Registration Dossier

Administrative data

Description of key information

In the key 90-day repeated dose oral toxicity study with 28-day recovery period with the test substance 2,4,6,8-tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane (Vi4D4), conducted according to OECD Test Guideline 408 and in compliance with GLP, the concluded systemic NOAEL for male rats was greater than 150 mg/kg bw/day based on no adverse effects observed. The systemic NOAEL for female rats was concluded to be equal to 15 mg/kg bw/day based on lower mean number of corpora lutea in females at 50 and 150 mg/kg bw/day, which correlated to lower mean ovary weights and a perturbation of ovarian cycling in these groups (Charles River Laboratories, 2020).

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:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25th of June 2019 to
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC, USA.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approximately 7 weeks
- Weight at study initiation: 178 - 266 gram
- Fasting period before study: No
- Housing: Polycarbonate, solid-bottom cages containing Bed-O-Cobs® or other bedding material.
- Diet: PMI Nutrition International, LLC Certified Rodent LabDiet 5002 meal, ad libitum.
- Water: Municipal tap water, treated by reverse osmosis and ultraviolet irradiation, ad libitum.
- Acclimation period: At least 7 days.

DETAILS OF FOOD AND WATER QUALITY: Results of analysis for nutritional components and environmental contaminants of the food as well as periodic analysis of the water were provided by the supplier. It was considered that no known contaminants in the feed or water were present that could interfere with the outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20°C to 26°C
- Humidity (%): 30% to 70%
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark

IN-LIFE DATES: From: June 2019 To: 4th of December 2019
Route of administration:
oral: gavage
Details on route of administration:
Each dose was administered via a syringe equipped with attached gavage cannula.
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Dosing formulations were prepared at least weekly at appropriate concentrations to meet the dose level requirements with the neat test substance container kept closed as much as possible during distribution activities.

VEHICLE
- Justification for use and choice of vehicle: The vehicle was selected based on the test substance’s characteristics and the subsequent relevant OECD testing guideline.
- Concentration in vehicle: 0, 3.75, 12.5, 37.5 mg/mL
- Amount of vehicle: 0, 3.75, 12.5, 37.5 mg/mL
- Lot/batch no.: 2IC0148, 2IH0387, and 1IG1538
- Purity: not specified
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples for concentration analysis were collected during the initial and last formulation preparations from all dose levels. Dose formulation samples for homogeneity analysis were collected during the initial formulation preparations from low and high dose levels. The analysis was utilising a gas chromatography method with flame ionisation.
Duration of treatment / exposure:
90 days
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control
Dose / conc.:
15 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 animals per sex per dose. 5 additional recovery animals were used for the vehicle and high dose groups.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The target exposure concentrations were selected by the sponsor representative in consultation with the study director based on a previous repeated dose study (Charles River Laboratories, 2019). In that study, the test substance was administered to 5 males and females via oral gavage for up to 14 consecutive days at dose levels of 0, 100, 500, 1000 and 300 mg/kg bw/day. See Charles River Laboratories (2019) for details on DRF study results.

- Rationale for animal assignment: Randomised scheme designed to achieve similar group mean body weights (within ± 20% of the mean for each).
- Fasting period before blood sampling for clinical biochemistry: at least 8 hours, however, no more than 24 hours.
- Rationale for selecting satellite groups: Randomised
- Post-exposure recovery period in satellite groups: 28 days.
- Section schedule rationale: randomised
Positive control:
N/a
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Cage-side observations were made at 0.5 - 3 hours post-dosing and at least once daily on non-dosing/recovery days. Cage side observations were not conducted on days when the detailed clinical observations were performed during the non-dosing and/or recovery period.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made outside the home cage in a standard arena once before the first administration, once a week (± 2 days) thereafter, and on the day of scheduled necropsy (for animals selected for necropsy). The observations focused on posture, convulsions/tremors, biting, faeces consistency, lacrimation/chromodacryorrhoea, salivation, piloerection, fur appearance, palpebral closure, respiratory rate/character, red/crusty deposits, mucous membranes/eye/skin colour, eye prominence and muscle tone.

BODY WEIGHT: Yes
- Time schedule for examinations: once before the first administration, once a week (± 2 days) and thereafter, and on the day of scheduled necropsy (for animals selected for necropsy).

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
Food consumption was measured weekly (± 2 days) throughout the study period.

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Once during the pre-treatment period
- Dose groups that were examined: All main study animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On the day of scheduled necropsy.
- Anaesthetic used for blood collection: No
- Animals fasted: Yes, at least 8 hours, however, not more than 24 hours.
- How many animals: All main and recovery study animals.
- 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.
- Animals fasted: Yes, at least 8 hours, however, not more than 24 hours.
- How many animals: All main and recovery study animals.
- Parameters checked in table were examined: Yes, see table 1.

URINALYSIS: Yes
- Time schedule for collection of urine: On the day of scheduled necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, at least 8 hours, however, not more than 24 hours.
- Parameters checked in table were examined: Yes, see table 1.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During the final week of the test substance administration and near the end of the recovery period. For the motor activity sessions, the examinations were performed in a sound-attenuated room equipped with a white-noise generation system to minimize environmental variations in the test conditions. Additionally, testing of treatment groups was balanced across the day of testing and executed by appropriately trained technicians. The test session was 60 minutes, consisted of twelve 5-minute intervals, and was reported in six 10-minute intervals. Motor activity evaluated total and ambulatory activity counts obtained for the 60 minute test session.
- Dose groups that were examined: All main study and recovery animals.
- Battery of functions tested: Home cage observations included posture, convulsions/tremors, biting, palpebral (eyelid) closure, faeces consistency. Handling observations included ease of removal from cage, ease of handling animal in hand, lacrimation/chromodacryorrhea, salivation, piloerection, fur appearance, palpebral closure, respiratory rate/character, red/crusty deposits, mucous membranes/eye/skin colour, eye prominence and muscle tone. Open field observations included time to first step (seconds), mobility, gait, convulsions/tremors, gait score, arousal, bizarre/stereotypic behaviour, rearing, backing, grooming, urination/defaecation. Sensory observations included approach response, touch response, startle response, olfactory orientation, pupil response, eyeblink response, forelimb extension, hindlimb extension and air righting reflex. Neuromuscular observations included hindlimb extensor strength, grip-strength-hind forelimb, rotarod performance and hindlimb foot splay. Physiological observations included catalepsy, body temperature and body weight.

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 2)

HISTOPATHOLOGY: Yes (see table 2)
Other examinations:
Hormone sample analysis: TSH, T3 and T4 hormone analysis were performed.
Statistics:
Data collected during the pre-treatment period were not tabulated, summarised, or statistically analysed. All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and are reported at the 1% and 5% levels, unless otherwise noted. Continuous functional observational battery data were analysed using parametric analysis. FOB parameters that yielded scalar or descriptive data were analysed using Fisher’s Exact.

Levene’s test was used to assess the homogeneity of group variances. The groups were compared using an overall one-way ANOVA F-test if Levene’s test was not significant or the Kruskal-Wallis test if it was significant. If the overall F-test or Kruskal-Wallis test was found to be significant, then pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively. Datasets with 2 groups were compared using a Dunnett’s test equivalent to t test in Nevis 2012 tables) or Dunn’s test.
Clinical signs:
no effects observed
Description (incidence and severity):
No test substance related clinical findings were observed. All clinical observations were incidental, occurred in single animals and/or were not noted in a dose response relationship.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred throughout the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related lower body weights were observed in both males and females at 150 mg/kg bw/day. The alteration in weight was noted on day 50 and continued until the terminal necropsy on day 90 and resulted in a lower mean body weight gain for the entire treatment period. The difference was statistically significant for males during days 50-57 and days 1-90. During days 57-90 slightly lower mean body weights were noted for males and females at 150 mg/kg bw/day dose group, resulting in 7.3% reduction in mean body weights for males and 3.4% reduction in mean body weights for females than those of control animals.

During the recovery period (Days 90–118), mean body weights and body weight gains for males and females in the 150 mg/kg bw/day group were generally similar to or slightly higher than the control group.
No test substance-related effects were observed on body weight at 15 and 50 mg/kg bw/day.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was unaffected by test substance administration. Differences from the control group were slight and/or not statistically significant.
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 ophthalmic lesions indicative of toxicity were observed in any of the test substance treated groups. All findings observed were typical in prevalence and appearance for laboratory rats of this age and strain.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
In the 150 mg/kg bw/day group males, lower mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC) and haemoglobin (HGB) concentration were observed and considered as test substance-related. Moreover, higher mean reticulocyte (RETIC) value were observed in the 150 mg/kg bw/day group males. However, this change was due to one male shifting the mean value and when this individual was excluded, the concentration was lower in a dose-dependent manner. Additionally, test-substance related lower HGB concentration was noted in females at all dose levels in a dose-dependent manner. Females in the 50 and 150 mg/kg bw/day dose group exhibited lower mean haematocrit (HCT), MCH, MCHC and mean corpuscular volume (MCV) concentrations.
At the recovery necropsy, no test substance-related changes were observed.

Coagulation parameters were unaffected by test substance administration. Differences from the control group were slight, not statistically significant, lacked a dose-response, and/or were attributed to biological variability.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Lower mean alkaline phosphatase (ALP) and triglycerides (TRIG) were observed in all test substance treated males and females. These changes were considered as test substance-related although they did not occur in a dose-related manner. Non-statistically significant lower mean aspartate aminotransferase (AST) values were observed in the 150 mg/kg bw/day dose group males and females. Generally statistically significantly higher mean cholesterol ( CHOL, HDL, and LDL) values were noted in the 50 or 150 mg/kg bw/day group females. The lower mean ALP and AST were not associated with toxicologic effects in the liver. Statistically significantly lower mean total bilirubin (TBIL) concentrations were noted in all test substance-treated females. Test substance-related increases in mean total protein (TPROT), albumin (ALB), and globulin (GLOB) concentrations were noted in the 50 mg/kg/day and 150 mg/kg/day group females.
At the recovery necropsy, no changes considered as test substance-related were noted in males. However, non-statistically significantly lower AST, ALT, and TBIL concentrations were noted in the 150 mg/kg bw/day group females than those in the control group.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Urinalysis parameters were unaffected by test substance administration. No statistically significant differences occurred when the control and test substance treated groups were compared.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Home cage observations were unaffected by test substance administration. Handling, open field, sensory, neuromuscular or physiological observations were unaffected by test substance administration. Motor activity patterns (total and ambulatory activity counts) were also unaffected by test substance administration.

Statistically significantly lower mean hindlimb grip strength was noted for the 150 mg/kg/day group males compared to the control group at the Week 16 evaluation; however, this difference was noted in a single sex and a similar effect was not noted during the Week 13 evaluation and therefore this difference was not considered test substance-related.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Lower mean ovary weights were noted in the 50 and 150 mg/kg bw/day group females, which microscopically correlated to decreased number of corpora lutea. Higher mean thyroid weights were noted in the 50 and 150 mg/kg bw/day group females and 150 mg/kg bw/day group males, which microscopically correlated to follicular cell hypertrophy. Higher mean and relative to body weight and brain weight liver weights were noted in the 50 and 150 mg/kg bw/day groups correlated to centrilobular hepatocellular hypertrophy in the 50 and 150 mg/kg bw/day group females and 150 mg/kg bw/day group males. There were no microscopic effects to account for the higher mean liver weight in the 50 mg/kg bw/day group males.

No other test substance-related organ weight changes were noted. Other differences had no patterns, trends, or correlating data to suggest toxicological relevance. Thus, other organ weight differences observed were considered incidental and/or related to difference of sexual maturity and unrelated to administration of the test substance.

In the recovery group, higher mean liver weights were noted in the 150 mg/kg bw/day groups. Relative liver weight to body weight and brain weight ratio were statistically significantly higher in recovery males at 150 mg/kg bw/day. These changes in liver weight did not correlate to any microscopic effects.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No test substance-related gross findings were noted. The gross findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence in control and treated group animals and, therefore, were considered unrelated to administration of the test substance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Minimal or mild hepatocellular centrilobular hypertrophy and thyroid follicular cell hypertrophy were observed in the 50 mg/kg bw/day females and 150 mg/kg bw/day males and females. These microscopic changes correlated with increased organ weights and were considered most likely to be adaptive and non-adverse responses to test substance-induced enzyme induction.

Minimal decreased lymphoid cellularity of the thymus, characterized by slightly thinner thymic cortices was noted in the 150 mg/kg bw/day dose group males and females. No other microscopic findings in the lymphoid system were observed and therefore, this change was considered non-adverse.

Decreased number of corpora lutea, characterised by loss of total number of corpora lutea of all stages, was observed in the 50 and 150 mg/kg bw/day dose group females,. The change, which was severe in some animals and was consistent with a perturbation of ovarian cycling also correlated with lower mean ovarian weights. Based on the likely effect on reproduction in female animals, the ovarian findings were considered adverse.

Other microscopic findings observed were considered incidental, commonly observed in this strain/ age of rats and/or were of similar incidence and severity in control and treated group animals and consequently, considered as not test substance-related.

In the recovery group, no test substance-related microscopic findings were noted. While there were decreased corpora lutea in the 150 mg/kg bw/day dose group females, the severity and incidence were similar to controls. Decreased ovarian corpora lutea can be observed in control 6-month Sprague Dawley rats, the approximate age of the rats at the end of the recovery period.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Thyroid Hormone Analysis: Statistically significantly lower mean T3 and T4 levels were observed in the 50 and 150 mg/kg bw/day group females which was considered as test substance-related. Additionally, test substance-related statistically significantly higher mean TSH values were noted in the 150 mg/kg bw/day group females.

At the recovery evaluation on day 119, mean T3 and TSH hormone values for females at 150 mg/kg bw/day were similar to the control group. Statistically significantly higher (T4) levels were observed for females at 150 mg/kg bw/day than the control group.

There were no test substance-related effects on thyroid hormone analysis noted for males and females at 15 mg/kg bw/day and males at 50 and 150 mg/kg bw/day at the primary and recovery necropsy evaluations.

Oestrous Cycle Determination: No test substance-related effect on the stage of oestrous prior to necropsy occurred for females at any dose level.
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No adverse effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
15 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Critical effects observed:
yes
Lowest effective dose / conc.:
50 mg/kg bw/day (nominal)
System:
female reproductive system
Organ:
ovary
Treatment related:
yes

Table 1 - Summary of Organ Weight Data - Terminal Euthanasia (Day 91/92)a

  Males Males Males Females Females Females
Group 2 3 4 2 3 4
Dose (mg/kg/day) 15 50 150 15 50 150
No. Animals per Group 10 10 10 10 10 10
Ovary/Oviduct (No. Weighed)b N/A N/A N/A 10 10 10
Absolute value N/A N/A N/A -0.28 -8.94 -20.43
% of body weight N/A N/A N/A -1.97 -11.32 -16.88
% of brain weight N/A N/A N/A 0.93 -8.21 -20.78
Thyroid/Parathyroid (No. Weighed)b 10 10 10 10 10 10
Absolute value 11.6 11.93 15.97 10.22 23.92 30.98
% of body weight 11.93 12.79 26.79 7.43 19.84 35.42
% of brain weight 13.85 12.84 20.29 11.32 24.5 30.56
Liver (No. Weighed)b 10 10 10 10 10 10
Absolute value 6.8 13.63 23.76 4.39 25.91 40.52
% of body weight 7.08 14.69 35.29 2.66 21.94 45.88
% of brain weight 10.09 16.08 29.02 5.51 26.59 39.68

a  Organ weight values rounded to 2 significant digits (values with all decimal places in Provantis tables).

b All values expressed as percent difference of control group means.

Based upon statistical analysis of group means, values highlighted in bold are significantly different from control group – P 0.05; refer to data tables for actual significance levels and tests used.

N/A = Not applicable.


Table 2 - Summary of Organ Weight Data – Recovery Euthanasia (Day 119/120)a

  Males Males Males Females Females Females
Group 2 3 4 2 3 4
Dose (mg/kg/day) 15 50 150 15 50 150
No. Animals per Group 0 0 5 0 0 5
Liver (No. Weighed)b N/A N/A 5 N/A N/A 5
Absolute value N/A N/A 9.27 N/A N/A 11.83
% of body weight N/A N/A 11.6 N/A N/A 11.76
% of brain weight N/A N/A 10.31 N/A N/A 16.06

a  Organ weight values rounded to 2 significant digits (values with all decimal places in Provantis tables).

b All values expressed as percent difference of control group means.

Based upon statistical analysis of group means, values highlighted in bold are significantly different from control group – P 0.05; refer to data tables for actual significance levels and tests used.

N/A = Not applicable.


Table 3 - Summary of Microscopic Findings – Terminal Euthanasia (Day 91/92)

  Males Males Males Males  Females Females Females  Females
Group 1 2 3 4 1 2 3 4
Dose (mg/kg/day) 0 15 50 150 0 15 50 150
No. Animals per Group 10 10 10 10 10 10 10 10
Liver (No. Examined) -10 -10 -10 -10 -10 -10 -9 -10
Hypertrophy; centrilobular, hepatocellular  0 0 0 5 0 0 4 8
Minimal - - - 4 - - 4 7
Mild - - - 1 - - 0 1
Thyroid Gland (No. Examined) -10 -10 -10 -10 -10 -10 -10 -10
Hypertrophy; follicular cell 0 0 0 7 0 0 4 8
Minimal - - - 6 - - 4 6
Mild - - - 1 - - 0 2
Thymus (No. Examined) -10 -10 -10 -10 -10 -10 -10 -10
Decreased cellularity; lymphoid 0 0 0 7 0 0 0 4
Minimal - - - 7 - - - 4
Ovary (No. Examined) N/A N/A N/A N/A -10 -10 -10 -10
Decreased number, corpora lutea N/A N/A N/A N/A 0 0 5 7
Minimal N/A N/A N/A N/A - - 1 1
Mild N/A N/A N/A N/A - - 1 1
Moderate N/A N/A N/A N/A - - 1 1
Marked N/A N/A N/A N/A - - 0 2
     Severe N/A N/A N/A N/A - - 2 2

- = No noteworthy findings.

N/A = not applicable.

Conclusions:
In the 90-day repeated dose oral toxicity study with 28-day recovery period with the test substance 2,4,6,8-tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane (Vi4D4), conducted according to OECD Test Guideline 408 and in compliance with GLP, the concluded systemic NOAEL for male rats was greater than 150 mg/kg bw/day based on no adverse effects observed. The systemic NOAEL for female rats was concluded to be equal to 15 mg/kg bw/day based on lower mean number of corpora lutea in females at 50 and 150 mg/kg bw/day, which correlated to lower mean ovary weights and a perturbation of ovarian cycling in these groups.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
15 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Klimisch score of 1.
System:
female reproductive system
Organ:
ovary

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

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In a 14-day dose range-finding study, which was conducted according to a protocol similar to OECD Test Guideline 407 with deviations, not in compliance with GLP, the registered substance 2,4,6,8tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane (Vi4D4), was administered daily by oral gavage to 5 male and 5 female rats at dose levels of 0, 100, 500 or 1000 mg/kg bw/day in corn oil for 14 days (Charles River Laboratories, 2019a). Based on lower body weight gains and/or body weight losses noted on days 1 to 6 in the 500 and 1000 mg/kg bw/day group males and females, the decision was made by the Sponsor to trigger dosing in a fifth group at a dose level of 300 mg/kg bw/day for up to 7 days.

The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, food consumption, clinical pathology parameters (serum chemistry), gross necropsy findings, and organ weights.

No test substance-related clinical findings or gross necropsy observations were noted. Test substance-related severe body weight losses and lower mean food consumption were noted in the 1000 mg/kg bw/day group males during days 1 to 7. Therefore, all males and females in this group were euthanised in extremis on day 7. All other animals survived to the scheduled necropsy. Test substance-related lower body weight gains and/or body weight losses were noted for all animals at 500 and 1000 mg/kg bw/day group, which led to lower mean day 7 body weights for the ≥ 300 mg/kg bw/day males and all test substance-treated females. Additionally, lower mean day 14 body weights were noted in the 100 and 500 mg/kg bw/day group females and the 500 mg/kg bw/day group males. Test substance-related lower mean food consumption was noted in the 500 mg/kg bw/day group males and the 1000 mg/kg bw/day group males and females during days 1 to 7; these results corresponded to mean body weight losses or lower mean body weight gains noted for these animals. Over the entire dosing duration (days 1 to 14), only the 500 mg/kg bw/day group males had lower food consumption. Statistically significantly lower albumin levels were noted for males at 500 mg/kg bw/day and lower glucose levels for males at 100 and 500 mg/kg bw/day. The lower glucose and albumin were attributed to lower food consumption and/or decreased body weight gains. Test substance-related increased liver weights (absolute and normalised) were noted in all test substance-treated males and females at the terminal necropsies on days 8 and 15. A NOAEL was not concluded in the range-finding study. Based on the results of the range-finding study the dose levels of 0, 15, 50 or 150 mg/kg bw/day were selected for the main 90-day repeated dose oral toxicity study.

In the main 90-day repeated dose oral toxicity study with 28-day recovery period, conducted according to OECD Test Guideline 408 and in compliance with GLP, the registered substance 2,4,6,8-tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane (Vi4D4), was administered daily by oral gavage to 10 male and 10 female Crl:CD(SD) rats at dose levels of 0, 15, 50 or 150 mg/kg bw/day in corn oil (Charles River Laboratories, 2020). The 10 male and 10 female control group animals received the vehicle, corn oil, only. There were 5 additional males and females in the control and high dose groups, which were used to assess reversibility of effects during the 28-day recovery period. The following parameters and endpoints were evaluated in the study: clinical signs, body weights, body weight gains, food consumption, functional observational battery, motor activity, ophthalmology, clinical pathology parameters (haematology, coagulation, clinical chemistry, and urinalysis), thyroid hormone analysis, gross necropsy findings, organ weights, and histopathologic examinations.

All animals survived until the scheduled necropsy. There were no test substance-related clinical observations or effects on food consumption, functional observational battery, motor activity, coagulation, or urinalysis. There were no test substance-related ophthalmic or macroscopic findings.

Test substance-related lower body weights were observed in both males and females at 150 mg/kg bw/day. By the end of the dosing period, body weights for males and females were 7.3% and 3.4% lower, respectively, compared to the control group. These effects were not observed following the 28-day recovery period. No test substance-related effects were observed on body weight at 15 and 50 mg/kg bw/day.

Test substance-related changes in haematology parameters were noted, which included lower mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), and reticulocyte count (RETIC) in the 150 mg/kg bw/day males, lower haematocrit (HCT), MCH, MCHC, and mean corpuscular volume (MCV) concentrations in the 50 mg/kg bw/day and 150 mg/kg bw/day females, and lower haemoglobin (HGB) concentration in the 150 mg/kg bw/day group males and in all females treated with the test substance. All affected haematology parameters were comparable to the control group at the recovery necropsy.

Test substance-related changes in clinical chemistry parameters were noted, which included lower aminotransferase (AST) in the 150 mg/kg bw/day group males and females, lower total bilirubin (TBIL) in all test substance-treated females, lower alkaline phosphatase (ALP) and lower triglycerides (TRIG) without dose dependency in all test substance treated males and females. Additionally, higher cholesterol (CHOL), total protein (TPROT), albumin (ALB), and globulin (GLOB) concentrations were noted in the 50 and 150 mg/kg bw/day group females. All affected clinical chemistry parameters were comparable to the control group at the recovery necropsy except for the non-statistically significant decrease of AST, alanine aminotransferase (ALT), and TBIL in the 150 mg/kg bw/day group females.

Thyroid hormone parameters evaluated included T3, T4, and thyroid stimulating hormone (TSH) concentrations. There were no changes to any of these parameters in males at any dose level, females at 15 mg/kg bw/day, or TSH levels for females at 50 mg/kg bw/day at the primary necropsy. Test substance-related statistically significantly lower T3 and T4 concentrations were noted in females dosed with 50 and 150 mg/kg bw/ day. A statistically significantly higher TSH value was observed for females in the 150 mg/kg bw/day group at primary necropsy, which was fully reversible by the time of the recovery necropsy. Higher T3 and T4 values, with T4 reaching statistical significance, were noted for females in the 150 mg/kg bw/day group at the recovery necropsy.

Lower mean ovary weights were noted in the 50 and 150 mg/kg bw/day group females. Higher mean thyroid weights were noted in the 50 and 150 mg/kg bw/day group females and 150 mg/kg bw/day group males. Higher mean and relative to body weight and brain weight liver weights were noted in the 50 and 150 mg/kg bw/day male and female groups. No other test substance-related organ weight changes were noted. In the recovery group, higher mean liver weights were noted in the 150 mg/kg bw/day groups. Relative liver weight to body weight and brain weight ratio were statistically significantly higher in recovery males at 150 mg/kg bw/day. These changes in liver weight did not correlate to any microscopic effects

At histopathology evaluation, minimal or mild hepatocellular centrilobular hypertrophy and thyroid follicular cell hypertrophy were observed in the 50 mg/kg bw/day females and 150 mg/kg bw/day males and females. These microscopic changes correlated with increased organ weights and were considered most likely to be adaptive and non-adverse responses to test substance-induced enzyme induction. Minimal decreased lymphoid cellularity of the thymus, characterized by slightly thinner thymic cortices was noted in the 150 mg/kg bw/day dose group males and females. Hepatocellular and thyroid follicular cell hypertrophy and thymus decreased lymphoid cellularity completely reversed after the recovery period. No other microscopic findings in the lymphoid system were observed and therefore, this change was considered non-adverse. Decreased number of corpora lutea, characterised by loss of total number of corpora lutea of all stages, was observed in the 50 and 150 mg/kg bw/day dose group females. The change, which was severe in some animals and was consistent with a perturbation of ovarian cycling also correlated with lower mean ovarian weights. Based on the likely effect on reproduction in female animals, the ovarian findings were considered adverse.

The systemic NOAEL for male rats was concluded to be greater than 150 mg/kg bw/day based on no adverse effects observed. The systemic NOAEL for female rats was concluded to be equal to 15 mg/kg bw/day based on lower mean number of corpora lutea in females at 50 and 150 mg/kg bw/day, which correlated to lower mean ovary weights and a perturbation of ovarian cycling in these groups.

Justification for classification or non-classification

Based on the available data, 2,4,6,8-tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane is not classified for systemic organ toxicity following repeated exposure according to Regulation (EC) No 1272/2008.