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

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

In the key one-generation reproductive toxicity study, conducted according to OECD Test Guideline 415 and in compliance with GLP, the parental NOAEL for systemic toxicity was concluded to be 0.8 mg/kg bw/day based on the mortality, clinical signs, food consumption, body weight effects, effects on organ weights noted at 20 and 50/35 mg/kg bw/day and pathology findings in the skeletal muscle at 20 and/or 50/35 mg/kg bw/day and heart at 4, 20 or 50/35 mg/kg bw/day (Dow Corning Corporation, 2002). The NOAEL for reproductive performance was concluded to be 20 mg/kg bw/day based on increased number of days between pairing and coitus and lower number of implantation sites at 50/35 mg/kg bw/day. The NOAEL for neonatal toxicity was concluded to be 4 mg/kg bw/day based on total litter losses, reduced postnatal survival, clinical signs and lower body weight gains at 20 and 50/35 mg/kg bw/day.

Link to relevant study records

Reference

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

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:

- Source: Charles River Laboratories, NC
- Age at study initiation: 6 to 7 weeks
- Weight at study initiation: Males 183 - 251g Females 146 - 194g
- Fasting period before study: No
- Housing: individually in wire mesh cages, females in plastic maternity cages with nesting material following positive evidence of mating
- Diet: Certified Rodent Lab Diet 5002, PMI Nutrition Inc., ad libitum
- Water: Reverse osmosis treated on-site drinking water ad libitum
- Acclimation period: 17 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.3 - 22.9
- Humidity (%): 31.5 - 59.1
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To: 27 October 2000 to 3 March 2001

Route of administration:

oral: gavage

Vehicle:

other: sesame oil

Details on 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

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: until positive evidence of mating or up to 14 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no, after 14 days with no evidence of mating, female placed in plastic cage with nesting material
- After successful mating each pregnant female was caged: individually in plastic cage with nesting material

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:

Administration for minimum of 70 days prior to mating.
Males dosed throughout mating and until scheduled necropsy.
Females dosed throughout mating, gestation and until lactation day 20.

Frequency of treatment:

Once daily

Details on study schedule:

- Age at mating of the mated animals in the study: approximately 17 weeks

Dose / conc.:

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

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:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: not stated

Parental animals: Observations and examinations:

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: males weekly, females weekly until positive evidence of mating then gestation days 0, 4, 7, 11, 14 and 20 and lactation days 1, 4, 7, 14 and 21

FOOD CONSUMPTION: Yes
- Time schedule for examinations: males weekly, females weekly until positive evidence of mating then gestation days 0, 4, 7, 11, 14 and 20 and lactation days 1, 4, 7, 14 and 21
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day and g food/kg body weight/day: Yes

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

Oestrous cyclicity (parental animals):

Vaginal smears were prepared daily to determine the stage of the estrous cycle beginning 14 days prior to pairing and continuing until evidence of mating was observed. For females with no evidence of mating, smearing continued until the end of the mating period. The average length was calculated for complete estrous cycles (i.e. total number of returns to metestrous (M) or diestrous (D) from estrous (E) or proestrous (P). Estrous cycle length was determined by counting the number of days from the first M or D in a cycle to the first M or D in the next cycle.

Sperm parameters (parental animals):

Parameters examined in P male parental generations:
right testis weight, right epididymis weight, sperm motility (CASA) sperm morphology (light microscopy).
left testis and epididymis weighed and homogenized - evaluated for homogenization resistant spermatid count and sperm production rate (CASA)

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Maximum of 8 pups/litter (4/sex/litter when possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, nipple retention (males)

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals following completion of mating period
- Maternal animals: All surviving animals on lactation day 21

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
Males - epididymides (total and cauda), pituitary, prostate, seminal vesicles with coagulating glands and testes were weighed.
Males - coagulating glands, pituitary, prostate, seminal vesicles, testes with epididymides and vas deferens and gross lesions were preserved for possible future examination.
Females - no organs weighed
Females - gross abnormalities retained for possible future examination
Females that did not deliver or with total litter loss - if evidence of macroscopic implantation then number of implantation sites and corpora lutea recorded. If no evidence of implantation then uteri placed in 10% ammonium sulfide solution for detection of early implantation loss.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed on post-natal day 21

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. Gross findings retained for possible future examination.

Statistics:

All analyses were conducted using two-tailed tests except as noted.
The litter was used as the experimental unit.
Chi-squared test with Yates correction factor - prenatal mating and fertility indices
One-way ANOVA with Dunnett's test - pre-coital intervals, estrous cycle lengths, parental weekly gestational and lactational body weight and food consumption data, gestation lengths, sperm numbers, sperm production rates, organ weights, pup numbers, live litter sizes, pup body weight
Kruskal-Wallis test with Mann-Whitney U test - sperm motility, sperm morphology, pup sexes at birth (% males per litter), post-natal survival

Reproductive indices:

Male Mating index
Female Fertility index
Male Fertility index

Offspring viability indices:

Mean litter size
Mean post-natal survival

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs noted at 50/35 and 20 mg/kg/day and attributable to treatment included impaired use of hindlimbs, reduced hindlimb resistance, hunched appearance, lacrimation, red material on nose, eyes, mouth and/or limbs, abdominal, urogenital and/or preputial areas or at the base of the tail, yellow material on various body surfaces and generally unkempt appearance. Also noted were abnormal gait, hypoactivity, shallow and/or laboured respiration and exophthalmus.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

At 50/35 mg/kg/day 5 males and 11 females were found dead and 5 males and 3 females were killed due to poor clinical condition, generally during the first 4 weeks of treatment. At 20 mg/kg/day 4 males and 7 females were found dead and 1 male and four females were killed due to poor clinical condition generally between weeks 10 and 11 of treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean weekly body weight gains in males at 50/35 mg/kg/day were lower up to week 4, comparable to control thereafter until the first week of mating when losses were noted. Overall, body weight gain for 50/35 mg/kg/day males was 13.7% lower than controls over the dosing period. Reduced body weight gain was also noted for 20 mg/kg/day males during the dosing period and the overall weight gain was 5% lower than controls.

For females at 50/35 mg/kg/day mean body weight gain was lower during the first 4 weeks of treatment. Mean overall body weight gain for the gestation period was also lower for this group, the difference occurring during the last week of gestation.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Weekly food consumption for 50/35 mg/kg/day males was lower during the first 2 weeks of dosing and for the first week of dosing for females in the same group.

For females during the lactation period, lower food consumption was noted in the 50/35 and 20 mg/kg/day groups.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

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:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Individual variation in estrous cycle was noted in all groups. Mean estrous cycle length in treated groups was comparable to control.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There was no effect of treatment.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

The mean number of days between pairing and coitus was higher in the 50/35 mg/kg bw/day group compared to control and historical control data (4.9 days vs 2.7 days).

There were no effects on mating and fertility indices.

The mean length of gestation at 50/35 mg/kg bw/day was higher than control and historical control (22.7 days vs 22.0 days or 22.3 days respectively).

Two females in the 50/35 mg/kg bw/day group died during delivery. While there were no other overt signs of dystocia, an number of females that died (see above) did so at or near the time of parturition.

Mean number of implantation sites was reduced in 50/35 mg/kg bw/day females

Dose descriptor:

NOAEL

Remarks:

Fertility

Effect level:

20 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: At 50/35 mg/kg/day increased number of days between pairing and coitus and increased mean gestation length, reduced mean number implantation sites, mean live litter size and number of pups born reduced.

Dose descriptor:

NOAEL

Remarks:

Parental

Effect level:

0.8 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: See "Remarks"

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:

not specified

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs of dehydrated appearance and bodies cool to touch were noted at a higher incidence at 50/35 and 30 mg/kg bw/day.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Total litter loss was noted for 4 females at 50/35 mg/kg bw/day and 1 female at 20 mg/kg bw/day. With the exception of 1 female at 50/35 mg/kg bw/day all litter losses occurred between lactation days 0 and 4. Furthermore, 2 females at 50/35 mg/kg bw/day and 1 at 20 mg/kg bw/day that were killed on gestation day 25 only had early resorptions in utero.

Mean live litter size and mean number of pups born in the 50/35 mg/kg bw/day group were lower than control and historical control data. A similar but less marked effect was also noted in the 20 mg/kg bw/day group.

The number of pups found dead or missing, presumed cannibalized, was higher in the 50/35 and 20 mg/kg bw/day groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weight gain was lower in 50/35 and 20 mg/kg bw/day groups from postnatal day 4.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No findings attributable to maternal treatment with the test article.

Histopathological findings:

not examined

Other effects:

not specified

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Remarks:

developmental

Generation:

F1

Effect level:

4 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Mean post-natal survival and pup weights reduced at 50/35 and 20 mg/kg/day

Critical effects observed:

not specified

Reproductive effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to other toxic effects:

reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

Table 1 - Survival

Dose (mg/kg/day)	Control	0.8	4	20	50/35
Males
Number in group	25	25	25	25	25
Found dead	0	0	0	4	5
Killed	0	0	0	1	5
Survived until termination	25	25	25	20	15
Females
Number in group	25	25	25	25	25
Found dead	0	0	0	7	11
Killed	0	0	0	4	3
Survived until termination	25	25	25	14	11

Table 2 - Mean Gestation length (days)

Dose (mg/kg/day)	Control	0.8	4	20	50/35
Number of animals	20	22	23	15	15
Gestation length	22.0	21.9	21.9	22.0	22.7**

** p<0.01

Table 3 - Summary of Implantation Sites

	Dose (mg/kg/day)	Control	0.8	4	20	50/35
Implantation sites	Mean SD N	15.4 2.54 20	16.4 1.68 22	17.0 1.98 23	14.4 3.31 15	12.1** 4.96 15
Number born	Mean SD N	14.8 2.69 20	15.3 1.58 22	16.0 2.20 23	13.5 3.54 15	8.7** 4.92 15
Unaccounted sites	Mean SD N	0.7 0.81 20	1.1 1.04 22	1.0 1.19 23	0.9 0.99 15	3.5** 5.42 15

** p<0.01

Only dams that delivered 1 or more pups included in above table

Table 4 - Absolute Organ Weight (g)

	Dose (mg/kg/day)	Control	0.8	4	20	50/35
Prostate	Mean SD N	1.10 0.225 25	1.08 0.244 25	1.04 0.193 25	0.93* 0.200 20	0.86** 0.188 15
Seminal vesicles	Mean SD N	1.95 0.384 25	1.91 0.308 25	1.87 0.299 25	1.86 0.308 20	1.58** 0.410 15
Left Testis	Mean SD N	1.83 0.157 25	1.77 0.280 25	1.82 0.343 25	1.77 0.132 20	1.68 0.219 15
Right Testis	Mean SD N	1.85 0.204 25	1.81 0.148 25	1.76 0.178 25	1.76 0.109 20	1.70 0.207 15
Left Epididymis	Mean SD N	0.70 0.068 25	0.69 0.100 25	0.68 0.064 25	0.68 0.079 20	0.63 0.082 15
Right Epididymis	Mean SD N	0.71 0.048 25	0.73 0.070 25	0.69 0.071 25	0.67 0.070 20	0.65* 0.084 15
Left Cauda Epididymis	Mean SD N	0.3065 0.03231 25	0.3088 0.05556 25	0.2933 0.03829 25	0.2893 0.03786 20	0.2756 0.05086 15
Right Cauda Epididymis	Mean SD N	0.2972 0.02999 25	0.3073 0.03786 25	0.2820 0.03070 25	0.2758 0.02949 20	0.2733 0.03788 15
Pituitary	Mean SD N	0.0152 0.00291 25	0.0139 0.00202 25	0.0145 0.00279 25	0.0134* 0.00216 20	0.0119** 0.00186 15

* p<0.05 ** p<0.01

Table 5 - Litter Data

	Dose (mg/kg/day)	Control	0.8	4	20	50/35
Number born	Mean SD N	14.8 2.69 20	15.3 1.58 22	16.0 2.20 23	14.0 3.57 17	8.7** 4.92 15
Sex at birth (% male)	Mean SD N	51.9 13.96 20	51.8 7.71 22	52.0 16.19 23	46.2 15.33 17	44.7 27.94 15
Live litter size (PND 0)	Mean SD N	14.6 2.54 20	14.9 1.63 22	15.6 2.45 23	13.1 3.06 17	7.4** 5.08 15

** p<0.01

Table 6 - Postnatal survival (% per litter)

	Dose (mg/kg/day)	Control	0.8	4	20	50/35
Birth to PND 4 (pre-selection)	Mean SD N	96.6 4.85 20	95.4 6.23 22	93.3 9.03 23	82.3 33.95 15	56.8** 43.91 14
PND 4 (post selection) to PND 21	Mean SD N	100.0 0.00 20	100.0 0.00 20	99.5 2.61 23	89.1** 21.59 8	77.8** 40.37 6

** p<0.01

N = number of litters

If dam died during interval the litter was excluded from viability calculation

Conclusions:

Following administration at 0, 0.8, 4, 20 or 50/35 mg/kg/day the parental NOAEL was considered to be 0.8 mg/kg/day based on the mortality, clinical signs, food consumption, body weight effects, effects on organ weights noted at 50/35 or 20 mg/kg bw/day and pathology findings in the skeletal muscle and heart at 4, 20 or 50/35 mg/kg bw/day. The NOAEL for reproductive performance was considered to be 20 mg/kg/day based on the increased number of days between pairing and coitus and lower number of implantation sites noted at 50/35 mg/kg/day. Based on the total litter losses, reduced postnatal survival and clinical signs and lower body weight gains noted at 50/35 or 20 mg/kg/day the NOAEL for neonatal toxicity was considered to be 4 mg/kg/day.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

20 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In the key one-generation reproductive toxicity study, conducted according to OECD Test Guideline 415 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 a minimum of 70 days prior to mating. Males were dosed throughout mating and until scheduled necropsy. Females were dosed throughout mating, gestation and until lactation day 20.

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 50/35 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.

The mean number of days between pairing and coitus and mean gestation length were increased in the 50/35 mg/kg bw/day group. No overt signs of dystocia were observed during the study, although several females of the 20 and 50/35 mg/kg bw/day group died at or near the time of parturition. Individual variation in oestrous cycle was noted in all groups. Mean oestrous cycle length in treated groups was comparable to control. There were no effects on mating and fertility indices. Pups from 50/35 and 20 mg/kg bw/day litters showed clinical signs of dehydrated appearance and bodies cool to touch at a higher incidence than those in the control group. Total litter loss was noted for 4 females at 50/35 mg/kg bw/day and 1 female at 20 mg/kg bw/day. With the exception of 1 female at 50/35 mg/kg bw/day all litter losses occurred between lactation days 0 and 4. Furthermore, 2 females at 50/35 mg/kg bw/day and 1 at 20 mg/kg bw/day that were killed on gestation day 25 had early resorptions in utero. Mean live litter size and mean number of pups born in the 50/35 mg/kg bw/day group were lower than control and historical control data. A similar but less marked effect was also noted in the 20 mg/kg bw/day group. The number of pups found dead or missing, presumed cannibalised, was higher in the 50/35 and 20 mg/kg bw/day groups. Mean litter body weight gain was lower in 50/35 and 20 mg/kg bw/day groups from postnatal day 4. There were no findings in the litter at gross necropsy. The NOAEL for reproductive performance was concluded to be 20 mg/kg bw/day based on the increased number of days between pairing and coitus and the lower number of implantation sites noted at 50/35 mg/kg bw/day. The NOAEL for neonatal toxicity was concluded to be 4 mg/kg bw/day based on total litter losses, reduced postnatal survival, clinical signs and lower body weight gains at 50/35 or 20 mg/kg bw/day.

Effects on developmental toxicity

Description of key information

In the key prenatal developmental toxicity study, conducted according to OECD Test Guideline 414 and in compliance with GLP, the concluded NOAEL for maternal and developmental toxicity was 10 mg/kg bw/day based on reduced body weight gain in maternal animals and reduced foetal body weight at high dose group females and litters (Eurofins, 2018). The reduced foetal weights are likely to be secondary to reduced maternal body weights.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 May 2017 to 27 October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2001

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: 12 weeks old females and from 19 to 26 weeks old males
- Weight at study initiation: 357 - 485 g males and 227 - 283 g females
- Fasting period before study: not specified
- Housing: Female animals were kept individually in IVC cages during most of the study period except during pre-mating period when females were kept in groups of two and the mating period when two females were paired with one male. Male rats were housed in groups of two during the pre-mating period and after mating period.
- Diet (e.g. ad libitum): Free access to Altromin 1324 maintenance diet for rats and mice, ad libitum.
- Water (e.g. ad libitum): Free access to tap water, sulphur acidified to a pH of approximately 2.8
- Acclimation period: at least 5 days

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

Route of administration:

oral: gavage

Vehicle:

other: sesame oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item and control formulations were prepared freshly once every 7-10 days based on the stability information. The prepared formulation was stored at room temperature. For the dose formulation the test substance was weighed and the vehicle was added to give the appropriate final concentration of the test item.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The vehicle, sesame oil, was selected as suggested by the sponsor based on test item's characteristics, hydrolysis in water and testing guideline.
- Lot/batch no. (if required): 16312506
- Concentration in vehicle: LD: 1.25; MD: 2.5; HD: 5

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before beginning of the treatment period, formulation samples were prepared and analysed in order to obtain knowledge about stability and homogeneity of the test item in the selected vehicle at Eurofins Munich as part of a separate GLP study. Samples for analysis of concentration of the test item in the dosing formulations were taken in the first and last week of the study for all doses.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1 male : 2 females
- Length of cohabitation: until pregnancy was confirmed by sperm in vaginal smear
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility. Not applicable
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: none

Duration of treatment / exposure:

between gestation day 5 and until gestation day 19.

Frequency of treatment:

7 days per week

Duration of test:

from pre-mating until necropsy of females

Dose / conc.:

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

Remarks:

low dose (LD)

Dose / conc.:

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

Remarks:

middle dose (MD)

Dose / conc.:

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

Remarks:

high dose (HD)

No. of animals per sex per dose:

25 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The test concentrations were selected based on a 90-day study, one generation reproductive toxicity study and in consultation with the sponsor. The highest dose level was chosen with the aim of inducing toxic effects, but not death or severe suffering. Thereafter, a descending sequence of dose levels was selected with the intention to demonstrate any dose-related response and a NOAEL.
- Rationale for animal assignment (if not random): random

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked included: health condition, morbidity and mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily checked for spontaneous activity, lethargy, recumbent position,convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin
and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed once before pairing, then sperm positive females were weighed on gestation days 0, 5, 8, 11, 14, 17 and 20. Males were not weighed in this study except once before initiation of pairing.

FOOD CONSUMPTION: Yes. Food consumption of sperm positive females was measured on gestation days 5, 8, 11, 14, 17 and 20. Food consumption was not measured for males.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: macroscopic examination of for structural abnormalities or pathological changes; any macroscopic findings were preserved in 4% neutral-buffered formaldehyde; uteri were removed and pregnancy status was confirmed; non-gravid uteri were further examined; each gravid uterus with the cervix was weighed; number of corpora lutea was counted; the uterine content was examined for embryonic or foetal deaths as well as the number of viable foetuses; degree of resorption was confirmed; position and number of foetuses in each uterine horn was recorded; males were sacrificed without any observations at any time after completion of mating procedure.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [the first 20 processed by Alizarin red staining from second half litter per group]
- Head examinations: Yes: [the first 20 processed by Alizarin red staining from second half litter per group]

Statistics:

A statistical assessment of the results of the body weight, food consumption was performed by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Foetal evaluation parameters like external, visceral, craniofacial and skeletal parameters were analysed using Fisher’s exact test. The litter were used as the unit for data analysis.The statistics were performed with GraphPad Prism V.6.01 software (p<0.05 is considered as statistically significant).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no clinical signs of toxicological relevance observed in the control, LD and MD dose groups. Specific clinical signs observed in dead/ moribund females were alopecia on right shoulder, moderate piloerection, reduced spontaneous activity, diarrhoea, apathy, spontaneous activity severely reduced, ataxia, , and hunched posture, piloerection (moderate), slow movements, moderately reduced spontaneous activity, half eyelid closure. Moreover, sunken flanks in female no. 93., exophthalmos in female no. 84 and slow movements in female no. 87 were observed. In terminally sacrificed females low incidences of clinical signs like alopecia were noted in the dose groups as well as in the control group. There were also occasional clinical signs like abnormal breathing in one HD female, red to dark red nasal discharge in one each female of the LD and HD groups, piloerection in 3 HD females and exophthalmos in one control female. Moving the bedding was also noted mainly immediately after administration and just for a short period in one control, 2 LD and 1 HD group females. This effect was considered to be mainly a local reaction to the test item rather than a systemic effect. All clinical signs observed in terminally sacrificed females were incidental or non adverse in nature. However, clinical signs observed in dead/moribund females could be attributed to the treatment with test item.

Mortality:

mortality observed, treatment-related

Description (incidence):

One mortality occurred at the HD group during the treatment period of the study, female no. 87 on gestation day 17. Two females (no. 84 and 93) of the HD group were euthanised in a moribund condition for animals welfare reasons on gestation days 9 and 19. These instances of mortality and morbidity were considered to be treatment-related. No other deaths were observed during the treatment period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean body weight remained unaffected and increased with the progress of the study in the control, LD, MD and HD groups. However, after initiation of treatment the
mean body weight gain was noted to be marginally but statistically significantly lower (p < 0.05) in the HD group during gestation days 8-11 (65.05 % of controls) when
compared to the controls. There was also marginally lower overall body weight gain (gestation day 0-20) observed in the HD group (89.24 % of controls) without achieving
statistical significance when compared to the controls. Although this effect on body weight gain in the HD group was marginal, it could be attributed to treatment with test
item.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was unaffected by the treatment in any of the dosed animals and food consumption of LD, MD and HD was comparable to the control group.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No effects were noted on gravid uterine weight.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

No gross pathological changes of toxicological relevance were observed during the macroscopic examination of the females of the control, LD and MD groups.
In the HD group found dead female (87), enlarged adrenal glands were observed during the gross pathological examination. This finding in the HD group dead female could be
considered as test item-related.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

no effects observed

Description (incidence and severity):

No test item-related effects of toxicological relevance.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

No test item-related effects of toxicological relevance were noted for implantation sites.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

No test item-related effects of toxicological relevance were noted for early and late resorptions.

Early or late resorptions:

no effects observed

Description (incidence and severity):

No test item-related effects of toxicological relevance were noted for early and late resorptions.

Dead fetuses:

no effects observed

Description (incidence and severity):

No test item-related effects of toxicological relevance were noted for number of live fetuses. No dead foetuses were noted in any of the groups.

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Successful mating resulted in 24/25 pregnancies in the LD group, 24/25 in the MD group and 19/22 in the HD group compared to 23/25 pregnancies in the control group. The marginally low pregnancy rates (no. of pregnancies / no. of females mated or sperm positive x 100) of 86.36 % in the HD group compared to 92% in the control group was considered to be a biological variation and well above standard pregnancy rate of minimum 80% in rat.

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Abnormalities:

effects observed, treatment-related

Localisation:

other: reduced body weight gain at high dose level

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean foetus weight (individual basis) of the HD group (3.26 g) was moderately, statistically significantly lower when compared to the control group (3.53 g) (92.35 % of controls, p< 0.001). The mean male and female foetus weight (Individual basis) of the HD group (male- 3.33 g and female -3.19 g) was also statistically significantly lower when compared to the control group (male- 3.60 g and female- 3.45 g) (92.5 % of controls in males and 92.46 % of controls in females, p< 0.001). The mean foetus weight (litter basis) of the HD group (3.30 g) was lower when compared to the control (3.54 g) but without achieving statistical significance (63.22 % of controls).
Moderately, statistically significantly lower male litter weight (litter basis) of the HD group (18.03 g) (73.35 % of controls, p< 0.05) was observed when compared to the control group (24.58 g). This effect on male litter weight could be attributed to relatively lower group mean male number in the HD group (5.42) when compared with the controls (6.52). There was also statistically significantly higher group mean foetus weight (3.64 g) and male foetus weight (3.73 g) (Individual basis) observed in the LD group when compared to the control which was not considered as test item-related.
The lower mean foetus weight (individual and litter basis) and male/female foetus weight (individual basis) of the HD group were considered to be related to the treatment with the test item.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

No test item-related effects of toxicological relevance were noted for number of live foetuses.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No test item-related effects of toxicological relevance were noted for sex ratio.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There were no test item-related effects of toxicological relevance for the total litter weight and female litter weight in any of the treatment groups when compared with the controls.

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

There were no external abnormalities considered to be of toxicological relevance in any of the dose groups. Statistical analysis of data revealed no significant differences compared to the control group. Low incidences of astomia (1 in MD), haematoma on various body parts in a few foetuses (1 in control, 1 in LD, 2 in MD and 1 in HD), lower jaw small (1 in control), small mandible (1 in MD), small foetus (1 in control) and small snout (1 in MD) were noted in isolated females of the control group and/or the dose groups without dose dependency. As these findings were observed mostly in single foetuses, they were considered to be incidental in nature and unrelated to the treatment.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

A statistically significant decrease in litter incidence for incomplete ossification of left and right parietal in the LD group and fused sacral vertebral centrum and arch in the MD group compared to the control group were considered to be incidental as frequencies were even less in numbers compared to the controls. Therefore, these findings are not to be considered as treatment-related and solely spontaneous in nature. A statistically significant increase for litter incidences of incomplete ossification of right squamosal (31.58 % compared to 5 % in controls, p< 0.05) was observed in the HD group when compared with the controls. Slightly higher litter incidences, but without achieving statistical significance for incomplete ossification were observed in the HD group when compared to the concurrent control group of basioccipital,frontal, hyoid body, parietal, squamosal, zygomatic arch, scapula, 2nd sternebra, 5th sternebra, cervical vertebral arch, pubis, humerus, misshapen humerus, irregularly ossified thoracic vertebral centrum, unossified 6th sternebra, unossified thoracic vertebral centrum, unossified forelimb metacarpals,full 14th rib, wavy ribs.
The observed statistically significantly reduced ossification without achieving statistical significance of several bones in the HD group that normally exhibit rapid ossification in the last days of gestation indicates a generalised skeletal delay in the HD group. This delayed ossification was considered to be associated with the observed slight maternal toxicity (lower body weight) and reduced foetal body weight of the HD group. Generally delayed ossification is not regarded to persist postnatally and not associated with long-term consequences on survival, general growth and development and therefore is not considered to be adverse.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There was higher litter incidence of umbilical artery transposed and bilateral dilatation of ureter observed in all treatment groups including control group. However, values were well within historical control data range (85.71 % - umbilical artery transposed and 63.64 % - dilated ureter). There was discolouration of a few organs like liver, spleen and thymus observed without dose dependency. Discolouration of organs was considered likely to reflect the consequence of a functional disorder and thus not strictly as developmental anomalies.
Visceral findings observed in the dose groups were at frequencies generally comparable to or in some cases slightly higher or lower in frequency compared to controls. As observed findings were either minor variations and/or due to a lack of dose dependency and consistency, no toxicological significance can be attributed to these findings and they were considered to be spontaneous in nature.

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

changes in litter size and weights

Abnormalities:

effects observed, treatment-related

Localisation:

other: reduced body weight in high dose group

Developmental effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to maternal toxicity:

developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

See attachment for result tables.

Conclusions:

In the prenatal developmental toxicity study, conducted according to OECD Test Guideline 414 and in compliance with GLP, the concluded NOAEL for maternal and developmental toxicity was 10 mg/kg bw/day based on reduced body weight gain in maternal animals and reduced foetal body weight at high dose group females and litters. The reduced foetal weights are likely to be secondary to reduced maternal body weights.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

10 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

In the key prenatal developmental toxicity study, conducted according to OECD Test Guideline 414 and in compliance with GLP, the concluded NOAEL for maternal and developmental toxicity was 10 mg/kg bw/day based on reduced body weight gain in maternal animals and reduced foetal body weight at high dose group females and litters (Eurofins, 2018).

Following mating, female rats were administered orally via gavage to 5, 10 or 20 mg/kg bw/day of 2,4,6-trimethyl-2,4,6-tris(3,3,3-trifluoropropyl)cyclotrisiloxane in sesame oil vehicle from gestation day 5 to gestation day 19. The control animals were administered the vehicle only. During the period of administration, the animals were observed precisely each day for signs of toxicity and mortality. Animals that died during the study were examined macroscopically. All surviving female animals were sacrificed on the respective gestation day 20. Following the gross necropsy, the uteri and ovaries were removed, weighed and examined for number of implantations, resorptions (early and late) live and dead foetuses. The uteri of the non-pregnant females were processed with 10 % ammonium sulphide solution and checked for the early embryonic deaths. Sperm positive females were weighed on gestation days 0, 5, 8, 11, 14, 17 and 20. Food consumption of sperm positive females was measured on gestation days 5, 8, 11, 14, 17 and 20.

Foetuses were identified, sexed and weighed. One half of each litter was examined for soft tissue anomalies. The remaining foetuses were processed by Alizarin red staining and the first 20 litters per group were examined for skeletal alterations. Craniofacial examination of the heads of the foetuses used for the soft tissue examination of at least 20 litters per group was performed for internal structure including the eyes, brain, nasal passage and tongue by razor blade serial sectioning technique.

One mortality occurred in the high dose group during the treatment period of this study. In addition, two females of the high dose group were euthanised in a moribund condition for animal welfare reasons. All clinical signs observed in terminally sacrificed females were incidental or non-adverse in nature. However, clinical signs observed in dead/moribund females could be attributed to the treatment with test item. Body weight gain was noted to be marginally but statistically significantly lower in the high dose group during gestation days 8-11 when compared to the controls. There was also marginally lower overall body weight gain (gestation day 0-20) observed in the high dose group without achieving statistical significance when compared to the controls. No treatment-related effect on food consumption, prenatal data parameters and gross pathology of terminally sacrificed females was observed up to highest dose tested. However, statistically significant lower mean foetus weight (individual and litter basis) and male/female foetus weight (individual basis) was observed in the high dose group when compared with the control. Furthermore, no treatment-related and toxicologically relevant external, visceral or craniofacial findings were observed in the high dose group. However, reduced ossification of some bones was observed which was indicative of a generalized delayed ossification associated with foetal growth retardation. Observed foetal effects in the high dose group are likely to be secondary to reduced maternal body weights and are not considered an adverse effect of the test item.

A supporting study is the one-generation reproductive study in rats (Dow Corning Corporation, 2002). In this study the NOAEL for general parental toxicity was 0.8 mg/kg bw/day 2,4,6-trimethyl-2,4,6-tris(3,3,3-trifluoropropyl)cyclotrisiloxane based on the mortality, clinical signs, food consumption, body weight effects, effects on organ weights noted at 50/35 or 20 mg/kg bw/day and pathology findings in the skeletal muscle and heart at 4, 20 or 50/35 mg/kg bw/day. Based on the total litter losses, reduced postnatal survival and clinical signs and lower body weight gains noted at 50/35 or 20 mg/kg bw/day the NOAEL for neonatal toxicity was considered to be 4 mg/kg bw/day.

Justification for classification or non-classification

Based on the available one-generation and prenatal developmental toxicity studies, 2,4,6-trimethyl-2,4,6-tris(3,3,3-trifluoropropyl)cyclotrisiloxane is classified for reproductive toxicity Category 2 (oral/dermal) according to Regulation (EC) No 1272/2008, with hazard statement H361fd: Suspected of damaging fertility. Suspected of damaging the unborn child.