2,4,6,8-tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3 Nov 2020 (study initiation) to 4 Nov 2021 (final report)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

29 Jul 2016

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2,4,6,8-tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane

EC Number:

219-863-1

EC Name:

2,4,6,8-tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane

Cas Number:

2554-06-5

Molecular formula:

C12H24O4Si4

IUPAC Name:

2,4,6,8-tetramethyl-2,4,6,8-tetravinyl-1,3,5,7,2,4,6,8-tetroxatetrasilocane

Test material form:

liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)

Details on species / strain selection:

This species and strain of rat is recognised as appropriate for reproduction studies and is susceptible to the effects of reproductive toxicants. Further, the laboratory has reproductive historical control data in this species from the same strain and source.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 11 weeks
- Weight at study initiation: Males: 279 and 353 g; Females: 199 and 260 g
- Fasting period before study: No
- Housing: Solid-bottom cages containing appropriate bedding material . From arrival until cohabitation: Group housed (up to 3 animals of the same sex). During cohabitation: Paired for mating in male home cage. Post breeding: individually housed. Following F1 weaning: Group housed (2 to 3 animals of the same sex) until euthanasia.
- Diet: Certified Rodent LabDiet 5002, PMI Nutrition International, ad libitum
- Water: Municipal tap water treated by reverse osmosis and ultraviolet irradiation, ad libitum
- Acclimation period: At least 7 days

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

IN-LIFE DATES: From: 10 Nov 2020 and 24 Nov 2020 To: 23 Feb 2021

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Remarks:

NF

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item dosing formulations were prepared approximately weekly and an adequate amount of each formulation was dispensed into daily aliquots, which were stored at room temperature (18°C to 24°C), protected from light, until use. The dosing formulations were stirred continuously during dosing.

The vehicle, corn oil, was dispensed approximately weekly for administration to Group 1 control animals and preparation of the test substance formulations. For administration to Group 1 control animals, an adequate amount of the vehicle was dispensed into daily aliquots, which were stored at room temperature (18°C to 24°C), protected from light, until use.

Following filling of the dosing syringe and feeding tube with the required amount of test substance formulation, the outside of the feeding tube was wiped clean of residual test substance formulation. The feeding tube was then lightly tapped onto a clean towel to remove any remaining droplet(s) from the tip of the tubing.

VEHICLE
- Justification for use and choice of vehicle: Also used in the OECD Test Guideline 408 and 414 studies used for dose selection
- Concentration in vehicle: 0. 6.25. 37.5 and 100 mg test item per mL vehicle
- Amount of formulation: 4 mL/kg bw (test item and vehicle dosing volume)

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: Up to 14 days
- Proof of pregnancy: Vaginal copulatory plug or sperm in a vaginal lavage, with latter performed daily during cohabitation
- After 14 days of unsuccessful pairing, pair separated with no further opportunity for mating

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Duplicate 1.0 mL formulation samples were collected and analyzed by a gas chromatography method with flame ionization detection using a validated analytical procedure as follows:
- 20 Nov 2020: all groups analyzed for concentration, Groups 2 and 4 for homogeneity
- 8 Feb 2021: Groups 1-3 for concentration; All Group 4 females were dead or euthanized by Lactation Day (LD) 9

Test substance formulations have been previously shown to be stable and homogeneous over the range of concentrations used in the current study and for at least 8 days at room temperature or refrigerated storage. Therefore, formulation stability and resuspension homogeneity were not re-assessed.

Duration of treatment / exposure:

- F0 males: At least 14 days pre-mating up to 14 days cohabitation, for a minimum of 28 days total
- F0 females surviving through scheduled necropsy: At least 14 days prior to mating, mating, gestation, and lactation through LD 21, for a total of at least 58 days.
- Females with no evidence of mating: Pre-mating through the day prior to euthanasia
- All F1 offspring: Potentially in utero and while nursing
- Selected F1 generation: From weaning (Postnatal Day (PND) 21) until the day prior to euthanasia (PND 35), with 14 days of direct exposure

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

corn oil vehicle

Doses / concentrations 2

Dose / conc.:

25 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

150 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

400 mg/kg bw/day (nominal)

No. of animals per sex per dose:

F0: 10M/10F
F1 litters: As born per dam, per report results tables
F1 generation: 10M/10F, as available

Control animals:

yes

Details on study design:

- Dose route rationale: Oral (gavage) is an accepted route of exposure in OECD Test Guideline 421, and has been used extensively for studies of this nature.
- Dose selection rationale: Existing OECD Test Guideline 408 and 414 data
- Rationale for animal assignment: Random
- Fasting period before blood sampling for clinical biochemistry: No

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, once in the morning and once in the afternoon.

DETAILED CLINICAL OBSERVATIONS: Yes
For all animals beginning the first day of dose administration (at least 14 days pre-mating), animals were removed from their cage and a detailed clinical observation was performed prior to dosing, once daily during the first week and twice weekly thereafter, beginning for males on the first day of test administration and for females on the first day of vaginal lavage. Following evidence of mating, detailed clinical observations were recorded on GDs 0, 4, 7, 10, 14, and 20 and LDs 0 (or 1), 4, 7, 10, 14, and 21. If a female was determined to have completed parturition by the morning parturition examinations, detailed clinical observations were recorded for this female on LD 0; otherwise, these observations were recorded on LD 1. In addition, clinical observations were recorded approximately 1 h post-dosing.

BODY WEIGHT: Yes
For all animals beginning the first day of dose administration (at least 14 days pre-mating), animals were weighed individually on Study Days 0, 3, and 7 during the first week and at least weekly, thereafter, until evidence of mating was observed or until euthanasia (for females without evidence of mating). Once evidence of mating was observed, female body weights were recorded on GDs 0, 4, 7, 10, 14, and 20 and on LD 0 (when possible), 1, 4, 7, 10, 14, and 21. Body weight gains were then determined for the intervals: GDs 0–7, 7–14, 14–20, and 0–20, and LD 1–4, 4–7, 7–14, 14–21, and 1–21. All animals had a final body weight recorded on the day of necropsy.

FOOD CONSUMPTION: Yes
For all animals beginning the first day of dose administration (at least 14 days pre-mating), food consumption was measured on Study Days 0, 3, 7, and 13 during the first week and at least weekly, thereafter, until mating. Once evidence of mating was observed, female food consumption was recorded on GDs 0, 7, 14, and 20 and LD 1, 4, 7, 10, 14, and 21.

WATER CONSUMPTION: No

CLINICAL CHEMISTRY: No

ENDOCRINE: Yes
Blood samples for thyroid hormone (total T4) analyses were collected from a jugular vein into sample tubes without anticoagulants for all dose groups on Study Day 28. Blood samples were maintained at room temperature and allowed to clot. Serum was isolated in a refrigerated centrifuge and stored in a freezer set to maintain a target of -70°C. The male samples were analysed using validated ultra-high performance liquid chromatography with dual mass spectroscopy assays. The female samples for Groups 1-3 (all Group 4 females died or were euthanized by LD 9) were retained, but not analysed.

Oestrous cyclicity (parental animals):

For all females, vaginal lavages were performed daily for 14 days prior to randomization and continuing until evidence of mating was observed or until the end of the mating period. The slides were microscopically examined to determine the stage of the oestrous cycle, with the average cycle length calculated for complete oestrous cycles. Vaginal lavages to determine the oestrous cycle stage also were performed at necropsy.

Sperm parameters (parental animals):

Observed effects on sperm, if any, were noted during the histopathologic examination.

Litter observations:

STANDARDISATION OF LITTER: Yes, for the F1 generation
To reduce variability among the F1 generation (pups treated during PNDs 21-35), 10 pups/litter of equal sex distribution, if possible, were randomly selected for this group on PND 4.

PARAMETERS EXAMINED: F1 litters (PNDs 0-21)
- Number of still and live births
- Sex of pups, determined on PNDs 0, 4, 14, and 21
- Postnatal mortality and a daily record of litter size
- Cageside observations, performed twice daily (morning and afternoon)
- Detailed clinical signs, assessed once daily on PNDs 1, 4, 7, 10, and 14, see F1 generation below for PND 21
- Pup body weight, measured on PNDs 1, 4, 7, 10, 14, and 21
- Anogenital distance (AGD), measured on PND 1
- Presence / absence of nipples/areolae in male pups on PND 13.
- T4 hormone analysis, PND 4 blood samples were collected for all litters (at least 2 culled pups/litter) via cardiac puncture from animals anesthetized with isoflurane into tubes without anticoagulants. PND 21 samples were collected for Groups 1-3 (1 pup/sex/litter from animals) via the vena cava following euthanasia by carbon dioxide inhalation and into tubes without anticoagulants. See the parental animal “observations and examinations” for the processing and analysis details. The PND 4 blood samples were retained, but not analysed.

PARAMETERS EXAMINED: F1 generation (PNDs 21-35)
- A daily record of litter size
- Cageside observations, twice daily (morning and afternoon)
- Detailed clinical signs, assessed once daily on PND 35, and twice daily on PNDs 21–34 (pre-dosing and roughly 1 h post-dosing)
- Pup body weight, measured daily during PNDs 21-35
- Food consumption, quantitatively measured daily during PNDs 21-35
- T4 hormone analysis, PND 35 blood samples were collected for Groups 1-3 (10 pups/sex/group, as available) via the vena cava following euthanasia by carbon dioxide inhalation and into tubes without anticoagulants. See parental animal observations for the processing and analysis details. These PND 35 blood samples were retained, but not analysed.

Postmortem examinations (parental animals):

SACRIFICE:
All surviving animals, including females that failed to deliver or with total litter loss, were euthanized by carbon dioxide inhalation. Females that were found dead or sacrificed (moribund or due to Group 4 termination by LD 9) prior to scheduled termination were euthanized per Test Facility SOPs. For all parental animals, the termination procedures are summarized in Table 1 below.

ORGAN WEIGHT: Yes
Organ weights were not obtained for the females found dead or euthanized.

For the surviving animals, organ weights were measured at necropsy, with the organs identified Table 2 below.

GROSS PATHOLOGY: Yes
For the female deaths (found dead or euthanized), a necropsy was performed. For females found dead or euthanized during gestation, the number and location of foetuses, corpora lutea, and implantation sites were recorded. If during lactation, the number of corpora lutea and former implantation sites were recorded.

Surviving animals were subjected to a complete necropsy examination, including external surface, all orifices, cranial cavity, external surfaces of the brain and spinal cord, and the thoracic, abdominal, and pelvic cavities, including viscera. For females that delivered or had macroscopic evidence of implantation, the numbers of implantation sites and former implantation sites were recorded, with the number of unaccounted-for sites calculated. Uteri of females without macroscopic evidence of implantation were opened and placed in 10% ammonium sulfide solution for detection of early implantation loss.

Collective uterine examinations:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Number of unborn foetuses: Yes
- Number of pups born dead / live: Yes

The tissues listed in Table 3 below were collected and preserved.

HISTOPATHOLOGY: Yes
For all animals (surviving or the females found dead / euthanized) in all dose groups, histology and histopathology were performed for “selected” tissues (testes, epididymis, and ovaries plus all identified gross lesions).
- For the histology, the selected tissues in all animals found dead or euthanized and in the surviving animals for groups 1 and 4 were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin (PAS staining for the testes and epididymis).
- A histopathologic examination of the selected tissues was performed for same groups of animals.

Postmortem examinations (offspring):

SACRIFICE:
Pups surviving to scheduled termination (PND 35 for the selected F1 generation, and PND 21 for the remaining pups) were sacrificed via carbon dioxide inhalation. Pups found dead or euthanized (moribund or due to Group 4 termination by PND 9) were euthanized per Test Facility SOPs.

For all pups, the termination procedures are summarized in Table 4 below.

ORGAN WEIGHT: Yes
Organ weights for pups surviving to scheduled termination (PND 35 for the selected F1 generation, and PND 21 for the remaining pups) were determined for the organs listed in Table 5 below. Organ weights were not obtained for pups found dead or euthanized.

GROSS PATHOLOGY: Yes
On PND 21 and 35, surviving pups were subjected to a complete necropsy examination, with emphasis on developmental morphology and organs of the reproductive system. Necropsy was also undertaken for pups found or were euthanized, with specified tissues retained. For pup deaths between PND 0-4, a fresh dissection technique was used, which included examination of the heart and major vessels. For pup deaths after PND 4, a gross examination was performed.

The tissues listed in Table 6 below were collected and preserved.

HISTOPATHOLOGY: No

Statistics:

All statistical tests were performed by computer and were two-tailed for significance levels of 5% and 1%. Following completion of the mating period, data from non-gravid females were excluded from calculation of means and from comparative statistics. All means are presented with standard deviations. The litter, rather than the pup, was considered the experimental unit.

Continuous data variables (body weights, body weight gains, and food consumption at each interval), oestrous cycle length, pre-coital intervals, gestation length, former implantation sites, unaccounted-for sites, thyroid hormone values, anogenital distance (absolute and relative to cube root of body weight), and number of nipples/areolae (only when nipples are present) were subjected to a parametric one-way analysis of variance (ANOVA). If the results of the ANOVA were significant (p<0.05), Dunnett’s test was applied to the data to compare the treated groups to the control group.

Male copulation, female conception, mating and fertility indices (male and female) of the treated groups were compared to the control group using the Chi-square test with Yates’ correction factor.

F1 ANIMALS:
The mean litter proportions (% per litter) of pup viability during the postnatal period and sex ratio at birth were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup difference. If the results of the ANOVA were significant (p<0.05), Dunn’s test was applied to the data to compare the treated groups to the control group. Mean litter weights, live litter size, and number of pups born per litter were subjected to the parametric ANOVA test and Dunnett’s test as described for parental animals.

Reproductive indices:

Male (Female) Mating Index (%) = (No. of Males with Evidence of Mating (or Females with Confirmed Pregnancy)) / (Total No. Males (Females) Used for Mating) x 100

Male Fertility Index (%) = (No. of Males Siring a Litter) / (Total No. Males Used for Mating) x 100

Female Fertility Index (%) = (No. of Females with a Confirmed Pregnancy) / (Total No. Females Used for Mating) x 100

Male Copulation Index (%) = (No. of Males Siring a Litter) / (No. of Males with Evidence of Mating (or Females with Confirmed Pregnancy)) x 100

Female Copulation Index (%) = (No. of Females with a Confirmed Pregnancy) / (No. of Females with Evidence of Mating (or Confirmed Pregnancy)) x 100

Offspring viability indices:

Mean Live Litter Size (No. per litter) = Total No. Viable Pups on PND 0 / No. Litters with Viable Pups PND 0

Postnatal Survival Between Birth and PND 0 (or PND 4; % per litter) = Sum of (Viable Pups Per Litter on PND 0 (or PND 4) / No. of Pups Born Per Litter) / No. of Litters Per Group x 100

Postnatal Survival for All Other Intervals (% per litter) = Sum of (Viable Pups Per Litter at End of Interval N / Viable Pups per Litter at Start of Interval N) / No. of Litters Per Group x 100
- Where N = PNDs 0-1, 1-4 (pre-selection), 4 (post-selection)-7, 7-10, 10-14, 14-21, and 4 (post-selection)-21

Total litter loss was determined when the last pup in the litter was found dead or euthanized in extremis prior to the scheduled euthanasia.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test item-related and adverse clinical signs were observed in the females that died (or were moribund euthanized) at 150 and 400 mg/kg bw/day. No test item clinical signs were seen (before dosing or 1 h post-dosing) for males up to 400 mg/kg bw/day, in surviving females at 25 or 150 mg/kg bw/day, or in the three females surviving at 400 mg/kg bw/day (until their euthanasia in poor condition on LDs 7 or 9).

At 400 mg/kg bw/day, the non-gravid female that died on GD 14 exhibited a thin body and pale faeces (GDs 7, 10, and/or 14) and hunched posture (veterinary examination). In the 10 females that were found dead or were euthanized at 400 mg/kg bw/day (GD 23 through LD 1, or LDs 7 or 9), the common clinical signs noted just before being found dead or euthanised included piloerection, hunched posture, and/or pale and cool body and/or extremities.

At 150 mg/kg bw/day, similar symptoms were observed in the single female found dead on GD 23.

Mortality:

mortality observed, treatment-related

Description (incidence):

Test-item related mortality (found dead or euthanised in extremis) in one female at 150 and six females at 400 mg/kg bw/day. The three surviving females at 400 mg/kg bw/day were euthanized in poor condition on LDs 7 or 9 (and effecting group termination). No deaths occurred in males up to 400 mg/kg bw/day or females at 25 or 150 mg/kg bw/day.

At 400 mg/kg bw/day, six females were found dead (two), euthanized in extremis (three), or suffered a total litter loss (one), with these deaths occurring on GDs 23 to LD 1. These deaths were attributed to prolonged gestation and, as assessed by the study veterinarian, dystocia. The seventh female that died in this group was euthanized in extremis on GD 14 following marked body weight loss and no food consumption from GDs 0–14, with this death attributed to hepatocellular degeneration/necrosis in the liver.

At 150 mg/kg bw/day, a single female was found dead on GD 23, with this death also considered related to prolonged gestation and the assessed dystocia.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Effects body weight or body weight gain were considered test item-related and adverse for males at 400 mg/kg bw/day, and for females during lactation at this dose. No test item-related and adverse body weight or body weight gain findings were identified for females during pre-mating or gestation, or for males or females at 25 and 150 mg/kg bw/day.

For males at 400 mg/kg bw/day, lower mean body weight gains were noted throughout the entire generation (Study Days (SDs) 0–28) and during pre-mating (SDs 0-13), both statistically significant. Consequently, mean absolute body weights in this group were lower (5.1% to 11.3%) than the control group during SDs 7–28 (statistically significant on SDs 13, 20, and 28).

For the three females that survived at 400 mg/kg bw/day (until LD 7 or 9 when euthanised in poor condition, group terminated), lower mean maternal body weight gains were noted during LDs 1–7, resulting in lower (7.0 to 8.0%) mean absolute body weights when compared to the control group (not statistically significant).

The single non-gravid female at 400 mg/kg bw/day that was euthanized in extremis on GD 14 had marked body weight loss (48g, 21.8%).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Test item-related and adverse effects on food consumption were identified for females during lactation at 400 mg/kg bw/day. Test item-related but non-adverse effects on this parameter were observed for males at this dose. At 25 and 150 mg/kg bw/day (SDs 0-28), no effects were considered related to test item administration for males or lactating females. During pre-mating or gestation, no test item-related effects on food consumption were noted at any dose.

For males at 400 mg/kg bw/day, the slightly lower (but not statistically significant) mean food consumption during premating (SDs 0–13) was considered test item-related but non-adverse due to the small magnitude of changes.

For the three surviving females at 400 mg/kg bw/day (until LD 7 or 9 when euthanised in poor condition, group terminated), lower mean food consumption was noted during LDs 1–7 (21 g/animal/day for LDs 1-4, 29 g/animal/day for LDs 4-7, both statistically significant).

The single non-gravid female at 400 mg/kg bw/day that was euthanized in extremis on GD 14 had no food consumption (0g GDs 0-14).

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

Endocrine findings:

effects observed, treatment-related

Description (incidence and severity):

Test item-related but non-adverse lower mean T4 concentrations were determined for F0 males at 150 and 400 mg/kg bw/day, with no test item-related effects on T4 levels were at 25 mg/kg bw/day. The blood samples collected from females were retained but not analyzed.

Lower mean T4 concentrations were noted in 150 and 400 mg/kg bw/day group F0 males in a dose-related manner (statistically significant at 400 mg/kg bw/day compared to the control group). However, in the absence of a histopathologic examination for this tissue (per study protocol), this finding is considered non-adverse based on the lack of effects on mean thyroid weights (absolute and/or relative), and due to the values being within background historical control for the laboratory.

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:

effects observed, treatment-related

Description (incidence and severity):

Test item-related and adverse histopathology was identified in the 400 mg/kg bw/day female euthanized in extremis on GD 14. Test item-related but non-adverse microscopic findings at this dose were seen for two females euthanized in extremis on GD 23 and total litter loss on LD 1. No test item-related histopathology (epididymis, testes, or ovaries) was observed for the animals examined at terminal sacrifice or for the ovaries in females that died (found dead or euthanised GD 23 though LD 1, or euthanised LD 7 or 9), with the findings considered incidental based on the strain/age of rat and/or comparison to the control group.

The specific test item-related histopathology at 400 mg/kg bw/day included:
* Hepatocellular degeneration/necrosis in the liver, the histopathologic cause of death for the 400 mg/kg bw/day female euthanised in extremis on GD 14, considered adverse, also exhibited marked decreases in body weight and food consumption
* Decreased lymphoid cellularity in the thymus, observed in two females at this dose (both euthanised, one in extremis on GD 23 and one due to total litter loss on LD 1), considered an adaptive change due to enzyme induction, correlated with a small thymus at gross examination, assessed as non-adverse.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No test item-related effect on oestrus cycle length or stage was observed up to 400 mg/kg bw/day.

Based on vaginal lavage 14 days prior to randomization until evidence of mating was observed (or until the end of the mating period), there was no effect related to test item administration on mean oestrus cycle length.

Based on vaginal lavage prior to necropsy, the females were determined to be in dioestrus, proestrus or oestrus. With one exception, more females were in dioestrus than in oestrus than in proestrus prior to necropsy, for both the treated groups and the control group. However, no gravid females were in oestrus at 400 mg/kg bw/day prior to necropsy, noting that all females in this group were found dead or euthanised by LD 9.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No test item-related sperm histopathology was noted at 25 mg/kg bw/day, but not at 400 mg/kg bw/day. No epididymis at 150 mg/kg bw/day were examined.

Mild sperm granuloma in the epididymis was indentified in one of two males examined at 25 mg/kg bw/day, but ithe lack of a dose-response. Not discussed in the study report text.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Increased gestation length and the veterinary diagnosis of dystocia were considered test item-related and adverse at 400 mg/kg bw/day, but not at 25 or 150 mg/kg bw/day. In addition, there was a failure of the gravid females at 400 mg/kg bw/day to birth fully viable litters. However, there were no test item-related effects on mean precoital intervals, male and female reproductive performance (mating, fertility, and copulation indices) or, as discussed above, mean oestrous cycle length / stage or sperm histopathology.

Gestation length was increased and dystocia was diagnosed for the 400 mg/kg bw/day dose group. Of the nine gravid females, only two had gestation lengths of 22 days, with the other lengths equal to 23 to 24 days, yielding a group mean of 22.8 days. This mean value is greater than the means for the control group (22.0 days) and the background historical control (22.1 days). The veterinary diagnosis of dystocia was based on various adverse clinical signs seen by clinical and veterinary staff during parturition. The single 150 mg/kg bw/day female that was euthanised on GD 23 also exhibited adverse clinical signs during parturition, with this dose group as a whole not assigned a diagnosis of dystocia.

Of the six females gravid females that littered at 400 mg/kg bw/day, only two birthed all live pups. The other four dams birthed either all dead pups (one litter) or a mix of alive and dead pups (three litters). In addition, three females died (found dead or euthanised on GDs 23-24) before littering, with two containing dead foetuses (one with a single resorption) and the third resorptions only. Collectively, these findings reflect a failure of the gravid dams to birth fully viable litters.

Please see Table 9 (reproductive performance) and Table 10 (adverse parturition findings) below.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

25 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Remarks on result:

other: test item-related and adverse findings from 150 mg/kg bw/day; the organ weight changes were assessed as test item-related but likely non-adverse in the study report, however the tissues in question were not examined microscopically per protocol

2

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no test item-related and adverse findings observed

Remarks on result:

other: highest dose tested

3

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive performance

other: increased gestation length, veterinarian diagnosis of dystocia, failure to produce wholly live litters

Remarks on result:

other: test item-related and adverse findings at 400 mg/kg bw/day

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

400 mg/kg bw/day (nominal)

System:

female reproductive system

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

F1 LITTERS (PNDs 0-21):
Test item-related and adverse clinical signs were observed in pups at 400 mg/kg bw/day of parental treatment up until group termination by PND 9, but no observations related to parent test item administration were identified at 25 or 150 mg/kg bw/day through PND 21.

The test-item related and adverse clinical observations for F1 pups through PND 9 at 400 mg/kg bw/day included small stature and/or cardio-pulmonary findings of pale body, cool body, cool extremities, and/or gasping. The reviewer considers the exact cause of the F1 clinical signs unknown, but could be related to the dam deaths (three GD 23 through LD 1 and three on PNDs 7 or 9), other dam findings (e.g., clinical signs, prolonged gestation, dystocia diagnosis), and/or a direct effect of the test item in utero or during lactation. See also body weight and gross pathology.

F1 GENERATION (PNDs 21-35):
There were no test item-related clinical signs identified in the F1 generation during detailed clinical examination or 1 h post-dosing up to 150 mg/kg bw/day. Due to 400 mg/kg bw/day group termination by PND 9, pups at this dose were not available for post-weaning treatment and examination.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

F1 LITTERS (PNDs 0-21):
Test item-related and adverse lower mean live litter size on PND 0 and lower postnatal survival (on PND 0 and during PNDs 0–1) were noted at 400 mg/kg bw/day of parental treatment compared to the control group. The differences at this dose were partially attributed to one dam with a total litter loss and two litters that were euthanized in extremis along with their dams on PND 0 or 1. Overall, the reviewer considers that these pup deaths (those found death or euthanised in extremis) could be related to the dam findings (deaths, clinical signs, increased gestation length, dystocia veterinary diagnosis), a direct effect of the test item in utero or during lactation, and/or the lack of milk in the stomach of pups that died. Forty (40) pups in five litters were found dead or euthanised in extremis at 400 mg/kg bw/day. For the same parental dose group, 10 pups in five litters were missing and presumed cannibalised. While postnatal survival of 400 mg/kg bw/day pups from PNDs 1-4 and PNDs 4-7 was comparable to control, 21 pups in three litters were euthanised at 400 mg/kg bw/day due to termination of their dams on PNDs 7 or 9 (effective group termination by PND 9). No pup deaths occurred with parental treatment at 25 or 150 mg/kg bw/day.

F1 GENERATION (PNDs 21-35):
In the F1 generation, all pups survived until terminal sacrifice on PND 35 up to 150 mg/kg bw/day. Due to 400 mg/kg bw/day group termination by PND 9, pups at this dose were not available for post-weaning treatment and examination.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

F1 LITTERS (PNDs 0-21):
Test item-related and adverse lower mean birth weights on PND 1 and lower mean body weights (and gains) for PNDs 1-7 were noted at 400 mg/kg bw/day of parental treatment (statistically significant for PNDs 4-7), with the litter as the experimental unit. The findings at 150 mg/kg bw/day were related to parent test item administration, but assessed as non-adverse. Body weights (gains) were considered unaffected by parental treatment at 25 mg/kg bw/day.

At 400 mg/kg bw/day of parental treatment, mean male and female pup birth weights (PND 1) were 25.0 and 28.8% lower, respectively (statistically significant), than the control group. Mean pup body weight gains at this dose were lower than the control group during PND 1–7 (statistically significant PNDs 4-7), with lower male and female pup body weights on PND 7 (25.8% and 28.5%, respectively, statistically significant) than the control group.

At 150 mg/kg bw/day of parental treatment, mean pup body weights (and gains) were slightly lower than the control group for PNDs 1-21 (not statistically significant). However, the differences in mean pup body weights remained within 6% of controls and hence considered test substance-related but non-adverse.

F1 GENERATION (PNDs 21-35):
Test item-related but non-adverse lower mean body weights (and gains) were identified in F1 generation males at 150 mg/kg bw/day. No body weight (gain) findings were considered related to test item administration in male pups at 25 mg/kg bw/day, or in female pups up to 150 mg/kg bw/day. Due to 400 mg/kg bw/day group termination by PND 9, pups at this dose were not available for post-weaning treatment and examination.

In the 150 mg/kg bw/day males, test item-related lower mean body weight gains were noted for entire pup treatment period (PNDs 21–35, statistically significant). Consequently, the mean male body weight was 9.6% lower than the control group on PND 35 (statistically significant on PND 25 and from PND 29). However, since the mean male body weight on PND 21 (prior to direct treatment) was 5.4% lower than the control group, the body weight (gain) effects following treatment on PNDs 21-34 was correspondingly assessed as mild and non-adverse.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

F1 LITTERS (PNDs 0-21):
Not applicable.

F1 GENERATION (PNDs 21-35):
Test item-related but non-adverse lower food consumption identified was in F1 generation males at 150 mg/kg bw/day. No food consumption findings were considered related to test item administration in male pups at 25 mg/kg bw/day, or in female pups up to 150 mg/kg bw/day. Due to 400 mg/kg bw/day group termination by PND 9, pups at this dose were not available for post-weaning treatment and examination.

In 150 mg/kg bw/day males, sporadically lower mean food consumption, evaluated as g/animal/day, was noted in the during the direct treatment period (per the report text, statistically significant on PNDs 27–28 and 30–34; not flagged as significant on Table 4.47). While these decrements corresponded with the non-adverse lower mean body weights (gains) for F1 generation males, the food consumption findings also were considered non-adverse due to the small magnitude of change.

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

Description (incidence and severity):

See Other effects: Endocrine findings below.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

F1 LITTERS (PNDs 0-21):
There were effects on anogenital distance (AGD) on PND 1 at 400 mg/kg bw/day of parental treatment; however, based on a comparison the absolute and relative AGD findings, these were considered to be secondary to the small stature of these pups (i.e., not a direct effect of the test item on AGD). There were no test item-related AGD findings at 25 or 150 mg/kg bw/day.

While mean absolute AGDs on PND 1 at 400 mg/kg bw/day of parental treatment were lower (statistically significant) than the control group, the mean AGDs relative to cube root of body weight values for this group were comparable to control, indicating that the lower absolute AGD findings were secondary to the small stature of these pups and not a direct effect of the test item.

F1 GENERATION (PNDs 21-35):
Not applicable.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

F1 LITTERS (PNDs 0-21):
There were no retained nipples in male pups on PND 13 up to 150 mg/kg bw/day of parental treatment. Due to 400 mg/kg bw/day group termination by PND 9, no pups were available for examination on PND 13.

F1 GENERATION (PNDs 21-35):
Not applicable.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

F1 LITTERS (PNDs 0-21):
Test item-related but likely non-adverse lower mean spleen, thymus, and thyroid/parathyroid weights were observed on PND 21 at 150 mg/kg bw/day of parental treatment, with no organ weight changes related to parental test item administration at 25 mg/kg bw/day. Due to 400 mg/kg bw/day group termination by PND 9, pups at this dose were not available for post-weaning treatment and examination.

In F1 litters on PND 21, test item-related but likely non-adverse organ weight changes (absolute and relative to body and brain weights) were observed compared to the control group:
* Spleen, lower mean spleen weights (up to 15.3 and 26.9% for males and females, respectively), statistically significant for females
* Thymus, lower mean thymus weights (up to 19.4 and 19.6% for males and females, respectively), not statistically significant
* Thyroid/parathyroid, lower mean weights (up to 21.7% for females), not statistically significant

In the absence of pup microscopic examination (per study protocol), these organ weight findings were considered likely non-adverse due to the relatively low magnitude of effect and the lack of related gross pathology findings.

F1 GENERATION (PNDs 21-35):
Test item-related but likely non-adverse lower mean thyroid/parathyroid weights in males and females and higher mean female kidney and liver weights in females were observed on PND 35 at 150 mg/kg bw/day. The lower mean kidney and liver weights in males at this dose were considered secondary to the lower mean body weights at this dose and therefore assessed as non-adverse. The higher mean epididymis weight at 150 mg/kg bw/day was considered unilateral and not test item-related. No organ weight effects related to test item administration at 25 mg/kg bw/day. Due to 400 mg/kg bw/day group termination by PND 9, pups organ weights could not be evaluated on PND 35.

The test substance-related but likely non-adverse alterations in organ weights in the F1 generation at 150 mg/kg bw/day (all statistically significant except as clarified) included:
• Thyroid/parathyroid, lower mean weights (absolute and relative to brain weight) in males and females (up to 22.1% and 14.2%, respectively)
• Kidney (female), higher mean weights (absolute and relative to body and brain weights, up to 10.6%)
• Liver (female), higher mean weights (absolute and relative to body and brain weights, up to 20.1%).

In the absence of pup microscopic examination (per study protocol), these alternations were considered likely non-adverse given the relatively low magnitude of effect. There also were no test item-related necropsy findings in the F1 generation at 150 mg/kg bw/day.

Gross pathological findings:

no effects observed

Description (incidence and severity):

F1 LITTERS (PNDs 0-21):
No macroscopic findings related to parental test item treatment were noted at weaning (PND 21 for parental treatment at 25 and 150 mg/kg bw/day, and up to PND 9 at 400 mg/kg bw/day) or in pups found dead or euthanized in extremis. Aside from the presence or absence of milk in the stomach, no other internal findings were observed.

F1 GENERATION (PNDs 21-35):
No macroscopic findings related to treatment of the F1 generation were observed up to 150 mg/kg bw/day. Due to 400 mg/kg bw/day group termination by PND 9, pups at this dose were not available for post-weaning treatment and examination.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

**Endocrine findings** effects observed, non-treatment-related
F1 LITTERS (PNDs 0-21):
There were no test item-related effects on thyroid hormone values (total T4) in pups on PND 21 at 25 or 150 mg/kg bw/day of parental exposure. PND 4 blood samples were collected, but not analysed. No pups at 400 mg/kg bw/day survived to PND 21 (found dead or euthanised by PND 9, effective group termination).

F1 GENERATION (PNDs 21-35):
PND 35 blood samples for T4 analysis were retained but not analysed for the F1 generation.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

1

Key result

Dose descriptor:

NOAEL

Remarks:

F1 litters

Generation:

F1

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

clinical signs

mortality

body weight and weight gain

gross pathology

Remarks on result:

other: test item-related and adverse findings at 400 mg/kg bw/day

2

Key result

Dose descriptor:

NOAEL

Remarks:

F1 generation

Generation:

F1

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no test item-related and adverse findings observed

Remarks on result:

other: highest dose tested due to 400 mg/kg bw/day group termination by LD 9

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

400 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

not specified

Dose response relationship:

yes

Relevant for humans:

not specified

Any other information on results incl. tables

Table 7. F0 Summary of organ weight data at scheduled necropsy ^a

	Males				Females
Group	1	2	3	4	1	2	3
Dose (mg/kg bw/day)	0	25	150	400	0	25	150
No. Animals per Group	10	10	10	10	9	9	7
Liver (No. Weighed)	(10)	(10)	(10)	(10)	(9)	(9)	(7)
Absolute value	17.58	19.67	20.91**	21.43**	15.91	17.04	19.07**
% of body weight	3.75	4.13*	4.63**	5.17**	4.86	5.10	5.72**
% of brain weight	846	923	1021**	1055**	829	865	962**
Thymus (No. Weighed)	(10)	(10)	(10)	(10)	(9)	(9)	(7)
Absolute value	0.43	0.49	0.37	0.27**	0.16	0.16	0.19
% of body weight	0.092	0.104	0.083	0.064*	0.049	0.047	0.055
% of brain weight	20.8	23.0	18.2	13.3**	8.43	7.96	9.29
Prostate (No. Weighed)	(10)	(10)	(10)	(10)	N/A	N/A	N/A
Absolute value	0.97	1.09	0.92	0.75*	N/A	N/A	N/A
% of body weight	0.209	0.230	0.204	0.181	N/A	N/A	N/A
% of brain weight	47.0	51.2	44.8	36.9*	N/A	N/A	N/A

N/A = Not applicable.

^a Organ weight values rounded to the appropriate decimal place to account for the size of the organ

* = Statistically significantly different from the control group at 0.05 using Dunnett’s test.

** = Statistically significantly different from the control group at 0.01 using Dunnett’s test.

 

Table 8. F0 Summary of gross pathology findings at scheduled necropsy

	Males				Females
Group	1	2	3	4	1	2	3	4 ^a
Dose (mg/kg bw/day)	0	25	150	400	0	25	150	400
No. Animals per Group	10	10	10	10	10	10	10	4
Prostate (No. Examined)	(10)	(10)	(10)	(10)	N/A	N/A	N/A	N/A
Small	0	0	0	1	N/A	N/A	N/A	N/A
Thymus (No. Examined)	(10)	(10)	(10)	(10)	(10)	(10)	(10)	(4)
Small	0	0	0	0	0	0	0	1

N/A = Not applicable.

^a Includes failed to deliver, total litter loss, early termination necropsy, and scheduled necropsy females.

 

Table 9. F0 Results of reproductive performance

Parameter	Dose Level (mg/kg bw/day)				HC^a Mean (Range)
	0	25	150	400
Male Mating Index (%)	100.0	100.0	90.0	100.0	98.0 (83.3–100.0)
Female Mating Index (%)	100.0	100.0	90.0	90.0	98.0 (83.3–100.0)
Male Fertility Index (%)	90.0	90.0	80.0	90.0	93.9 (80.0–100.0)
Female Fertility Index (%)	90.0	90.0	80.0	90.0	93.9 (80.0–100.0)
Male Copulation Index (%)	90.0	90.0	88.9	90.0	95.7 (80.0–100.0)
Female Conception Index (%)	90.0	90.0	88.9	90.0	95.7 (80.0–100.0)
Estrous Cycle Length (days)	4.3	4.1	4.2	4.7	4.2 (3.9–5.2)
Precoital Interval (days)	2.0	4.0*	2.0	3.0	2.7 (1.4–4.5)

^a Laboratory historical control data (version 2019.05).

* = Statistically significant at 0.05 compared to the control group.

 

Table 10.F0 Females found dead or euthanizedin extremiswith adverse parturition findings

Female No.	Type of Death/Day	Clinical and Veterinary Observations
150 mg/kg bw/day
7813	FD/GD 23	GD 22: cool body, pale body and extremities, swollen urogenital area GD 23: pale*, cold to touch*, piloerection*, low carriage*, hypoactive*, reluctant to move in cage*, brown discharge coming from vulva*, very flat body lying in cage*
400 mg/kg bw/day
7809	EE/LD 1	LD 0: Pup No. 07 noted with pale body*, cool body*, gasping; Pup No. 05 noted with pale body, cool body*, gasping; Pup No. 06 noted with pale body*, cool body*, gasping LD 1: piloerection, pale and cool body, hunched*, decreased stool*, all pups were cold*, bruising present on abdomen*,slightly cool extremities*, piloerection*, increased respirations*
7812	EE/GD 23	GD 22: piloerection, pale body, pale extremities, partial closure of eyes*, slight delay in skin turgor*, pale*, decreased activity*, elevated respiratory effort*, decreased small stool* GD 23: piloerection, pale body and extremities, cool body, slight delay in skin turgor*, pale*, hunched*, slightly cool*, piloerection*, blood on ventrum and vulva when abdomen being palpated*
7827	EE/LD 0	LD 0: piloerection; hunched posture; pale body and extremities; slight delay in skin turgor*; piloerection*; elevated respiratory effort*; all pups were cold, pink with blue hue
7829	TLL/LD 1	LD0: piloerection; hunched posture; pale body; decreased activity*, and mildly weak with a mildly elevated respiratory rate and effort*

FD = Found dead; EE = Euthanized in extremis; TLL = Total litter loss; GD = Gestation Day; LD = Lactation Day.

* Observations recorded by Veterinary Staff.

 

Table 11. F0 F1 Postnatal Survival (% per litter)

Day	Dose Level (mg/kg bw/day)				CRL HC^a Mean (Range)
	0	25	150	400
PND 0 (relative to # born)	100.0	100.0	100.0	65.9	98.3 (88.1–100.0)
PND 0–1	100.0	100.0	97.1	43.0++	98.9 (89.1–100.0)
PND 1–4	100.0	100.0	96.8	96.7	98.3 (87.5–100.0)
PND 4–7	100.0	100.0	100.0	100.0	99.5 (96.7–100.0)
PND 7–14	100.0	100.0	100.0	0.0	-
PND 14–21	100.0	100.0	100.0	0.0	-
Birth to PND 4 (preselection)	100.0	100.0	94.0	31.1++	95.7 (84.5–100.0)
PND 4 (postselection) to 21	100.0	100.0	100.0	0.0	-

PND = Postnatal Day; - = not applicable.

^a Laboratory historical control data.

++ =Statistically significant at 0.01 compared to the control group.

Applicant's summary and conclusion

Conclusions:

In a study conducted according to OECD Test Guideline 421 and in compliance with GLP (reliability score 1), Sprague-Dawley rats were administered 2,4,6,8-tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane at 0, 25, 150, and 400 mg/kg bw/day via oral gavage (corn oil vehicle). F0 males were exposed at least 14 days pre-mating up to 14 days cohabitation (minimum 28 days total), while mated F0 females were exposed at least 14 days prior to mating and through mating and, as survived, through gestation and lactation until LD 21 (minimum 58 days total). Females with no evidence of mating were treated pre-mating through the day prior to their euthanasia. All F1 offspring were potentially exposed in utero and, as survived, while nursing until PND 21, with the selected F1 generation directly administered the test item from weaning (PNDs 21-34, euthanised PND 35). The identified NOAELs are as follows:
* F0 female NOAEL for systemic effects was identified as 25 mg/kg bw/day, based on findings considered to be test item-related and adverse (mortality and associated clinical signs at 150 and 400 mg/kg bw/day, and adverse effects on body weights and body weight gains, food consumption, and for the one non-gravid female euthanised on GD 14, liver histopathology at 400 mg/kg bw/day).
* The reviewer considers the F0 male NOAEL for systemic effects to be 150 mg/kg bw/day, based on test item-related and adverse effects at 400 mg/kg bw/day (decreased body weights and body weight gains). The male organ weight effects at 150 and 400 mg/kg bw/day were reported as “likely non-adverse”).
* The female reproductive NOAEL was considered to be 150 mg/kg bw/day, based on test item-related and adverse findings at 400 mg/kg bw/day (increased gestation length, a veterinary diagnosis of dystocia, and failure to birth fully viable litters)
* The male reproductive NOAEL was considered to be greater than or equal to 400 mg/kg bw/day, with no test item-related and adverse effects observed up to this dose.
* The NOAEL for the F1 litters during PNDs 0-21 was identified as 150 mg/kg bw/day based on test item-related adverse findings at 400 mg/kg bw/day (lower mean live litter size, lower mean postnatal survival, clinical signs, lower pup weights at birth and lower pup body weights and gains for PNDs 1-7. Most of these pup findings occurred temporally close to the maternal deaths (three of the six dams that littered at this dose were found dead or euthanised in extremis on GD 23 to LD1). The reviewer considers the exact cause of the F1 litter effects unknown, but these findings could be related to the toxicity in the dams (deaths, clinical signs, increased gestation length, dystocia veterinary diagnosis), a direct effect of the test item in utero or during lactation, and/or the lack of milk in the stomachs of pups that died.
* The NOAEL for the F1 generation treated on PNDs 21-34 was considered to be 150 mg/kg bw/day (highest dose tested), with no test item-related and adverse effects observed at this dose. Due to 400 mg/kg bw/day group termination by PND 9, pups at this dose were not available for post-weaning treatment and examination.