2,4,6-trimethyl-2,4,6-tris(3,3,3-trifluoropropyl)cyclotrisiloxane

Endpoint summary

Description of key information

In the key oral 90-day repeated dose toxicity study (Dow Corning Corporation, 2002) conducted according to EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents) and in compliance with GLP, the systemic NOAEL for 2,4,6-trimethyl-2,4,6-tris(3,3,3-trifluoropropyl)cyclotrisiloxane was concluded to be 0.8 mg/kg bw/day based on mortality at 20 or 50/35 mg/kg bw/day, clinical signs, body weight, food consumption, clinical chemistry, functional observation battery effects and organ weight effects at 20 and/or 50/35 mg/kg bw/day and pathology findings in the skeletal muscle at 20 and 50/35 mg/kg/day groups and heart at 4, 20 or 50/35 mg/kg bw/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Crl:CD(SD)IGS BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, NC
- Age at study initiation: approximately 7 weeks
- Weight at study initiation: Males 225 - 309g Females 160 - 214g
- Fasting period before study: No
- Housing: individually in wire mesh cages
- Diet: Certified Rodent Lab Diet 5002, PMI Nutrition Inc., ad libitum except overnight prior to blood collection
- Water: Reverse osmosis treated on-site drinking water ad libitum
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.1 - 22.7
- Humidity (%): 33.3 - 68.1
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To: 24 October 2000 to 25 January 2001

Route of administration:

oral: gavage

Vehicle:

other: sesame oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: prepared weekly by weighing appropriate amount of test article and adding sufficient vehicle

VEHICLE
- Concentration in vehicle: 0, 0.4, 2, 10, 25/17.5 mg/mL
- Amount of vehicle: 2mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity and stability established before start of study.
Weekly for first 4 weeks then monthly analysis of formulations for test article concentration during the study

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

5 days/week

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

20 per sex in Control and high dose (50/35 mg/kg/day) groups
10 per sex in intermediate dose groups

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: not stated
Initial high dose of 50 mg/kg/day not tolerated and reduced to 35 mg/kg/day from Day 5.
- Post-exposure recovery period in satellite groups: 28-day recovery period initially included but due to excessive mortality at high dose, no recovery period conducted.

Observations and examinations performed and frequency:

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pre-dose and Week 12
- Dose groups that were examined: All

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 4 and 13
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table No. 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 4 and 13
- Animals fasted: Yes / No / No data
- How many animals: all
- Parameters checked in table No. 2 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Week 12
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity ( see table No. 3)

LOCOMOTOR ACTIVITY: Yes
- Time schedule for examinations: Week 12
- Dose groups that were examined: all
- Parameters: ambulatory and total motor activity

OTHER:
BODY TEMPERATURES: recorded from 5 animals/sex/group approx 1 hour after dosing during first 2 weeks of treatment.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 3)
ORGAN WEIGHT : Yes (see table 3)
HISTOPATHOLOGY: Yes (see table 3)

Statistics:

All analyses were two-tailed for significance levels of 5% and 1%. Body weights, body weight changes, food consumption, clinical pathology values and organ weights were subjected to a one-way analysis of variance (ANOVA) followed by Dunnett's Test if the ANOVA revealed statistical significance (p<0.05). Continuous Functional Observational Battery (FOB) and Locomotor Activity data were analysed using one-way ANOVA. If significant effects were observed at a given timepoint, Dunnett's test was conducted to determine significant treatment differences from the control group. Discontinuous (ordinal or descriptive) FOB data were analysed using Fisher's Exact test to determine significant treatment differences from the control group.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test-article related clinical signs were noted at 50/35 and 20 mg/kg/day and primarily included ataxia, impaired use of hindlimbs, hindlimb tremors, walking on metatarsus, increased salivation and red or yellow material on body surfaces. Other clinical signs noted during the study included prostration, head bobbing, body cool to touch, shallow respiration, lethargy and piloerection.

During detailed clinical observations slight to total impaired mobility was observed at 50/35 mg/kg/day generally throughout the study. Abnormal gait was also noted and included ataxia, dragging hindlimbs, walking on metatarsus and hunched gait.
Mean body temperature was lower for 50/35 mg/kg/day females on Day 2.

Mortality:

mortality observed, treatment-related

Description (incidence):

Twenty-two animals were found dead or killed due to poor clinical condition during the study. One male and 3 females were found dead or killed following administration of 50 mg/kg/day. After reduction in the dose to 35 mg/kg/day, 7 males and 7 females were killed or found dead. At 20 mg/kg/day 1 female was found dead and 1 killed. The majority of animals died during the first 5 weeks of dosing and the deaths were considered test-article related. Two control males were also found dead in weeks 4 and 5 respectively.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weight and body weight gain were lower than control for 50/35 mg/kg/day animals and 20 mg/kg/day males generally throughout the study. Overall weight gain was up to 16% lower than control during the dosing period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean food consumption was lower than control for the 50/35 mg/kg/day group generally throughout the study.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No effect of treatment.

Haematological findings:

no effects observed

Description (incidence and severity):

No effect of treatment.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Test-article related increases in urea nitrogen were noted at 50/35 and 20 mg/kg/day at weeks 4 (males only) and 13 (males and females). Mean phosphorus levels were increased for 20 mg/kg/day males and 50/35 mg/kg/day animals at both evaluations during the study. Mean potassium levels were increased for 20 mg/kg/day and 50/35 mg/kg/day males during the study and for 50/35 mg/kg/day females in Week 13. Mean cholesterol was lower in 50/35 mg/kg/day males at Week 13.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

When evaluating results it should be noted that many of the high dose animals with the most severe hindlimb effects did not survive until the evaluation in week 12.
There were no test-article effects on home cage observations.
An increased number of animals at 50/35 mg/kg/day were noted to have hard and tense muscle tone during the handling observations.
Slightly impaired mobility and abnormal gait was noted for 50/35 mg/kg/day females during the open field observations.
There were no test-article effects on sensory observations.
One female at 50/35 mg/kg/day was noted with exceptionally low hindlimb grip strength during the neuromuscular observations.
There were no remarkable differences apparent between control and treated groups for physiological parameters.
When evaluating results, it should be noted that many of the high dose animals with the most severe hindlimb effects did not survive until the evaluation in week 12.
Overall no remarkable differences between control and treated animals, one female at 50/35 mg/kg/day was noted with the lowest combined motor activity counts.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Increased mean absolute and relative (to final body weight and brain weight) liver weights were noted at 20 and 50/35 mg/kg/day. Lower seminal vesicle (absolute and relative) weights were also noted for males in these groups and lower prostate (absolute and relative) weights were noted in 50/35 mg/kg/day males.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No findings related to treatment.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test-article related microscopic changes were noted in the liver, skeletal muscle and heart and were generally comparable between decedent animals and those surviving to scheduled necropsy.

In the liver, periportal vacuolar change (hepatocytes enlarged by a variably distinct micro- to macro vacuolated cytoplasm) was noted with dose-related incidence in all treated groups. In the absence of correlating clinical chemistry findings, these hepatic changes was considered not to be adverse.

In the skeletal muscle (rectus femoris), minimal to moderate degeneration (hyaline and basophilic cytoplasm, shape and size variations of myocytes, loss of cross striations and increased cellularity) was noted in the 20 and 50/35 mg/kg/day groups.

In the heart, treatment-related or treatment-exacerbated subacute (accumulations of mononuclear cells, primarily macrophages within and surrounding degenerative myocytes) or chronic (mononuclear inflammatory cells and/or fibroplasia and scattered degeneration and necrosis of myocytes) myopathy was noted in 4, 20 and 50/35 mg/kg/day groups. This finding was morphologically similar to spontaneously developing cardiomyopathy typically seen in older rats, however, it was considered to be test-article related in this case.

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

0.8 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

other: gastrointestinal; cardiovascular; musculoskeletal

Organ:

heart

liver

other: skeletal muscle

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Table 1 - survival

Dose (mg/kg/day)	Control	0.8	4	20	50/35
Males
Number in group	20	10	10	10	20
Found dead	2	0	0	0	4
Killed	0	0	0	0	4
Survived until scheduled termination	18	10	10	10	12
Females
Number in group	20	10	10	10	20
Found dead	0	0	0	1	4
Killed	0	0	0	1	6
Survived until scheduled termination	20	10	10	8	10

Conclusions:

Oral administration of 2,4,6-trimethyl-2,4,6-tris(3,3,3-trifluoropropyl)cyclotrisiloxane to rats at 0.8, 4, 20 or 50/35 mg/kg/day for 13 weeks was not tolerated at the highest dose and resulted in a number of mortalities, 2 animals also died at 20 mg/kg/day. Pathological changes were noted in the liver, heart and skeletal muscle and several clinical findings were noted consistent with the skeletal muscle toxicity. Generally, effects were primarily noted at 20 or 50 mg/kg/day and also included body weight, food consumption, clinical chemistry and organ weight effects.

Based on mortality at 20 or 50/35 mg/kg/day, clinical signs, body weight, food consumption, clinical chemistry, FOB and organ weight effects at 20 and/or 50/35 mg/kg/day and pathology findings in the skeletal muscle and heart at 4, 20 or 50/35 mg/kg/day, the NOAEL was considered to be 0.8 mg/kg/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

0.8 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: dermal

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

no guideline followed

Principles of method if other than guideline:

Standard tox study design

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Langshaw Farms, Augusta, Michigan
- Age at study initiation: approx 5 weeks old
- Weight at study initiation: not stated
- Housing: standard stainless steel cages
- Diet: PURINA Rabbit Chow (ad libitum)
- Water: fresh water (ad libitum)
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 50-60
- Air changes (per hr): not stated
- Photoperiod (hrs dark / hrs light): not stated

IN-LIFE DATES: From: To: not stated

Type of coverage:

occlusive

Vehicle:

unchanged (no vehicle)

Details on exposure:

TEST SITE
- Area of exposure: not stated
- % coverage: not stated
- Type of wrap if used: impervious sleeve of plastic material
- Time intervals for shavings or clippings: at least 24 hours before initial application, as needed thereafter

REMOVAL OF TEST SUBSTANCE
- Washing: lukewarm water
- Time after start of exposure: 6 hours

TEST MATERIAL
- Amount applied (volume or weight with unit): not stated
- Concentration: 100%
- Constant volume or concentration used: not stated

USE OF RESTRAINERS FOR PREVENTING INGESTION: no

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

6 hours per day

Frequency of treatment:

5 days per week for 3 weeks

Dose / conc.:

40 mg/kg bw/day (nominal)

Dose / conc.:

200 mg/kg bw/day (nominal)

Dose / conc.:

400 mg/kg bw/day (nominal)

No. of animals per sex per dose:

6

Control animals:

yes, sham-exposed

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily after application of test material

DETAILED CLINICAL OBSERVATIONS: No data

DERMAL IRRITATION (if dermal study): No data

BODY WEIGHT: Yes
- Time schedule for examinations: Day 1, 4, 8, 12, 16 and 21

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Day 1, 4, 8, 12, 16 and 21

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: Yes (methoxyflurane)
- Animals fasted: No data
- How many animals: All
- Parameters not stated

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination
- Animals fasted: No data
- How many animals: All
- Parameters not stated

URINALYSIS: Yes
- Time schedule for collection of urine: for 16 hours prior to termination
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters not stated

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, performed on all major tissues, organs, orifices, cranial, thoracic, abdominal and pelvic cavities and their viscera.
ORGAN WEIGHT: A complete set of organs and tissues were weighed
HISTOPATHOLOGY: Yes, a complete set of organs and tissues were removed and preserved.

Statistics:

Statistical comparisons between control and test groups were carried out where applicable. The data were analysed by employing Dunnett multiple t-test. Where appropriate, a non-parametric analysis of variance by ranks was used to evaluate these parameters. The 95% (p<0.05) confidence level was used as the criteria of significance.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no remarkable clinical signs. Three male and 2 females died during the treatment period at 400 mg/kg/day. One female at 200 mg/kg/day died as a result of acute pneumonia.

Dermal irritation:

not specified

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Significant reduction in body weight gain was noted for females at 200 and 400 mg/kg/day, similar, but less marked, effects on body weight were noted for males at the same doses.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Lower food consumption was recorded at 200 and 400 mg/kg/day during the treatment period.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There was no effect of treatment.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Serum alkaline phosphatase activity was significantly lower at 200 or 400 mg/kg/day. Significantly higher serum glutamic-pyruvic transaminase and serum glutamic oxalacetic transaminase were noted in males at 200 or 400 mg/kg/day. For females, only serum glutamic oxalacetic transaminase was higher at 200 mg/kg/day.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There was no effect of treatment.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Relative liver weight was lower in females at all doses and to a lesser extent males.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No remarkable findings.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No effect of treatment.

Histopathological findings: neoplastic:

no effects observed

Dose descriptor:

NOAEL

Effect level:

40 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

gastrointestinal tract

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

Dermal administration of 2,4,6-trimethyl-2,4,6-tris(3,3,3-trifluoropropyl)cyclotrisiloxane to rabbits at 40, 200 or 400 mg/kg bw/day (5 days a week for 3 weeks) resulted in mortality at 400 mg/kg bw/day and lower body weight gain, food consumption, serum alkaline phosphatase activity and relative organ weight and increased serum glutamic-pyruvic transaminase and glutamic oxalacetic transaminase at 200 and 400 mg/kg bw/day. The no-observed-adverse-effect-level was considered to be 40 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

40 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Additional information

In the key oral 90-day repeated dose toxicity study (Dow Corning Corporation, 2002) conducted according to EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents) and in compliance with GLP, 2,4,6-tris(3,3,3-trifluoropropyl)cyclotrisiloxane was administered orally by gavage to rats at 0.8, 4, 20 or 50/35 mg/kg bw/day for 13 weeks. The test substance was not tolerated at the highest dose and resulted in a number of mortalities. Two animals also died at 20 mg/kg bw/day. Pathological changes were noted in the liver, heart and skeletal muscle and several clinical findings were noted consistent with the skeletal muscle toxicity. In the liver a periportal vacuolar change (hepatocyte enlarged by a variably distinct micro- to macro vacuolated cytoplasm) was recorded. In skeletal muscle minimal to moderate degeneration consisting of hyaline and basophilic cytoplasm, shape and size variations of myocytes, loss of cross striations and increased cellularity was noted in the 20 and 50/35 mg/kg/day groups. In the heart subacute (accumulations of mononuclear cells, primary macrophages within and surrounding degenerative myocytes) or chronic (mononuclear inflammatory cells and/or fibroplasia and scattered degeneration and necrosis of myocytes) myopathy was recorded at 4, 20 or 50/35 mg/kg bw/day. Generally, effects were primarily noted at 20 or 50 mg/kg bw/day and included body weight, food consumption, clinical chemistry and organ weight effects. Based on mortality at 20 or 50/35 mg/kg bw/day, clinical signs, body weight, food consumption, clinical chemistry, functional observation battery and organ weight effects at 20 and/or 50/35 mg/kg bw/day and pathology findings in the skeletal muscle at 20 and 50/35 mg/kg/day and heart at 4, 20 or 50/35 mg/kg bw/day, the NOAEL was concluded to be 0.8 mg/kg bw/day.

Dermal administration of 2,4,6-trimethyl-2,4,6-tris(3,3,3-trifluoropropyl)cyclotrisiloxane to rabbits at 40, 200 or 400 mg/kg/day (5 days a week for 3 weeks) resulted in mortality at 400 mg/kg/day and lower body weight gain, food consumption, serum alkaline phosphatase activity and relative organ weight and increased serum glutamic-pyruvic transaminase and glutamic oxaloacetic transaminase at 200 and 400 mg/kg/day. The NOAEL was considered to be 40 mg/kg/day.

Justification for classification or non-classification

Based on the existing data, 2,4,6-trimethyl-2,4,6-tris(3,3,3-trifluoropropyl)cyclotrisiloxane is classified as STOT-RE 1 (oral, liver, heart, skeletal muscle) and STOT-RE 2 (dermal, liver) according to Regulation EC (No) 1272/2008.