals) and in the recovery period thereafter (Recovery animals only).

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Hematological parameters of treated rats were considered unaffected by treatment with the test item.
No statistically significant changes were recorded at the end of the treatment period. Any statistically significant differences recorded at the end of the recovery period (but not at the end of the treatment period) were considered not related to treatment with the test item.
Coagulation parameters of treated rats were considered not to have been affected by treatment with the test item.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes were noted in clinical biochemistry parameters after treatment with the test item up to 1000 mg/kg/day.
Any (statistically significant) changes noted were considered to be unrelated to treatment based on the small magnitude of change and/or as these changes occurred in the absence of a dose-related trend, or were recorded at the end of the recovery period only.
Thyroid hormone analyses: Serum levels of T4 in Main males (F0-generation) were considered unaffected by treatment with the test item up to 1000 mg/kg/day.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all examined animals.
Motor activity in males at end of treatment was similar between control and treated animals.
Motor activity in Main females at end of treatment was increased at 300 and 1000 mg/kg/day, for total movements as well as and ambulations. As this difference was caused by one animal in each group (Nos. 84 and 88), this was considered not toxicologically relevant.
In Recovery females at the end of treatment total movements and ambulations were similar between treated and control animals.
A similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period was noted in all groups and sexes.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related alterations in organ weights.
In Main females lower absolute thyroid gland weights were recorded at the end of treatment starting at 100 mg/kg/day. These were considered the result of relatively high control values and not related to treatment with the test item.
Statistically significant changes in Main animals at the end of treatment were noted in seminal vesicles in males (lower relative to body weight at 100 and 300 mg/kg/day) and liver of females (higher relative to body weight at 300 mg/kg/day). These weight changes were considered to be of no toxicological significance as they occurred in the absence of a dose related trend.
Statistically significant changes in Recovery animals at the end of recovery were noted in the adrenal gland of females (lower absolute and relative to body weight at 1000 mg/kg/day) and kidney of females (lower relative to body weight at 1000 mg/kg/day). These weight changes were considered to be of no toxicological significance as they occurred in recovery animals only.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

There were gross observations in the lungs of test item-treated animals at the end of the treatment period. Gray-white focus/foci were recorded in the lungs of 3/10 males at 100 mg/kg/day, in 1/10 males at 300 mg/kg/day and in 2/10 females at 1000 mg/kg/day. In 1/10 males at 300 mg/kg/day a hardened, discolored lung lobe was recorded. The microscopic correlate for these necropsy findings was multifocal granulomatous inflammation.
The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

There were microscopic observations of note in the lungs of some animals after treatment with the test item at all dose levels.
At the end of the treatment period, main finding in the lungs of both sexes consisted of granulomatous inflammation (varying from minimal to marked degree). This was recorded in three males and two females at 100 mg/kg/day, two males and two females at 300 mg/kg/day and in one male and three females at 1000 mg/kg/day. The multifocal granulomatous inflammatory processes contained multinucleated foreign body giant cells, a varying amount of vacuolated material (mainly in macrophages) and granulocytic inflammatory cells. This inflammatory process was seen in one or more lung lobes and was in most cases located close to the main stem bronchi.
Additional findings in the lungs included, alveolar (vacuolated) macrophage aggregations (varying from minimal to marked degree) and an increased incidence and severity of perivascular/peribronchial mixed inflammatory cell infiltrate (minimal to slight degree). There was no dose-dependent relationship for the incidences and severities of these lung alterations.
After the 14-day treatment-free recovery period two males and three females of the 1000 mg/kg/day group showed granulomatous inflammation (minimal-slight). The alveolar (vacuolated) macrophage aggregations and/or perivascular/peribronchial mixed inflammatory cell infiltrates were recorded for some animals but didn’t exceed a minimal degree. The alterations in the lungs of the recovery animals were recorded at lower severities compared to the animals at the end of the treatment period, suggesting partial, ongoing recovery. These alterations were attributed to aspiration of non-lethal amounts of test item formulation resulting in local inflammatory lesions in the lungs.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Key result

Dose descriptor:

NOAEL

Remarks:

systemic effects

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No systemic parental toxicity was observed up to 1000 mg/kg/day

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

inflammatory lesions in the lungs (granulomatous inflammation, alveolar (vacuolated) macrophage aggregates and perivascular/peribronchial mixed inflammatory cell infiltrates)

Conclusions:

In an oral combined repeated dose toxicity study with the reproduction/developmental toxicity screening test conducted according to OECD Test Guideline 422 and in compliance with GLP, the NOAEL for 1,1,1,5,5,5-hexamethyl-3,3-bis[(trimethylsilyl)oxy]trisiloxane for systemic parental toxicity in males and females rats was at least 1000 mg/kg bw/day based on no adverse systemic effects observed. Local effects, aspiration-related alterations were present in the lungs of some males and females at 100, 300 and 1000 mg/kg/day and consisted of granulomatous inflammation, alveolar (vacuolated) macrophage aggregates and perivascular/peribronchial mixed inflammatory cell infiltrates correlating to macroscopic gray-white foci or lobar discoloration and hardening, with partial recovery.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

subacute

Species:

rat

Additional information

In the key combined repeated dose toxicity study with the reproduction/developmental toxicity screening test conducted according to OECD Test Guideline 422 and in compliance with GLP, 1,1,1,5,5,5-hexamethyl-3,3-bis[(trimethylsilyl)oxy]trisiloxane (CAS 3555-47-3, EC 222-613-4) was administered orally by gavage to Wistar Han rats at dosages of 0, 100, 300 and 1000 mg/kg bw/day followed by a 14-day treatment-free recovery period. The rats of the control group received water. Main males and Recovery males were treated for 29 days, including a minimum of 14 days prior to mating and during the mating period for Main males. Females that delivered offspring were treated for two weeks prior to mating, during mating, during post-coitum, and at least 13-15 days of lactation (for 52-65 days). Females that failed to deliver pups were treated for 41-52 days. Recovery females were treated during the same period as Main females, until at least the first scheduled necropsy of Main females (55 days).

No systemic parental toxicity was observed up to the highest dose level tested (1000 mg/kg bw/day). No toxicologically significant changes were noted in any of the parameters investigated in this study (i.e. clinical appearance, functional observations (motor activity, grip strength, hearing ability, pupillary reflex and static righting reflex), body weight, food consumption, hematology, clotting parameters, clinical biochemistry (including male T4 thyroid hormone levels), macroscopic examination and organ weights. Granulomatous inflammation was observed in the lungs of several test item-treated animals, most likely resulting from aspiration of test item formulation. The granulomas contained multinucleated foreign body giant cells and vacuolated material was present in macrophages in the granulomas and in alveolar macrophage aggregates. There was no dose-dependent relationship for the incidences and severities of these lung alterations and the vacuolated material most likely represented the test item. In one animal, foamy vacuolated material was present in the alveoli. The location of the inflammatory process close to the bifurcation further supports aspiration of test item as the cause of the inflammatory processes. Subacute manifestations after prolonged aspiration of small amounts of material are described to include pulmonary alveolar histiocytosis and granuloma formation in the lungs (Damsch et al., 2011). The formation of granulomas in the lungs and accompanying inflammatory lesions in test item-treated animals was therefore not regarded to be a systemic test item effect, but most likely resulted from accidental aspiration of non-lethal amounts of test item, resulting in local inflammatory lesions in the lungs which partially recovered after the 14-day treatment-free period (Charles River Laboratories, 2021, reliability 1).

Justification for classification or non-classification

The available data suggests that 1,1,1,5,5,5-hexamethyl-3,3-bis[(trimethylsilyl)oxy]trisiloxane should not be classified for specific target organ toxicity following repeated oral exposure according to Regulation (EC) No. 1272/2008