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Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

In the key reproduction/developmental toxicity screening test conducted with 2,4,6,8-tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane (Vi4-D4; CAS No. 2554-06-5, EC No. 219-863-1) according to OECD Test Guideline 421 (with an extended post-weaning exposure period) and in compliance with GLP (Charles River Laboratories, 2021; Klimisch score 1), the F0 systemic NOAEL was 25 mg/kg bw/day in males and females, based on organ weight changes in males from 150 mg/kg bw/day and on mortality and adverse clinical signs in females from 150 mg/kg bw/day (organ weight findings at 150 mg/kg bw/day). Additional effects were reported in both sexes at 400 mg/kg bw/day. The F0 reproductive NOAELs were 150 mg/kg bw/day in females, based on the adverse increased gestation length, a veterinary diagnosis of dystocia and related clinical signs in the dams, and a failure to produce fully viable litters, and greater than or equal to 400 mg/kg bw/day for males. As potentially related to the dam toxicity (systemic or reproductive), the F1 litter NOAEL was 150 mg/kg bw/day, based on lower mean live litter size on PND 0, lower postnatal survival (PNDs 0-1), adverse clinical signs on PND 1, lower birth weights on PND 1 at 400 mg/kg bw/day, and lower weight gain PNDs 1 -7.

Link to relevant study records
Reference
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
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 Jul 2016
GLP compliance:
yes
Limit test:
no
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
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
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
corn oil vehicle
Dose / conc.:
25 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
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
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.
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: 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.
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
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
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
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
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.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
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
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
Key result
Critical effects observed:
no
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

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)

HCa

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 HCa

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.

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.
Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Based on Klimisch score 1 study
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 study conducted according to OECD Test Guideline 421 and in compliance with GLP (reliability score 1), Sprague-Dawley rats were administered Vi4-D4 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 through 14 days of 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 up to 400 mg/kg bw/day of dam exposure and, as survived, while nursing until PND 21 (“F1 litters”), with the selected F1 generation also directly administered the test item at 25 and 150 mg/kg bw/day from weaning (PNDs 21-34, euthanised PND 35). 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.

 

The F0 parameters evaluated included: mortality, cage side observations, detailed clinical signs, body weight, food consumption, male blood T4 analysis on Study Day 28, female oestrous cycle stage and length prior to randomisation, and oestrus cycle stage at necropsy, reproductive indices (mating, copulation, and fertility indices), organ weights (if survived to terminal sacrifice) and, for surviving animals or those found dead or euthanised, gross pathology and testis, epididymis, ovary, and gross lesion histopathology. Blood samples were collected from surviving females on LD 21 up to 150 mg/kg bw/day for possible future T4 analysis (no 400 mg/kg bw/day females survived past PND 9). For the dams that survived to terminal sacrifice or those that died (GD 23 to LD 1, or on LDs 7 or 9), the reproductive-related gross pathology examined included gravid uterine weight, the number and location of foetuses, corpora lutea, implantation sites and/or former implantation sites, number unborn foetuses, number of pups born live / dead, as relevant to when dam death occurred (gestation or lactation) and whether the dam littered. For females without macroscopic evidence of implantation, early implantation loss was determined.

 

Except for the neonatal F1 litter parameters, the F1 parameters evaluated and corresponding results are discussed under effects on developmental toxicity. For the F1 litters, the neonatal parameters assessed were the number of still and live births, sex of pups (and sex ratio) on PND 0, postnatal morality and viability indices (PNDs 0-1), cageside observations (mortality and moribundity; PNDs 0-1), detailed clinical signs (PND 1), pup birth weights (PND 1).

 

Test item-related and adverse findings in the F0 animals were identified for these parameters:

* Female mortality and related clinical signs at 150 and 400 mg/kg bw/day

* Lower body weights and/or body gains in males and in females during lactation, both at 400 mg/kg bw/day

* Lower food consumption in females at 400 mg/kg bw/day

* Liver degenerative histopathology in the single non-gravid female at 400 mg/kg bw/day, euthanised in extremis on GD 14

* Female reproductive performance at 400 mg/kg bw/day, based on increased gestation length, veterinary diagnosed dystocia and associated clinical signs, and a failure to produce fully viable litters at 400 mg/kg bw/day. Noting there were no test item-related effects on mean precoital intervals, male and female reproductive indices (mating, copulation, fertility) or on the female oestrus cycle stage / length. Further, no sperm histopathology related to test item administration was observed.

 

Test item-related and adverse neonatal findings in the F1 litters at 400 mg/kg bw/day of parental treatment included:

* Lower mean live litter size on PND 0 and lower postnatal survival on PNDs 0-1

* Clinical signs on PND 1

* Lower mean birth weights on PND 1.

 

For all other F0 and neonatal F1 litter parameters, there were no effects observed, the findings were considered unrelated to the test-item, or the findings were assessed as test item-related but non-adverse (or "likely non-adverse"). The F0 organ weight changes at scheduled necropsy at 150 mg/kg bw/day were considered test-item related but likely non-adverse due to the low magnitude of change and, except for the male prostate, no macroscopic correlates. In the single male at 400 mg/kg bw/day with a macroscopic finding (small prostate), there was no microscopic correlate. Per study protocol, tissues besides the testes, epididymis, and ovaries were not examined histopathologically except in the case of gross lesions.

 

The F0 and neonatal F1 litter findings considered test item-related and adverse, and the organ weight findings reported as likely non-adverse, are discussed below.

 

For the F0 animals, test item-related and adverse clinical signs were observed in the females that died (or were euthanized) at 150 (one) and 400 mg/kg bw/day (10). The single non-gravid female that died on GD 14 at 400 mg/kg bw/day exhibited a thin body, pale faeces, and hunched posture. In the nine females that were found dead or euthanized at 400 mg/kg bw/day (GD 23 through LD 1, or on LDs 7 or 9), the common clinical signs noted just before death included piloerection, hunched posture, cool and pale body and/or extremities. Similar symptoms were observed in the female that was found dead on GD 23 at 150 mg/kg bw/day.

 

Effects on F0 body weight and body weight gain were considered test item-related and adverse for males at 400 mg/kg bw/day and, for females, mostly during lactation at this dose. For males, 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. 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), lower mean maternal body weight gains were noted during LDs 1–7, with lower (7.0 to 8.0%) mean absolute body weights when compared to the control group (not statistically significant). In addition, the single non-gravid female that was euthanized in extremis on GD 14 at 400 mg/kg bw/day had marked body weight loss (48g, 21.8%).

 

Test item-related and adverse effects on food consumption were identified for F0 females mostly during lactation at 400 mg/kg bw/day. 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). In addition, the single non-gravid female that was euthanized in extremis on GD 14 at 400 mg/kg bw/day had no food consumption reported (0g GDs 0 -14).

Test-item related organ weight changes were observed for the liver in 150 and 400 mg/kg bw/day males and in 150 mg/kg bw/day females (not determined for the 400 mg/kg bw/day group since terminated by LD 9), and for thymus and prostate in 400 mg/kg bw/day males. However, per the study report, the findings are likely non-adverse due to the low magnitude of change and, except for the prostate, a lack of macroscopic correlates. In a single male at 400 mg/kg bw/day, the macroscopic finding (small prostate) did not have a microscopic correlate. Per study protocol, tissues besides the testes, epididymis, and ovaries were not examined histopathologically except in the case of gross lesions.

 

Test item-related and adverse histopathology (hepatocellular degeneration/necrosis in the liver, the histopathologic cause of death) was identified in the single F0 female euthanized in extremis on GD 14 at 400 mg/kg bw/day.

 

Increased gestation length and a veterinary diagnosis of dystocia were considered test item-related and adverse for F0 females at 400 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. Of the nine gravid females this dose, 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 is higher than the control group mean (22.0 days) and the background historical control mean (22.1 days). The veterinary diagnosis of dystocia at 400 mg/kg bw/day was based on various adverse clinical signs as 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 also with a single resorption) and the third containing resorptions only.

 

For the F1 litters, item-related and adverse neonatal findings were identified. 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. These differences were partially attributed to one dam with a total litter loss and two litters that were euthanised in extremis along with their dams on PND 0 or 1. Test item-related and adverse neonatal clinical signs were observed in the F1 litters at 400 mg/kg bw/day of parental treatment on PND 1. These clinical signs included small stature and/or cardio-pulmonary findings of pale body, cool body, cool extremities, and/or gasping. In addition, test item-related and adverse lower mean pup birth weights on PND 1 were noted 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.

The identified NOAELs for F0 systemic and reproductive effects and the neonatal F1 litter NOAEL for this study are as follows:

* The parental systemic NOAEL was 25 mg/kg bw/day in males and females, based on organ weight changes in males from 150 mg/kg bw/day and on mortality and adverse clinical signs in females from 150 mg/kg bw/day (organ weight findings at 150 mg/kg bw/day). Additional effects were reported for both sexes at 400 mg/kg bw/day: Lower male body weights (gains), lower female body weight gains / food consumption and, for the one non-gravid female euthanised on GD 14, liver histopathology. While the organ weight findings are characterized as likely non-adverse in the study report, the relevant tissues (thyroid / parathyroid, kidney, and liver) were not examined microscopically, per protocol. Thus, the organ weight findings were used by the laboratory in establishing the systemic NOAEL.

* 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 neonatal NOAEL for the F1 litters 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 on PND 0, lower mean postnatal survival on PNDs 0-1, clinical signs on PND 1, and lower pup birth weights on PND 1. Noting that three of the six dams that littered at this dose were found dead or euthanised in extremis on GD 23 to LD1.

 

In the available 90-day oral repeated dose toxicity study (Charles River Laboratories, 2020), conducted according to OECD Test Guideline 408 and in compliance with GLP, the registered substance 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).  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 reproductive-related findings in this study were not confirmed in the OECD Test Guideline 421 study.

Effects on developmental toxicity

Description of key information

In the key oral prenatal developmental toxicity study conducted with Vi4-D4 according to OECD Test Guideline 414 and in compliance with GLP (Charles River Laboratories, 2020; Klimisch score 1), the development NOAELs (maternal and prenatal) were determined to be 100 mg/kg bw/day based on the adverse lower maternal and foetal body weights at 400 mg/kg bw/day.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10th September 2019 to 16th Dec 2019
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:
June, 2018
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
August, 1998
Deviations:
not specified
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD(SD)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: 10-12 weeks old
- Weight at study initiation: 200-250 g (Gestation day 0).
- Fasting period before study: not specified
- Housing: Animals were individually housed in clean, solid-bottom cages with appropriate bedding and equipped with an automatic watering valve.
- Diet: Certified Rodent LabDiet 5002 meal, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 2-6 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 26 °C
- Humidity (%): 30 to 70%
- Air changes (per hr): ≥10 per hour
- Photoperiod (hrs dark / hrs light): 12 hours light /12 hours dark
- Other: 100 % fresh air with no air recirculation was maintained.

IN-LIFE DATES: not specified
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Preparation occurred approximately weekly with an adequate amount of each formulation dispensed into daily aliquots. These aliquots were stored in a room with controls set to maintain a temperature between 18 to 24°C and protected from light until use.

VEHICLE
- Justification for use and choice of vehicle: the vehicle has been selected as suggested by the sponsor based on the test item's characteristics and testing guideline. The selected vehicle was corn oil.
- Concentration in vehicle: 0; 6.25; 25; 100 mg/mL
- Amount of vehicle: 4 mL/kg
- Lot/batch no.: 2IC0148, 1IG1538, and 2IH0387
- Purity: not specified
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Duplicate sets of samples (1.0 mL) for concentration analysis were collected twice during the study for all dose levels. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% of theoretical concentration.

Duplicate sets of samples (1.0 mL) for homogeneity analysis were collected once during the study from low dose and high dose groups. Homogeneity results were considered acceptable if the relative standard deviation of the mean concentration was ≤ 10% and if mean sample concentration results were within or equal to ± 15% of theoretical concentration.

Stability analysis was not performed since the test substance formulations have been previously shown to be stable over the range of concentrations used for this study for at least 8 days.

Analyses were performed by gas chromatography method using flame ionization detection using a validated analytical procedure.
Details on mating procedure:
Time-mated females were received from Charles River Laboratories on gestation days 1, 2, 3 or 4.
Duration of treatment / exposure:
From gestation day 6 to 20.
Frequency of treatment:
Single daily dose
Duration of test:
From Gestation day 6 to 20.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control
Dose / conc.:
25 mg/kg bw/day (nominal)
Remarks:
Low dose (LD)
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Middle dose (MD)
Dose / conc.:
400 mg/kg bw/day (nominal)
Remarks:
High dose (HD)
No. of animals per sex per dose:
25 females per group.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose selection was based on two previous dose range finding studies. The highest dose of 400 mg/kg bw/day was targeted to cause reduced body weight gains and feed consumption although not causing death or any obvious suffering. The lower doses were selected to provide dose response data for any observed toxicity and to establish a NOAEL value.
In a dose range-finding study, the test substance was administered to 5 male and 5 female (nonpregnant) Sprague Dawley rats per dose group via oral gavage for up to 14 consecutive days at dose levels of 0, 100, 300, 500, and 1000 mg/kg bw/day. All animals in the 1000 mg/kg bw/day group were euthanized in extremis on Day 8 following significant body weight losses in treated males during the first week of dosing. Mean body weights for the males was 15.9% lower and for females it was 5.2% lower than the concurrent control groups on Day 7. All other animals survived to the scheduled euthanasia. Test substance-related body weight losses or lower body weight gains, with corresponding lower food consumption, were generally noted for males and females in the 500 mg/kg bw/day group throughout the dosing period (Days 1–14), resulting in lower mean body weights than the control groups on Day 14. Lower body weight gains were also noted for males at 300 mg/kg bw/day and females at 100 and 300 mg/kg bw/day during Days 1–7, without corresponding lower food consumption or effects on mean body weights. Test substance-related increased liver weights (absolute and normalised) were noted in all test substance-treated males and females at the day 8/15 terminal necropsy. There were no correlations to differences in serum chemistry parameters.
In a dose range-finding prenatal developmental toxicity study, the test substance was administered to 5 pregnant Sprague Dawley females per dose group via oral gavage from Gestation Days 6–20 at dose levels of 0, 200, 400, and 600 mg/kg bw/day. 5 Females in the 400 and 600 mg/kg bw/day dose groups had decreased body weights gains (19% and 39%,respectively) and feed consumption (9% and 13%, respectively) over the Gestation Days 6–21 treatment period, which resulted in body weight decreases of 7% and 13%, respectively, on Gestation Day 21. All treated groups had increased absolute liver weights. A higher mean litter proportion of postimplantation loss (19.6% per litter) was noted in the 600 mg/kg bw/day group compared to the control group (3.2% per litter). A corresponding lower mean number of viable fetuses (8.5 per dam) was also observed in this group compared to the control group (12.6 per dam). The differences in this group were primarily attributed to 1 female, with 42.9% postimplantation loss and only 4 viable foetuses. Intrauterine survival was unaffected by test substance administration at dosage levels of 200 and 400 mg/kg bw/day.
- Rationale for animal assignment: randomised.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily, in the morning and in the afternoon.
- Cage side observations checked included: The general health/mortality and moribundity was recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: During the dosing period, observations were done once prior to dosing and once 1–2 hours postdose.

BODY WEIGHT: Yes
- Time schedule for examinations: on gestation day 0 and thereafter, daily during days 5-21.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption was quantitatively measured on Gestation Days 5–21 (daily).

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: Animals were subjected to a complete necropsy examination, which included evaluation of the thoracic, abdominal, and pelvic cavities with their associated organs and tissues. The liver, thyroid gland and kidneys were weighed at necropsy for all scheduled euthanasia animals. Paired organs were weighed together. Tissues were collected from liver, kidneys, thyroid gland and all gross lesions. The thyroid gland from all females was embedded in paraffin, sectioned, mounted on glass slides, and stained with haematoxylin and eosin. The thyroid gland was examined microscopically from all females in all groups.

Thyroid hormones: Yes
- Blood samples for thyroid hormone analyses were collected (prior to noon in order to avoid diurnal fluctuations in thyroid hormone levels) via a jugular vein into tubes without anticoagulant on GD 21. The blood samples were analysed for triiodothyronine (total T3), thyroxine (total T4) and thyroid-stimulating hormone (TSH).
Ovaries and uterine content:
The ovaries and uterine content were 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
- Other: the placentae were also examined.
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: approximately one half per litter
- Skeletal examinations: Yes: approximately one half per litter (those not used for soft tissue examinations)
- Head examinations: Yes: approximately one half per litter (those used in soft tissue examinations)
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analysed as indicated by sex and occasion or by litter. Mean, standard deviation, percentage, and/or incidence were reported whenever possible.
Indices:
No data
Historical control data:
yes, for the background incidence of fetal malformations and developmental variations (from Charles River Laboratories).
Clinical signs:
no effects observed
Description (incidence and severity):
No test substance-related clinical observations were noted at the daily examinations at any dosage level. Observations noted in the test substance-treated groups occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose related. No clinical observations were noted 1-2 hours postdosing at any dosage level. With the exception of 1 female in the control group, all females were gravid.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
All females in the control, 25, 100, and 400 mg/kg bw/day groups survived to the scheduled necropsy on gestation days 21.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean maternal body weight losses were noted in the 400 mg/kg bw/day group following the initiation of dosing (gestation days 6–7 and 7–8), resulting in a statistically significant mean body weight loss in this group during gestation days 6–9. The only other statistically significant difference from the control group was a lower mean body weight gain in the 400 mg/kg bw/day group during gestation days 20–21. Although mean body weight gains in the 400 mg/kg/day group were generally comparable to the control during the remainder of the treatment period (gestation days 9–12, 12–15, and 15–21), the initial decrement resulted in a non-statistically significantly lower (11.4%) mean body weight gain in this group when the overall treatment period (gestation days 6–21) was evaluated.
Mean absolute body weights in the 400 mg/kg bw/day group were statistically significantly (6.1% and 5.7%) lower than the control group on gestation days 8 and 9. Thereafter, mean absolute body weight were generally comparable to the control group throughout the treatment period. However, mean corrected body weight and mean corrected body weight gain were statistically significantly (6.4% and 28.7%, respectively) lower in the 400 mg/kg bw/day group compared to the control group. The body weight effects in this group were considered test substance related and adverse.
Mean maternal body weights, body weight gains, corrected body weights, corrected body weight gains in the 25 and 100 mg/kg bw/day groups were unaffected by test article administration. Differences from the control group were slight and not statistically significant.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In the 400 mg/kg bw/day dose group, lower mean food consumption (33.5% compared to control) was observed during gestation days 6-9. No other differences in the other dose groups occurred when compared to the control group.
Food efficiency:
not specified
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
Description (incidence and severity):
Mean absolute liver weights in the 25, 100, and 400 mg/kg bw/day groups were statistically significantly higher (12.4% to 29.2%) than the control group. These effects were considered test substance related. A statistically significantly higher (9.6%) mean absolute kidneys weight was also noted in the 400 mg/kg bw/day group compared to the control group; this finding was not considered test substance related due to the magnitude of the change. No other statistically significant differences were noted at any dosage level.
Gross pathological findings:
no effects observed
Description (incidence and severity):
At the scheduled necropsy on gestation day 21, no test substance-related internal findings were observed at dosage levels of 25, 100, and 400 mg/kg bw/day. Macroscopic findings observed in the test substance-treated groups occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test substance-related histologic changes in the thyroid gland. All histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than administration of the test substance. There was no test substance-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Statistically significantly lower mean T3 (44.4%) and T4 (38.5%) concentrations were noted in the 400 mg/kg bw/day group compared to the control group. In addition, statistically significantly higher (58.9% to 76.8%) mean TSH concentrations were noted in the 25, 100, and 400 mg/kg bw/day groups compared to the control group, albeit in a manner that was not clearly dose responsive and there was no corresponding test substance-related or dose-responsive change in mean T3 and T4 concentrations in the 25 and 100 mg/kg bw/day groups. These findings were considered test substance-related, but not adverse, due to lack of effects on the thyroid gland weight and histopathology.
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
The mean numbers of corpora lutea was comparable to the control group.
Details on maternal toxic effects:
Intrauterine survival was unaffected by test substance administration at all dosage levels. Parameters evaluated included postimplantation loss, live litter size, and foetal sex ratios. Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups.
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
Abnormalities:
effects observed, treatment-related
Localisation:
other: Body weight
Description (incidence and severity):
Decreased body weight at 400 mg/kg bw/day
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean foetal body weights (male, female, and combined sexes) in the 400 mg/kg bw/day group were statistically significantly lower (14.9% to 16.2%) than the concurrent control group. The values were also below the respective minimum mean values in the Charles River Ashland developmental historical control data (version 2019.01). Therefore, the lower mean foetal body weights in this group were considered test substance-related and adverse. Mean foetal body weights in the 25 and 100 mg/kg bw/day groups were unaffected by test substance administration.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
No test substance-related external malformations were observed in foetuses in this study. One foetus in the 100 mg/kg bw/day group had a small left eye bulge and one foetus in the 25 mg/kg bw/day group had a cleft palate (entire length). In the absence of a dose response, these malformations were not considered test substance-related.
No external developmental variations were observed in foetuses in this study.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
No test substance-related foetal malformations were observed at any dosage levels.
In the 25 mg/kg bw/day, one female was observed with split palatine plates, corresponding to cleft palate noted externally. However, this was considered as not test-item related in the absence of a dose response occurring.
At 400 mg/kg bw/day, test substance-related skeletal developmental variations were noted. They included the findings of delayed ossification which correspond to the lower foetal weights in the same group and were considered nonadverse as they were expected to resolve over time.
Visceral malformations:
no effects observed
Description (incidence and severity):
No test substance-related visceral malformations were observed in foetuses in this study. One foetus in the 400 mg/kg bw/day group had a retroesophageal right subclavian artery; however, this was noted for a single foetus, the mean litter proportion was not statistically significant compared to the control group, and the value was within the Charles River Ashland developmental historical control data range. Therefore, this malformation was not considered test substance-related. No test substance-related visceral developmental variations were observed in foetuses in the study. Findings observed in the test substance-treated groups were noted infrequently, similarly in the control group, were not observed in a dose related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the Charles River Ashland developmental historical control data.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant shorter mean absolute anogenital distance (11.3%) was noted in the 400 mg/kg bw/day group males compared to the control group; however, mean anogenital distance relative to the cube root of the body weight in these males was only 6.3% lower than the control group and not statistically significant. Because the mean anogenital distance in this group was only slightly shorter than noted in the control group and was not statistically significant when normalized to body weight, the difference in mean absolute anogenital distance in the 400 mg/kg bw/day group males was considered secondary to the lower mean foetal body weights. Mean absolute and relative anogenital distances for female foetuses in this group were comparable to the control group. Mean anogenital distances in the 25 and 100 mg/kg bw/day groups were comparable to the control group.
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Remarks on result:
other: test item-related and adverse findings at 400 mg/kg bw/day
Key result
Abnormalities:
effects observed, treatment-related
Description (incidence and severity):
see foetal NOAEL above
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
400 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
not specified
Dose response relationship:
yes
Relevant for humans:
yes

Relation to maternal toxicity: Uncertain at this time if the developmental effects are a secondary non-specific consequence of maternal toxicity effects.

Conclusions:
In an oral prenatal developmental toxicity study, conducted according to OECD Test Guideline 414 and in compliance with GLP, a maternal and prenatal development NOAEL was determined to be 100 mg/kg bw/day based on lower maternal and foetal body weights at 400 mg/kg bw/day.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Based on reliable OECD Test Guideline 414 study
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 main prenatal developmental toxicity study, conducted according to OECD Test Guideline 414 and in compliance with GLP (Charles River Laboratories, 2020; Klimisch score 1), Crl:CD(SD) female rats were exposed to 0, 25, 100, and 400 mg/kg bw/day of Vi4-D4 in corn oil from gestation days 6 to 20. The following parameters and end points were evaluated in the study: clinical signs, body weights, body weight gains, gravid uterine weights, food consumption, thyroid hormones, gross necropsy findings, organ weights and histopathology, intrauterine growth and survival, anogenital distance, and foetal morphology.

 

All females in the control, 25, 100, and 400 mg/kg bw/day groups survived to the scheduled necropsy on gestation day 21. No test substance-related clinical observations were noted at the daily examinations at any dose level, and no clinical observations were noted 1-2 hours following dose administration at any dose level.

 

In the 400 mg/kg bw/day group, mean maternal body weight losses were noted following the initiation of dosing (gestation days 6–8) and lower mean food consumption was noted during gestation days 6– 9, resulting in a mean body weight loss in this group during gestation days 6-9 and lower (6.1% and 5.7%) mean absolute body weights on gestation days 8 and 9, respectively. Although mean body weight gains in the 400 mg/kg bw/day groups were comparable to the control group during the remainder of the treatment period, the initial decrements resulted in a lower (11.4%) mean body weight gain when the entire treatment period (gestation days 6-21) was evaluated. Mean maternal body weights at the end of gestation were comparable to the control group. In addition, mean corrected body weight and corrected body weight gain in this group were lower (6.4% and 28.7%, respectively) than the control group, although there was no difference in mean gravid uterine weights versus controls. These body weight effects were considered test substance related and adverse. Mean maternal body weights, body weight gains, corrected body weights, corrected body weight gains, food consumption and gravid uterine weights in the 25 and 100 mg/kg bw/day groups were unaffected by test article administration.

 

Lower mean T3 and T4 concentrations (44.4% and 38.5%, respectively) compared to controls were noted in the 400 mg/kg bw/day group; higher mean TSH concentrations (58.9% to 76.8%) were noted in all test substance-treated groups, although there was no corresponding test substance-related or dose-responsive change in mean T3 and T4 concentrations in the 25 and 100 mg/kg bw/day groups. These findings were considered test substance-related, but not adverse, due to lack of effects on the thyroid gland weight and histopathology.

 

There were no test substance-related macroscopic findings noted at any dose level. Mean absolute liver weights in the 25, 100, and 400 mg/kg bw/day groups were 12.4% to 29.2% higher than the control group. These effects were considered test substance-related but not adverse. There were no test substance-related histologic changes in the liver noted at any dose level.

 

Mean foetal body weights (male, female, and combined sexes) in the 400 mg/kg bw/day group were 14.9% to 16.2% lower than the concurrent control group and were below the minimum mean values in the developmental historical control data; these differences were considered test substance-related and adverse, and it is uncertain whether these are a secondary non-specific consequence of maternal toxicity effects. A statistically significant shorter mean absolute anogenital distance (11.3%) was noted in the 400 mg/kg bw/day group males compared to the control group; however, mean anogenital distance relative to the cube root of the body weight in these males was only 6.3% lower than the control group and not statistically significant. Because the mean anogenital distance in this group was only slightly shorter than noted in the control group and was not statistically significant when normalised to body weight, the difference in mean absolute anogenital distance in the 400 mg/kg bw/day group males was considered secondary to the lower mean foetal body weights. Intrauterine growth in the 25 and 100 mg/kg bw/day groups and intrauterine survival at all dose levels were unaffected by test substance administration. Mean absolute and relative anogenital distances for female foetuses in this group were comparable to the control group.

 

There were no test substance-related foetal malformations noted at any dosage level. Test substance-related skeletal developmental variations were noted at 400 mg/kg bw/day and consisted of findings of delayed ossification (wavy ribs, incomplete ossification of the sternebrae, supernumerary cervical ribs, short thoracolumbar supernumerary ribs, and incomplete ossification of the vertebral thoracic centrum). These findings corresponded to lower foetal weights in this group and were considered non-adverse as they were expected to resolve over time.

 

In the study, the maternal and prenatal development NOAELs were determined to be 100 mg/kg bw/day based on lower maternal and foetal body weights at 400 mg/kg bw/day (Charles River Laboratories, 2020; Klimisch score = 1).

 

In the dose range-finding prenatal developmental toxicity study, which was conducted according to a protocol similar to OECD Test Guideline 414 with deviations, not in compliance with GLP (Charles River Laboratories, 2019b; Klimisch score 2), the registered substance Vi4-D4, was administered daily by oral gavage to 5 pregnant female rats during gestation days 6-20 at dose levels of 0, 200, 400 or 600 mg/kg bw/day in corn oil. The following parameters and end points were evaluated in this study:  clinical signs, body weights, body weight gains, food consumption, gross necropsy, organ weights, intrauterine survival, and external foetal morphology.

 

Test substance-related lower mean body weight gains were noted in females at 400 and 600 mg/kg bw/day. Additionally, at the scheduled necropsy on gestation day 21, enlarged placenta(e) for 1, 2, and 3 females in the 200, 400, and 600 mg/kg bw/day groups, respectively, were observed. Higher mean absolute kidney and liver weights were noted in the 200, 400, and 600 mg/kg bw/day groups compared to the control group. Higher mean post-implantation loss, with a corresponding lower mean number of live foetuses, were noted in the 600 mg/kg bw/day group when compared to the control group; the difference was primarily attributed to a single female with 42.9% post-implantation loss and only 4 live foetuses. Intrauterine survival at 200 and 400 mg/kg bw/day and external foetal morphology at 200, 400, and 600 mg/kg bw/day were unaffected by test substance administration. A NOAEL was not concluded in the study. Based on the results, dose levels of 25, 100, and 400 mg/kg bw/day were selected for the main prenatal developmental toxicity study.

 

In a supporting study conducted according to OECD Test Guideline 421 and in compliance with GLP (reliability score 1), Sprague-Dawley rats were administered Vi4-D4 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 through 14 days of 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 up to 400 mg/kg bw/day of dam exposure and, as survived, while nursing until PND 21 (“F1 litters”), with the selected F1 generation directly administered the test item at 25 and 150 mg/kg bw/day from weaning (PNDs 21-34, euthanised PND 35). 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.

 

The F0 systemic and reproductive parameters and corresponding results are discussed under Effects on fertility, with those for the F1 pups (litter and F1 generation) discussed here.

 

For the F1 litters (PNDs 0-21), the number of still and live births, sex of pups (and sex ratio), postnatal morality, viability indices, daily litter size, cageside observations (mortality and moribundity), detailed clinical signs, pup body weight, anogenital distance (AGD), presence / absence of nipples/areolae in male pups, blood analysis for thyroxin (T4, if survived to PND 21), organ weights (if survived to PND 21), and gross pathology (for pups that died or survived to PND 21) were assessed. PND 4 blood samples were collected for possible future T4 analysis.

 

In the F1 generation (directly treated on PNDs 21-35), evaluations were conducted for daily litter size, cageside observations (mortality and moribundity), detailed clinical signs, pup body weight, food consumption, organ weights (if survived to PND 35), and gross pathology (for pups that died or survived to PND 35). For pups surviving to PND 35, blood samples were obtained for possible future T4 analysis.

 

Test item-related and adverse findings in the F1 litters included:

* Lower mean live litter size and postnatal survival at 400 mg/kg bw/day, only relevant to the F1 litters

* Clinical signs at 400 mg/kg bw/day

* Lower mean birth weights on PND 1 and lower mean body weights (and gains) for PNDs 1-7, both at 400 mg/kg bw/day, only relevant to the F1 litters. In the F1 generation, no adverse body weight (or gain) effects were identified.

 

No findings in the F1 generation were considered test item-related and adverse up to 150 mg/kg bw/day. Due to 400 mg/kg bw/day group termination by PND 9, no pups were available for post-weaning treatment and examination. 

 

For all other F1 litter parameters and for the F1 generation, there were no effects observed, the findings were considered unrelated to the test-item, or the findings were assessed as test item-related but non-adverse (or "likely non-adverse"). In the absence of pup microscopic examination, the test item-related but likely non-adverse assessment for the F1 litter and F1 generation organ weight changes seen at scheduled necropsy at 150 mg/kg bw/day were based on the relatively low magnitude of change and the lack of macroscopic correlates.

 

The F1 litter findings considered test item-related and adverse are discussed below.

 

For the F1 litters (PNDs 0-21), the following test item-related and adverse findings were identified. As relevant only to the F1 litters, 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. These differences were partially attributed to one dam with a total litter loss and two litters that were euthanised in extremis along with their dams on PND 0 or 1. In total, forty (40) pups in five litters were found dead or euthanised in extremis at 400 mg/kg bw/day. Further, 10 pups in five litters were missing and presumed cannibalised. In addition, 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).

 

Test item-related and adverse clinical signs were observed in the F1 litters at 400 mg/kg bw/day of parental treatment, up until group termination by PND 9. These clinical signs included small stature and/or cardio-pulmonary findings of pale body, cool body, cool extremities, and/or gasping.

 

In the F1 litters, test item-related and adverse lower mean pup 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. 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.

 

The identified F1 NOAELs (litter and F1 generation) for this study are as follows:

* 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 at birth, lower mean postnatal survival, clinical signs, lower pup weights at birth and lower pup weights and gains. Noting that three of six dams that littered at this dose were found dead or euthanised in extremis on GD 23 to LD1.

* 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 the 400 mg/kg bw/day group termination by PND 9, pups at this dose were not available for post-weaning treatment and examination.

 

The F0 systemic NOAELs were 25 mg/kg bw/day for males and females, with the F0 reproductive NOAELs equal to 150 mg/kg bw/day for females and greater than or equal to 400 mg/kg bw/day for males (see Effects on fertility).

Justification for classification or non-classification

Based on the available data, 2,4,6,8-tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane is classified as Category 1B (H360F, May damage fertility) for effects on fertility and Category 2 (H361d, Suspected of damaging the unborn child) for developmental toxicity according to Regulation (EC) No. 1272/2008. Thus, the combined hazard code H360Fd is assigned to the registered substance.

Additional information